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Detection and possible significance of a common leukemia-associated antigen, CAMAL, in human myeloid… Logan, Patricia Marie 1987

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DETECTION AND POSSIBLE SIGNIFICANCE OF A COMMON  LEUKEMIA-ASSOCIATED  ANTIGEN, CAMAL, IN HUMAN MYELOID LEUKEMIA  By  P a t r i c i a M a r i e Logan  D.V.M., The U n i v e r s i t y o f Guelph, 1980  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Department o f M i c r o b i o l o g y )  We a c c e p t t h i s t h e s i s as conforming to t h e required  standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1987 © P a t r i c i a M a r i e Logan, 1987  In  presenting  degree  this  thesis  in partial fulfilment of  requirements  for  an  of  department  this thesis for scholarly or  by  his  or  her  I further agree that permission for  purposes  permission.  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  extensive  may be granted by the head of  representatives.  It  is  understood  that  publication of this thesis for financial gain shall not be allowed without  DE-6(3/81)  advanced  at the University of British Columbia, I agree that the Library shall make it  freely available for reference and study. copying  the  copying  my or  my written  ABSTRACT  Human acute n o n l y m p h o b l a s t i c o r myelogenous leukemia a m a l i g n a n t d i s e a s e o f t h e bone marrow i n v o l v i n g (blood-forming) c e l l s of t h e myeloid lineage. n e o p l a s t i c d i s e a s e , whose fundamental despite intensive research.  (ANLL o r AML) i s  hemopoietic  ANLL i s a complex  nature i s only p a r t i a l l y  understood  The d i s e a s e i s c o m p l i c a t e d by i t s apparent  h e t e r o g e n e i t y i n terms o f t h e degree o f d i f f e r e n t i a t i o n o f h e m o p o i e t i c stem c e l l  involvement i n d i f f e r e n t p a t i e n t s and t h e c e l l u l a r e x p r e s s i o n o f  i m m u n o l o g i c a l l y d e f i n e d s u r f a c e markers. i n myelogenous leukemia  The p r e s e n c e o f a common a n t i g e n  (CAMAL) has been p r e v i o u s l y i d e n t i f i e d .  This  t h e s i s examines t h e e x p r e s s i o n o f t h e CAMAL marker i n o r on bone marrow (BM)  and p e r i p h e r a l b l o o d (PB) c e l l s u s i n g a monoclonal  i n d i r e c t immunoperoxidase s l i d e t e s t .  antibody-based  I n c r e a s e d numbers of CAMAL-positive  c e l l s were found i n o r on BM and PB o f m y e l o i d leukemia p a t i e n t s ( w i t h a c u t e o r c h r o n i c forms o f t h e d i s e a s e ) compared w i t h those found i n normals  o r most lymphoid m a l i g n a n c i e s .  demonstrated  R e s u l t s p r e s e n t e d h e r e i n have  t h a t f l u c t u a t i o n s i n CAMAL BM v a l u e s (% p o s i t i v e  c o r r e l a t e d w i t h s u r v i v a l time p r i o r t o r e l a p s e .  I n a b l i n d study, ANLL  p a t i e n t s Whose CAMAL BM v a l u e s decreased post-chemotherapy significantly  cells)  (p < 0.025) l o n g e r f i r s t r e m i s s i o n times  had  (x = 19.2 months)  t h a n p a t i e n t s w i t h i n c r e a s i n g o r s t a t i c CAMAL BM v a l u e s (x = 6.8 months). CAMAL BM v a l u e s were o f t e n observed t o i n c r e a s e d u r i n g r e m i s s i o n , p r i o r t o r e l a p s e , suggesting the presence of r e s i d u a l s u b c l i n i c a l disease. A d d i t i o n o f excess p u r i f i e d l e u k e m i a - d e r i v e d CAMAL t o an i n v i t r o m y e l o i d progenitor c e l l  assay caused p r o f o u n d i n h i b i t i o n o f normal  but had no i n h i b i t o r y e f f e c t on CFU-c growth from m y e l o i d  CFU-c growth leukemia  iii  patients i n active disease states.  D e p l e t i o n o f CAHAL from normal  plasma  and c o n d i t i o n e d media ( s o u r c e s of numerous h e m o p o i e t i c growth r e g u l a t o r y f a c t o r s ) caused s i g n i f i c a n t i n h i b i t i o n of normal, but n o t m y e l o i d leukemic, CFU-c growth.  These r e s u l t s i n d i c a t e d t h a t m y e l o i d  leukemic  c e l l s p o s s e s s e d apparent d i f f e r e n c e s i n r e s p o n s i v e n e s s t o CAMAL-mediated h e m o p o i e t i c r e g u l a t i o n compared t o normal to  cells.  i n h i b i t i o n by l e u k e m i a - d e r i v e d CAMAL may  m a l i g n a n t c l o n e over normal  cells.  Lack of r e s p o n s i v e n e s s  f a c i l i t a t e dominance o f the  iv  TABLE OF CONTENTS Page  CHAPTER I . I.  INTRODUCTION  1  Hemopoiesis  1  A.  Introduction  1  B.  Assays f o r hemopoietic stem c e l l s 1. I n v i v o assays 2. I n v i t r o assays . . .  4 4 8  R e g u l a t i o n o f hemopoiesis 1. The hemopoietic c o l o n y - s t i m u l a t i n g f a c t o r s a. Murine C o l o n y - S t i m u l a t i n g F a c t o r s M u l t i p o t e n t i a l Colony-Stimulating F a c t o r (Multi-CSF) Granulocyte-Macrophage ColonyS t i m u l a t i n g F a c t o r (GM-CSF) .' Macrophage C o l o n y - S t i m u l a t i n g F a c t o r (M-CSF) Granulocyte Colony-Stimulating Factor (G-CSF) Other Murine R e g u l a t o r y M o l e c u l e s w i t h GM-CSF A c t i v i t y Megakaryocyte Colony S t i m u l a t i o n Eosinophil D i f f e r e n t i a t i o n Factor Erythroid Colony-Stimulating Factors Lymphocyte Growth F a c t o r s i. T C e l l Growth F a c t o r (TCGF) o r I n t e r l e u k i n 2 (IL-2) ii. B C e l l S t i m u l a t i n g F a c t o r s (BSF) . . . . b. Human C o l o n y - S t i m u l a t i n g F a c t o r s Pluripotent Colony-Stimulating Factor GM-CSF (CSF-a) G-CSF (CSFfl)) M-CSF (Human U r i n a r y C o l o n y - S t i m u l a t i n g Factor E r y t h r o p o i e t i n (epo) E r y t h r o i d P o t e n t i a t i n g A c t i v i t y (EPA) Leukemic B l a s t Growth F a c t o r (LBGF) T C e l l Growth F a c t o r (TCGF) o r I n t e r l e u k i n 2 (IL-2) B C e l l S t i m u l a t i n g F a c t o r s (BSF) c. D i s t i n c t Membrane Receptors f o r ColonyStimulating Factors d. Other F u n c t i o n s o f t h e C o l o n y - S t i m u l a t i n g Factors  15 16 16  T  C.  2.  N o n s p e c i f i c Substances w i t h Hemopoietic Growth Promoting A c t i v i t y  16 23 24 24 25 26 26 26 27 27 27 28 28 29 29 30 30 31 31 31 32 32 33  34  V  Page 3.  4. II.  Negative G r a n u l o p o i e t i c Regulators Lactoferrin Acidic Isoferritins . Interferons T r a n s f e r r i n .. E-Type P r o s t a g l a n d i n s Leukemia-Associated Inhibitor Tumor N e c r o s i s F a c t o r G r a n u l o c y t i c Chalone Other Reported I n h i b i t o r s o f M y e l o p o i e s i s Microenvironmental  I n f l u e n c e s on Hemopoiesis  47 50  A.  . . . .  50 50 51 52 54  . .  56 56 58 58  C.  C h r o n i c G r a n u l o c y t i c Leukemia C l i n i c a l Course and Pathogenesis Stem C e l l O r i g i n Chromosomal Changes and t h e i r P o s s i b l e S i g n i f i c a n c e R e l a t i o n s h i p Between CGL C e l l s and Growth R e g u l a t o r s Acute Nonlymphoblastic Leukemia C l i n i c a l Course and Pathogenesis Stem C e l l O r i g i n Chromosomal Changes and T h e i r P o s s i b l e S i g n i f i c a n c e R e l a t i o n s h i p Between ANLL C e l l s and Growth Regulators D i f f e r e n t i a t i o n I n d u c t i o n i n M y e l o i d Leukemia  I I I . Human M y e l o i d Leukemia-Associated  Antigens  Thesis Objectives  CHAPTER I I . I.  . . . .  THE HUMAN MYELOID LEUKEMIAS  B.  IV.  . . . . .  35 36 40 41 42 43 43 44 45 45  MATERIALS AND METHODS  . .  60 61 63 72  75  D e t a i l e d Procedures  75  A.  Monoclonal A n t i b o d i e s  75  1. 2. 3.  75 77 77  Production Purification Immunoadsorbent p r e p a r a t i o n  B.  P o l y c l o n a l (Rabbit) Antibodies  78  C.  Secondary A n t i b o d i e s  79  vi  Page D.  Antigens 1. CAMAL 2. Negative c o n t r o l p r o t e i n antigens  80 80 81  E.  Polyacrylamide  81  F.  P a t i e n t Samples  G.  C e l l Preparations  H.  F l u o r e s c e n c e - A c t i v a t e d C e l l S o r t e r (FACS) S t u d i e s 1. 2. 3.  I.  G e l E l e c t r o p h o r e s i s (PAGE) '  81 82 . . . .  C e l l Labeling FACS A n a l y s i s o f L a b e l e d C e l l Samples FACS C e l l S o r t i n g Technique  83 83 83 84  I n d i r e c t Immunoperoxidase S t a i n i n g o f S i n g l e C e l l Preparations  87  J.  B l i n d Study P r o t o c o l 1. P a t i e n t Group 2. A n a l y s i s Groups 3. S t a t i s t i c a l Analysis  88 88 89 90  K.  M y e l o i d P r o g e n i t o r C e l l Assay 1. General P r o t o c o l 2. P r e p a r a t i o n o f C o n d i t i o n e d Medium a. P l a c e n t a l C o n d i t i o n e d Medium (PCM) b. L e u c o c y t e C o n d i t i o n e d Medium (PHA-LCM) 3. P r e p a r a t i o n o f 2% Stock M e t h y l c e l l u l o s e 4. P r e p a r a t i o n o f Human Plasma 5. P r e p a r a t i o n o f C e l l Samples 6. P l u c k i n g and S t a i n i n g o f C o l o n i e s 7. P r e p a r a t i o n o f Human Plasma f o r CAMAL Depletion Studies 8. P r e p a r a t i o n o f C o n d i t i o n e d Medium f o r CAMAL Depletion Studies 9. CAMAL A d d i t i o n S t u d i e s  91 91 92 93 93 94 94 96 96  S t a t i s t i c a l Analyses  98  L.  CHAPTER I I I .  97 97 98  EVALUATION AND DIAGNOSTIC IMPLICATIONS OF A RAPID  SLIDE TEST FOR CAMAL  100  I.  Introduction  100  II.  Results  102  A.  Comparative Immunoperoxidase R e a c t i v i t y Between Monoclonal and R a b b i t A n t i b o d i e s  102  vii  Page B.  CAMAL-1 Immunoperoxidase S l i d e T e s t  105  G.  A p p l i c a t i o n o f t h e I n d i r e c t Immunoperoxidase S l i d e T e s t t o t h e Study o f Other M y e l o i d C e l l Markers  122  CHAPTER IV.  CAMAL EXPRESSION IN LEUKEMIA  126  I.  Introduction  126  II.  Results  126  A.  Morphology o f CAMAL-positive C e l l s 1. FACS S o r t i n g S t u d i e s 2. Immunoperoxidase S t u d i e s a. Lack o f C o r r e l a t i o n Between BM B l a s t C e l l Numbers and CAMAL BM V a l u e b. Lack o f C o r r e l a t i o n Between R e g e n e r a t i v e o r A p l a s t i c BM and CAMAL BM Value c. The Morphology o f CAMAL-positive C e l l s by Immunoperoxidase  .  126 127 136 136 136 138  B.  ANLL Remission P a t h o l o g y  146  C.  The Presence o f CAMAL i n Plasma  150  D.  CAMAL A d s o r p t i o n S t u d i e s  152  CHAPTER V.  SIGNIFICANCE OF CAMAL AS A PROGNOSTIC MARKER FOR  REMISSION IN ACUTE N0NLYMPH0BLASTIC LEUKEMIA  163  I.  Introduction  163  II.  Results  165  A.  R e l a t i o n s h i p Between S u r v i v a l Time P r i o r t o R e l a p s e and Change i n CAMAL BM V a l u e s  166  B.  C o r r e l a t i o n Between D e c r e a s i n g CAMAL BM V a l u e s P o s t chemotherapy and Longer Remission Times  166  C.  CAMAL BM V a l u e s and Simultaneous BM Morphology  173  D.  S u p p o r t i v e Data from Other ANLL P a t i e n t s  173  E.  R e l a t i o n s h i p Between F i r s t Remission Length and CAMAL BM V a l u e a t D i a g n o s i s  174  I n c r e a s i n g CAMAL BM V a l u e s and R e l a p s e  174  F.  viii  Page CHAPTER V I .  THE POSSIBLE ROLE OF CAMAL IN MYELOPOIESIS  182  I.  Introduction  182  II.  Results  182  A.  CAMAL-1 P o s i t i v e C o l o n i e s i n CGL  182  B.  Presence o f CAMAL i n C o n d i t i o n e d Medium  183  C.  CAMAL D e p l e t i o n S t u d i e s  189  1. 2.  190  3. 4. 5. D.  E.  CAMAL A d d i t i o n S t u d i e s 1. E f f e c t o f CAMAL A d d i t i o n on Normal M y e l o i d Colony Growth 2. E f f e c t o f CAMAL A d d i t i o n on M y e l o i d Leukemia P a t i e n t s ' Colony Growth Evidence t h a t Normal and Leukemia-derived CAMAL May Not Be t h e Same  CHAPTER V I I . I.  DISCUSSION  194 194 197 197 199 203 205  210  213  Discussion A.  213  E v a l u a t i o n and D i a g n o s t i c I m p l i c a t i o n s o f a Rapid S l i d e T e s t f o r CAMAL  213  B.  CAMAL E x p r e s s i o n  217  C.  S i g n i f i c a n c e o f CAMAL as a P r o g n o s t i c Marker f o r  D. II.  E f f e c t o f CAMAL D e p l e t i o n on Normal Bone Marrow . . . E f f e c t o f CAMAL D e p l e t i o n on Normal P e r i p h e r a l Blood E f f e c t o f CAMAL D e p l e t i o n on ALL Bone Marrow . . . . E f f e c t o f CAMAL D e p l e t i o n on C h r o n i c G r a n u l o c y t i c Leukemia P e r i p h e r a l B l o o d E f f e c t o f CAMAL D e p l e t i o n on ANLL  i n Leukemia  R e m i s s i o n i n ANLL  219  The P o s s i b l e Role o f CAMAL i n M y e l o p o i e s i s  224  Summary and C o n c l u s i o n s  232  Appendix  234  References  239  ix  LIST OF TABLES  Page I  The murine hemopoietic  II  The human hemopoietic  III  Negative g r a n u l o p o i e t i c r e g u l a t o r s  IV  Comparison o f myelogenous leukemia c e l l r e a c t i v i t y w i t h r a b b i t anti-CAMAL and CAMAL-1 u s i n g immunoperoxidase . . . .  106  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s from n o n l y m p h o b l a s t i c leukemia p a t i e n t s l a b e l e d w i t h CAMAL-1  114  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s from p a t i e n t s w i t h c h r o n i c g r a n u l o c y t i c leukemia  116  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s from p a t i e n t s w i t h preleukemia/MDS i n c l u d i n g RAEBIT . . . .  117  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s from p a t i e n t s w i t h lymphoid m a l i g n a n c i e s o r normals  119  FACS s o r t i n g r e s u l t s from two newly diagnosed p a t i e n t s ' p e r i p h e r a l b l o o d samples  128  V  VI  VII  VIII  IX  X  XI  XII  colony-stimulating factors colony-stimulating factors  17-19 20-22 38  ANLL  FACS s o r t i n g r e s u l t s from t h r e e ANLL r e m i s s i o n p a t i e n t s ' p e r i p h e r a l b l o o d samples  131  FACS s o r t i n g r e s u l t s f o r CGL p a t i e n t s ' p e r i p h e r a l blood c e l l s  133  Lack o f c o r r e l a t i o n between t h e number o f b l a s t  cells  p r e s e n t i n bone marrow and t h e CAMAL BM v a l u e  137  CAMAL BM v a l u e s i n r e g e n e r a t i n g bone marrows  139  XIV  CAMAL BM v a l u e s i n n o n - r e g e n e r a t i n g bone marrows  140  XV  D e t e c t i o n o f CAMAL i n plasma by i m m u n o a f f i n i t y chromatography  151  Examples o f ANLL p a t i e n t s whose CAMAL BM v a l u e s d e c r e a s e d post-chemotherapy  168  Examples o f ANLL p a t i e n t s whose CAMAL BM v a l u e s i n c r e a s e d o r remained t h e same post-chemotherapy  169  ANLL p a t i e n t s w i t h i n c r e a s i n g CAMAL BM v a l u e s d u r i n g remission  176  XIII  XVI  XVII  XVIII  X  Page  XIX  E f f e c t o f CAMAL d e p l e t i o n on normal bone marrow  XX  E f f e c t o f CAMAL d e p l e t i o n on normal p e r i p h e r a l b l o o d  XXI  E f f e c t o f CAMAL d e p l e t i o n on acute leukemia (ALL) bone marrow  XXII  E f f e c t o f CAMAL d e p l e t i o n on c h r o n i c  191 . . . .  lymphoblastic 196 granulocytic  leukemia p e r i p h e r a l b l o o d  . . .  XXIII  E f f e c t o f CAMAL d e p l e t i o n on ANLL  XXIV  Summary o f CAMAL d e p l e t i o n on m y e l o i d c o l o n y  XXV  E f f e c t o f CAMAL a d d i t i o n on normal m y e l o i d c o l o n y  XXVI  E f f e c t o f CAMAL a d d i t i o n on m y e l o i d leukemic c o l o n y growth E f f e c t o f CAMAL a d d i t i o n on normal p e r i p h e r a l b l o o d CAMAL d e p l e t e d c u l t u r e s  XXVII  195  198 200  growth growth  201 . .  204  207 211  xi  LIST OF FIGURES Page 1.1  Schematic diagram r e p r e s e n t i n g  1.2  I n t e r a c t i o n s between t h e n e g a t i v e  2.1  C e l l p o p u l a t i o n s c o l l e c t e d by t h e f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t i n g technique  2.2  3.1  hemopoiesis  3  granulopoietic regulators  Dose response curve f o r c o n d i t i o n e d on normal p e r i p h e r a l b l o o d CFU-c  medium  . .  37  85-86  (PHA-LCM) 95  Comparative immunoperoxidase r e a c t i v i t y between r a b b i t anti-CAMAL serum and CAMAL-1 monoclonal a n t i b o d y on ANLL bone marrow c e l l s  103-104  3.2  Immunoperoxidase s i n g l e c e l l  107-111  3.3  ALL r e m i s s i o n bone marrow c e l l s monoclonal a n t i b o d y  3.4  4.1  s l i d e t e s t w i t h CAMAL-1 l a b e l e d by CAMAL-1  Immunoperoxidase s i n g l e c e l l s l i d e t e s t w i t h m y e l o i d - s p e c i f i c monoclonal a n t i b o d i e s  121 other 124-125  P e r i p h e r a l b l o o d c e l l s from an ANLL p a t i e n t a t d i a g n o s i s s o r t e d on t h e b a s i s o f s t r o n g r e a c t i v i t y w i t h r a b b i t anti-CAMAL serum  130  4.2  P e r i p h e r a l b l o o d c e l l s from two CGL p a t i e n t s s o r t e d on the b a s i s o f s t r o n g r e a c t i v i t y w i t h r a b b i t anti-CAMAL serum . . 134-135  4.3  Morphology o f C A M A L - l - p o s i t i v e m y e l o i d c e l l s i n ANLL remission patients  4.4  4.5  4.6  4.7  4.8a  4.8b  142-143  Lymphocytes from an ANLL r e m i s s i o n p a t i e n t s ' p e r i p h e r a l b l o o d p o s i t i v e l y l a b e l e d by CAMAL-1  144  Diffuse granular by CAMAL-1  145  s t a i n i n g o f CGL p e r i p h e r a l b l o o d  cells  FACS s o r t i n g study o f an ANLL post-bone marrow t r a n s p l a n t p a t i e n t ' s p e r i p h e r a l blood c e l l s  148-149  E l u t i o n p r o f i l e o f m a t e r i a l from normal human plasma bound by a CAMAL-1 immunoadsorbent column  153  ELISA r e a c t i v i t y o f p l a s m a - d e r i v e d p u r i f i e d CAMAL w i t h CAMAL-1 monoclonal a n t i b o d y  154  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f CAMAL from human plasma p u r i f i e d by a f f i n i t y chromatography  154b  xii  Page 4.9  4.10  4.11  5.1  5.2  5.3  5.4  5.5  5.6  6.1  6.2  6.3  6.4  6.5  6.6  6.7  Immunoperoxidase s t a i n i n g o f an ANLL post-bone marrow transplant patient's p e r i p h e r a l blood c e l l s , labeled with CAMAL-1, one month p r i o r t o r e l a p s e  155  Normal p e r i p h e r a l b l o o d c e l l s , i n c u b a t e d w i t h i n c r e a s i n g amounts o f CAMAL and l a b e l e d by immunoperoxidase  157-160  Morphology o f CAMAL-1 r e a c t i v e c e l l s from a normal p e r i p h e r a l b l o o d sample a f t e r i n c u b a t i o n w i t h o r w i t h o u t CAMAL  161-162  Kaplan-Meier s u r v i v a l curve showing s u r v i v a l time t o r e l a p s e f o r ANLL p a t i e n t s i n b l i n d study  prior 170  CAMAL BM v a l u e s over time f o r two ANLL p a t i e n t s w i t h s i g n i f i c a n t l y decreased v a l u e s post-chemotherapy  171  CAMAL BM v a l u e s over time f o r t h r e e ANLL p a t i e n t s whose v a l u e s i n c r e a s e d o r were unchanged f o l l o w i n g treatment . . . .  172  CAMAL BM v a l u e s f o r one ANLL p a t i e n t over t h e course of h i s d i s e a s e  177  CAMAL-1 immunoperoxidase s l i d e t e s t r e s u l t s o f p e r i p h e r a l b l o o d c e l l s from an ANLL r e m i s s i o n p a t i e n t showing i n c r e a s i n g r e a c t i v i t y  178-179  CAMAL-1 immunoperoxidase s l i d e t e s t r e s u l t s o f p e r i p h e r a l b l o o d c e l l s from an ANLL r e m i s s i o n p a t i e n t showing d e c r e a s i n g r e a c t i v i t y  181  C e l l s from CGL p e r i p h e r a l b l o o d CFU-c l a b e l e d w i t h CAMAL-1 by immunoperoxidase  184-185  E l u t i o n p r o f i l e o f m a t e r i a l bound t o a CAMAL-1 immunoadsorbent column from p l a c e n t a l c o n d i t i o n e d medium  . . .  187  B i n d i n g o f m a t e r i a l from two sources o f c o n d i t i o n e d media t o CAMAL-1 and n e g a t i v e c o n t r o l i m m u n o a f f i n i t y columns . . . .  188  Normal marrow CFU-c from c o n t r o l and CAMAL d e p l e t e d cultures  193  Summary o f t h e e f f e c t o f CAMAL d e p l e t i o n on CFU-c normals and m y e l o i d leukemics  202  from  I n h i b i t i o n o f normal marrow CFU-c by a d d i t i o n o f p u r i f i e d l e u k e m i a - d e r i v e d CAMAL  206  E f f e c t of a d d i t i o n of p u r i f i e d leukemia-derived on CFU-c from ANLL p a t i e n t s  208  CAMAL  xiii  Page 6.8  Lack o f i n h i b i t i o n of CGL p e r i p h e r a l b l o o d CFU-c by a d d i t i o n o f p u r i f i e d l e u k e m i a - d e r i v e d CAMAL  209  xiv  ACKNOWLEDGEMENTS The a u t h o r i s g r a t e f u l f o r t h e p o s t - d o c t o r a l f e l l o w s h i p support g i v e n throughout t h i s p r o j e c t by t h e M e d i c a l Research C o u n c i l o f Canada. S p e c i a l thanks i s a l s o due Dr. J u l i a Levy f o r h e r guidance, wisdom, and encouragement. A v a i l a b i l i t y o f p a t i e n t samples used throughout t h e s e s t u d i e s depended l a r g e l y on t h e enthusiasm and c o o p e r a t i o n r e c e i v e d from collaborators at various i n s t i t u t i o n s . Acknowledgement o f t h i s a s s i s t a n c e goes t o t h e f o l l o w i n g : The Department o f Hematology, C e l l S e p a r a t o r U n i t , and Dr. J . D e n e g r i and t h e Immunotransplant L a b o r a t o r y (Vancouver G e n e r a l H o s p i t a l ) and Drs. A.C. and C. Eaves ( T e r r y Fox L a b o r a t o r y ) f o r k i n d l y p r o v i d i n g p a t i e n t samples; Dr. M a r t i n ( S t . P a u l ' s H o s p i t a l ) f o r p r o v i d i n g t h e p l a c e n t a f o r t h e p r e p a r a t i o n o f c o n d i t i o n e d media; Dr. Noel Buskard (Vancouver G e n e r a l H o s p i t a l and Canadian Red C r o s s ) f o r h i s c o n t i n u e d support and f o r p r o v i d i n g samples from p a t i e n t s and normals; The Department o f P a t h o l o g y and D i v i s i o n o f Hematopathology (Vancouver G e n e r a l H o s p i t a l ) and, i n p a r t i c u l a r , Dr. Sheldon Naiman, f o r h i s e x p e r t i s e i n c o n f i r m i n g s l i d e e v a l u a t i o n s and f o r t h e many o t h e r ways i n which he p r o v i d e d support and c o o p e r a t i o n f o r t h i s project; P r o f e s s o r P e t e r I s a a c s o n and K e i t h M i l l e r (Morbid Anatomy, U n i v e r s i t y C o l l e g e , London), Dr. David Swirsky (Cambridge), Dr. Ray Powles (Royal Marsden H o s p i t a l , London), Dr. Dorothy Crawford (Department o f Hematology, School o f M e d i c i n e , U n i v e r s i t y C o l l e g e , London), Dr. Melvyn Greaves and Lynn A l t a s s (ICRF, London), and Dr. A l i s o n Buchan (Department o f P h y s i o l o g y , UBC, Vancouver) f o r t h e i r c o o p e r a t i o n and a d v i c e ; Dr. I a n McDonald f o r a s s i s t a n c e i n o b t a i n i n g numerous p e r i p h e r a l b l o o d samples from normal v o l u n t e r s ; The a u t h o r wishes a l s o t o thank t h e f o l l o w i n g i n d i v i d u a l s f o r providing training or technical assistance: Dr. Hans Messner and h i s l a b o r a t o r y s t a f f and r e s e a r c h e r s , w i t h s p e c i a l thanks t o N a z i r Jamal ( O n t a r i o Cancer I n s t i t u t e , T o r o n t o , O n t a r i o ) , and Dr. C. Eaves and h e r s t a f f ( T e r r y Fox L a b o r a t o r y ) f o r t h e i r assistance i n developing the i n v i t r o myeloid progenitor c e l l assay; Dr. M a r s h a l K a d i n (Hematopathology, U n i v e r s i t y o f Washington) and Dr. Hugh Freeman ( G a s t r o e n t e r o l o g y , Department o f M e d i c i n e , UBC, Vancouver) f o r p r o v i d i n g l a b o r a t o r y space and equipment f o r c a r r y i n g out i n i t i a l immunoperoxidase s t a i n i n g p r o c e d u r e s ;  XV  Dr. Dagmar Kalousek ( T e r r y Fox L a b o r a t o r y , Vancouver) f o r h e r cytogenetic analyses; Dan Z e c h i n i f o r h i s e x p e r t i s e and commitment i n h e l p i n g t o p e r f o r m the FACS a n a l y s e s and c e l l s o r t i n g p r o c e d u r e s ; Stephen Whitney, f o r h i s t e c h n i c a l h e l p i n c a r r y i n g out the immunoperoxidase s l i d e t e s t p r o c e d u r e s ; Other members o f t h i s p r o j e c t , who have h e l p e d me by t h e i r support, a d v i c e and c o o p e r a t i o n , i n c l u d i n g Dr. Andrew Malcolm, Dr. Robert Shipman, and Joan S h e l l a r d . Research s u p p o r t was a l s o p r o v i d e d by the N a t i o n a l Cancer of Canada and the M e d i c a l Research C o u n c i l of Canada.  Institute  xvi  LIST OF ABBREVIATIONS  the  A b b r e v i a t i o n s below a r e l i s t e d thesis.  ANLL CAMAL BM PB CAMAL BM v a l u e NK NC CFU-S CFU-c BFU-E CFU-E CFU-mega CFU-GM CFU-eos CFU-mast CFU-TL CFU-BL CFU-GEMM CGL Ph G-6-PD CSF IL PSF HCGF BPA MCGF SAF 1  MG1 CSA DF BSF BCGF BGDF BCDF TRF GM G M KD SCM epo NIF-T TCGF GM-EA IFN  i n t h e o r d e r i n which they appear i n  acute n o n l y m p h o b l a s t i c leukemia common a n t i g e n i n myelogenous (acute) leukemia bone marrow p e r i p h e r a l blood % CAMAL-1 p o s i t i v e c e l l s natural k i l l e r natural cytotoxic colony-forming unit-spleen colony-forming u n i t - c u l t u r e burst-forming u n i t , erythroid colony-forming u n i t , e r y t h r o i d c o l o n y - f o r m i n g u n i t , megakaryocyte c o l o n y - f o r m i n g u n i t , granulocyte-macrophage colony-forming u n i t , eosinophil c o l o n y - f o r m i n g u n i t , mast c e l l c o l o n y - f o r m i n g u n i t , T lymphocyte c o l o n y - f o r m i n g u n i t , B lymphocyte mixed c o l o n i e s c o n t a i n i n g g r a n u l o c y t e s , macrophages, megakaryocytes and e r y t h r o i d c h r o n i c g r a n u l o c y t i c leukemia P h i l a d e l p h i a chromosome glucose-6-phosphate dehydrogenase colony-stimulating factor Interleukin persisting c e l l stimulating factor hemopoietic c e l l growth f a c t o r b u r s t promoting a c t i v i t y mast c e l l growth f a c t o r stem c e l l a c t i v a t i n g f a c t o r macrophage-granulocyte i n d u c e r colony-stimulating activity differentiation factor B c e l l stimulatory factor B c e l l growth f a c t o r B c e l l growth and d i f f e r e n t i a t i o n f a c t o r B c e l l differentiation factor T c e l l replacing factor granulocyte-macrophage granulocyte macrophage kilodalton s p l e e n c o n d i t i o n e d medium erythropoietin n e u t r o p h i l i n h i b i t o r y f a c t o r , d e r i v e d from T c e l l s T c e l l growth f a c t o r g r a n u l o m o n o p o i e t i c enhancing a c t i v i t y interferon  xvii  LF TF TNF AIF LIA PGE LAI CIL PMN MHC LAA FAB ALL CD FAL LFA CGL MBC/LBC ELISA FACS MAb CAMAL-1 PAGE BLV PEG DEAE Ig FIT C HRP FCS SEM CR PCM PHA-LCM MC BMT MDS RAEBIT CLL rbc PBS LDH L R (R) D T (in Fig 5 . 4 ) CM  lactoferrin transferrin tumor necrosis factor acidic isoferritin leukemia-associated inhibitory activity prostaglandin E leukemia-associated inhibitor colony-inhibiting lymphokine polymorphonuclear leucocyte major histocompatability complex leukemia-associated antigens French-American-British acute lymphoblastic leukemia cluster determinant 3-a-fucosyl-N-acetyllactosamine leucocyte function antigen chronic granulocytic leukemia, myeloid blast crisis/lymphoid blast crisis enzyme linked immunosorbent assay fluorescence-activated c e l l sorter monoclonal antibody monoclonal antibody specific for CAMAL polyacrylamide gel electrophoresis bovine leukosis virus polyethylene glycol diethyl amino ethyl immunoglobulin fluorescein isothiocyanate horseradish peroxidase fetal calf serum standard error of the mean complete remission placental conditioned medium • phytohemagglutin-lymphocyte conditioned medium methylcellulose bone marrow transplant myelodysplastic syndrome refractory anemia with excess blasts in transformation chronic lymphocytic leukemia erythrocytes phosphate buffered saline lactic dehydrogenase acute leukemia remission relapse dead bone marrow transplant conditioned medium  1  CHAPTER I  INTRODUCTION  I.  HEMOPOIESIS A.  Introduction Hemopoiesis  cells.  r e f e r s t o t h e f o r m a t i o n and development o f b l o o d  The c o n t r o l o f t h i s p r o c e s s i n v o l v e s an i n t r i c a t e l y b a l a n c e d  s e t o f i n t e r a c t i o n s between v a r i o u s c e l l s and growth r e g u l a t o r y f a c t o r s whose u l t i m a t e r o l e i s t h e maintenance o f a h o m e o s t a t i c s i t u a t i o n within the i n d i v i d u a l .  The changing  requirements  ( i n c r e a s e d o r decreased demands) f o r any p a r t i c u l a r type o f b l o o d c e l l may thus be met i n any number o f s i t u a t i o n s i n c l u d i n g c e l l d e a t h o r consumption/loss  r e s u l t i n g from  i n f l a m m a t i o n , hemorrhage o r o t h e r .  normal  infection,  Under normal  conditions, the  9 p r o d u c t i o n r a t e f o r t h e n e u t r o p h i l i c l e u c o c y t e a l o n e i s 1.6 x 10 c e l l s / k g / d a y i n t h e human ( 1 ) .  T h i s huge number r e l a t e s d i r e c t l y t o  the f a c t t h a t t h e h a l f - l i f e o f mature c i r c u l a t i n g n e u t r o p h i l s i s o n l y 6-7  hours  (2,3).  There a r e two broad groups o f h e m o p o i e t i c c e l l p o p u l a t i o n s . These a r e : 1.  2.  myeloid c e l l s ,  including granulocytic,  megakaryocytic,  and e r y t h r o i d  lymphoid  i n c l u d i n g B and T  cells,  monocytic,  cells, lymphocytes.  Some c e l l s which r e s i d e i n v a r i o u s organs and t i s s u e s ( n o n - c i r c u l a t i n g ) were a l s o o r i g i n a l l y d e r i v e d from h e m o p o i e t i c  2  precursors; these c e l l s progeny  of the monocytic  Langerhans c e l l s  i n c l u d e the f i x e d t i s s u e mast c e l l s series, i e .  (skin), osteoclasts  a l v e o l a r macrophages ( l u n g ) .  Kupffer c e l l s  and  (liver),  (bone), m i c r o g l i a ( b r a i n ) and  N a t u r a l k i l l e r (NK), n a t u r a l  cytotoxic  (NC), non-B non-T ( n u l l ) c e l l s and d e n d r i t i c r e t i c u l a r c e l l s lymphoid  t i s s u e s a r e l i k e l y the progeny  found i n  of h e m o p o i e t i c c e l l s as w e l l .  From the y o l k sac b l o o d i s l a n d s o f the embryo, h e m o p o i e t i c c e l l s m i g r a t e t o the f e t a l and d i f f e r e n t i a t i o n . of  a l l bones.  A f t e r b i r t h , hemopoiesis  The demands f o r hemopoiesis  As a r e s u l t , hemopoiesis locations  l i v e r where they undergo r a p i d  stem  expansion  o c c u r s i n the marrow  decreases with maturity.  i n a d u l t s takes p l a c e i n more r e s t r i c t e d  ( p r i m a r i l y w i t h i n the m e d u l l a r y space of f l a t bones and  the  e p i p h y s e s o f l o n g bones), a l t h o u g h i n s i t u a t i o n s o f extreme need, e x t r a m e d u l l a r y hemopoiesis may  o c c u r i n the s p l e e n , l i v e r and  lymph  nodes. The m a j o r i t y o f c i r c u l a t i n g b l o o d c e l l s r e p r e s e n t the mature o r end stages of d i f f e r e n t i a t i o n of h e m o p o i e t i c c e l l s . cells  I n i t i a l l y these  a r e thought t o have a r i s e n from p r e c u r s o r s w i t h i n the bone  marrow known as stem c e l l s . u n d e r s t a n d i n g of hemopoiesis.  The stem c e l l concept i s c e n t r a l t o our Stem c e l l s have the c a p a c i t y t o  p r o l i f e r a t e e x t e n s i v e l y and t o s e l f - r e p l i c a t e as w e l l as t o produce more d i f f e r e n t i a t e d daughter c e l l s .  Their p o t e n t i a l i t i e s for  m a t u r a t i o n a r e d i f f e r e n t , hence some a r e more "committed" t h a n o t h e r s to development a l o n g a p a r t i c u l a r pathway.  A  schematic  r e p r e s e n t a t i o n of t h i s concept i s shown i n F i g u r e 1.1.  The most  p r i m i t i v e u n d i f f e r e n t i a t e d stem c e l l s a r e r e f e r r e d t o as p l u r i p o t e n t stem c e l l s  s i n c e they a r e c a p a b l e of p r o d u c i n g stem c e l l progeny  of  3  F i g u r e 1.1.  Schematic  diagram r e p r e s e n t i n g  M o d i f i e d from r e f e r e n c e s  hemopoiesis.  4,5.  CFU-S I = c o l o n y - f o r m i n g u n i t —  s p l e e n which form b l a s t c e l l c o l o n i e s  CFU-S I I = c o l o n y - f o r m i n g u n i t — s p l e e n which form mixed c o l o n i e s CFU-c = c o l o n y - f o r m i n g u n i t ( c u l t u r e ) BFU-E = b u r s t - f o r m i n g u n i t , e r y t h r o i d CFU-E = c o l o n y - f o r m i n g u n i t , e r y t h r o i d CFU-mega = c o l o n y - f o r m i n g u n i t , megakaryocyte CFU-GM = c o l o n y - f o r m i n g u n i t , granulocyte-macrophage CFU-eos = c o l o n y - f o r m i n g u n i t , e o s i n o p h i l CFU-mast = c o l o n y - f o r m i n g u n i t , mast c e l l  4  b o t h t h e m y e l o i d and lymphoid  lineages.  These i n t u r n g i v e r i s e t o  more d i f f e r e n t i a t e d p r o g e n i t o r c e l l s w i t h i n each l i n e a g e .  As  d i f f e r e n t i a t i o n a l o n g t h e developmental pathway o c c u r s , t h e r e i s a concomitant decrease i n t h e c a p a c i t y f o r s e l f - r e n e w a l u n t i l capacity i s lost  and f u n c t i o n a l end-stage  scheme o f hemopoiesis  this  c e l l s a r e produced.  i s thought t o be p y r a m i d a l i n n a t u r e ,  This  involving  an a m p l i f i c a t i o n o f c e l l numbers a t s u c c e s s i v e s t a g e s o f differentiation.  B.  ASSAYS FOR HEMOPOIETIC STEM CELLS 1.  IN VIVO ASSAYS The h e m o p o i e t i c stem c e l l concept as o u t l i n e d i n F i g u r e 1.1  has a r i s e n from b o t h s p e c u l a t i o n and e x p e r i m e n t a t i o n . S p e c u l a t i o n t h a t the mature f u n c t i o n a l end c e l l s  i n the  c i r c u l a t i n g b l o o d s t r e a m a r o s e from the d i f f e r e n t i a t i o n of primitive ancestral c e l l s originally (6-8).  l o c a t e d i n the bone marrow (BM) came  from p a t h o l o g i s t s such as Maximow, Downey and Osgood  The groundwork f o r t h e s c i e n t i f i c  hemopoiesis was l a i d  i n 1961 by T i l l  i n v e s t i g a t i o n of  and M c C u l l o c h (9) when they  d e s c r i b e d t h e s p l e e n c o l o n y - f o r m i n g assay.  In t h i s technique,  s u p r a l e t h a l l y - i r r a d i a t e d mice were i n f u s e d i n t r a v e n o u s l y w i t h BM cells  and, w i t h i n 7 - 1 4  days, v i s i b l e  d i s c r e t e c o l o n i e s of  x h e m o p o i e t i c c e l l s were observed i n t h e s p l e e n s o f t h e s e mice. The c e l l s presumed t o be r e s p o n s i b l e f o r the p r o d u c t i o n o f t h e s e c o l o n i e s were named " c o l o n y - f o r m i n g u n i t — That the CFU-S were c l o n a l i n o r i g i n c e l l ) was demonstrated  s p l e e n " o r CFU-S. ( d e r i v e d from a s i n g l e  by t h e use of donor bone marrow w i t h  5  radiation-induced  g r o s s chromosomal markers (10,11).  I n these  i n v e s t i g a t i o n s , a l l o f t h e progeny o f a s i n g l e CFU-S  contained  the same g e n e t i c marker, p r o v i n g s i n g l e clone.  t h a t they o r i g i n a t e d from a  When t h e c e l l s w i t h i n i n d i v i d u a l c o l o n i e s were  examined m i c r o s c o p i c a l l y , i t was found t h a t some CFU-S formed multilineage and  c o l o n i e s c o n s i s t i n g of e r y t h r o i d , megakaryocytic  granulocytic c e l l s  ( i n d i c a t i n g that the karyotypic  a r o s e i n a v e r y p r i m i t i v e stem c e l l ) w h i l e o t h e r s  markers  formed  only  g r a n u l o c y t i c o r e r y t h r o i d progeny ( i n d i c a t i n g t h a t t h e markers originated i n less primitive c e l l s ) . The  self-renewal  c a p a c i t y o f CFU-S was shown by  retransplantation studies morphologically  (12).  Even s i n g l e CFU-S c o n s i s t i n g  o f e r y t h r o i d progeny c o u l d , upon  r e t r a n s p l a n t a t i o n , form c o l o n i e s c o n t a i n i n g progeny o f s i n g l e o r mixed l i n e a g e . population  The f a c t t h a t t h e t o t a l h e m o p o i e t i c  cell  c o u l d be r e c o n s t i t u t e d from t h e progeny o f a s i n g l e  CFU-S (more l i k e l y a subset o f CFU-S) was s t r o n g  indication for  the p l u r i - o r m u l t i p o t e n t i a l i t y o f t h i s p r i m i t i v e stem  cell  (13,14). While some o f t h e i n v i v o CFU-S s t u d i e s have s t r o n g l y suggested t h a t a t l e a s t some CFU-S may be p l u r i p o t e n t i a l , has  always been much c o n t r o v e r s y  concerning  there  this interpretation  and many i n v e s t i g a t o r s b e l i e v e t h a t CFU-S a r e m y e l o p o i e t i c c e l l s without lymphopoietic  potential.  r e f l e c t e d i n F i g u r e 1.1 by a d o t t e d lymphoid stem c e l l .  stem  This uncertainty i s  l i n e from t h e p l u r i p o t e n t t o  I t i s u n l i k e l y t h a t t h e standard  assay a l o n e w i l l e v e r r e s o l v e t h i s p o i n t .  CFU-S  The f a c t t h a t  this  6  assay i n v o l v e s a r e l a t i v e l y s h o r t d u r a t i o n and i n a s p l e n i c microenvironment may  one which  occurs  h e l p to e x p l a i n the l a c k of  observed lymphoid c e l l s w i t h i n s p l e e n c o l o n i e s .  The  absence of  a thymic microenvironment c o u l d c e r t a i n l y e x p l a i n the l a c k of T-lymphocytes i n t h e s e c o l o n i e s . have n o t been r e p o r t e d  While B-lymphocytes themselves  i n s p l e n i c c o l o n i e s , c e l l s c a p a b l e of  forming B c e l l progeny have been (15). disagreed  w i t h t h e s e f i n d i n g s (16).  Other i n v e s t i g a t o r s have  I t has  a l s o been shown t h a t  most of the stem c e l l s i n j e c t e d i n t o l e t h a l l y - i r r a d i a t e d mice seed and  grow i n the bone marrow r a t h e r than the s p l e e n  Most l i k e l y a v e r y p r i m i t i v e subset  of CFU-S has  p l u r i p o t e n t i a l i t y w h i l e most CFU-S are r e s t r i c t e d myelopoiesis,  assay.  Evidence supporting  p o s s i b i l i t y came from s t u d i e s of r a d i a t i o n - i n d u c e d markers i n r a t s , where the the same marker was t o be p r e s e n t  the  this chromosomal  sometimes found  i n both r e c i p i e n t e r y t h r o i d spleen colonies  donor p e r i p h e r a l b l o o d  lymphocytes, i n d i c a t i n g t h a t b o t h  types were the progeny of the same donor stem c e l l Convincing  and cell  (17).*  e v i d e n c e e x i s t s i n d i c a t i n g t h a t t h e r e i s a good  d e a l of h e t e r o g e n e i t y easy t o imagine i f one as a m u l t i t u d e  to  a t l e a s t under the c o n d i t i o n s imposed by  r o u t i n e spleen colony  (5).  w i t h i n CFU-S p o p u l a t i o n s views the e n t i r e p r o c e s s  (18-20).  This i s  of hemopoiesis  of i n t e r a c t i n g c e l l u l a r d i v i s i o n s and  differentiations.  The  earliest  (most p r i m i t i v e ^ or  u n d i f f e r e n t i a t e d ) c e l l s would have the g r e a t e s t c a p a c i t y f o r s e l f - r e n e w a l and would produce g r e a t e r numbers of progeny w i t h l i m i t e d degrees of commitment a l o n g  a specified lineage  and  7  hence i n c r e a s e d p r o l i f e r a t i v e c a p a c i t y . hemopoietic  C e l l s f u r t h e r a l o n g the  pathway would g r a d u a l l y l o s e t h e i r a b i l i t y  s e l f - g e n e r a t e as they g a i n e d g r e a t e r degrees  of  u n t i l becoming mature f u n c t i o n a l b l o o d c e l l s .  to  differentiation The  developmental  pathway from stem c e l l t o p r o g e n i t o r c e l l t o m o r p h o l o g i c a l l y r e c o g n i z a b l e immature t o mature b l o o d c e l l 1.1)  (as shown i n F i g u r e  would thus o c c u r i n a g r a d u a l s e r i e s of " s t e p s " .  u s e f u l t o c o n s i d e r when examining  F i g u r e 1.1  l i t e r a t u r e p e r t a i n i n g to hemopoietic In  colony  This i s  as w e l l as the assays.  v i v o s t u d i e s u s i n g the g e n e t i c a l l y anemic (stem  cell  v d e f i c i e n t ) mouse s t r a i n W/W  , which have v e r y low numbers of  CFU-S, showed t h a t t h i s d e f e c t c o u l d be cured by of  reconstitution  t h e s e mice w i t h normal syngeneic marrow c o n t a i n i n g  r a d i a t i o n - i n d u c e d markers o r w i t h marrow from another mutant strain, Sl/Sl  , which had normal hemopoietic  abnormal "microenvironment"  (21,22).  paramount i n our u n d e r s t a n d i n g  stem c e l l s but  an  These s t u d i e s were  of the stem c e l l concept  as w e l l as some of the f a c t o r s t h a t i n f l u e n c e stem  itself  cell  regulation. Another i n v i v o system, the d i f f u s i o n chamber c u l t u r e , been developed  i n o r d e r t o i n v e s t i g a t e the e f f e c t of  humoral f a c t o r s on hemopoiesis (or  (23-28).  o t h e r s p e c i e s ) a r e implanted  with  c e l l s i n medium w i t h  serum o r plasma i n a p l a s m a - c l o t o r s e m i - s o l i d agar The  long-range  In t h i s system, mice  intraperitoneally  d i f f u s i o n chambers c o n t a i n i n g hemopoietic  has  support.  chambers a l l o w f r e e d i f f u s i o n of humoral f a c t o r s t o the  i n s i d e w i t h o u t p e r m i t t i n g t r a n s f e r of c e l l s .  Normal  and  8  m a l i g n a n t h e m o p o i e t i c c e l l s from t i s s u e c u l t u r e , bone marrow, p e r i p h e r a l b l o o d , s p l e e n , y o l k sac, and f e t a l c u l t u r e d i n t h i s manner.  After 7 - 1 4  l i v e r have been  days of growth, d i f f u s i o n  chambers a r e removed and the c e l l s i n s i d e examined f o r c o l o n y (CFU-d) f o r m a t i o n , enumeration,  or subculture i n t o i n v i t r o  p r o g e n i t o r c e l l assay systems (the l a t t e r assay w i l l d i s c u s s e d i n the f o l l o w i n g s e c t i o n ) .  Similarities  be  and  d i f f e r e n c e s i n r e s p o n s i v e n e s s t o a number of substances a f f e c t i n g h e m o p o i e t i c c e l l s have been observed between bone marrow i n s i t u o r i n v i t r o and w i t h i n d i f f u s i o n chamber c u l t u r e s (29-34).  Moreover i t i s thought t h a t d i f f u s i o n  chamber  g r a n u l o c y t i c p r o g e n i t o r s a r e d i f f e r e n t , p o s s i b l y more p r i m i t i v e , than p r o g e n i t o r s forming g r a n u l o c y t i c c o l o n i e s i n v i t r o  (35).  While t h e s e d i f f e r e n c e s between d i f f u s i o n chamber c u l t u r e s o t h e r i n v i v o o r i n v i t r o h e m o p o i e t i c stem c e l l assays  and  clearly  e x i s t , the d i f f u s i o n chamber c u l t u r e t e c h n i q u e i s s t i l l  regarded  by many t o be the b e s t system i n which t o study the e f f e c t of long-range s o l u b l e f a c t o r s on hemopoiesis The  i n vivo.  i n v i v o assays f o r h e m o p o i e t i c stem c e l l s l a i d  the  groundwork f o r our u n d e r s t a n d i n g of the complex n a t u r e of hemopoiesis.  More r e c e n t l y , i n v i t r o s t u d i e s have been  developed which have r a p i d l y expanded our c o n c e p t u a l view o f hemopoiesis 2.  as o u t l i n e d i n F i g u r e  1.1.  IN VITRO ASSAYS In 1966,  two groups  of i n v e s t i g a t o r s developed an i n v i t r o  t e c h n i q u e c a p a b l e of growing murine h e m o p o i e t i c  colonies  (34,35).  t o the study o f  T h i s methodology was  r a p i d l y extended  9  human h e m o p o i e t i c c o l o n i e s i n v i t r o  (36).  The  technique  i n v o l v e s the growth o f d i s c r e t e c o l o n i e s , d e r i v e d from i n d i v i d u a l hemopoietic precursor c e l l s ,  i n a semi-solid  medium (agar, m e t h y l c e l l u l o s e o r plasma c l o t - b a s e d ) growth and serum f a c t o r s . maintains  and  containing  The v i s c o s i t y of t h e support medium  c e l l s d e r i v e d from an i n d i v i d u a l c l o n o g e n i c  i n close proximity, analyzed.  support  allowing separate  precursor  c o l o n i e s t o be enumerated,  I n v i t r o c u l t u r e methods f o r a l l of the s i n g l e  and m u l t i l i n e a g e - a s s o c i a t e d p r o g e n i t o r s have been developed (17-34).  i n t h e mouse and human  Colony-forming p r o g e n i t o r  have been g i v e n t h e g e n e r a l name CFU-c  cells  (colony-forming  u n i t - c u l t u r e ) and i n c l u d e e r y t h r o i d and g r a n u l o c y t i c p r o g e n i t o r s ( F i g u r e 1.1).  Large e r y t h r o i d p r o g e n i t o r s or the c o l o n i e s  d e r i v e d from them have been termed BFU-E ( b u r s t - f o r m i n g u n i t s  —  e r y t h r o i d ) ; t h e i r s m a l l e r , more mature p r o g e n i t o r s have been termed CFU-E ( c o l o n y - f o r m i n g  unit —  erythroid).  Other  myeloid  p r o g e n i t o r s have been named CFU-mega; CFU-GM, CFU-eos o r CFU-mast t o d e s c r i b e t h e i r p a r t i c u l a r l i n e a g e commitment F i g u r e 1.1). use,  While the t e r m i n o l o g y  (see  j u s t d e s c r i b e d i s i n common  d i f f e r e n t terms have been proposed by v a r i o u s  laboratories. progenitors  The c u l t u r e of T and B-lymphoid-committed  (CFU-TL, CFU-BL) has a l s o been developed  (42,43,49,51) b u t most i n v e s t i g a t o r s b e l i e v e t h e m a j o r i t y of c e l l s forming  these  than t h e myeloid  lymphoid c o l o n i e s i n v i t r o a r e more mature  progenitor c e l l s described (52).  M u l t i p o t e n t i a l stem c e l l s grown i n v i t r o form mixed c o l o n i e s v a r i o u s l y composed of g r a n u l o c y t e s , macrophages, megakaryocytes,  10  and  erythroid cells  cells,  (CFU-GEMM).  Mixed c o l o n i e s c o n t a i n i n g mast  e o s i n o p h i l s , and lymphoid c e l l s  have a l s o been r e p o r t e d  (53-58).  ( u s u a l l y T-lymphocyte)  These mixed c o l o n i e s may  c o n t a i n CFU-S as w e l l as m u l t i p o t e n t i a l and s i n g l e - l i n e a g e progenitors  o f a v a r i e t y o f commitment pathways (44,45,59,60).  E x t r e m e l y p r i m i t i v e human and murine m u l t i p o t e n t i a l stem  cells  w i t h b l a s t c e l l morphology have a l s o been grown i n v i t r o (61-63); t h e s e c o l o n i e s c o n t a i n a l a r g e number o f CFU-S as w e l l as m u l t i p o t e n t i a l and more l i n e a g e - r e s t r i c t e d p r o g e n i t o r Evidence f o r the existence  cells.  of a p l u r i p o t e n t i a l (with both  m y e l o i d and lymphoid p o t e n t i a l ) stem c e l l i n t h e mouse has a l r e a d y been d i s c u s s e d  i n the previous  section.  I n t h e human,  e v i d e n c e t o support t h i s concept has come from s t u d i e s i n v o l v i n g neoplasms o f h e m a t o p o i e t i c c e l l s .  In 90% o f p a t i e n t s w i t h  c h r o n i c g r a n u l o c y t i c leukemia (CGL), BM c e l l s  contain a  c h a r a c t e r i s t i c g r o s s chromosomal t r a n s l o c a t i o n t ( 9 ; 2 2 ) r e f e r r e d t o as t h e P h i l a d e l p h i a ( P h ) chromosome (66,67). 1  chromosome has been demonstrated i n n u c l e a t e d granulocytes,  The P h  erythroid  cells,  monocytes, megakaryocytes and p r o b a b l y  B-lymphocytes (68-71) i n d i c a t i n g t h a t t h e k a r y o t y p i c  abnormality  a r o s e i n a stem c e l l common t o b o t h m y e l o i d and lymphoid Further  1  cells.  e v i d e n c e f o r t h e c l o n a l i t y o f t h i s d i s o r d e r as w e l l as  i t s p l u r i p o t e n t stem c e l l o r i g i n came from enzymatic s t u d i e s . Glucose-6-phosphate dehydrogenase (G-6-PD) i s a polymorphic intracellular  enzyme w i t h two isoenzyme t y p e s .  The l o c u s f o r  G-6-PD i s on t h e X chromosome and s i n c e one X chromosome undergoes random i n a c t i v a t i o n d u r i n g  embryogenesis i n females,  11  individual cells  from G-6-PD heterozygous  females w i l l  produce  one o r t h e o t h e r G-6-PD isoenzyme b u t n o t both. The t o t a l  cell  p o p u l a t i o n i n such a G-6-PD h e t e r o z y g o t e w i l l , t h e r e f o r e , r e v e a l a 50:50 isoenzyme s y n t h e s i s p r o f i l e F i a l k o w e t a l . have demonstrated  under normal c i r c u m s t a n c e s .  t h a t female G-6-PD  h e t e r o z y g o t e s w i t h CGL (and some w i t h acute  nonlymphoblastic  leukemia) had a s i n g l e isoenzyme type i n many o f t h e i r b l o o d cells  (72-74).  I n c h r o n i c g r a n u l o c y t i c leukemia, t h e s e  were shown t o i n c l u d e m y e l o i d and lymphoid  (B) c e l l s .  cells I n acute  myelogenous leukemia, a s i n g l e G-6-PD i s o t y p e was found i n b o t h leukemic  l e u c o c y t e s and e r y t h r o c y t e s .  I n t e r e s t i n g l y , w h i l e CGL  has been shown t o o r i g i n a t e i n a p l u r i p o t e n t i a l stem c e l l , t h e massive  expansion o f h e m o p o i e t i c  the granulocyte-monocyte  c e l l s occurs p r i m a r i l y w i t h i n  lineage.  F u r t h e r evidence f o r the  e x i s t e n c e o f a common stem c e l l f o r b o t h t h e m y e l o i d and lymphoid cells  l i n e a g e s i n t h e human came from G-6-PD a n a l y s i s o f such  from mixed c o l o n i e s (75). In  a d d i t i o n to the evidence already presented f o r  multipotential colonies i n vitro, for (a)  there i s indisputable  evidence  the c l o n a l i t y of colonies: m i c r o m a n i p u l a t i o n and t i m e - l a p s e cinematography  techniques  have shown t h a t m u l t i p o t e n t i a l and s i n g l e l i n e a g e c o l o n i e s of  a v a r i e t y o f types can be grown from s i n g l e  cells  (46,76,77). (b)  granulocyte-macrophage  and e r y t h r o i d c o l o n i e s from G-6-PD  h e t e r o z y g o t e s were shown t o p o s s e s s o n l y a s i n g l e type  (78,79).  isoenzyme  12  (c)  mixed c u l t u r e s o f male and female c e l l s were shown by karyotypic colonies The  a n a l y s i s t o form e i t h e r "male" o r "female"  (80,81).  evidence that p r o g e n i t o r  i n v i t r o a r e indeed  c e l l s which form c o l o n i e s  t h e progeny o f stem c e l l s observed i n t h e  i n v i v o assays i s as f o l l o w s . (a)  I n d i v i d u a l CFU-S form s p l e e n c o l o n i e s c o n t a i n i n g  progenitor  c e l l s (5). (b)  D e n s i t y - f r a c t i o n a t i o n o f murine BM c e l l s populations cells.  t h a t c o n t a i n stem c e l l s b u t no p r o g e n i t o r  T h i s f r a c t i o n c o u l d , however, form s p l e e n  containing progenitor (c)  can i s o l a t e  c e l l s (82).  I t i s p o s s i b l e t o generate murine p r o g e n i t o r fractionated fetal  progenitor  techniques  techniques gradient  (54).  (stem and  S p l e e n and  (PB) a l s o c o n t a i n c o l o n y - f o r m i n g  cells,  albeit  Numerous c e l l f r a c t i o n a t i o n  have been employed t o s e p a r a t e  and p r o g e n i t o r  cells  a t a frequency o f about 1.5% i n  BM, t h e r i c h e s t source o f such c e l l s  at much lower f r e q u e n c i e s .  contain  c e l l s (83).  hemopoietic precursor  c e l l s ) are present  p e r i p h e r a l blood  cells in  l i v e r suspension c u l t u r e s that  stem c e l l s b u t no p r o g e n i t o r Murine c l o n o g e n i c  colonies  c e l l populations  and c h a r a c t e r i z e stem  and subsets  thereof.  These  have i n c l u d e d adherence t o g l a s s beads ( 8 4 ) , d e n s i t y  separation  (84-89), v e l o c i t y s e d i m e n t a t i o n  (90-95),  e l e c t r o p h o r e s i s ( 9 6 ) , f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t i n g (97) and m i t o g e n - b i n d i n g (19,20,83,98). c h a r a c t e r i z a t i o n o f CFU-S s u b s e t s ,  S e p a r a t i o n and progenitor  c e l l s and CFU-E  13  have been p o s s i b l e u t i l i z i n g these t e c h n i q u e s . the c l o n o g e n i c p r o g e n i t o r c e l l s i d e n t i f i e d fact,  Evidence  i n vitro  that  are, i n  t h e c e l l s r e s p o n s i b l e f o r t h e p r o d u c t i o n o f mature,  f u n c t i o n a l end-stage b l o o d c e l l s comes from a number o f studies. (a)  granulocyte-macrophage and e r y t h r o i d c o l o n y - f o r m i n g are i n a c t i v e c e l l c y c l e i n v i v o  (b)  t h e frequency  and p r o l i f e r a t i v e  (54). capacity of  granulocyte-macrophage and e r y t h r o i d c o l o n y - f o r m i n g is sufficient  cells  cells  t o account f o r t h e r e q u i r e d number o f  e r y t h r o i d c e l l s and polymorphonuclear l e u c o c y t e s i n v i v o under s t e a d y - s t a t e c o n d i t i o n s . (c)  during states of p h y s i o l o g i c a l disturbance a c t i v e regeneration of granulocytes irradiation  and monocytes f o l l o w i n g  o r o t h e r c y t o t o x i c events,  hypertransfusion) colony l e v e l s  ( f o r example,  and f o l l o w i n g  f l u c t u a t e i n corresponding  fashion. The  i n vitro  c l o n o g e n i c p r o g e n i t o r c e l l assay  just  d e s c r i b e d has enormously i n c r e a s e d our u n d e r s t a n d i n g o f hemopoiesis and i t s r e g u l a t o r y f a c t o r s .  The v a s t m a j o r i t y o f  c o l o n i e s formed i n t h e s e m i - s o l i d c u l t u r e t e c h n i q u e from p r o g e n i t o r s w i t h r e l a t i v e l y  are derived  advanced l i n e a g e commitment  ( f o r example, CFU-GM, CFU-E, GM c l u s t e r - f o r m i n g c e l l s ) . CFU-GEMM (mixed) c o l o n y - f o r m i n g  c e l l s are c e r t a i n l y  and t h e s e c e l l s a r e l i k e l y t o be t h e i n v i t r o CFU-S subset  (CFU-S-II) (54,61,98-100).  detectable  counterpart of a  The c e l l thought t o be  the most p r i m i t i v e CFU-S (CFU-S-I) has been i d e n t i f i e d  i n vitro  14  i n the mouse (63,64) and i n human u m b i l i c a l c o r d b l o o d (65). With the development of the l o n g term bone marrow c u l t u r e t e c h n i q u e , i t became e v i d e n t t h a t p l u r i p o t e n t stem c e l l s  that  c o u l d form CFU-S c o u l d be m a i n t a i n e d and were c a p a b l e of r e c o n s t i t u t i n g the e n t i r e h e m o p o i e t i c system i n i r r a d i a t e d mice (101-105).  The b a s i s f o r the l o n g term maintenance o f p r i m i t i v e  h e m o p o i e t i c c e l l s l a y i n the f o r m a t i o n i n c u l t u r e of a s t r o m a l adherent c e l l  l a y e r (106,107).  This stromal l a y e r contained  (pre-)adipocytes, f i b r o b l a s t - l i k e endothelial  cells,  o s t e o b l a s t s , r e t i c u l u m and mesenchymal c e l l s and macrophages w i t h a s s o c i a t e d h e m o p o i e t i c c e l l s o f a l l degrees o f differentiation.  P r o l i f e r a t i o n o f stem c e l l s i n the adherent  l a y e r c o u l d be m a i n t a i n e d f o r up t o months under c o n t r o l l e d conditions.  The a p p l i c a t i o n s of the continuous bone-marrow  c u l t u r e t e c h n i q u e have been reviewed (a)  (108) and  include:  i n v e s t i g a t i o n o f the e f f e c t of chemotherapeutic i r r a d i a t i o n on s t r o m a l c e l l  drugs  and  exposure p r i o r t o bone marrow  transplantation (b)  the study o f hormonal i n f l u e n c e s p e p t i d e s , growth f a c t o r s ) on  (c)  (corticosteroids,  hemopoiesis  the c a r c i n o g e n i c e f f e c t o f v i r u s e s , i r r a d i a t i o n c h e m i c a l c a r c i n o g e n s on  hemopoiesis  (d)  i n v e s t i g a t i o n o f the i n f l u e n c e o f g e n e t i c s on  (e)  the e s t a b l i s h m e n t o f f a c t o r - d e p e n d e n t and hemopoietic c e l l  hemopoiesis  -independent  lines.  E v i d e n c e s u p p o r t i n g the concept of hemopoiesis i n F i g u r e 1.1  and  has been p r e s e n t e d .  as o u t l i n e d  At t h i s p o i n t i t i s  15  appropriate t o d e s c r i b e the nature of the r e g u l a t o r y molecules or factors that influence t h i s  C.  process.  REGULATION OF HEMOPOIESIS During normal hemopoiesis,  i n t e r a c t i o n s o c c u r between  s t i m u l a t o r y and i n h i b i t o r y m o l e c u l e s ,  t h e c e l l s t h a t produce them and  t h e i r t a r g e t c e l l s , so t h a t p h y s i o l o g i c a l l y a p p r o p r i a t e numbers o f blood c e l l s  a r e produced.  d u r i n g a c t i v e leukemic hemopoiesis.  T h i s complex i n t e r p l a y i s o f t e n d i s r u p t e d  s t a t e s , r e s u l t i n g i n t h e s u p p r e s s i o n o f normal  A t p r e s e n t , t h e r e i s no t o t a l l y  satisfactory  e x p l a n a t i o n f o r t h e complete s u p p r e s s i o n o f normal m y e l o i d c e l l s t h a t o c c u r s i n acute n o n l y m p h o b l a s t i c  leukemia  (ANLL).  s u p p r e s s i o n takes p l a c e i n s p i t e o f t h e f a c t t h a t leukemic l o n g e r c e l l c y c l e times than do normal c e l l s .  Simple  progenitor This  c e l l s have  explanations  i n v o l v i n g t h e "crowding o u t " o f normal m y e l o i d p r o g e n i t o r s by t h e leukemic  c l o n e cannot answer t h e fundamental q u e s t i o n o f how t h e  leukemic  c e l l s g a i n e d a c o m p e t i t i v e advantage i n t h e f i r s t p l a c e .  T h i s i s an a c t i v e a r e a o f r e s e a r c h a t t h e p r e s e n t  time.  I n o r d e r t o a p p r e c i a t e t h e s i g n i f i c a n c e o f t h e many f a c t o r s r e g u l a t i n g hemopoiesis,  i t i s necessary  to consider the r o l e s played  by 1) t h e hemopoietic  c o l o n y - s t i m u l a t i n g f a c t o r s , 2) o t h e r  n o n s p e c i f i c molecules  i n c l u d i n g serum components, 3) n e g a t i v e  r e g u l a t o r s , and 4) m i c r o e n v i r o n m e n t a l r e g u l a t i n g lymphopoiesis  influences.  While some f a c t o r s  w i l l be d e s c r i b e d f o r t h e sake o f  completeness, i t i s n o t w i t h i n t h e scope o f t h e o b j e c t i v e s o f t h i s t h e s i s t o cover t h i s a s p e c t o f hemopoiesis i n d e t a i l . the emphasis w i l l f a l l  on r e g u l a t i o n o f m y e l o p o i e s i s .  Consequently,  16  1.  THE HEMOPOIETIC COLONY-STIMULATING FACTORS The  term " c o l o n y - s t i m u l a t i n g f a c t o r " o r CSF d e r i v e s from  the i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f r e g u l a t o r y m o l e c u l e s shown t o cause p r o l i f e r a t i o n o f h e m o p o i e t i c  cells resulting i n  the f o r m a t i o n o f r e c o g n i z a b l e c o l o n i e s i n v i t r o . Once i t had been r e c o g n i z e d t h a t a number o f CSFs e x i s t e d w i t h a c t i v i t y on d i f f e r e n t p r o g e n i t o r c e l l  l i n e a g e s , terminology  a r o s e designed t o i d e n t i f y t h e s e v a r i o u s CSFs.  Often a p r e f i x  or  c e l l lineage(s)  s u f f i x i s used  t o i n d i c a t e which h e m o p o i e t i c  i s s t i m u l a t e d by t h e CSF ( T a b l e s I and I I ) .  A l l of the w e l l  c h a r a c t e r i z e d CSFs a r e g l y c o p r o t e i n s w i t h r e p o r t e d m o l e c u l a r weights  r a n g i n g from 15 - 70 KD.  They a r e b i o l o g i c a l l y  -11 at  extremely  produced  low m o l a r i t i e s  (10  by a v a r i e t y o f c e l l  active  -13 t o 10  M).  A l l are  types and have f u n c t i o n s o t h e r  than s t i m u l a t i n g t h e p r o l i f e r a t i o n o f hemopoietic  precursor  cells.  survival,  These o t h e r f u n c t i o n s i n c l u d e t a r g e t c e l l  in vitro  i n d u c t i o n of d i f f e r e n t i a t i o n  particular cell  l i n e a g e ) and s t i m u l a t i o n o f a v a r i e t y o f  f u n c t i o n s i n mature end stage a)  (commitment t o a  cells.  MURINE COLONY-STIMULATING FACTORS M u l t i p o t e n t i a l Colony-Stimulating Factor Multi-CSF  T cell  i s produced  (Multi-CSF)  by m i t o g e n - s t i m u l a t e d  T-cells,  cloned  l i n e s , c e r t a i n T lymphomas and hybridomas as w e l l as by  the murine myelomonocytic leukemia  cell  l i n e , WEHI-3B.  The most  common s o u r c e f o r t h i s f a c t o r had been c o n d i t i o n e d medium from pokeweed m i t o g e n - s t i m u l a t e d  mouse s p l e n i c lymphocytes (SCM) o r  T a b l e I . The murine c o l o n y - s t i m u l a t i n g f a c t o r s  Colony-stimulating factor  Alternate nomenclature  Multi-CSF  IL-3.PSF.HCGF, BPA,MCGF,SAF  GM-CSF  MGI-1GM.CSAGM  M-CSF  CSF-1,MGI-1M, CSA-M  G-CSF  MGI-1G.DF  Progenitor target c e l l ( s )  Molecular weight  Purified  23 KD  \/  GM,G,M p r o g e n i t o r s , 23 KD i n i t i a l stages o f erythroid precursors, fetal eosinophil progenitors*  C F U - S , b l a s t (stem) cells.multipotent stem cells,GM,G,M, eosinophil,mast °cell.megakaryocyte and e r y t h r o i d p r o genitors  M (primarily) pro. g e n i t o r s , some GM, G progenitors  G (primarily) prog e n i t o r s , M only at high concentrat i o n s , weak a c t i v i t y on i n i t i a l s t a g e s o f m u l t i p o t e n t i a l and erythroid precursors  60-70 KD (dimer) 28-35 KD (monomer) 25 KD  Cloned  A c t i v i t y on human c e l l s  References  y/  X"  109-118  </  \/  *  119-123  y/  X  X  123-129  X  (G)  123,130133  T a b l e I . The murine  Colony-stimulating factor  Alternate nomenclature  colony-stimulating  Progenitor target c e l l ( s ) eosinophil precursors  factors  Molecular weight  EDF  Erythropoietin  epo  CFU-E  40-45 KD  TCGF, I L - 2  activated T cells  21-30 KD  a)IL-4,BCGF-l, BSF p i  activated B cells (proliferation)  b)BGDF,BCGF I I  activated B cells (prolifera t i o n and I g secretion)  c)BCDF,TRF  induces d i f f e r e n t i a t i o n ( I g production) i n activated, proliferating B cells  ND  induction of Ig secretion i n B cells  56 KD  growth  B c e l l stimulatory f a c t o r s (BSF)  Neuroleukin  Purified  46 KD  Eosinophil differentiation factor  T cell factor  (cont'd)  A c t i v i t y on human c e l l s  References  134  ND  135  partial  136-141  11-22 KD 20 KD (recombinant IL-4) 40-55 KD  Cloned  X partial  partial  X V  142-150  X  151,152  !—  1  Table  *  r e c o m b i n a n t m u r i n e GM-CSF and e r y t h r o i d p r e c u r s o r s .  Abbreviations:  at  I.  The murine  very  high  colony-stimulating  concentrations  factors  stimulates  the  (cont'd)  proliferation  of  eosinophil,  megakaryocyt  '•. I  I L , i n t e r l e u k i n ; P S F , p e r s i s t i n g c e l l s t i m u l a t i n g f a c t o r ; HCGF, h e m o p o i e t i c c e l l g r o w t h f a c t o r ; B P A , b u r s t promo t i n g a c t i v i t y ; MCGF, mast c e l l g r o w t h f a c t o r ; S A F , stem c e l l a c t i v a t i n g f a c t o r ; M G I , m a c r o p h a g e - g r a n u l o c y t e i n d u c e r ; C S A , c o l o n y - s t i m u l a t i n g a c t i v i t y ; D F , d i f f e r e n t i a t i o n f a c t o r ; BCGF, B c e l l g r o w t h f a c t o r ; BGDF, B c e l l g r o w t h a n d d i f f e r e n t i a t i o n f a c t o r ; B C D F , B c e l l d i f f e r e n t i a t i o n f a c t o r ; T R F , T c e l l r e p l a c i n g f a c t o r ; GM, g r a n u l o cyte-macrophage; G , g r a n u l o c y t e ; M , macrophage.  T a b l e I I . The human c o l o n y - s t i m u l a t i n g  factors  Colony-stimulating factor  Alternate nomenclature  Progenitor target c e l l ( s )  IL-3  Multi-CSF  B l a s t c e l l s , m u l t i - 14-28 KD lineage myeloid colonies, erythroid, G, M, GM, e o s i n o p h i l , b a s o p h i l , megakaryocyte progenitors  Pluripoietin  Pluripotent CSF  Multipotential stem c e l l s , GM and e r y t h r o i d progenitors  GM-CSF  CSF<*, GMCSF-<x, NIF-T  B l a s t c e l l s , GM and e o s i n o p h i l i c p r o g e n i t o r s **  14-35 KD  y  y  GM p r o g e n i t o r s ( p r i m a r i l y G)  18-22 KD  y  y  G-CSF  CSFyg  M-CSF  CSF-1, human u r i n e CSF  GM p r o g e n i t o r s ( p r i m a r i l y M) i n mouse, i n d i r e c t e f f e c t on human CFU-GM  Leukemic b l a s t growth f a c t o r  LBGF  Blast  cells  Molecular weight  18 KD  Purified  A c t i v i t y on murine c e l l s  References  ND  y  y  35-45 KD, 18-26 KD horaodimers  30 KD  Cloned  (differentiat i o n o f WEHI3B c e l l s )  y  partial  153  154-162  y 154,156,163 (G, d i f f e r e n *** t i a t i o n of WEHI-3B c e l l s )  y  ND  164-166  162,167  to  O  T a b l e I I . The human c o l o n y - s t i m u l a t i n g f a c t o r s ( c o n t ' d )  Colony-stimulating factor  Alternate nomenclature  Molecular weight  Progenitor target c e l l ( s )  Purified  Cloned  Erythropoietin  epo  CFU-E  39 KD  y  y  Erythroid potentiating activity  EPA  BFU-E, CFU-E  28 KD  y  y  TCGF, I L - 2  activated T cells  15 KD  a)BSF 1,BCGFI  activated B cells (proliferation)  150-200 KD 60-80 KD (reported)  partial  *  b)BGDF,BCGFII  activated B cells ( p r o l i f e r a t i o n and Ig secretion)  > 60 KD  partial  X  c)BCDF,TRF  Ig s e c r e t i o n i n activated, p r o l i ferating B cells  35 KD  partial  X  T cell factor  growth  B c e l l stimulatory f a c t o r s (BSF)  Neuroleukin  induction of Ig secretion i n B cells  A c t i v i t y on murine c e l l s  (CFU-E) (BFU-E,CFU-E)  168-170 171,172 136,  y/ •  173-176  177-181  y 56 KD  References  151,152  (murine)  * Yang Y-C, C i a r l e t t a AB, Temple PA, Chung MP, K o v a c i c S, W i t e k - G i a n n o t t i J S , L e a r y AC, K r i z R, Donahue RE, Wong GG, C l a r k SC: Human I L - 3 ( m u l t i - C S F ) : I d e n t i f i c a t i o n by e x p r e s s i o n c l o n i n g o f a n o v e l h e m a t o p o i e t i c g r o w t h f a c t o r r e l a t e d t o m u r i n e I L - 3 . C e l l 47:3, 1986. ** r e c o m b i n a n t human GM-CSF a t h i g h c o n c e n t r a t i o n s  s t i m u l a t e s m u l t i p o t e n t i a l and e r y t h r o i d p r o g e n i t o r s .  *** Nagata S, T s u c h i y a M, Asano S, K a z i r o Y, Yamazaki T, Yamamoto 0, H i r a t a Y, K u b o t a N, Oheda M, Nomura H, Ono M: M o l e c u l a r c l o n i n g and e x p r e s s i o n o f cDNA f o r human g r a n u l o c y t e c o l o n y - s t i m u l a t i n g f a c t o r . N a t u r e 319:415, 1986.  Table  I I . The human c o l o n y - s t i m u l a t i n g  factors  (cont'd)  Abbreviations: CSF, colony-stimulating factor; NIF-T, neutrophil inhibitory factor - derived from T c e l l s ; IL, i n t e r l e u k i n ; BCGF, B c e l l growth factor; BCDF, B c e l l d i f f e r e n t i a t i o n factor; BGDF, B c e l l growth and d i f f e r e n t i a t i o n factor; TRF, T c e l l replacing factor; GM, granulocytemacrophage; G, granulocyte; M, macrophage.  23  WEHI-3B c e l l s u n t i l t h e a v a i l a b i l t i y o f t h e c l o n e d molecule  recombinant  (115).  Multi-CSF stimulates  the i n v i t r o formation of b l a s t  cell,  g r a n u l o c y t e (CFU-G), macrophage (CFU-M), granulocyte-macrophage (CFU-GM), e o s i n o p h i l  (CFU-eos), mast c e l l  megakaryocyte (CFU-mega), e r y t h r o i d (CFU-GEMM) c o l o n i e s (epo)  from murine BM.  (CFU-mast),  (BFU-E and CFU-E), and mixed A d d i t i o n of e r y t h r o p o i e t i n  enhances t h e f o r m a t i o n and s i z e o f e r y t h r o i d c o l o n i e s i n  the p r e s e n c e o f m u l t i - C S F Of  great  (100).  i n t e r e s t was t h e d i s c o v e r y  that  leukemia-derived  (WEHI-3B) m u l t i - C S F was d i f f e r e n t than m u l t i - C S F from SCM.  A  s i n g l e amino a c i d s u b s t i t u t i o n (114,115) appeared t o e x i s t i n the  leukemia-derived f a c t o r .  Minor b i o l o g i c a l d i f f e r e n c e s  between t h e s e m u l t i - C S F s has a l s o been observed, r e l a t i n g t o decreased f u n c t i o n a l a c t i v i t y o f t h e l e u k e m i a - d e r i v e d f a c t o r with regard  t o s t i m u l a t i o n o f e r y t h r o i d and mast  proliferation  (182).  Granulocyte-Macrophage Colony S t i m u l a t i n g GM-CSF may be d e r i v e d lung  cell  Factor  (GM-CSF)  from medium c o n d i t i o n e d  (most commonly), h e a r t ,  spleen,  by murine  s a l i v a r y glands,  thigh  muscle, bone s h a f t , k i d n e y , thymus o r b r a i n as w e l l as from concanavalin A-stimulated (119-121,183,184).  T19.1 hybridoma  cells  As i t s name i m p l i e s , GM-CSF s t i m u l a t e s t h e  p r o l i f e r a t i o n o f CFU-G, CFU-M, o r CFU-GM.  In f e t a l l i v e r  c u l t u r e s , GM-CSF has a l s o been shown t o be c a p a b l e o f stimulating the production  of eosinophil colonies.  The  cell  24  p r o p o r t i o n of each d e v e l o p i n g  c o l o n y type i s r e l a t e d t o  c o n c e n t r a t i o n of GM-CSF used i n v i t r o .  the  When bone marrow c e l l s  were c u l t u r e d w i t h h i g h c o n c e n t r a t i o n s of GM-CSF,  extremely  l a r g e (> 5000 c e l l s ) pure g r a n u l o c y t i c c o l o n i e s developed  (92);  a t low c o n c e n t r a t i o n s of GM-CSF, o n l y macrophage c o l o n i e s developed declined  (185,186).  The number of CFU-GM c o l o n i e s d e v e l o p i n g  (as expected) w i t h d e c r e a s i n g GM-CSF c o n c e n t r a t i o n s .  A t v e r y h i g h c o n c e n t r a t i o n s , recombinant GM-CSF has been shown t o s t i m u l a t e p r o l i f e r a t i o n of e o s i n o p h i l , megakaryocyte e r y t h r o i d p r e c u r s o r s as w e l l  and  (187-190).  Macrophage C o l o n y - S t i m u l a t i n g  F a c t o r (M-CSF)  M-CSF has been c h a r a c t e r i z e d from t h r e e s o u r c e s :  L-cell  c o n d i t i o n e d medium (124), y o l k sac c o n d i t i o n e d medium (125) pregnant mouse u t e r i n e e x t r a c t (191).  and  M-CSF p r i m a r i l y  s t i m u l a t e s the p r o l i f e r a t i o n of macrophage c o l o n i e s from bone marrow a l t h o u g h G-containing  i t can a l s o s t i m u l a t e a s m a l l number of  colonies i n culture.  Synergism between M-CSF and  h e m o p o i e t i n 1 (IL-1) has been shown t o r e s u l t i n the  formation  of v e r y l a r g e macrophage c o l o n i e s , by a mechanism of i n d u c t i o n of M-CSF r e c e p t o r s on p r i m i t i v e hemopoietic  Granulocyte  Colony-Stimulating  Factor  cells  (192).  (G-CSF)  G-CSF, l i k e GM-CSF, has been d e r i v e d from medium from e n d o t o x i n - t r e a t e d mice (132).  lung-conditioned  G-CSF has  a l s o been  d e r i v e d from medium c o n d i t i o n e d by thymus, h e a r t , muscle, s a l i v a r y g l a n d , bone s h a f t , k i d n e y ,  s p l e e n , and  normal  25  p e r i t o n e a l c e l l s (131).  T h i s CSF has t h e c a p a c i t y t o s t i m u l a t e  the growth o f s m a l l pure G c o l o n i e s from murine bone marrow and a t h i g h c o n c e n t r a t i o n s , a s m a l l number o f GM and o c c a s i o n a l l y M c o l o n i e s form (133).  G-CSF, u n l i k e t h e p r e v i o u s l y d e s c r i b e d  murine CSFs, can a l s o s t i m u l a t e t h e p r o l i f e r a t i o n  o f human G  colonies i n v i t r o .  Other Murine R e g u l a t o r y M o l e c u l e s w i t h GM-CSF A c t i v i t y There have been r e p o r t s o f o t h e r murine CSFs w i t h a c t i v i t y on GM p r o g e n i t o r c e l l s i n v i t r o .  However, u n l i k e t h e p r e v i o u s l y  d e s c r i b e d CSFs, none o f t h e s e p u t a t i v e CSFs has been p u r i f i e d t o homogeneity. is unclear. (1)  As a r e s u l t  their precise biological  Three such f a c t o r s w i l l be mentioned:  C o n d i t i o n e d medium from a c l o n e d embryonic c e l l c o n t a i n e d a 65 - 70 KD molecule  Post-endotoxin  mouse serum c o n t a i n s a m o l e c u l e  M-CSF b u t o f lower clear  line  which s t i m u l a t e d  g r a n u l o c y t e and macrophage c o l o n y development (2)  significance  (193). s i m i l a r to  (< 23 KD) m o l e c u l a r weight.  i f t h i s serum a c t u a l l y  I t i s not  c o n t a i n s GM-CSF o r an a b e r r a n t  M-CSF i n a d d i t i o n t o G-CSF. (3)  A molecule  o f m o l e c u l a r weight 65 KD, p r e s e n t i n  inflammatory  exudate c e l l c o n d i t i o n e d medium, r e p o r t e d l y  s t i m u l a t e d t h e p r o l i f e r a t i o n o f CFU-GM and G c o l o n i e s (194). molecule  Again,  i ti s difficult  t o determine i f t h i s  represents contamination  o t h e r known CSF m o l e c u l e s .  of the preparation with  26  Megakaryocyte Colony S t i m u l a t i o n Murine m u l t i - C S F  appears t o be t h e p r i m a r y  f o r m a t i o n o f CFU-Mega.  Two o t h e r molecules  stimulus f o r the  have a l s o been  d e s c r i b e d w i t h a c t i v i t y on murine megakaryocyte p r o g e n i t o r s . megakaryocyte enhancing f a c t o r has been i d e n t i f i e d  A  i n medium  c o n d i t i o n e d by WEHI-3B, lung, bone s h a f t , p e r i t o n e a l c e l l s and the macrophage c e l l  l i n e P388 (195), b u t i t i s u n c l e a r i f t h i s  f a c t o r i s a d i s t i n c t e n t i t y or the r e s u l t of the s y n e r g i s t i c e f f e c t o f some o t h e r known m e d i a t o r w i t h m u l t i - C S F .  Human u r i n e  from p a t i e n t s w i t h a p l a s t i c anemia o r i d i o p a t h i c thrombocytopenic purpura  has been shown t o c o n t a i n a MEG-CSF  w i t h a c t i v i t y on mouse megakaryocyte p r o g e n i t o r s  (196).  Eosinophil D i f f e r e n t i a t i o n Factor A n o v e l lymphokine was r e c e n t l y d e s c r i b e d which appears t o be a r e g u l a t o r y m o l e c u l e f o r e o s i n o p h i l p r o d u c t i o n  (134).  This  e o s i n o p h i l d i f f e r e n t i a t i o n f a c t o r (EDF), produced by T c e l l c l o n e s r e a c t i v e t o a l i o - and p a r a s i t e a n t i g e n s d e r i v e d from p a r a s i t i z e d mice, s t i m u l a t e d e o s i n o p h i l d i f f e r e n t i a t i o n i n liquid EDF  (but n o t s e m i - s o l i d ) bone marrow c u l t u r e s .  Presumably,  a c t s on committed bone marrow p r e c u r s o r s t o s t i m u l a t e t h e  p r o d u c t i o n o f normal, f u n c t i o n a l e o s i n o p h i l s .  Erythroid Colony-Stimulating  Factors  Anemic mouse serum c o n t a i n s a m o l e c u l e thought t o be e r y t h r o p o e t i n (epo) from i t s n e u t r a l i z a t i o n by anti-human epo serum (182). T h i s f a c t o r s t i m u l a t e s t h e p r o l i f e r a t i o n o f l a t e r  27  stages  of e r y t h r o i d c o l o n y f o r m a t i o n  (CFU-E).  Erythroid  p r o g e n i t o r s are s t i m u l a t e d by m u l t i - C S F ( i n which case, r e f e r r e d to as " b u r s t promoting a c t i v i t y " or BPA),  and  often t o some  e x t e n t by GM-CSF and G-CSF a c t i n g w i t h e r y t h r o p o i e t i n . Recombinant murine GM-CSF by concentrations,  itself,  at very  high  s t i m u l a t e s p r o l i f e r a t i o n of e r y t h r o i d  precursors.  Lymphocyte Growth F a c t o r s While i t i s not p a r t of the o b j e c t i v e s of t h i s t h e s i s t o i n v e s t i g a t e the lymphoid arm detail,  of the h e m o p o i e t i c pathway i n  lymphocyte growth f a c t o r s w i l l be b r i e f l y mentioned f o r  the sake of p r e s e n t i n g a complete overview of hemopoiesis. (i)  T - C e l l Growth F a c t o r  (TCGF) or I n t e r l e u k i n 2  (IL-2)  TCGF or IL-2 has been d e r i v e d from murine T-lymphoma or hybridoma c e l l  l i n e s and  spleen c e l l  c o n d i t i o n e d media.  This molecule stimulates p r o l i f e r a t i o n of antigenmitogen-primed T  lymphocytes.  ( i i ) B C e l l Stimulating Factors B cell  growth and  identified as y e t .  (BSF)  d i f f e r e n t i a t i o n f a c t o r s have been  ( T a b l e I) a l t h o u g h  not c o m p l e t e l y  characterized  Interleukin 4 (IL-4), a putative B c e l l  f a c t o r (BCGF), has been m o l e c u l a r l y c l o n e d and to have p r o l i f e r a t i v e and  reported B c e l l  growth  demonstrated  immunoglobulin-secretion  i n d u c t i v e e f f e c t s on a c t i v a t e d B c e l l s lists  or  (150).  Table  I  growth and d i f f e r e n t i a t i o n f a c t o r s  which have been p a r t i a l l y p u r i f i e d and  characterized.  28  N e u r o l e u k i n , produced by l e c t i n - s t i m u l a t e d T c e l l s , has  a l s o been shown t o induce immunoglobulin s e c r e t i o n i n  c u l t u r e d p e r i p h e r a l b l o o d mononuclear  b)  cells.  HUMAN COLONY-STIMULATING FACTORS  Interleukin-3 IL-3  (IL-3)  o r m u l t i - C S F has v e r y r e c e n t l y been i d e n t i f i e d and 2  molecularly  cloned.  I t shares many s i m i l a r i t i e s t o human  GM-CSF, which i s b e l i e v e d t o have a r i s e n from a common a n c e s t r a l gene.  Both a r e produced by a c t i v a t e d T lymphocytes and t h e  genes f o r b o t h a r e t i g h t l y l i n k e d (9 k i l o b a s e s  a p a r t ) on  3 chromosome 5. progenitors  IL-3 appears t o r e a c t w i t h more p r i m i t i v e  o r stem c e l l s than does GM-CSF and g i v e s r i s e t o  g r e a t e r numbers o f b l a s t c e l l c o l o n i e s w i t h h i g h e r s e l f - g e n e r a t i v e p o t e n t i a l than does GM-CSF. myeloid progenitors IL-3  (Table  II).  Essentially a l l  and stem c e l l s a r e a p p a r e n t l y  s t i m u l a t e d by  The marked degree o f h e t e r o g e n e i t y i n  m o l e c u l a r weight o f IL-3 i s due t o i t s l a r g e c o n t e n t o f c a r b o h y d r a t e , a s i t u a t i o n s i m i l a r t o GM-CSF.  Pluripotent Colony-Stimulating  Factor  Human p l u r i p o t e n t CSF ( p l u r i p o i e t i n ) , d e r i v e d conditioned  from medium  by human b l a d d e r c e l l cancer l i n e 5637 and p u r i f i e d  to homogeneity, has been r e p o r t e d  t o be c a p a b l e o f s t i m u l a t i n g  the p r o l i f e r a t i o n o f m u l t i p o t e n t i a l stem c e l l s , GM and e r y t h r o i d progenitors  (153).  T h i s CSF appears t o p o s s e s s t h e combined  29  p r o p e r t i e s o f human GM-CSF and G-CSF, d i s c u s s e d next. or not p l u r i p o i e t i n  Whether  i s t h e same as IL-3 has n o t been r e p o r t e d .  GM-CSF (CSFa) Two CSFs w i t h GM c o l o n y - s t i m u l a t i n g a c t i v i t y , GM-CSF and G-CSF, have been i d e n t i f i e d from a v a r i e t y o f human s o u r c e s : c o n d i t i o n e d media from p e r i p h e r a l b l o o d  (197), l u n g (198), and  p l a c e n t a (154,199) as w e l l as from c e l l  lines  number o f organ c u l t u r e s (203).  (200-202) and a  GM and G-CSF have been  s e p a r a t e d from each o t h e r , m o l e c u l a r l y c l o n e d , and shown t o s t i m u l a t e d i s t i n c t CFU-GM s u b s e t s colony formation, peaking c o l o n i e s a t t h a t time  (154).  GM-CSF s t i m u l a t e s GM  a t 14 days, w i t h a predominance o f M  (155,161,204).  E o s i n o p h i l colony  f o r m a t i o n i s a l s o s t i m u l a t e d d i r e c t l y by GM-CSF.  I n combination  w i t h e r y t h r o p o i e t i n , GM-CSF has been shown t o s t i m u l a t e m u l t i p o t e n t i a l and e r y t h r o i d c o l o n y f o r m a t i o n , p o s s i b l y by an i n d i r e c t mechanism.  R e c e n t l y , recombinant human GM-CSF has been  shown t o s t i m u l a t e t h e p r o l i f e r a t i o n  of b l a s t  p a t i e n t s w i t h acute m y e l o b l a s t i c leukemia  cell  c o l o n i e s from  (162) and induce  macrophage p r o d u c t i o n o f I L - 1 , TNF-a, IL-2 r e c e p t o r s and mRNA f o r G-CSF (CSFB) and M-CSF  (CSF-1).  G-CSF (CSFB) G-CSF p r e f e r e n t i a l l y  s t i m u l a t e s G c o l o n y f o r m a t i o n b u t can  a l s o s t i m u l a t e some GM and o c c a s i o n a l M c o l o n i e s p e a k i n g days i n v i t r o .  at 7  I t has been demonstrated t h a t human G-CSF has  30  b i o c h e m i c a l and b i o l o g i c a l p r o p e r t i e s comparable t o murine G-CSF. R e c e n t l y a f a c t o r w i t h granulomonopoietic activity  (GM-EA) has been i d e n t i f i e d  (205).  enhancing  T h i s f a c t o r had no  c o l o n y - s t i m u l a t i n g a c t i v i t y by i t s e l f but, when added t o human bone marrow c u l t u r e s c o n t a i n i n g a source o f GM c o l o n y - s t i m u l a t i n g a c t i v i t y , GM-EA caused  an enhancement o f GM  c o l o n y numbers a t b o t h day 7 and day 14 i n c u b a t i o n . shown t o be produced by human monocyte-derived  GM-EA was  lipid-containing  cells.  M-CSF (Human U r i n a r y C o l o n y - S t i m u l a t i n g Although  Factor)  o f human o r i g i n , M-CSF has no s t r o n g d i r e c t  s t i m u l a t o r y e f f e c t on t h e p r o l i f e r a t i o n o f human c o l o n i e s . However, on mouse bone marrow, t h i s molecule  has e f f e c t s  similar  to murine M-CSF, c a u s i n g s e l e c t i v e p r o l i f e r a t i o n o f macrophage colony formation  (164).  Monocytes, f i b r o b l a s t s and e n d o t h e l i a l  c e l l s a r e a l l sources o f M-CSF.  Human M-CSF i s thought  t o be  i n v o l v e d i n t h e s u r v i v a l and a c t i v a t i o n o f monocytes and macrophages.  E r y t h r o p o i e t i n (epo) Human u r i n e has been u t i l i z e d as a source f o r e r y t h r o p o i e t i n (168,206). e r y t h r o i d colony  P u r i f i e d epo s t i m u l a t e s mature  (CFU-E) f o r m a t i o n .  p l u r i p o i e t i n o r epo i n combination  As mentioned p r e v i o u s l y , w i t h CSFa w i l l support t h e  31  s u r v i v a l and p r o l i f e r a t i o n o f m u l t i l i n e a g e and e a r l y  erythroid  (BFU-E) c o l o n i e s .  Erythroid Potentiating Activity  (EPA)  E r y t h r o i d - p o t e n t i a t i n g a c t i v i t y stimulates both e a r l y (BFU-E) and l a t e  (CFU-E) e r y t h r o i d c o l o n y f o r m a t i o n i n humans  and mice but has no e f f e c t on o t h e r m y e l o i d c o l o n y growth (171,172).  EPA has been p u r i f i e d from medium c o n d i t i o n e d by the  human T-lymphoblast  cell  l i n e Mo,  lymphotropic v i r u s HTLV-II.  EPA  i n f e c t e d w i t h the human T - c e l l i s a l s o produced by most o t h e r  HTLV-II i n f e c t e d mature T-lymphoblast cell  l i n e s and by some monocyte  lines.  Leukemic B l a s t Growth F a c t o r (LBGF) R e c e n t l y , a h e m o p o i e t i c growth f a c t o r termed growth f a c t o r o r LBGF was b l a d d e r carcinoma c e l l  p a r t i a l l y p u r i f i e d from a human  l i n e HTB9 (167).  s t i m u l a t i o n of b l a s t c e l l  leukemic b l a s t  LBGF was  shown t o cause  growth from a c u t e m y e l o b l a s t i c  leukemia p a t i e n t s ' p e r i p h e r a l b l o o d . s e p a r a t e d from GM-CSF a c t i v i t y ,  LBGF a c t i v i t y c o u l d  i n d i c a t i n g t h a t LBGF may  n o v e l , d i s t i n c t h e m o p o i e t i c growth f a c t o r .  be be a  I t s r e l a t i o n s h i p or  i d e n t i t y w i t h IL-3 has n o t been d e s c r i b e d .  T C e l l Growth F a c t o r (TCGF) o r I n t e r l e u k i n - 2  (IL-2)  As i n the mouse, human IL-2 has been d e r i v e d from a n t i g e n or m i t o g e n - s t i m u l a t e d T lymphocytes or  lymphomas ( J u r k a t ) .  and from primed T c e l l  Human IL-2 w i l l n o t cause  lines  proliferation  32  of unprimed T lymphocytes, a c t i n g o n l y once these c e l l s have been "primed" by a n t i g e n , mitogen, o r o t h e r a c t i v a t i n g  agents.  B C e l l S t i m u l a t i n g F a c t o r s (BSF) A s i m i l a r s i t u a t i o n w i t h r e g a r d t o B c e l l s t i m u l a t o r y and d i f f e r e n t i a t i o n - i n d u c i n g f a c t o r s e x i s t s i n humans as t h a t i n t h e murine system.  F a c t o r s c a u s i n g p r o l i f e r a t i o n and  d i f f e r e n t i a t i o n o f a c t i v a t e d B c e l l s have been d e s c r i b e d and p a r t i a l l y c h a r a c t e r i z e d (Table I I ) .  Similarly,  neuroleukin,  capable o f i n d u c i n g immunoglobulin s e c r e t i o n i n B c e l l s , has been i d e n t i f i e d  i n humans.  Enhanced p r o l i f e r a t i o n o f a c t i v a t e d  B c e l l s has a l s o been r e p o r t e d t o be an e f f e c t o f an i n t e r f e r o n - a l p h a (IFNa.) A  c)  (207).  DISTINCT MEMBRANE RECEPTORS FOR COLONY-STIMULATING FACTORS Membrane r e c e p t o r s f o r CSFs have been i d e n t i f i e d  mouse.  i n the  The murine CSFs t h a t r e g u l a t e granulocyte-macrophage  p r o d u c t i o n have d i s t i n c t , coexpressed their target c e l l s .  membrane r e c e p t o r s on  This coincides with the observation that  the CSFs appear t o have e v o l v e d s e p a r a t e l y ( n o t from a common a n c e s t r a l gene), based on d i s s i m i l a r i t i e s o f amino a c i d analyses  o f CSFs w i t h i n a s p e c i e s .  Most murine  macrophage (GM) c e l l s s i m u l t a n e o u s l y o r f o u r CSFs (multi-CSF,  express  sequence  granulocyte-  receptors f o r three  GM-CSF, M-CSF and G-CSF).  Each o f  these CSFs presumably has r e c e p t o r s on c e l l s o f o t h e r hemopoietic  l i n e a g e s as evidenced  p r o l i f e r a t i o n i n these c e l l s  by t h e i r a b i l i t y t o s t i m u l a t e  (see Table I ) .  While no apparent  33  d i r e c t competition  f o r r e c e p t o r b i n d i n g e x i s t s between  these  CSFs, t h e b i n d i n g o f a p a r t i c u l a r CSF o f t e n r e s u l t s i n t h e down-modulation and a c t i v a t i o n o f r e c e p t o r s f o r o t h e r CSF types (208): 1)  Receptor b i n d i n g o f m u l t i - C S F r e s u l t e d i n down-modulation of r e c e p t o r s f o r a l l o t h e r CSFs w i t h GM  2)  activity;  Receptor b i n d i n g o f GM-CSF caused down-modulation o f r e c e p t o r s f o r G-CSF and M-CSF (but n o t m u l t i - C S F ) ;  3)  GM-CSF r e c e p t o r s were down-modulated by h i g h  concentrations  of M-CSF; and 4)  M-CSF r e c e p t o r s were down-modulated by h i g h  concentrations  of G-CSF. Of g r e a t i n t e r e s t was t h e d i s c o v e r y t h a t t h e murine M-CSF receptor  (MW 165 KD) i s s t r u c t u r a l l y r e l a t e d and may be  i d e n t i c a l t o the product  d)  o f t h e c-fms oncogene  (209).  OTHER FUNCTIONS OF THE COLONY-STIMULATING FACTORS As mentioned p r e v i o u s l y , i n a d d i t i o n t o t h e i r  proliferative  a c t i v i t y on h e m o p o i e t i c p r e c u r s o r c e l l s , t h e myeloid o t h e r demonstrated f u n c t i o n s . 1)  These i n c l u d e :  maintenance o f t h e i n v i t r o s u r v i v a l o f h e m o p o i e t i c from p r o g e n i t o r c e l l  cells,  l e v e l up t o mature p o s t - m i t o t i c  polymorphonuclear l e u c o c y t e s 2)  CSF's have  (210-213).  an a b i l i t y t o i r r e v e r s i b l y commit m u l t i - o r b i p o t e n t i a l hemopoietic precursors  to a p a r t i c u l a r  differentiation  pathway (214,215). 3)  s t i m u l a t i o n o f v a r i o u s f u n c t i o n s i n mature (end) c e l l s .  34  The  kinds  o f f u n c t i o n a l a c t i v i t i e s s t i m u l a t e d by CSFs have  been r e p o r t e d t o i n c l u d e i n c r e a s e d antibody-dependent n e u t r o p h i l and  eosinophil-mediated  c y t o t o x i c i t y and i n c r e a s e d  phagocytosis  by n e u t r o p h i l s and macrophages. I t has been demonstrated t h a t GM-CSF and G-CSF  levels  i n c r e a s e d r a m a t i c a l l y f o l l o w i n g exposure t o b a c t e r i a l p r o d u c t s (227-229) and r e t u r n t o normal l e v e l s f o l l o w i n g e l i m i n a t i o n o f the b a c t e r i a ( o r t h e i r p r o d u c t s ) .  I n s i t u a t i o n s o f acute  b a c t e r i a l i n f e c t i o n o f a non-immune h o s t ,  such r a p i d  increases  i n serum l e v e l s o f f a c t o r s known t o s t i m u l a t e p h a g o c y t o s i s k i l l i n g might be a c r i t i c a l  f a c t o r i n the a b i l i t y of a host to  combat such i n f e c t i o n i n i t s e a r l y  2.  and  stages.  NONSPECIFIC SUBSTANCES WITH HEMOPOIETIC GROWTH PROMOTING ACTIVITY In a d d i t i o n t o t h e h e m o p o i e t i c c o l o n y - s t i m u l a t i n g  already discussed,  factors  t h e r e a r e a number o f m o l e c u l e s which p o s s e s s  p o o r l y c h a r a c t e r i z e d growth promoting a c t i v i t y f o r h e m o p o i e t i c cells.  These substances a r e o f n o n s p e c i f i c n a t u r e ,  t h e i r a c t i v i t y by i n d i r e c t means.  exerting  Some o f t h e s e m o l e c u l e s a r e  mentioned h e r e t o i l l u s t r a t e f u r t h e r t h e complex n a t u r e o f t h e i n f l u e n c e s on h e m o p o i e t i c r e g u l a t i o n . P h o r b o l e s t e r s and l i t h i u m s a l t s were shown t o induce increased  l e v e l s o f GM-CSFs i n d i r e c t l y through a c t i v i t y on  macrophages o r o t h e r mononuclear c e l l s  (230-233).  There a r e a  l a r g e number o f serum components w i t h r e p o r t e d growth promoting a c t i v i t i e s f o r cultured c e l l s .  Indeed serum c o n t a i n s  over  five  35  hundred d i f f e r e n t p r o t e i n s whose f u n c t i o n s a r e , i n g e n e r a l , poorly characterized at best. growth promoting  Serum components w i t h known  a c t i v i t i e s i n c l u d e t r a n s f e r r i n , albumin  and  numerous hormones ( t h e CSFs, p l a t e l e t - d e r i v e d growth f a c t o r , i n s u l i n and  i n s u l i n - l i k e growth f a c t o r s , t h y r o i d hormones,  t e s t o s t e r o n e , e s t r a d i o l and g l u c o c o r t i c o i d s , among many others).  The  importance  of t h e hemopoietic  colony s t i m u l a t i n g  f a c t o r s has been d i s c u s s e d , but many of the o t h e r serum components l i s t e d can p l a y a major r o l e i n hemopoietic r e g u l a t i o n i n i n v i t r o hemopoietic batches  cell  assays.  Different  of f e t a l c a l f serum o r plasma have been demonstrated t o  c o n t a i n markedly d i f f e r e n t amounts of t h y r o i d hormones, g l u c o c o r t i c o i d s , t e s t o s t e r o n e , e s t r a d i o l and  insulin  (234).  A  p a r t i c u l a r serum b a t c h can p r o f o u n d l y e f f e c t the a c t i v i t y of known CSFs.  I t has been observed  batches, only M  t h a t w i t h c e r t a i n serum  ( o r G) c o l o n i e s developed  (or at l e a s t  predominated) u s i n g the same source of GM-CSFs (235). Furthermore,  s e l e c t i v e d e l e t i o n has been shown t o be the o n l y  e f f e c t i v e method t o c o n s i s t e n t l y demonstrate an e f f e c t by g i v e n serum component on c o l o n y f o r m a t i o n i n v i t r o  (234).  a These  p o i n t s can c o m p l i c a t e i n t e r p r e t a t i o n s of r e p o r t e d e f f e c t s of p a r t i c u l a r substances  on h e m o p o i e t i c  cell  s t i m u l a t i o n or  i n h i b i t i o n and s h o u l d always be c o n s i d e r e d i n such  3.  assays.  NEGATIVE GRANULOPOIETIC REGULATORS Negative  feedback  i s an important  component i n the  p h y s i o l o g i c a l r e g u l a t i o n of many hormone systems.  Cortisol,  an  36  adrenal  s t e r o i d , a c t s as a n e g a t i v e  feedback r e g u l a t o r i n the  hypothalamic-pituitary-adrenocortical production  axis to l i m i t  of p i t u i t a r y a d r e n o c o r t i c o t r o p i n  granulopoiesis, negative e x t e n s i v e l y and i n v i t r o and  the  (ACTH).  In  r e g u l a t o r s have been s t u d i e d  c o n c l u s i v e evidence e x i s t s f o r t h e i r r o l e both  in vivo.  While t h e r e have been many r e p o r t s  g r a n u l o p o i e t i c i n h i b i t o r s (236-238, r e v i e w s ) ,  t h e r e are  of  fewer  i n h i b i t o r y m o l e c u l e s t h a t have been i n v e s t i g a t e d t h o r o u g h l y (239).  Furthermore, i t i s apparent from r e v i e w i n g  the w e l l c h a r a c t e r i z e d n e g a t i v e  r e g u l a t o r s of  reports  of  granulopoiesis  t h a t t h e r e are a myriad of i n t e r a c t i o n s o c c u r r i n g between these m o l e c u l e s and the end  the c e l l s t h a t produce them.  A c l e a r p i c t u r e of  r e s u l t i n terms of i n h i b i t i o n of g r a n u l o p o i e s i s  r e l y on an u n d e r s t a n d i n g of t h e s e i n t e r a c t i o n s . created  will  F i g u r e 1.2  was  t o emphasize the enormous c o m p l e x i t y of i n t e r p l a y  between c e l l s and (specifically,  inhibitory factors in  i n the p r o d u c t i o n  of  the  neutrophil/monocyte/macrophage l i n e a g e ) . r e g u l a t o r s t h a t w i l l be d i s c u s s e d  granulopoiesis  The  negative  are l i s t e d i n T a b l e I I I .  Lactoferrin Lactoferrin  ( L F ) , an i r o n - b i n d i n g g l y c o p r o t e i n , has  number of important p h y s i o l o g i c a l r o l e s . form, LF has  radicals. stored  In i t s i r o n - d e p l e t e d  b a c t e r i c i d a l or b a c t e r i o s t a t i c p r o p e r t i e s .  augments n e u t r o p h i l adhesiveness and I t i s synthesized  a  production  of  LF  hydroxyl  i n immature g r a n u l o c y t i c c e l l s  i n the secondary g r a n u l e s of mature c e l l s  (325).  and  37  i  O  polymorphonuclear leucocyte  F i g u r e 1.2.  I n t e r a c t i o n s between t h e n e g a t i v e g r a n u l o p o i e t i c r e g u l a t o r s .  T8 , T4 , T lymphocytes b e a r i n g T8 o r T4 c e l l s u r f a c e a n t i g e n s ; LF, l a c t o f e r r i n ; IL-1, i n t e r l e u k i n 1; TF, t r a n s f e r r i n ; a l F N , f l F N , i n t e r f e r o n a o r y; TNF, tumor n e c r o s i s f a c t o r ; GM-CSF, g r a n u l o c y t e macrophage c o l o n y - s t i m u l a t i n g f a c t o r ; CFU-GM, c o l o n y - f o r m i n g u n i t , granulocyte-macrophage; AIF, a c i d i c i s o f e r r i t i n ; L I A , l e u k e m i a - a s s o c i a t e d i n h i b i t o r y a c t i v i t y ; PGE, E-type p r o s t a g l a n d i n s ; +  +  a = l a r g e , n o n - p h a g o c y t i c , non-adherent, F c bone marrow c e l l i n normals and m y e l o i d leukemics b = LIA = leukemia-associated i n h i b i t o r y a c t i v i t y c = n e g a t i v e r e g u l a t o r produced by normal adherent BM c e l l s * s i g n i f i e s d i f f e r e n c e i n r e s p o n s i v e n e s s t o n e g a t i v e r e g u l a t i o n by t h e f a c t o r i n m y e l o i d leukemia p a t i e n t s v e r s u s normals. -» = p o s i t i v e ( s t i m u l a t o r y ) s i g n a l -» = n e g a t i v e ( s u p p r e s s i v e o r i n h i b i t o r y ) s i g n a l +  38  Table I I I .  Negative g r a n u l o p o i e t i c  Regulator (abbreviation), A l t e r n a t e Name  Molecular  regulators  Weight  References  85-100  KD  240-259  A c i d i c i s o f e r r i t i n s (AIF), Leukemia-associated i n h i b i t o r y a c t i v i t y (LIA)  19-  21  KD  260-272  Interferons  (IFN-a, IFN-B, IFN-y)  15-  40  KD  273-284  Transferrin  (TF)  L a c t o f e r r i n (LF), colony i n h i b i t i n g a c t i v i t y (CIA)  E-type p r o s t a g l a n d i n s Leukemia-associated (LAI)  80 (PGEi,PGE2) inhibitor  KD  286-300  350 Native: Active:  239,255,285  > 500 KD 150-170 KD  301-303  Tumor n e c r o s i s f a c t o r (TNF), LuKII  Native: 40-45 KD SDS-PAGE: 17-18 KD components  304-310  Granulocytic  4 p e p t i d e s of 20-30 amino a c i d s each  311-314  chalone  Other: a) C o l o n y - i n h i b i t i n g lymphokine (CIL) b) n e g a t i v e r e g u l a t o r produced by normal adherent BM c e l l s c) u n s p e c i f i e d c e l l p r o d u c t s from leukemics o r a p l a s t i c anemics  ND  = not  described  85 KD ND ND  315 316 317-324  39  There a r e numerous r e p o r t s c o n c e r n i n g the r o l e o f LF i n the i n h i b i t i o n o f granulocyte/macrophage  c o l o n y forming c e l l s  (CFU-GM) ( T a b l e I I I ) .  i l l u s t r a t e s , the cumulative  As F i g u r e 1.2  e f f e c t of LF i n t h i s r e g a r d i s the r e s u l t o f many i n t e r a c t i o n s w i t h o t h e r r e g u l a t o r y m o l e c u l e s and w i t h i t s t a r g e t p o p u l a t i o n , a l l of which may  cell  v a r y i n amount a t any g i v e n time.  LF has been shown t o e f f e c t the f o l l o w i n g : 1)  b i n d i n g t o s p e c i f i c r e c e p t o r s on HLA-DR-positive human monocytes (246,253) and I a (I-A and I-E/C) a n t i g e n - p o s i t i v e murine macrophages (258), i n h i b i t i n g the r e l e a s e o f GM-CSF from t h e s e c e l l s  2)  (248,258).  i n h i b i t i o n of p r o d u c t i o n and/or s e c r e t i o n of a from human monocytes which causes o t h e r c e l l  monokine(s)  types (T  lymphocytes, f i b r o b l a s t s , e n d o t h e l i a l c e l l s ) t o r e l e a s e GM-CSF 3)  (250,256,257).  i n h i b i t i o n of t h e p r o d u c t i o n and/or r e l e a s e by human monocytes of two o t h e r i n h i b i t o r y m o l e c u l e s , a c i d i c isoferritins F i g u r e 1.2  (251) and p r o s t a g l a n d i n E (247).  shows t h a t the e f f e c t of LF i s not the same i n  m y e l o i d leukemia p a t i e n t s as t h a t seen i n normals. d i f f e r e n c e i s m a n i f e s t i n two ways.  This  I n the f i r s t p l a c e ,  polymorphonuclear l e u c o c y t e s (PMN's) from leukemia p a t i e n t s have lower l e v e l s o f endogenous LF than normals, and  (perhaps of  g r e a t e r s i g n i f i c a n c e ) t h e i r PMN's c o n t a i n low o r u n d e t e c t a b l e l e v e l s o f the a c t i v e form o f LF (240,245,254,326,327). the  Secondly  LF-mediated s u p p r e s s i o n o f GM-CSF from monocytes o f leukemia  p a t i e n t s i s d e c r e a s e d compared t o t h a t o f normals  40  (253,328,329).  While p a r t o f t h e e x p l a n a t i o n f o r t h i s may  r e s i d e i n t h e decreased number o f PMN's p r e s e n t i n a c u t e leukemics  i n the a c t i v e s t a t e of t h e i r disease, t h i s  a l o n e cannot e x p l a i n t h e s i t u a t i o n and/or amount o f L F , e s p e c i a l l y leukemics. of  effects  factor  responsiveness  i n t h e case o f c h r o n i c m y e l o i d  As F i g u r e 1.2 p o i n t s out (* = d i f f e r e n c e i n e f f e c t  inhibitors  situation  o f decreased  myeloid  i n myeloid  leukemics  compared t o n o r m a l s ) ,  i s b u t t h e f i r s t o f many such s i t u a t i o n s  this  i n which t h e  seen by a n e g a t i v e r e g u l a t o r o f normal m y e l o p o i e s i s a r e  different  i n t h e case o f leukemics.  The c u m u l a t i v e  e f f e c t s of  t h e s e d i f f e r e n c e s may be o f tantamount importance i n t h e e x p l a n a t i o n o f how t h e leukemic normal m y e l o i d p r o g e n i t o r c e l l s ,  c l o n e g a i n s a f o o t h o l d over enabling the malignant  c e l l s to  a t t a i n a s m a l l b u t d i s t i n c t growth advantage over t h e i r normal counterparts.  Acidic  Isoferritins  The  i s o f e r r i t i n s a r e comprised  o f two types o f s u b u n i t s , H  ( m o l e c u l a r weight 21 KD) which i s a s s o c i a t e d w i t h h e a r t  tissue  and L ( m o l e c u l a r weight 19 KD) which i s a s s o c i a t e d w i t h  liver.  The  a c i d i c i s o f e r r i t i n s (AIF) a r e composed m a i n l y  and  a r e produced by HLA-DR-positive monocytes/macrophages  (268,330).  of H subunits  T a r g e t c e l l s a r e s i m i l a r l y r e s t r i c t e d by C l a s s I I  antigenic expression.  A I F have been shown t o cause a  s u p p r e s s i o n o f i n v i t r o CFU-GM c o l o n y f o r m a t i o n i n t h e mouse and of CFU-GEMM, BFU-E and CFU-GM c o l o n y f o r m a t i o n i n humans (266,270).  Both i n t e r f e r o n - a l p h a and p r o s t a g l a n d i n E can  41  augment t h e e f f e c t o f AIF w h i l e b o t h LF and interferon-gamma can inhibit i t s effect  ( F i g u r e 1.2).  As w i t h LF, t h e r e i s a d i f f e r e n c e between normals and myeloid  leukemics  w i t h r e s p e c t t o t h e q u a n t i t y o f and  s e n s i t i v i t y t o AIF. cells  AIF l e v e l s a r e h i g h e r i n leukemia p a t i e n t s *  (261,268) b u t , s i n c e t h e r e i s a d e f i c i e n c y o f I a a n t i g e n s  on t h e m y e l o i d p r o g e n i t o r c e l l s o f leukemia p a t i e n t s , they l a c k s e n s i t i v i t y t o the i n h i b i t o r y population of r a p i d l y  effects  o f AIF.  p r o l i f e r a t i n g myeloid  A study o f a  leukemic  blast  cells  i n v i t r o a l s o demonstrated t h e i r l a c k o f s e n s i t i v i t y t o i n h i b i t i o n by A I F (263).  Interferons Interferons  (IFN) a r e comprised  o f a group o f g l y c o p r o t e i n s  which a r e produced and s e c r e t e d by c e l l s i n response t o i n f e c t i o n w i t h v i r u s e s and o t h e r s t i m u l i . of  interferons —  by f i b r o b l a s t s ,  myelopoiesis  types  IFN-beta ( f i b r o b l a s t IFN) which i s produced IFN-gamma (immune IFN) which i s produced by T  lymphocytes, and IFN-alpha It i s clear  There a r e t h r e e  ( l e u c o c y t e IFN), o f l e u c o c y t e o r i g i n .  t h a t IFNs p l a y an i n h i b i t o r y r o l e i n  (Table I I I ) .  Colony  f o r m a t i o n by m u l t i p o t e n t i a l  (CFU-GEMM) and e r y t h r o i d (BFU-E) p r o g e n i t o r s i s suppressed by all  t h r e e IFN's t o t h e same degree (239).  effective IFN-B). (not  IFN-gamma i s most  i n t h e s u p p r e s s i o n o f CFU-GM (IFN-y > IFN-o > The i n h i b i t o r y  e f f e c t on c o l o n y f o r m a t i o n i s d i r e c t  a r e s u l t o f a c t i o n upon an i n t e r m e d i a r y c e l l type o r i t s  products).  I t has been shown t h a t IFN-alpha  blocks  42  d i f f e r e n t i a t i o n beyond t h e myelocyte l e v e l  (277,280).  As F i g u r e  1.2 i l l u s t r a t e s , t h e r e a r e a number o f i n t e r a c t i o n s t h a t occur between IFNs and o t h e r n e g a t i v e and  IFN-gamma s y n e r g i z e  concentrations  regulatory molecules.  t o suppress c o l o n y  formation  IFN-alpha i n vitro at  o f each t h a t have no e f f e c t whatsoever were each  to be used a l o n e (310).  The p r o d u c t i o n  and/or r e l e a s e o f  IFN-alpha i s suppressed by t r a n s f e r r i n and  prostaglandin  E and  augmented by IL-1. There appears t o be no d i f f e r e n c e between t h e s e n s i t i v i t i e s of m y e l o i d p r o g e n i t o r respect  c e l l s o f leukemia p a t i e n t s o r normals w i t h  t o t h e IFN's, w i t h t h e e x c e p t i o n  suppresses IFN-alpha-induced e x p r e s s i o n  that prostaglandin of I a antigens,  s i t u a t i o n that could e f f e c t the actions of other r e g u l a t o r s known t o be so r e s t r i c t e d  E  a  negative  (LF, A I F , T F ) .  Transferrin T r a n s f e r r i n ( T F ) , l i k e L F , i s an i r o n - b i n d i n g  glycoprotein,  p l a y s a b a c t e r i o s t a t i c r o l e and has an i n h i b i t o r y e f f e c t on myelopoiesis.  TF i s r e l e a s e d by a p o p u l a t i o n  of T 8 - p o s i t i v e T  lymphocytes and suppresses t h e r e l e a s e o f GM-CSF from lectin-stimulated T4-positive, Class I I antigen-positive  cells  (285). In normals, TF i s r e l e a s e d T8  cells.  from C l a s s I I a n t i g e n - p o s i t i v e  However, i n leukemia p a t i e n t s , TF i s r e l e a s e d  Class I I antigen-negative  from  T8 c e l l s and t h e TF-mediated  i n h i b i t o r y a c t i v i t y derived  from t h e s e c e l l s i s i n c r e a s e d  (239).  43  E-Type P r o s t a g l a n d i n s E-type p r o s t a g l a n d i n s  (PGE^  and PGE^)  a r e produced  human b l o o d monocytes and murine p e r i t o n e a l macrophages  by (288).  PGEs a c t by s e l e c t i v e i n h i b i t i o n of I a a n t i g e n - p o s i t i v e monocyte-macrophage p r o g e n i t o r s (267,299).  Their  inhibitory  e f f e c t i s augmented by t h e i r a b i l i t y t o s t i m u l a t e I a a n t i g e n e x p r e s s i o n on m y e l o i d responsiveness  p r o g e n i t o r c e l l s , thereby  i n c r e a s i n g the  of these c e l l s to n e g a t i v e r e g u l a t i o n by AIF  the PGEs themselves (239).  and  I n t e r e s t i n g l y , PGEs can cause a 30  -  70% enhancement of BFU-E p r o d u c t i o n i n v i t r o which i s T cell-dependent  and d i r e c t l y r e l a t e d t o the e x p r e s s i o n of C l a s s  I I a n t i g e n s on the BFU-E p r o g e n i t o r s d e r i v e d CSF  (331).  s t i m u l a t e s the p r o d u c t i o n of PGE.  Monocyte/macrophageLF can  inhibit  PGE  p r o d u c t i o n . These i n t e r a c t i o n s are summarized i n F i g u r e  and  f u r t h e r s e r v e t o i l l u s t r a t e the complex n a t u r e  s t i m u l a t o r y and  of  1.2  the  i n h i b i t o r y i n f l u e n c e s of the r e g u l a t o r s of  myelopoiesis. Again,  an abnormal r e s p o n s i v e n e s s  been i d e n t i f i e d (293-296).  i n myeloid  to the e f f e c t of PGE  leukemia p a t i e n t s * p r o g e n i t o r  Decreased s e n s i t i v i t y to PGE-mediated  Leukemia-Associated  observed  cells  inhibition,  r e l a t e d to an i n s u f f i c i e n c y of C l a s s I I a n t i g e n s on p r o g e n i t o r c e l l s , was  has  the  compared t o normal c o n t r o l s .  Inhibitor  Leukemia-associated  inhibitor  (LAI) has been r e p o r t e d to  reduce the number of normal CFU-c i n S-phase but has no on c l o n o g e n i c c e l l s from a c u t e o r c h r o n i c m y e l o i d  effect  leukemics  44  (301) .  The i n h i b i t o r y a c t i v i t y o f t h i s m o l e c u l e was shown t o be  p r e s e n t i n a s u b u n i t (150-170 KD) o f i t s n a t i v e form (> 500 KD) (302) .  LAI i s produced by c e l l s p r e s e n t i n b o t h normals and  m y e l o i d leukemics i n d i c a t i n g t h a t i t may p l a y a r e g u l a t o r y i n normal m y e l o p o i e s i s . the same i n b o t h normal  The phenotype o f L A I - p r o d u c i n g and leukemic s t a t e s  h o n - p h a g o c y t i c , non-adherent, cell.  role  cells,  (303), i s a l a r g e ,  F c - r e c e p t o r p o s i t i v e bone marrow  I n c r e a s e d p r o d u c t i o n o f such an i n h i b i t o r , c a p a b l e o f  r e d u c i n g t h e p r o l i f e r a t i v e r a t e o f normal  granulopoietic  p r o g e n i t o r s , c o u l d h e l p t o e x p l a i n t h e growth advantage o f m a l i g n a n t m y e l o i d c e l l s over  normals.  Tumor N e c r o s i s F a c t o r Tumor n e c r o s i s f a c t o r  (TNF) was named over a decade ago t o  d e s c r i b e a serum substance induced by e n d o t o x i n t h a t was c a p a b l e of  causing s e l e c t i v e t o x i c i t y f o r malignant c e l l s  (304).  Since  t h a t time, TNF has been p u r i f i e d from r a b b i t and mouse serum (332) and from human c e l l cloned  lines  (305,306).  Human TNF has been  (305,333) and i t i s now apparent t h a t a m o l e c u l e  i n t h e case o f t h e i n t e r f e r o n s , a f a m i l y o f r e l a t e d  ( o r , as  molecules)  i d e n t i c a l t o t h a t d e s c r i b e d as TNF i s produced by human monocytes (334) and n a t u r a l k i l l e r myeloid  cells  (308) as w e l l as by  (eg. HL-60) and l y m p h o b l a s t o i d (eg. LuKII) c e l l  Human TNF has been shown t o cause s i g n i f i c a n t  lines.  (50 - 98%)  i n h i b i t i o n o f CFU-GM as w e l l as i n h i b i t i o n o f BFU-E and CFU-GEMM (310).  U n l i k e the p r e v i o u s l y d e s c r i b e d negative r e g u l a t o r s of  m y e l o p o i e s i s , TNF causes more i n h i b i t i o n o f CFU-c ( c l u s t e r s < 40  45  c e l l s ) i n n o n - r e m i s s i o n ANLL p a t i e n t s than i n those i n r e m i s s i o n o r normals  (310).  Granulocytic  Chalone  The concept o f g r a n u l o c y t i c " c h a l o n e s " dates back over two decades.  Chalones a r e d e s c r i b e d as t i s s u e - s p e c i f i c ,  s p e c i e s - n o n s p e c i f i c n e g a t i v e feedback r e g u l a t o r substances  that  a r e produced by mature c e l l s w i t h i n t h e same l i n e a g e as t h a t i n which t h e i r e f f e c t s a r e m a n i f e s t i n immature c e l l s  (335).  The  g r a n u l o c y t i c chalone has been r e p o r t e d t o be a p o l y p e p t i d e o f 20 - 30 amino a c i d s (311,312).  The e x i s t e n c e o f t h e  g r a n u l o c y t i c chalone as o r i g i n a l l y d e f i n e d has been q u e s t i o n e d (314), a l t h o u g h many i n v e s t i g a t i o n s have concluded t h a t mature polymorphonuclear  leucocytes (neutrophils) contain negative  feedback r e g u l a t o r y s u b s t a n c e s .  These m o l e c u l e s have been  r e p o r t e d t o suppress t h e p r o l i f e r a t i o n o f g r a n u l o c y t i c p r e c u r s o r cells.  In l i g h t of the i n v e s t i g a t i o n s concerning the r e l e a s e of  lactoferrin  (LF) from t h e s e c e l l s ,  i t i s very p o s s i b l e that  this  substance was r e s p o n s i b l e f o r t h e i n h i b i t o r y e f f e c t s observed by s o l u b l e p r o d u c t s d e r i v e d from polymorphonuclear l e u c o c y t e s , a l t h o u g h i t i s d i f f i c u l t t o r e c o n c i l e the r e p o r t e d d i f f e r e n c e s i n m o l e c u l a r weight  (LF = 85 - 100 KD, chalone = 25 amino a c i d s )  of t h e s e s u b s t a n c e s .  Other Reported  I n h i b i t o r s of Myelopoiesis  A number o f o t h e r l e s s w e l l c h a r a c t e r i z e d i n h i b i t o r s o f g r a n u l o p o i e s i s have been r e p o r t e d (315-324).  Whether o r n o t  46  t h e s e substances a r e d i f f e r e n t from t h o s e a l r e a d y or  a)  investigated  have r e l e v a n c e i n v i v o has y e t t o be proven.  Colony-inhibiting  lymphokine  C o l o n y - i n h i b i t i n g lymphokine  (CIL) i s a substance o f  r e p o r t e d m o l e c u l a r weight 85 KD which i s s y n t h e s i z e d and r e l e a s e d from a human hybridoma  cell  line  (6TM), a f u s i o n  between PHA-stimulated normal lymphocytes and a mutant J u r k a t T lymphoma c e l l  line  (315).  A c o r r e l a t i o n between e x p r e s s i o n o f  C l a s s I I a n t i g e n s and s e n s i t i v i t y t o CIL was found i n human c e l l l i n e s and bone marrow p r o g e n i t o r s (336).  CIL caused a massive  i n h i b i t i o n o f CFU-GM as w e l l as CFU-GEMM, BFU-E and CFU-E.  This  v e r y i n t e r e s t i n g lymphokine may prove t o have s i g n i f i c a n c e i n the  r e g u l a t i o n o f normal and/or abnormal m y e l o p o i e s i s .  Recent  e v i d e n c e i n d i c a t e s t h a t CIL s p e c i f i c a l l y r e c o g n i z e s a m o l e c u l a r s t r u c t u r e common t o b o t h human and murine I a m o l e c u l e s (336) and i s capable of b l o c k i n g stimulator c e l l lymphocyte r e a c t i o n s  (MLR).  r e c o g n i t i o n i n mixed  The g e n e r a t i o n o f immune responses  and r e g u l a t i o n o f hemopoiesis must be l i n k e d i n a g r e a t many ways; CIL may p r o v e t o be one o f t h e r e g u l a t o r y m o l e c u l e s involved i n this b)  interaction.  An u n d e f i n e d n e g a t i v e r e g u l a t o r y substance produced by  normal adherent marrow c e l l s i n v i t r o causes p r i m i t i v e p r o g e n i t o r c e l l s t o go i n and o u t o f c e l l  cycle.  normal  This negative  c o n t r o l on t h e p r o l i f e r a t i v e c a p a c i t y o f normal b l o o d p r o g e n i t o r s had no e f f e c t on CGL p r o g e n i t o r s o f marrow o r b l o o d o r i g i n , which remained c o n t i n u o u s l y i n c y c l e i n t h e p r e s e n c e o f  47  the normal marrow adherent c e l l s another case i n which normal  (316).  T h i s appears t o be  i n h i b i t o r y signals are i n e f f e c t i v e  i n t h e c o n t r o l o f leukemic p r o g e n i t o r c e l l s , c)  U n s p e c i f i e d c e l l p r o d u c t s from p a t i e n t s w i t h h e m o p o i e t i c disorders There have been a number o f r e p o r t s which have i n d i c a t e d  t h a t m y e l o i d leukemic c e l l s can suppress t h e p r o l i f e r a t i o n o f c o - c u l t u r e d normal CFU-GM i n v i t r o  (317-319,321,323,324).  S i m i l a r l y , t h e c o - c u l t u r e o f marrow o r b l o o d c e l l s from  aplastic  anemics w i t h normals has been shown t o cause i n h i b i t i o n o f normal CFU-c and e r y t h r o i d c o l o n y p r o d u c t i o n (320,322).  The  p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f t h e f a c t o r s i n v o l v e d i n t h i s s u p p r e s s i o n o f normal hemopoiesis awaits f u r t h e r study and may prove t o i n c l u d e some o f t h e n e g a t i v e r e g u l a t o r s p r e v i o u s l y described.  4.  MICROENVIRONMENTAL INFLUENCES ON HEMOPOIESIS The r o l e o f s p e c i f i c t i s s u e microenvironments  i n the  r e g u l a t i o n o f hemopoiesis  i s c l e a r l y e s t a b l i s h e d , although i n  most c a s e s , n o t p r e c i s e l y  characterized.  In t h e case o f l y m p h o p o i e s i s , m i c r o e n v i r o n m e n t a l r e g u l a t i o n appears t o be an a b s o l u t e requirement f o r t h e complete development  o f b o t h T and B lymphocytes.  Pre-T c e l l s from t h e  bone marrow m i g r a t e t o t h e thymus and become "educated" w i t h i n t h i s organ t o become mature a n t i g e n - s p e c i f i c , M H C - r e s t r i c t e d f u n c t i o n a l end c e l l s .  Thymic e p i t h e l i a l and n u r s e c e l l s  the c o r t e x a r e thought t o be r e s p o n s i b l e f o r t h i s  within  regulation  48  through the p r o d u c t i o n  of T c e l l  developmental f a c t o r s .  B  lymphocytes from the bone marrow complete t h e i r development w i t h i n secondary lymphoid organs, n o t a b l y  the s p l e e n  and  lymph  nodes. M i c r o e n v i r o n m e n t a l i n f l u e n c e s on m y e l o p o i e s i s been e s t a b l i s h e d .  Most e v i d e n c e has  CFU-S i n i r r a d i a t e d r e c i p i e n t mice. spleens CFU  have a l s o  come from the growth of CFU-S d e v e l o p i n g  i n the  of such r e c i p i e n t s were p r e d o m i n a n t l y e r y t h r o i d , w h i l e  developing  i n the BM  of the same mice were composed  p r i m a r i l y of g r a n u l o c y t i c c e l l s  (5,337).  i m p l i e d t h a t " e r y t h r o p o i e t i c " and  This  "granulopoietic"  e x i s t e d w i t h i n d i f f e r e n t h e m o p o i e t i c organs. were g r a f t e d i n t o the s p l e e n , granulocytic  developing  ( i n the marrow c a v i t y ) and  splenic tissue) lineage r e s t r i c t i o n . c o l o n i e s has,  observation niches  When bone s h a f t s  colonies displayed erythroid (in  adjacent  A n a l y s i s of s p l e n i c  however, i n d i c a t e d t h a t m u l t i p o t e n t i a l  d i f f e r e n t i a t i o n e x i s t s w i t h i n most c o l o n i e s . requirement f o r m i c r o e n v i r o n m e n t a l c e l l always a b s o l u t e ,  as i t has  Moreover,  i n f l u e n c e s are  the not  been e s t a b l i s h e d t h a t a s i n g l e  colony-forming c e l l  i n the absence of o t h e r  i n t o a mixed c o l o n y  i n v i t r o containing various progenitor  p o t e n t i a l i t i e s w i t h i n i t s own The  both  c e l l s can  develop cell  milieu (5).  Dexter long-term bone marrow c u l t u r e system  has  demonstrated t h a t l i n e a g e commitment by m u l t i p o t e n t i a l stem c e l l s does o c c u r i n the p r e s e n c e of adherent s t r o m a l layers.  The  cell  p r e c i s e mechanisms c o n t r o l l i n g such commitment  w e l l as stem c e l l  s u r v i v a l i n t h i s system are n o t  well  as  49  c h a r a c t e r i z e d a t p r e s e n t and may cloned stromal c e l l  r e q u i r e the e s t a b l i s h m e n t of  l i n e s i n o r d e r t o d e f i n e the i n f l u e n c e s of  c e l l u l a r m a t r i c e s and/or humoral r e g u l a t o r s w i t h i n the microenvironment. The c l a s s i c a l  example p e r t a i n i n g t o the importance o f the  bone marrow s t r o m a l microenvironment hemopoiesis  i n the r e g u l a t i o n o f  comes from the study of S l / S l  mice, which p o s s e s s d  a g e n e t i c a l l y - d e t e r m i n e d m a c r o c y t i c anemia t r a i t . have normal  Sl/Sl  mice  stem c e l l s , as e v i d e n c e d by the a b i l i t y of t h e i r  bone marrow t o r e c o n s t i t u t e hemopoiesis i n the stem v c e l l - d e f i c i e n t W/W mouse s t r a i n . T r a n s p l a n t a t i o n o f mouse d stem c e l l s from normal BM f a i l e d t o cure the S l / S l defect, i n d i c a t i n g t h a t i n S l / S l * * mice,  the h e m o p o i e t i c  deficiency  r e l a t e d t o an abnormal s t r o m a l microenvironment,  poorly  c o n d u c i v e t o the s u r v i v a l and p r o l i f e r a t i o n of h e m o p o i e t i c cells Sl/Sl  (22).  Furthermore,  stem  i n long-term "Dexter" marrow c u l t u r e s ,  adherent s t r o m a l c e l l s f a i l e d t o support  hemopoiesis  d but S l / S l  h e m o p o i e t i c stem c e l l s p r o l i f e r a t e d n o r m a l l y when  grown on normal BM proposed  stromal c e l l s  (338).  Finally,  Zipori  t h a t , f o r any i n d i v i d u a l CFU-c, i t s r e l a t i v e  position  w i t h i n o r nearby the marrow s t r o m a l c e l l p o p u l a t i o n would determine  i t s fate.  I t was  demonstrated  t h a t exogenous crude o r  pure sources of CSF had decreased a c t i v i t y  i n the presence of  i n c r e a s i n g numbers of adherent murine marrow s t r o m a l c e l l s (339).  This r e s t r a i n t i n d i f f e r e n t i a t i o n occurred concomitantly  w i t h i n c r e a s e d s e l f renewal p r o g e n i t o r CFU-c  maintenance.  Obviously both microenvironmental c e l l s w i t h i n hemopoietic  50  t i s s u e s and humoral f a c t o r s p l a y important r o l e s i n t h e b a s i c r e g u l a t i o n o f hemopoiesis, b o t h l i k e l y o p e r a t i n g a t a l l c e l l levels  (stem t o p r o g e n i t o r c e l l p o p u l a t i o n s ) .  THE HUMAN MYELOID LEUKEMIAS There a r e two major forms o f human m y e l o i d leukemia based on t h e i r c l i n i c a l nature —  c h r o n i c g r a n u l o c y t i c leukemia  a c u t e n o n l y m p h o b l a s t i c leukemia  (ANLL).  o r i g i n a t i n g i n h e m o p o i e t i c stem c e l l s .  (CGL) and  Both a r e c l o n a l m a l i g n a n c i e s Such c e l l s a r e l i k e l y o p t i m a l  t a r g e t s f o r n e o p l a s t i c t r a n s f o r m a t i o n due t o t h e i r n o r m a l l y e x t e n s i v e potential for proliferation.  A.  CHRONIC GRANULOCYTIC  LEUKEMIA  C l i n i c a l Course and P a t h o g e n e s i s C h r o n i c g r a n u l o c y t i c leukemia i s a m y e l o i d c e l l malignancy runs a c h r o n i c c o u r s e o f two t o t h r e e y e a r s d u r a t i o n . c h a r a c t e r i z e d by a massive  which  CGL i s  expansion o f m y e l o i d p r o g e n i t o r c e l l  p o p u l a t i o n s , r e s u l t i n g i n e x c e s s i v e numbers o f (predominantly) g r a n u l o c y t e s a t a l l s t a g e s o f m a t u r a t i o n which i n f i l t r a t e t h e c i r c u l a t i o n , t h e s p l e e n and o t h e r t i s s u e s  (340-342).  Evidence shows t h a t CGL may develop as a r e s u l t o f r a d i a t i o n exposure,  a notable i n c r e a s e i n the i n c i d e n c e of the disease being  observed i n s u r v i v o r s o f t h e atomic bomb a t t a c k on H i r o s h i m a (343,344).  However i n t h e m a j o r i t y o f c a s e s , no s p e c i f i c agent has  been i d e n t i f i e d .  I n t e r e s t i n g l y , i n a p a t i e n t h a v i n g undergone  a l l o g e n e i c bone marrow t r a n s p l a n t a t i o n , r e l a p s e o f t h e d i s e a s e i n donor c e l l s  ( w i t h a c q u i s i t i o n o f t h e Ph' chromsome) was r e c e n t l y  51  reported  (345) i n d i c a t i n g t h e p o s s i b i l i t y t h a t a p e r s i s t i n g  oncogenic  agent o r event c o u l d be i n v o l v e d i n some c a s e s . Over 90% o f CGL p a t i e n t s demonstrate  t h e Ph' chromosome, a  r e c i p r o c a l t r a n s l o c a t i o n t (9;22) between t h e l o n g arms o f chromosomes 9 and 22, i n the leukemic c l o n e (66,67).  During  p r o g r e s s i o n o f t h e d i s e a s e , t h e Ph' chromosome-positive  c e l l s become  more numerous due t o t h e i r growth advantage  cells.  over normal  E v e n t u a l l y g r e a t e r than 99% o f t h e d i v i d i n g c e l l s i n t h e bone marrow are Ph'-positive.  U l t i m a t e l y most p a t i e n t s e n t e r an " a c c e l e r a t e d "  phase o r an acute leukemia s i t u a t i o n  (blast c r i s i s ) .  In t h e b l a s t  c r i s i s s t a g e , a p p r o x i m a t e l y 65 - 75% o f p a t i e n t s develop an acute m y e l o i d - t y p e leukemia o f t e n r e f r a c t o r y t o treatment.  The r e m a i n i n g  25 - 35% develop an acute l y m p h o b l a s t i c leukemia, o f t e n r e s p o n s i v e t o treatment w i t h v i n c r i s t i n e and p r e d n i s o n e .  Stem C e l l  Origin  Karyotypic  (Ph* chromosome) and enzymatic  (G-6-PD) s t u d i e s have  p r o v i d e d c o n c l u s i v e e v i d e n c e t h a t CGL i s a c l o n a l d i s e a s e o f p l u r i p o t e n t stem c e l l s .  Indeed,  as mentioned  previously,  this  d i s e a s e has g i v e n credence t o t h e e x i s t e n c e o f a common lymphoid-myeloid  stem c e l l  i n humans.  The Ph' chromosome has been found i n g r a n u l o c y t e s , monocytes, macrophages, n u c l e a t e d e r y t h r o i d c e l l s , megakaryocytes,  basophils,  e o s i n o p h i l s and t h e i r p r o g e n i t o r s as w e l l as B c e l l s (68-70,346,347).  Sex chromosome mosaicism  (46 XY Ph'/47 XXY) i n 2  CGL p a t i e n t s i n d i c a t e d t h e m o n o c l o n a l i t y o f t h e d i s e a s e  (348,349).  52  Glucose-6-phosphate dehydrogenase isoenzyme s t u d i e s heterozygous females w i t h CGL  a l s o i n d i c a t e d t h a t the d i s e a s e was  c l o n a l nature, o r i g i n a t i n g i n a p l u r i p o t e n t discussed  previously  stem c e l l .  has  T h i s has  c r i s i s phase of the d i s e a s e . c e l l s have been shown to be  While most CGL  in blast  blast crisis  patients*  of m y e l o i d o r i g i n (350), a number of  cases have shown lymphoid c h a r a c t e r i s t i c s (350,351). (352-354) have demonstrated u s i n g  markers, t h a t a p p r o x i m a t e l y o n e - t h i r d  of CGL  Greaves  blast crisis  patients the  common (c)ALL v a r i e t y , l e s s f r e q u e n t l y  of n u l l c e l l phenotype.  l e s s f r e q u e n t have been r e p o r t s  blast crises involving  of CGL  w i t h T-lymphocyte phenotypes (355-357).  c l o n e as t h a t  and  immunological membrane  examined p o s s e s s e d a lymphoid c e l l phenotype, most o f t e n of  c e l l s were Ph'  been  stem c e l l o r i g i n of  come from i n v e s t i g a t i o n s of the c e l l s i n v o l v e d  colleagues  of  (72-74).  S t r o n g e v i d e n c e s u p p o r t i n g the p l u r i p o t e n t CGL  in  Much cells  A l l of these lymphoid b l a s t  p o s i t i v e , i n d i c a t i n g t h a t they a r o s e from the same involved  i n the  chronic  " g r a n u l o c y t i c " phase of  the  disease.  Chromosomal Changes and Two  known c e l l u l a r  Their Possible  ( p r o t o - ) oncogenes, c - s i s and  been i d e n t i f i e d as b e i n g i n v o l v e d t r a n s l o c a t i o n of the Ph'  Significance  i n the r e c i p r o c a l t  chromosome (358-361).  normal l o c a t i o n of c - s i s , the  c - a b l , have (9;22)  Chromosome 22  c e l l u l a r homologue of the  is  the  Simian  sarcoma v i r u s oncogene, which encodes p l a t e l e t - d e r i v e d growth f a c t o r (362).  Chromosome 9 i s the normal l o c a t i o n of c - a b l , the  cellular  53  homologue of the murine A b e l s o n leukemia v i r u s oncogene.  The  Ph*  chromosome i n v o l v e s r e c i p r o c a l t r a n s l o c a t i o n of b o t h proto-oncogenes. E x p r e s s i o n of c - s i s i s not g e n e r a l l y d e t e c t a b l e i n CGL, it  making  l i k e l y t h a t t h i s proto-oncogene p l a y s a l e s s e r r o l e , i f any,  the p a t h o g e n e s i s of CGL.  in  However, t h e c o n s i s t e n t t r a n s l o c a t i o n of  c-abl to a s p e c i f i c breakpoint  a r e a on chromosome 22 near the  lambda  l i g h t c h a i n immunoglobulin gene l o c u s r e s u l t s i n a m p l i f i c a t i o n and anomalous t r a n s c r i p t i o n of the c - a b l gene (359,361,363,364). abnormal gene p r o d u c t  has been shown t o have t y r o s i n e k i n a s e  s i m i l a r t o t h a t of many known v i r a l t r a n s f o r m i n g u n l i k e t h a t of i t s normal c e l l u l a r homologue. expression  of c - a b l may  of hemopoiesis i n Considerable  This activity  p r o t e i n s (365)  This  and  aberrant  be of s i g n i f i c a n c e i n the abnormal  expression  CGL. evidence  p a t h o g e n e s i s f o r CGL.  e x i s t s which s u p p o r t s  In many c a s e s ,  a multistep  a c q u i s i t i o n of the  Ph'  chromosome appeared t o be a secondary event i n the p r o g r e s s i o n of disease.  A number of p a t i e n t s p r e s e n t i n g w i t h c l i n i c a l CGL  that  the was  Ph' n e g a t i v e were l a t e r shown t o become Ph' p o s i t i v e (366-368).  The  p r e s e n c e of Ph' n e g a t i v e b l a s t c e l l c r i s i s f o l l o w i n g Ph' p o s i t i v e c h r o n i c phase and  the d i s c o v e r y of Ph' n e g a t i v e B-lymphocytes d e r i v e d  from the same G-6-PD isoenzyme c l o n e as the Ph' c e l l s provided (369-371).  f u r t h e r evidence  t h a t CGL  had  p o s i t i v e leukemia  a multistep pathogenesis  Many i n v e s t i g a t o r s b e l i e v e t h a t CGL,  p l u r i p o t e n t stem c e l l ,  originating in a  i n v o l v e s a s u c c e s s i o n of g e n e t i c changes  of which i s a c q u i s i t i o n of the Ph'  chromosome).  These  (one  successive  a l t e r a t i o n s , t o genes t h a t a r e l i k e l y c l o s e l y t i e d t o r e g u l a t i o n of the b a s i c c e l l u l a r p r o c e s s e s  of p r o l i f e r a t i o n and  differentiation,  54  a r e thought t o r e s u l t i n a p r o g r e s s i v e u n c o u p l i n g o f the normal s i g n a l s c o n t r o l l i n g p r o l i f e r a t i o n and subsequent (354-372).  maturation  I n l i g h t of t h i s view, the c h r o n i c phase of CGL  j u s t i f i a b l y be c o n s i d e r e d as a " p r e m a l i g n a n t " o r a t l e a s t l e u k e m i c " phase.  The b l a s t c r i s i s  stage of CGL  commonly i n c l u d e t r i s o m y 8, isochromosome 17 o r Ph' (373,374).  "pre-acute  i s associated with  a d d i t i o n a l chromosomal changes i n over 75% of p a t i e n t s .  duplication  may  These  chromosome  I f the genes a f f e c t e d by t h e s e a d d i t i o n a l  chromosomal changes were i n v o l v e d i n c o u p l i n g p r o l i f e r a t i o n  and  m a t u r a t i o n , the r e s u l t a n t p r o g r e s s i v e u n c o u p l i n g of t h e s e p r o c e s s e s c o u l d e f f e c t the e v o l u t i o n of a subclone w i t h i n c r e a s i n g l y  malignant  c h a r a c t e r i s t i c s , i n c l u d i n g "maturation a r r e s t " .  be  d i s c u s s e d f u r t h e r i n acute n o n l y m p h o b l a s t i c  R e l a t i o n s h i p Between CGL The  This w i l l  leukemia.  C e l l s and Growth R e g u l a t o r s  frequency of i n v i t r o c o l o n y - f o r m i n g c e l l s i n CGL i s  extremely e l e v a t e d —  1 0 0 - f o l d f o r bone marrow and 1,000  60,000-fold f o r p e r i p h e r a l b l o o d i n u n t r e a t e d p a t i e n t s That t h e s e c o l o n y - f o r m i n g c e l l s belonged demonstrated  by the presence o f the Ph*  (375-377).  t o the m a l i g n a n t  clone  was  chromosome w i t h i n the  d i v i d i n g c e l l s of most c o l o n i e s (378-380).  Long term bone marrow  c u l t u r e s o f CGL  t h a t r e s i d u a l normal  c e l l s r e c e n t l y demonstrated  p r o g e n i t o r s were indeed p r e s e n t and, a f t e r 3 weeks i n c u l t u r e  (381).  i n most c a s e s , e a s i l y d e t e c t a b l e  There i s u n i f o r m expansion o f a l l  m y e l o i d p r o g e n i t o r compartments d u r i n g the c h r o n i c phase of the disease.  With the onset of b l a s t c r i s i s , major changes o c c u r i n t h i s  p a t t e r n of i n v i t r o growth, u n t i l a p a t t e r n s i m i l a r t o t h a t seen i n  55  acute n o n l y m p h o b l a s t i c  leukemia i s a t t a i n e d  ( t o be d i s c u s s e d ) .  Throughout the e n t i r e course of the d i s e a s e , CGL r e t a i n an a b s o l u t e dependency on CSF  f o r growth.  colony-forming CFU-c from  CGL  c h r o n i c phase p a t i e n t s e x h i b i t normal morphology and degrees maturation.  There have been r e p o r t s t h a t CGL  s t i m u l a t i o n by CSF  a l r e a d y d e s c r i b e d , the r e s p o n s i v e n e s s o f m y e l o i d  as normal c e l l s .  of  c e l l responsiveness to  i s s l i g h t l y l e s s than normal (382,383).  many n e g a t i v e r e g u l a t o r s (LF, AIF, TF, PGE,  cells  leukemia c e l l s t o  LAI, TNF)  In most c a s e s , a s i g n i f i c a n t l y  As  i s n o t the same  decreased  s e n s i t i v i t y t o i n h i b i t i o n has been documented which may  be of  s i g n i f i c a n c e w i t h r e g a r d t o the p r o l i f e r a t i v e advantage o f the malignant The  c l o n e over normal h e m o p o i e t i c  cells.  s i g n i f i c a n c e of a u t o s t i m u l a t i o n of leukemia c e l l s  p r o d u c t i o n o f t h e i r own controversy.  by  growth f a c t o r s has been an a r e a of  S u p p o r t i n g e v i d e n c e i n c l u d e s the o b s e r v a t i o n t h a t  m u l t i - C S F dependent h e m o p o i e t i c  cell  l i n e s became leukemogenic  a c q u i r e d the a b i l i t y t o s e c r e t e m u l t i - C S F When one o f t h e s e CSF-dependent c e l l  simultaneously  l i n e s was  and  (384-385).  transfected with a  r e t r o v i r u s c o n s t r u c t c o n t a i n i n g the gene f o r GM-CSF, subclones became leukemogenic (386).  a t the same time as autonomously-producing  GM-CSF  F u r t h e r e v i d e n c e showed t h a t when c h i c k e n macrophages  i m m o r t a l i z e d w i t h v-myc were s u b s e q u e n t l y i n f e c t e d w i t h the v - m i l gene, leukemic c e l l s were produced  that autostimulated t h e i r  p r o l i f e r a t i o n through s y n t h e s i s of a v i a n M-CSF (387).  T cell  lines  t r a n s f o r m e d by HTLV became TCGF s e c r e t o r s and had a decreased dependency on exogenous TCGF (388).  However, t h e r e a r e o t h e r cases  whereby CSF-dependent c e l l l i n e s were i n f e c t e d w i t h Abelson  leukemia  56  i v i r u s , became leukemic and d i d n o t s y n t h e s i z e  o r express  increased  r e c e p t o r numbers f o r CSF (389,390). There i s c e r t a i n l y e v i d e n c e t h a t CGL monocytic c e l l s can s e c r e t e t h e i r own growth f a c t o r s (CSFs), g e n e r a l l y normal.  and i n c h r o n i c phase, CSF l e v e l s a r e  This i s u n l i k e l y to explain the p r o l i f e r a t i v e  advantage o f t h e leukemic c l o n e however, s i n c e b o t h normal and CGL c e l l s are s i m i l a r l y responsive CSF  i n vitro.  The f a c t t h a t normal c e l l s have s h o r t e r c e l l  times than leukemic c e l l s (391).  t o , and a b s o l u t e l y r e q u i r e , exogenous  f u r t h e r complicates  cycle  this interpretation  Furthermore, i t i s l i k e l y t h a t a l a r g e number o f c e l l s  t h a n normal o r leukemic monocytic c e l l s  other  can produce CSF w i t h i n t h e  body, and t h e c u m u l a t i v e i n f l u e n c e s o f such p r o d u c t i o n ,  although not  c h a r a c t e r i z e d , make i t h i g h l y u n l i k e l y t h a t a u t o c r i n e  s t i m u l a t i o n of  growth by m y e l o i d leukemic c e l l s  can be o f c r i t i c a l  importance i n t h e  i n i t i a t i o n o f leukemia i n a s i n g l e c e l l .  B.  ACUTE NONLYMPHOBLASTIC LEUKEMIA C l i n i c a l Course and P a t h o g e n e s i s Acute n o n l y m p h o b l a s t i c leukemia (ANLL) o r acute myelogenous  leukemia (AML) i s a r a p i d l y f a t a l d i s e a s e  i f untreated.  Patients  e x h i b i t an i n c r e a s i n g number o f immature, p o o r l y o r n o n - d i f f e r e n t i a t e d b l a s t c e l l s o f t h e granulocyte-monocyte which e v e n t u a l l y  s p i l l over i n t o t h e p e r i p h e r a l  Maturation of c e l l s  circulation.  i n ANLL i s d e f e c t i v e and, as a r e s u l t ,  immature c e l l s do n o t d i f f e r e n t i a t e t o any g r e a t e x t e n t increase  i n number.  Clinical  i n normal h e m o p o i e t i c c e l l s ,  signs  lineage  early  and hence  involve the associated  decrease  i n c l u d i n g anemia, thrombocytopenia and  57  mature granulocytopenia/monocytopenia.  In approximately one-third of  ANLL p a t i e n t s , a p r e l e u k e m i c o r m y e l o d y s p l a s t i c s t a t e preceeds t h e development o f acute leukemia  (392).  Chemotherapy treatment a l l o w s  a p p r o x i m a t e l y 75% o f a d u l t ANLL p a t i e n t s t o a c h i e v e remission.  complete  The d u r a t i o n o f r e m i s s i o n i s v a r i a b l e and leukemic  r e l a p s e c o n t i n u e s t o be a s e r i o u s problem  i n ANLL.  In most cases o f ANLL no s p e c i f i c e t i o l o g i c a l e v e n t ( s ) have been i d e n t i f i e d , chemotherapeutic  agent(s) or  a l t h o u g h r a d i a t i o n exposure and  ( a l k y l a t i n g ) agents a r e known t o be c a p a b l e o f  c a u s i n g damage t o g e n e t i c m a t e r i a l which may be a s s o c i a t e d w i t h t h e development o f acute leukemia  (393-398).  ANLL i s an extremely heterogeneous m o r p h o l o g i c a l l y - d e f i n e d subgroups c e l l u l a r markers. group  d i s e a s e i n terms o f  and i m m u n o l o g i c a l l y - d e f i n e d  The F r e n c h - A m e r i c a n - B r i t i s h (FAB) c o - o p e r a t i v e  o r i g i n a l l y proposed  a c l a s s i f i c a t i o n system f o r ANLL as f o l l o w s  (399-400): Ml:  m y e l o b l a s t i c leukemia w i t h o u t m a t u r a t i o n ,  M2:  m y e l o b l a s t i c leukemia w i t h m a t u r a t i o n ,  M3:  h y p e r g r a n u l a r p r o m y e l o c y t i c leukemia,  M4:  myelomonocytic  M5:  monocytic  M6:  erythroleukemia.  leukemia,  leukemia, and  While d i f f e r i n g responses t o v a r i o u s chemotherapeutic been r e p o r t e d between t h e s e subgroups, p a t h o g e n e s i s have been e s t a b l i s h e d .  agents have  no s p e c i f i c d i f f e r e n c e s i n  58  Stem C e l l  Origin  Most, i f n o t a l l , cases o f ANLL a r e thought t o be monoclonal i n o r i g i n , based on chromosomal and G-6-PD s t u d i e s . G-6-PD s t u d i e s have i n d i c a t e d t h a t , w h i l e ANLL i s a d i s e a s e  o f stem c e l l  appears t o be a degree o f h e t e r o g e n e i t y  present with regard  l e v e l o f stem c e l l  involvement.  p a t i e n t s t h e stem c e l l precursor be  (401).  involved  there to the  I n some, p o s s i b l y most, ANLL seems t o be t h e granulocyte-monocyte  Other p a t i e n t s have demonstrated leukemic o r i g i n t o  i n more p r i m i t i v e (GM and e r y t h r o i d )  only) progenitors  origin,  (73,402,403).  o r more r e s t r i c t e d  (monocyte  Chromosomal s t u d i e s have a l s o shown  t h a t , i n some ANLL p a t i e n t s , c l o n a l markers were p r e s e n t i n b o t h erythroid precursors  and t h e leukemic c e l l s  (404).  Recently, B  lymphocyte involvement was a l s o demonstrated i n some b u t n o t most ANLL p a t i e n t s by s t u d i e s  i n c l u d i n g i d e n t i f i c a t i o n o f heavy  immunoglobulin gene rearrangements and G-6-PD a n a l y s i s Whether t h i s h e t e r o g e n e i t y  chain  (405,406).  r e f l e c t s t r u e d i f f e r e n c e s i n stem  cell  o r i g i n o r a d i f f e r e n c e i n r e s t r i c t i o n o f d i f f e r e n t i a t i o n has n o t been e s t a b l i s h e d , n o r have these d i f f e r e n c e s been c o r r e l a t e d w i t h pathogenetic, prognostic patients.  or therapeutic  d i f f e r e n c e s between ANLL  These p o s s i b i l i t i e s await f u r t h e r i n v e s t i g a t i o n s .  Chromosomal Changes and T h e i r P o s s i b l e Cytogenetic studies  Significance  o f ANLL c e l l s have i n d i c a t e d t h a t w i t h t h e  advent o f new procedures ( i n p a r t i c u l a r , i n c u b a t i o n for  o f marrow c e l l s  s e v e r a l days) a l l cases o f ANLL may i n v o l v e chromosomal changes.  Commonly r e p o r t e d  karyotypic  nonrandom t r a n s l o c a t i o n s  abnormalities  i n ANLL have  included  (between chromosomes 8 and 21 i n M2 ANLL,  59  and between chromosomes 15 and  17 i n M3  ANLL), p a r t i a l o r  total  t r i s o m y of chromosome 1, l o s s of chromosome 7, 21, X o r Y, t r i s o m y of chromosome 8 or 21 d e l e t i o n of the l o n g arm  (reviewed  i n 407).  of chromosome 5 (5q ) has  t o be a s s o c i a t e d w i t h a number of hematologic ANLL (408).  Of  An  and  acquired  a l s o been shown  dysplasias including  i n t e r e s t i n t h i s l a t t e r chromosomal anomaly i s the  r e c e n t d i s c o v e r y t h a t the proto-oncogene c-fms ( t h e c e l l u l a r homologue of v-fms, the t r a n s f o r m i n g gene of the McDonough s t r a i n of t h e f e l i n e sarcoma v i r u s ) was chromosome 5 and was, (409).  The  l o c a t e d near the b r e a k p o i n t ( s )  i n f a c t , d e l e t e d from the 5q  c-fms gene i s n o r m a l l y  expressed  on  chromosome  i n hemopoietic  cells  and  has been shown t o be s t r u c t u r a l l y r e l a t e d , i f not i d e n t i c a l t o the M-CSF (CSF-1) r e c e p t o r (209).  I n the 5q  c e l l s a r e hemizygous f o r c-fms. r e c e p t o r numbers may  syndrome,  T h i s suggests  hemopoietic  t h a t a l t e r e d M-CSF  be capable of c a u s i n g an abnormal response t o  normal growth f a c t o r - m e d i a t e d  p r o l i f e r a t i o n and/or d i f f e r e n t i a t i o n .  I t i s c l e a r t h a t i n o t h e r systems, a l t e r i n g the normal p a t t e r n of e x p r e s s i o n of proto-oncogenes can c o n t r i b u t e to transformation (411,412) and  (410).  malignant  In ANLL, t h e a m p l i f i c a t i o n of the c-myc gene  the c-myb gene (413) have been r e p o r t e d .  Abnormal  e x p r e s s i o n of proto-oncogenes, whose normal r o l e i s thought t o be i n the r e g u l a t i o n of b a s i c c e l l u l a r p r o l i f e r a t i o n and p r o c e s s e s , may L i k e CGL, (414).  The  differentiation  w e l l be p a r t of leukemogenesis i n ANLL. ANLL i s presumed t o be a d i s e a s e of m u l t i - s t a g e  first  stage  (which may  a l s o c o n s i s t of m u l t i p l e  can be equated t o the p r e l e u k e m i c / m y e l o d y s p l a s t i c c o u l d i n c l u d e the c h r o n i c phase of CGL.  nature  "steps")  s i t u a t i o n , which  Here, stem c e l l s  are  60  a f f e c t e d i n such a way t h a t , a l t h o u g h g e n e t i c a l l y abnormal, they a r e still  c a p a b l e o f e x p r e s s i n g some c o n t r o l over  programs.  differentiation  In t h i s s i t u a t i o n , q u a n t i t a t i v e d i f f e r e n c e s i n c e l l s at  some m a t u r a t i o n l e v e l s would o c c u r b u t s i m i l a r types o f d i f f e r e n t i a t i o n programs would s t i l l be f o l l o w e d .  I n t h e second  stage ( a g a i n , p o s s i b l y m u l t i - s t e p i t s e l f ) , t h e d i s e a s e advances as g e n e t i c c o n t r o l s l i n k i n g p r o l i f e r a t i o n and d i f f e r e n t i a t i o n p r o g r e s s i v e l y uncouple.  The e n s u i n g d y s r e g u l a t i o n o f hemopoiesis  now  a l l o w s t h e e x p r e s s i o n o f f o r m e r l y " f o r b i d d e n " programs. P r o l i f e r a t i o n c o n t i n u e s b u t d i f f e r e n t i a t i o n does n o t f o l l o w , as t h e l i n k between t h e s e p r o c e s s e s has been a l t e r e d o r broken.  A  m a t u r a t i o n " a r r e s t " o c c u r s which now m a n i f e s t s c l i n i c a l l y  as ANLL o r  CGL b l a s t  crisis.  R e l a t i o n s h i p Between ANLL C e l l s and Growth R e g u l a t o r s ANLL c e l l s c u l t u r e d i n v i t r o  i n the progenitor c e l l  assay behave  i n a t o t a l l y d i f f e r e n t manner than c h r o n i c phase CGL c e l l s i n t h e same system.  C l o n i n g e f f i c i e n c y and growth p a t t e r n s v a r y  c o n s i d e r a b l y between ANLL p a t i e n t s .  T y p i c a l growth b e h a v i o r can  range from no growth, s m a l l - l a r g e c l u s t e r  (4 - 39 c e l l  f o r m a t i o n o n l y , t o an abnormally h i g h c l u s t e r : c o l o n y  clones)  ratio  (341,415,416) w i t h c e l l s o f t e n r e v e a l i n g l a c k o f normal m a t u r a t i o n , or  i n some systems,  leukemic b l a s t c e l l morphology (417,418).  i s c y t o g e n e t i c e v i d e n c e t h a t c l o n e s produced  i n v i t r o by ANLL c e l l s  a r e d e r i v e d from t h e o r i g i n a l i n v i v o leukemic c l o n e D u r i n g complete  There  (378,419).  r e m i s s i o n , i n v i t r o c o l o n y growth p a t t e r n s  r e t u r n t o normal i n terms o f b o t h numbers and a b i l i t y t o mature.  In  61  long-term ANLL bone marrow c u l t u r e s , i t was p o s s i b l e t o demonstrate t h a t most newly diagnosed ANLL p a t i e n t s s t i l l  r e t a i n e d a p o o l of  normal h e m o p o i e t i c stem c e l l s which were apparent by 4 - 6 weeks i n c u b a t i o n and had presumably  been suppressed i n v i v o  As i n t h e case o f CGL, t h e growth o f ANLL c e l l s  (381). i n vitro  remains  c o m p l e t e l y dependent on a source o f CSF through t h e course o f t h e disease.  I n g e n e r a l , ANLL and normal  c e l l s appear t o be s i m i l a r l y  s e n s i t i v e t o s t i m u l a t i o n by CSF, a l t h o u g h some i n v e s t i g a t o r s have r e p o r t e d i n c r e a s e d (382) and d e c r e a s e d cells.  (420) r e s p o n s i v e n e s s by ANLL  Some i n t e r e s t i n g e v i d e n c e c o n c e r n i n g subnormal CSF l e v e l s i n  t h e immediate v i c i n i t y reported.  o f ANLL c e l l s  i n t h e marrow has been  N o n - e x i s t e n t o r v e r y low CSF p r o d u c t i o n by adherent marrow  c e l l s has been observed i n p a t i e n t s w i t h l e s s d i f f e r e n t i a t e d forms o f ANLL (421).  A c o r r e s p o n d i n g l y poor response t o chemotherapy was a l s o  n o t e d i n t h e s e p a t i e n t s , a l t h o u g h i f complete normal  r e m i s s i o n was a t t a i n e d ,  l e v e l s o f CSF p r o d u c t i v i t y was now measured from  adherent  marrow c e l l s . As i n t h e case o f CGL, decreased r e s p o n s i v e n e s s o f ANLL c e l l s t o i n h i b i t o r y molecules  (LF,AIF,PGE,LAI) has been observed.  Possibly a  combination o f decreased amounts o f d i f f e r e n t i a t i o n - i n d u c i n g  factors  and l a c k o f n e g a t i v e r e g u l a t i o n a r e enough t o i n i t i a t e o r a t l e a s t promote, t h e emergence o f a c l o n e o f c e l l s p o s s e s s i n g a growth advantage  C.  over normal  c e l l populations.  DIFFERENTIATION INDUCTION IN MYELOID LEUKEMIA R e v e r s i o n o f t h e m a l i g n a n t phenotype i n m y e l o i d leukemia  cells  by t h e use o f v a r i o u s agents c a p a b l e o f i n d u c i n g d i f f e r e n t i a t i o n has  62  been a s u b j e c t of widespread r e s e a r c h . our u n d e r s t a n d i n g  of some a s p e c t s  d i s e a s e , and of t h e p r o c e s s  T h i s work has c o n t r i b u t e d t o  o f t h e o r i g i n and e v o l u t i o n of t h i s  of normal d i f f e r e n t i a t i o n ,  as w e l l as  defining p o s s i b i l i t i e s f o r novel therapeutic modalities. p r e v i o u s l y d i s c u s s e d , acute m y e l o i d little,  As  leukemic c e l l s s e l f - g e n e r a t e w i t h  i f any, concomitant d i f f e r e n t i a t i o n .  I t f o l l o w s t h a t the  r a t i o of s e l f - r e p l i c a t i v e to d i f f e r e n t i a t i v e c e l l d i v i s i o n plays a major r o l e  i n the progression of the n e o p l a s t i c c e l l  population.  Some time ago, Ichikawa observed t h a t murine Ml m y e l o i d cell  c u l t u r e s c o u l d be induced  to d i f f e r e n t i a t e  some types o f c o n d i t i o n e d media (422).  leukemic  by t h e a d d i t i o n o f  In other i n v e s t i g a t i o n s ,  murine WEHI-3B c e l l s were found t o be s i m i l a r l y i n d u c i b l e by adding a source  of CSF t o c u l t u r e d c e l l s (423,424).  Furthermore, i t was  d i s c o v e r e d t h a t , w i t h i n t h e c l o n e d Ml and WEHI-3B c e l l individual  c e l l s c o u l d form c l o n e s w i t h markedly  responsiveness  t o CSF-induced d i f f e r e n t i a t i o n .  lines,  different Subclones were  developed from human (425,426) and murine (422,427-431) m y e l o i d leukemia c e l l s t h a t c o u l d be induced mature macrophages o r g r a n u l o c y t e s e s t a b l i s h e d which were b l o c k e d differentiate.  I n these  were o f t e n observed.  to d i f f e r e n t i a t e  by CSF.  (D ) to +  Other c l o n e s  (D ) were  i n t h e i r a b i l i t y t o be induced t o  l a t t e r c l o n e s , s p e c i f i c chromosomal changes  Clonal differences i n differentiation  i n d u c i b i l i t y mediated by o t h e r compounds than CSF were a l s o reported. inhibited  Change i n c o n s t i t u t i v e p r o t e i n e x p r e s s i o n t h a t  apparently  normal i n d u c t i o n of d i f f e r e n t i a t i o n by c e r t a i n compounds  were i d e n t i f i e d i n D for individual  +  c e l l s (432).  These changes, o f t e n  different  i n d u c i n g compounds, were thought t o be due t o  63  c o n s t i t u t i v e e x p r e s s i o n of d i f f e r e n t s p e c i f i c gene e x p r e s s i o n pathways. examining  Indeed,  2-dimensional  gel electrophoretic  the e x p r e s s i o n of hundreds o f p r o t e i n s d u r i n g normal and  induced m y e l o i d d i f f e r e n t i a t i o n suggested parallel,  t h a t t h e r e were m u l t i p l e ,  i n d i v i d u a l l y programmed gene e x p r e s s i o n pathways i n v o l v e d  in differentiation the CSF  profiles  (433).  The d i f f e r e n t i a t i o n - i n d u c i n g components i n  s o u r c e s were shown t o be G-CSF and  (weakly) GM-CSF i n the  mouse, GM-CSF (CSFa) and G-CSF (CSFB) i n humans (391).  Other  compounds w i t h the a b i l i t y t o induce d i f f e r e n t i a t i o n i n m y e l o i d leukemia c e l l s i n c l u d e d i m e t h y l s u l p h o x i d e (DMSO), s t e r o i d s , p h o r b o l e s t e r s , b u t y r a t e and many c y t o t o x i c drugs used as agents  i n myeloid  chemotherapeutic  leukemia.  Numerous i n v e s t i g a t i o n s have been c a r r i e d out i n o r d e r t o determine  i f the d i f f e r e n t i a t i o n induced m y e l o i d leukemia c e l l s were  a c t u a l l y e q u i v a l e n t t o "normal"  cells.  While c e r t a i n c e l l membrane .  a n t i g e n i c markers of normal mature c e l l s have been i d e n t i f i e d induced c e l l s  (434), many s t u d i e s have r e p o r t e d abnormal r e a c t i v i t i e s  w i t h a number o f monoclonal mature c e l l s  on  (435).  a n t i b o d i e s known t o r e a c t w i t h normal  B i o c h e m i c a l (436) and f u n c t i o n a l membrane  markers (437) have a l s o been r e p o r t e d t o be a t y p i c a l o r d e f e c t i v e i n the induced c e l l s ,  i n d i c a t i n g t h a t leukemic c e l l s which have been  induced t o d i f f e r e n t i a t e a r e n o t e q u i v a l e n t t o t h e i r normal mature c e l l counterparts.  I I I . HUMAN MYELOID LEUKEMIA-ASSOCIATED ANTIGENS There has been i n t e n s e i n t e r e s t i n the study of human l e u k e m i a - a s s o c i a t e d a n t i g e n s (LAA)  o r markers o f leukemic  cells.  64  These i n v e s t i g a t i o n s have p r o v i d e d d i f f e r e n t i a t i o n and  insight into  leucocyte  the c e l l u l a r o r i g i n of leukemia as w e l l as  p r o v i d e d d i a g n o s t i c reagents from lymphoid leukemias.  u s e f u l i n the d i s c r i m i n a t i o n of  myeloid  I n i t i a l l y , many s t u d i e s f o c u s s e d on  the  i d e n t i f i c a t i o n of a n t i g e n i c markers t h a t would be s p e c i f i c f o r (unique  t o ) leukemic  blasts.  I t was  hoped t h a t such markers c o u l d  u t i l i z e d b o t h f o r i n i t i a l d i a g n o s i s and d i s e a s e d u r i n g r e m i s s i o n and  f o r d e t e c t i o n of  be  residual  f o l l o w i n g bone marrow t r a n s p l a n t a t i o n .  Upon i n t e n s i v e i n v e s t i g a t i o n , p u t a t i v e l e u k e m i a - s p e c i f i c a n t i g e n s have g e n e r a l l y been found LAA  to be  leukemia-associated  have been shown t o be p r e s e n t on normal m y e l o i d  antigens.  These  c e l l s at c e r t a i n  s t a g e s of d i f f e r e n t i a t i o n o r d u r i n g embryogenesis and,  i n many  i n s t a n c e s , on non-hemopoietic t i s s u e s as w e l l . E a r l y i n v e s t i g a t i o n s provided evidence human l e u k e m i a - a s s o c i a t e d formation) leukemic  antigens.  f o r the e x i s t e n c e of  C e l l u l a r immune responses  (blast  by r e m i s s i o n lymphocytes i n the p r e s e n c e of i n a c t i v a t e d  c e l l s from the same p a t i e n t s were r e p o r t e d  (438,439).  Lymphocytes from normal i d e n t i c a l twins were shown to respond to t h e i r twin's (440).  leukemic  c e l l s but not to t h e i r r e m i s s i o n  leucocytes  Delayed type h y p e r s e n s i t i v i t y r e a c t i o n s by p a t i e n t s were  e l i c i t e d by t h e i r leukemic  b l a s t c e l l membrane a n t i g e n s  Attempts by o t h e r i n v e s t i g a t o r s t o reproduce some of observations, u t i l i z i n g  (441,442).  these  s i m i l a r methodologies have f a i l e d  to  s u b s t a n t i a t e some of these f i n d i n g s (443,444). S e v e r a l examples of the p r o d u c t i o n of a u t o a n t i b o d i e s r e a c t i v e w i t h p a t i e n t s ' own  leukemic  b l a s t s have been r e p o r t e d .  In some  i n s t a n c e s , an i n c r e a s e i n these a u t o a n t i b o d i e s d u r i n g r e m i s s i o n  was  65  reported  (445,446).  Antibody dependent c y t o t o x i c i t y f o r leukemic  m y e l o b l a s t s by p a t i e n t s e r a has a l s o been d e s c r i b e d  (447,448).  Immunoglobulins have been i d e n t i f i e d on the s u r f a c e o f leukemic  cells  (449,450) and, a f t e r e l u t i o n from t h e s e c e l l s , were shown t o have c y t o t o x i c i t y a g a i n s t leukemic c e l l s LAA,  (450).  A humoral response t o  s e p a r a b l e from an a n t i - a l l o a n t i g e n response, was  demonstrated  many ANLL p a t i e n t s f o l l o w i n g immunotherapy w i t h i r r a d i a t e d leukemic b l a s t s  (451).  C h a r a c t e r i z a t i o n o f the p u t a t i v e  r e c o g n i z e d i n t h e s e s t u d i e s has n o t been c a r r i e d out and  in  allogeneic  LAA their  p o s s i b l e b i o l o g i c a l s i g n i f i c a n c e remains unknown. A n t i s e r a t o human leukemia c e l l s have been r a i s e d i n a number of non-human s p e c i e s ( x e n o - a n t i s e r a ) w i t h the aim o f d e f i n i n g human LAA.  I n most c a s e s , e x t e n s i v e a b s o r p t i o n of such h e t e r o a n t i s e r a w i t h  normal h e m o p o i e t i c c e l l s was  n e c e s s a r y t o r e n d e r the a n t i s e r a  s p e c i f i c f o r leukemic c e l l s .  H e t e r o a n t i s e r a i d e n t i f y i n g human  m y e l o i d LAA have been r a i s e d i n non-human p r i m a t e s (452-453), (453-456) and mice (457-458).  rabbits  By i n d u c i n g t o l e r a n c e i n mice t o  r e m i s s i o n l e u c o c y t e s , mice l a t e r immunized w i t h leukemic c e l l s the same p a t i e n t s produced type (ANLL vs ALL)  a n t i s e r a s p e c i f i c f o r the leukemic  (457-459).  The same i n v e s t i g a t o r s a l s o  monkey a n t i - m y e l o b l a s t serum w i t h m y e l o i d leukemic blast crisis) specificity, m o l e c u l a r weight  from cell  produced  (ANLL and  CGL  r e a c t i v e with a myeloblast antigen of  75 - 80 KD and p i 7.8  of leukemic m y e l o b l a s t s (460).  shed i n v i t r o from the s u r f a c e  T h i s compound was  c h a r a c t e r i s t i c compounds shed from the s u r f a c e of  one of a s e r i e s of leukemic  m y e l o b l a s t s t h a t d i f f e r e d q u a n t i t a t i v e l y and q u a l i t a t i v e l y compounds shed by o t h e r types o f leukemic o r nonleukemic  from  cells.  66  T h e i r murine h e t e r o a n t i s e r u m has been shown t o be c a p a b l e o f p r e d i c t i n g r e l a p s e i n ANLL r e m i s s i o n p a t i e n t s (461,462). i n v e s t i g a t o r s have r e p o r t e d s i m i a n anti-ANLL absorbed  serum which, when  a g a i n s t normal human b l o o d c e l l s , r e c o g n i z e d b l a s t  from an ANLL p a t i e n t b u t n o t b l o o d c e l l s from h i s normal twin.  Other  cells  identical  T h i s p a t i e n t ' s leukemic b l a s t s , when used as s t i m u l a t o r c e l l s  i n a mixed lymphocyte r e a c t i o n , caused normal twin's lymphocytes  s i g n i f i c a n t s t i m u l a t i o n of h i s  and h i s own r e m i s s i o n lymphocytes  (463).  That s e v e r a l LAA e x i s t e d on m y e l o i d leukemic c e l l s was i n d i c a t e d by h e t e r o a n t i s e r a a b s o r p t i o n s t u d i e s w i t h acute and c h r o n i c m y e l o i d leukemia c e l l s  (464).  When i n d i v i d u a l ANLL o r CGL p a t i e n t ' s c e l l s  were used f o r a b s o r p t i o n , i t was apparent by t h e s e r a were c r o s s - r e a c t i v e  (ANLL and CGL), some were unique t o  c e r t a i n donors, w h i l e some m y e l o i d to  t h a t some a n t i g e n s d e t e c t e d  leukemia p a t i e n t s ' b l a s t s  failed  r e a c t w i t h any o f a p a n e l o f s i m i a n a n t i s e r a t o m y e l o i d LAA. With t h e advent  o f monoclonal  a n t i b o d y t e c h n o l o g y i t became  p o s s i b l e t o i d e n t i f y i n d i v i d u a l a n t i g e n i c markers o f h e m o p o i e t i c c e l l s w i t h i n c r e a s i n g ease. Milstein lines,  O r i g i n a l l y d e s c r i b e d by K o h l e r and  (465), t h i s t e c h n i q u e a l l o w e d t h e p r o d u c t i o n o f h y b r i d c e l l  each p r o d u c i n g homogeneous monoclonal  s i n g l e a n t i g e n i c determinant a r e c a p a b l e o f b e i n g produced quantities.  of a s i n g l e antigen. i n vitro  Such a n t i b o d i e s  or i n vivo i n large  Much r e s e a r c h e f f o r t has been spent on attempts t o  produce monoclonal on m a l i g n a n t  antibodies directed to a  antibodies that react s p e c i f i c a l l y with antigens  c e l l s and n o t w i t h normal c e l l u l a r  d i s t i n g u i s h acute m y e l o i d from a c u t e lymphoid  antigens, or that  leukemia.  f r u i t f u l r e s u l t s o f t h e s e i n v e s t i g a t i o n s have been t h e  The most  67  c h a r a c t e r i z a t i o n o f human l e u c o c y t e l a r g e number o f r e p o r t e d  d i f f e r e n t i a t i o n antigens.  myeloid-specific  monoclonal  The  antibodies  (MAbs) emerging from t h e s e s t u d i e s prompted t h e e s t a b l i s h m e n t of annual " I n t e r n a t i o n a l Workshops on Human Leukocyte D i f f e r e n t i a t i o n A n t i g e n s " i n 1983 (466-468). standardization  These workshops have enabled t h e  o f nomenclature f o r l e u c o c y t e d i f f e r e n t i a t i o n  a n t i g e n s and a l l o w e d j o i n t a n a l y s i s o f hundreds o f MAbs and t h e i r r e l a t i o n s h i p t o each o t h e r , p r e v i o u s l y individual investigators  i m p o s s i b l e t o s o r t out by  ( o r groups) working i n d e p e n d e n t l y .  The  number o f " c l u s t e r d e t e r m i n a n t " (CD) groups o r subgroups distinguished  i n t h i s manner a r e now 50, i n c l u d i n g  antigens  i d e n t i f i e d on T and B c e l l s , m y e l o i d c e l l s and a c t i v a t e d Previous studies  cells.  (reviewed 469-472) d e s c r i b i n g marker p r o f i l e s o f  human m y e l o i d leukemia c e l l s have i n d i c a t e d t h a t no i n d i v i d u a l MAbs o r c y t o c h e m i c a l markers r e c o g n i z e d t h e m a l i g n a n t b l a s t c e l l s o f every ANLL p a t i e n t .  Moreover, w i t h i n  i n d i v i d u a l ANLL p a t i e n t s , terms o f t h e s e markers. true genetic  the b l a s t c e l l population of  considerable  heterogeneity also existed i n  I t i s not clear i f t h i s s i t u a t i o n r e f l e c t s  h e t e r o g e n e i t y o r asynchronous l i m i t e d d i f f e r e n t i a t i o n .  I t has been demonstrated t h a t t h e p r o p o r t i o n  of p a t i e n t b l a s t  cells  w i t h s p e c i f i c markers u s u a l l y v a r i e s from below 15% t o 90% (472).  A  number o f i n v e s t i g a r o r s have t r i e d t o c o r r e l a t e FAB c l a s s i f i c a t i o n o f ANLL w i t h r e a c t i v i t y t o d e f i n e d  m y e l o i d a n t i g e n s (472-476).  While i t  appears t h a t most o f t h e s e s t u d i e s have n o t shown any c l e a r c o r r e l a t i o n between ANLL c e l l s u r f a c e classes, recent  phenotypes and s p e c i f i c FAB  c o - o p e r a t i v e s t u d i e s have d e s c r i b e d  between c e r t a i n a n t i g e n i c markers and FAB c l a s s e s  an a s s o c i a t i o n (CD14 a n t i g e n w i t h  68  M4/M5) (477).  S i n c e markers o f leukemic  c e l l s appear t o be normal  gene p r o d u c t s , i t i s u s e f u l t o d i s c u s s t h e b e t t e r c h a r a c t e r i z e d molecules The  i n t h i s group. "Leukocyte  Common A n t i g e n " , f o r m e r l y r e f e r r e d t o as T200 o r  B220, i s now e n t i t l e d CD45.  The CD45 a n t i g e n i s expressed  d e n s i t y on t h e s u r f a c e o f hemopoietic While n o t l i n e a g e s p e c i f i c ,  cells  at high  ( l e u c o c y t e s ) (478).  i t has been demonstrated t o be c l o s e l y  i n v o l v e d i n l i n e a g e - s p e c i f i c f u n c t i o n s o f lymphocytes, T - c e l l mediated c y t o t o x i c i t y .  including  The T200 gene encodes f o u r forms o f  t h e same m o l e c u l e by a l t e r n a t e s p l i c i n g  (mol wt 220, 205, 190 and 180  KD) which appear t o be d i f f e r e n t i a l l y expressed on subsets o f T c e l l s , B c e l l s , monocytes and g r a n u l o c y t e s (reviewed 468). A l a r g e p a n e l o f g r a n u l o c y t e - r e a c t i v e MAbs have been i n v e s t i g a t e d and found t o i d e n t i f y a s e r i e s o f "granulocyte-associated" antigens.  CD16 a n t i g e n , t h e F c r e c e p t o r on  g r a n u l o c y t e s , has been r e p o r t e d t o range i n m o l e c u l a r weight  from  54 - 62 KD, presumably due t o m i c r o h e t e r o g e n e i t y o f the molecule (479).  A 2.2 - 3.5 f o l d i n c r e a s e i n b i n d i n g o f a n t i - F c MAbs was  demonstrated a f t e r induced a c t i v a t i o n o f n e u t r o p h i l s (480). carbohydrate  A  a n t i g e n , 3 - a - f u c o s y l - N - a c e t y l l a c t o s a m i n e (FAL) has  a l s o been i d e n t i f i e d  i n association with a series of granulocyte  membrane p r o t e i n s o f m o l e c u l a r weights (479,481,482).  220, 180, 155, 130 and 98 KD)  FAL ( o r X-hapten) i s now e n t i t l e d CD15 by  s t a n d a r d i z e d nomenclature.  FAL i s thought  t o be i n v o l v e d i n  n e u t r o p h i l f u n c t i o n s such as m a r g i n a t i o n , p h a g o c y t o s i s and r e s i s t a n c e to b a c t e r i a l hydrolases  (483).  In addition to being strongly  expressed by g r a n u l o c y t e s , CD15 has a l s o been i d e n t i f i e d on ANLL  69  p a t i e n t c e l l s , e s p e c i a l l y M4 and M5 c l a s s e s as w e l l as on CGL c e l l s (484).  S p r i n g e r and Anderson (479) a l s o i d e n t i f i e d t h r e e o t h e r 125  antigens i n  I-labeled granulocyte lysates.  These were  molecules  of 68, 65 (CAMAL) and 27 KD. "Monocyte-associated"  a n t i g e n s i n c l u d e CD14, s t r o n g l y expressed  on monocytes and found  i n a s s o c i a t i o n with  ( p r i m a r i l y ) M4 and M5  s t a g e s o f ANLL (486).  MAbs s p e c i f i c f o r pl50/95,  the least  c h a r a c t e r i z e d member o f t h e l e u c o c y t e f u n c t i o n a n t i g e n f a m i l y , a r e r e a c t i v e w i t h monocytes and were i n c l u d e d i n t h e CD14 group Most m y e l o i d - a s s o c i a t e d a n t i g e n s a r e expressed degrees on b o t h g r a n u l o c y t i c and monocytic c e l l l e u c o c y t e f u n c t i o n a n t i g e n (LFA)  to varying  lineages.  f a m i l y of heterodimers  t o a l s o i n c l u d e s CD11 (C3bi r e c e p t o r ) and LFA-1. g l y c o p r o t e i n s share a common B s u b u n i t linked with d i f f e r e n t a subunits.  (484).  The  just  These c e l l  referred surface  (mol wt 95 KD) n o n c o v a l e n t l y  A p r o v i s i o n a l marker group  e n t i t l e d CDwl8 has been proposed f o r t h e LFA f a m i l y .  In f a c t , the  s p e c i f i c a n t i g e n i d e n t i f i e d i s presumably t h e B s u b u n i t , s i n c e anti-CDwl8 MAbs can immunoprecipitate within this family —  the (at l e a s t ) 3  LFA-1, CD11/C3bi r e c e p t o r and pl50/95.  the C3bi r e c e p t o r (mol wt 170/95 KD) i s expressed monocytes and NK c e l l s  heterodimers  (reviewed  486),  i n g e s t i o n of C3bi-coated p a r t i c l e s  CD11,  on g r a n u l o c y t e s ,  and mediates t h e adherence and  (487).  The CD11 a n t i g e n has been  demonstrated on c e l l s from many cases o f M4 and M5 ANLL, M l , M2, and M3 t o a l e s s e r degree, and CGL (488). expressed LFA-1  LFA-1 (mol wt 180/95 KD) i s  on B and T c e l l s , NK c e l l s , monocytes and g r a n u l o c y t e s .  i s thought  facilitates  t o r e p r e s e n t an important  adhesion  l e u c o c y t e i n t e r a c t i o n v i a transmembrane  glycoprotein that signalling  70  (489) .  A myelomonocytic-associated  glycolipid  (CD17),  l a c t o s y l c e r a m i d e i s expressed on most g r a n u l o c y t e s , many monocytes, p l a t e l e t s and c e l l s (484).  ( o r c e l l l i n e s ) from ANLL and ALL p a t i e n t s  I t i s thought  that oligosaccharides l i k e lactosylceramide,  e x p r e s s e d i n normal and m a l i g n a n t h e m o p o i e t i c be i n v o l v e d i n some way regulation  (481-483).  demonstrated (490) .  c e l l p o p u l a t i o n s , may  i n c e l l - t o - c e l l i n t e r a c t i o n s and growth^ An i n c r e a s e i n l a c t o s y l c e r a m i d e c o n t e n t  was  t o be a s s o c i a t e d w i t h g r a n u l o c y t e d i f f e r e n t i a t i o n  I n t e r e s t i n g l y , a study of the e x p r e s s i o n o f 20  g l y c o l i p i d s i n acute leukemia c e l l s  different  i n d i c a t e d t h a t i t was  p o s s i b l e to  classify  even p o o r l y d i f f e r e n t i a t e d acute leukemias  lymphoid  on the b a s i s of t h e i r g l y c o l i p i d s and t h a t the s t r u c t u r e of  cell  s u r f a c e g l y c o l i p i d s became more complex w i t h  differentiation  as m y e l o i d  or  increasing  (491).  A n t i g e n s p r e s e n t on h i g h l y immature ANLL b l a s t c e l l s have been reported  (492-495).  These a n t i g e n s , absent from most mature m y e l o i d  i cells,  a r e l i k e l y most c l o s e l y a s s o c i a t e d w i t h c e l l s  stages of d i f f e r e n t i a t i o n . The My-10  o r CD34 a n t i g e n (mol wt 115  l i k e many o t h e r s i d e n t i f i e d by monoclonal hemopoietic p r o g e n i t o r c e l l s been demonstrated and  lymphoid  (494).  at p r i m i t i v e KD),  antibodies, reacts with  CD34  +  p r o g e n i t o r c e l l s have  t o be c a p a b l e of p r o d u c i n g progeny o f b o t h m y e l o i d  (B and T) l i n e a g e s , i n d i c a t i n g t h a t the e x p r e s s i o n of  t h i s a n t i g e n o c c u r s on extremely p r i m i t i v e m u l t i p o t e n t i a l stem cells.  A r e c e n t study by Lowenberg and Bauman o f the s u r f a c e  phenotypes o f ANLL c o l o n y forming c e l l s  (CFU)  i n d i c a t e d t h a t the  CFU  showed i n t e r p a t i e n t a n t i g e n i c h e t e r o g e n e i t y as w e l l as d i f f e r e n c e s between the leukemic c e l l p o p u l a t i o n s i n e x p r e s s i o n of a n t i g e n s i n  71  the same p a t i e n t (497) .  (496).  Other s t u d i e s have supported t h e s e f i n d i n g s  V a r i a b l e CFU e x p r e s s i o n ( i n c l u d i n g absence) o f a n t i g e n s  p r e s e n t i n t h e t o t a l leukemic c e l l p o p u l a t i o n has important i m p l i c a t i o n s f o r the c l i n i c a l d e t e c t i o n or purging of r e s i d u a l leukemic  cells.  S u r f a c e marker s t u d i e s a r e g e n e r a l l y n o t n e c e s s a r y f o r t h e d i a g n o s i s and m o n i t o r i n g o f c h r o n i c phase CGL, due t o t h e c h a r a c t e r i s t i c overproduction of recognizable granulocytic  cells.  The b l a s t c r i s i s phase (CGL BC) however, may be o f lymphoid o r myeloid type.  With d i f f e r i n g treatments and prognoses,  e s s e n t i a l t o attempt CGL  t o d i s t i n g u i s h between t h e s e two forms.  BC c e l l s have s u r f a c e marker phenotypes  ANLL.  i t is Myeloid  comparable t o those i n  Lymphoid CGL-BC c e l l s have t h e same phenotype as t h a t o f ALL  cells.  Immunophenotyping o f CGL-BC has i d e n t i f i e d o t h e r l e s s common  variants  ( e r y t h r o i d , mixed, megakaryocytic  and u n d i f f e r e n t i a t e d  (498) . From t h e numerous i n v e s t i g a t i o n s r e g a r d i n g a n t i g e n i c a n a l y s i s o f normal  and leukemic m y e l o p o i e s i s , t h e r e has emerged s t r o n g e v i d e n c e  t h a t leukemic c e l l phenotypes the e x p r e s s i o n o f normal f a s h i o n ) and abnormal,  p r o b a b l y r e s u l t from a combination o f  genes ( n o t always  asychronous  apparent m a t u r a t i o n a r r e s t i n ANLL.  i n normal  differentiation,  quantitative including  The study o f t h e e x p r e s s i o n of  normal m y e l o i d d i f f e r e n t i a t i o n a n t i g e n s ( f o r example, l o s s o f HLA-DR a n t i g e n and g a i n o r l o s s o f many o t h e r s w i t h i n c r e a s i n g s t a t e s o f d i f f e r e n t i a t i o n ) has enabled important comparisons and normal  gene e x p r e s s i o n i n hemopoiesis.  between m a l i g n a n t  72  THESIS OBJECTIVES The e x i s t e n c e o f a common a n t i g e n i n (myelogenous) acute leukemia,  (CAMAL), thought  t o be a c e l l  s u r f a c e p r o t e i n on BM and PB  c e l l s o f m y e l o i d leukemia p a t i e n t s , had been p r e v i o u s l y e s t a b l i s h e d . R a b b i t h e t e r o a n t i s e r a s p e c i f i c f o r t h e CAMAL a n t i g e n was to  demonstrated  r e a c t w i t h m y e l o i d leukemia c e l l membrane e x t r a c t s i n t h e ELISA  and w i t h BM o r PB c e l l s from m y e l o i d leukemia p a t i e n t s (ANLL a t d i a g n o s i s and d u r i n g r e m i s s i o n and CGL) i n f l u o r e s c e n c e - a c t i v a t e d cell  s o r t e r (FACS) a n a l y s i s b u t n o t a t d e t e c t a b l e l e v e l s w i t h  from normals o r most lymphoid I d e n t i f i c a t i o n of the c e l l  leukemia p a t i e n t s (501,513,514).  t y p e ( s ) e x p r e s s i n g t h i s marker,  p a r t i c u l a r l y i n r e m i s s i o n p a t i e n t s , had n o t been c a r r i e d out. monoclonal  a n t i b o d y r e f e r r e d t o as CAMAL-1 was s u b s e q u e n t l y  which demonstrated i n ELISA (515).  A  developed  s p e c i f i c i t y f o r polyacrylamide g e l p u r i f i e d  CAMAL  T h i s MAb, however, showed l i m i t e d and i n c o n s i s t e n t  r e a c t i v i t y w i t h BM, PB c e l l s and c e l l nonlymphoblastic  cells  l i n e s from p a t i e n t s w i t h  leukemia i n FACS a n a l y s i s f o r c e l l membrane CAMAL  (467,515). The  f o l l o w i n g o b j e c t i v e s o f t h i s t h e s i s p r o j e c t f o c u s on  e s t a b l i s h i n g i n s i g h t i n t o t h e n a t u r e o f CAMAL e x p r e s s i o n by c e l l s and the p o s s i b l e s i g n i f i c a n c e o f such e x p r e s s i o n . 1.  A simple r a p i d monoclonal  antibody-based  t e s t was r e q u i r e d t o  d e t e c t t h e presence o f t h e CAMAL marker i n BM and PB c e l l s .  The  n a t u r e o f the d i f f e r e n c e i n c e l l u l a r r e a c t i v i t i e s between the monoclonal  and h e t e r o a n t i b o d i e s needed t o be e s t a b l i s h e d .  73  The  p o s s i b l e p r e s e n c e o f CAMAL i n normal h e m o p o i e t i c c e l l s  needed t o be examined, and t h e i d e n t i f i c a t i o n o f c e l l expressing The  types  CAMAL was r e q u i r e d .  p o s s i b l e s i g n i f i c a n c e of the frequent  d e t e c t i o n o f CAMAL i n  BM o r PB o f ANLL r e m i s s i o n p a t i e n t s needed t o be i n v e s t i g a t e d . The be The  question  of i t s r e l a t i o n s h i p t o c l i n i c a l  prognosis  needed t o  answered. a u t h o r wished t o examine t h e p o s s i b i l i t y t h a t t h e CAMAL  p r o t e i n might p o s s e s s some f u n c t i o n a l a c t i v i t y r e l a t e d t o h e m o p o i e t i c c e l l growth, p r o l i f e r a t i o n The  i n v i t r o myeloid progenitor  cell  and/or  differentiation.  assay, a u s e f u l system f o r  t h i s e v a l u a t i o n , c o u l d be u t i l i z e d t o i n v e s t i g a t e t h i s . CAMAL c o u l d be s e l e c t i v e l y leukemia-derived  depleted  from t h e system and  CAMAL c o u l d be added t o t h e system,  subsequent e v a l u a t i o n o f m y e l o i d c o l o n y  growth.  with  Normal  74  Footnotes 1.  Recent e v i d e n c e f o r t h e e x i s t e n c e o f p l u r i p o t e n t i a l stem c e l l s has come from gene t r a n s f e r s t u d i e s .  R. M u l l i g a n p r e s e n t e d  data ( 4 t h  T e r r y Fox Cancer Conference, Stem C e l l s and Autologous Bone Marrow T r a n s p l a n t a t i o n , Vancouver, B.C., September 21-22, 1987) showing t h a t i d e n t i c a l genomic i n t e g r a t i o n p a t t e r n s were observed i n lymphocytes (B and T c e l l s ) and macrophages when murine bone marrow, i n f e c t e d w i t h a r e t r o v i r u s , was used t o r e c o n s t i t u t e i r r a d i a t e d r e c i p i e n t s . Mulligan's  research  i n d i c a t e d t h a t i t was p o s s i b l e t o i n f e c t  p l u r i p o t e n t stem c e l l s w i t h o u t a f f e c t i n g t h e i r f u n c t i o n and t h a t  very  l i m i t e d numbers o f c l o n e s gave r i s e t o t h e m a j o r i t y o f h e m o p o i e t i c c e l l s a t any g i v e n time, i m p l y i n g numbers o f marked stem c e l l 2.  C l a r k SC, Kamen R: factors. PA,  Science  clones.  The human h e m a t o p o i e t i c  colony-stimulating  236:1229, 1987, and Yang Y-C, C i a r l e t t a AB, Temple  Chung MP, K o v a c i c  S, W i t e k - G i a n n o t t i  Donahue RE, Wong GG, C l a r k SC: I d e n t i f i c a t i o n by e x p r e s s i o n  J S , L e a r y AC, K r i z R,  Human IL-3 ( m u l t i - C S F ) :  c l o n i n g o f a n o v e l h e m a t o p o i e t i c growth  f a c t o r r e l a t e d t o murine IL-3. 3.  a s e q u e n t i a l c o n t r i b u t i o n by s m a l l  C e l l 47:3, 1986.  B i o l o g i c a l p r o p e r t i e s o f human i n t e r l e u k i n - 3 , p r e s e n t e d  by S. C l a r k  a t t h e 4 t h T e r r y Fox Cancer Conference, Stem C e l l s and Autologous Bone Marrow T r a n s p l a n t a t i o n , Vancouver, B.C., September 21-22, 1987.  75  CHAPTER I I  MATERIALS AND  I.  METHODS  DETAILED PROCEDURES A.  Monoclonal A n t i b o d i e s 1.  Production The monoclonal a n t i b o d y  a n t i g e n was  s p e c i f i c f o r the CAMAL  p r e p a r e d by Dr. Andrew J . Malcolm i n Dr. J u l i a  Levy's l a b o r a t o r y . prepared  (MAb)  T h i s MAb,  from a f u s i o n of NS-1  CAMAL-1 ( s u b c l a s s IgG-1), myeloma c e l l s and  s p l e n o c y t e s from mice immunized w i t h p u r i f i e d g e l e l e c t r o p h o r e s i s (PAGE)] CAMAL a n t i g e n . procedure  G.  was  Balb/c  [by p o l y a c r y l a m i d e  The  hybridoma  f o l l o w e d t h a t d e s c r i b e d by O i and Herzenberg  (499)  w i t h m o d i f i c a t i o n s by p r e v i o u s r e s e a r c h e r s i n t h i s l a b o r a t o r y (500), and was  the same as t h a t now  d e s c r i b e d f o r the  p r e p a r a t i o n of a p p r o p r i a t e n e g a t i v e c o n t r o l MAbs. The n e g a t i v e c o n t r o l MAbs were chosen from a s e r i e s of MAbs produced which were s p e c i f i c f o r p24,  the major  p r o t e i n of the Bovine L e u k o s i s V i r u s (BLV). old  structural  F i v e t o s i x week  female B a l b / c mice were immunized subcutaneously  ug of g e l - p u r i f i e d adjuvant.  Two  BLV  One  weeks f o l l o w i n g a second immunization,  week l a t e r , a strongly-immune mouse was  of p u r i f i e d p24  i n 0.1  c o n s e c u t i v e days and  30  a n t i g e n i n 50% complete Freund's  t h e s e mice were t e s t e d f o r r e a c t i v i t y i n ELISA w i t h p24.  with  ml s t e r i l e PBS  s e r a from purified  g i v e n 20  ug  i n t r a v e n o u s l y on 3  2 days l a t e r the s p l e e n was  removed f o r  76  f u s i o n w i t h l o g phase NS-1 c e l l s . s p l e n o c y t e s : N S - l c e l l s was used. polyethylene glycol  A r a t i o o f 5:1 These c e l l s were f u s e d w i t h  (PEG) and p l a t e d i n t o 96 w e l l t i s s u e  culture  p l a t e s a t 1.6 x 10~* c e l l s / w e l l i n Dulbecco's m o d i f i e d minimal e s s e n t i a l medium w i t h 20% FCS, hypoxanthine  (2 yg/ml),  a m i n o p t e r i n (0.18 yg/ml), thymidine (0.4 yg/ml) and t r e a t e d 5 B a l b / c thymocytes cells/well.  from 5 week o l d mice a t 10  feeder  A f t e r i n c u b a t i o n a t 37°C and 10% C 0  2  f o r 10-14  days, 0.1 ml c u l t u r e s u p e r n a t a n t s were assayed f o r r e a c t i v i t y i n ELISA w i t h p24 ( c o a t e d a t 1.0 yg/ml).  Hybridomas t h a t showed  p o s i t i v e r e a c t i v i t y w i t h p24 (and n e g a t i v e r e a c t i v i t y w i t h a b a t t e r y o f i r r e l e v a n t a n t i g e n s , i n c l u d i n g CAMAL) were c l o n e d 3 times by l i m i t i n g d i l u t i o n , expanded and grown as a s c i t e s i n p r i s t a n e - t r e a t e d B a l b / c mice.  Monoclonal  a n t i b o d i e s from a  p a r t i c u l a r anti-p24 hybridoma (BLV-1) were chosen as an a p p r o p r i a t e c o n t r o l f o r CAMAL-1 s t u d i e s f o r t h e f o l l o w i n g reasons: (1) t h i s MAb was t h e same s u b c l a s s as CAMAL-1  (IgG-1);  (2) i t r e a c t e d i n i n d i r e c t immunoperoxidase t e s t s w i t h PHA-stimulated  lymphocytes  from B L V - i n f e c t e d c a t t l e ; and (3) i t  was c a p a b l e o f p e r f o r m i n g as an e f f e c t i v e  immunoadsorbent,  p u r i f y i n g p24 from a crude v i r u s p r e p a r a t i o n i n a f f i n i t y chromatography.  S i n c e t h e s e a r e a l l p r o p e r t i e s shared by  CAMAL-1, and s i n c e an a p p r o p r i a t e n e g a t i v e c o n t r o l MAb was critical BLV-1  f o r many o f t h e experiments  d e s c r i b e d i n Chapter V I ,  was chosen from 12 anti-p24 l i n e s f o r these purposes.  77  2.  Purification CAMAL-1 and BLV-1 were p u r i f i e d from t h e i r r e s p e c t i v e  ascites f l u i d  i n t h e same manner u s i n g d i e t h y l a m i n o e t h y l  chromatography.  (DEAE)  A s c i t e s was c e n t r i f u g e d f o r 10 min a t 400 g t o  remove f i b r i n c l o t s .  A 50% s a t u r a t e d  was added dropwise w i t h c o n s t a n t  (NH.KSO. s o l u t i o n 4 2 4 s t i r r i n g to the a s c i t e s f l u i d .  T h i s s a l t c u t was k e p t o v e r n i g h t  a t 4°C and c e n t r i f u g e d a t  15,000 rpm i n a S o r v a l l RC5B f o r 30 min.  The r e s u l t i n g  p r e c i p i t a t e was resuspended i n a volume o f 0.03M T r i s b u f f e r (pH 7.4)  e q u i v a l e n t t o t h e o r i g i n a l volume o f a s c i t e s f l u i d , and o  d i a l y z e d overnight  a t 4 C i n 2 changes o f t h e same b u f f e r .  DEAE-Sephacel was poured i n t o a chromatography column and e q u i l i b r a t e d i n 0.03M T r i s b u f f e r .  The p r e p a r e d  a s c i t e s was  passed over t h e DEAE-Sephacel and t h e beads were washed w i t h b u f f e r u n t i l OD  = 0.  The p u r i f i e d a n t i b o d y  the column u s i n g a continuous Tris).  gradient  was e l u t e d from  (0.05-0.2M NaCl i n 0.03M  C o l l e c t e d f r a c t i o n s were monitored f o r absorbance a t 280  nm and those  c o n t a i n i n g I g were p o o l e d ,  concentrated  over PEG,  d i a l y z e d e x t e n s i v e l y i n PBS, and t e s t e d i n t h e ELISA f o r r e a c t i v i t y w i t h CAMAL.  Protein concentrations  of the p u r i f i e d  monoclonal a n t i b o d i e s were determined u s i n g t h e B i o r a d p r o t e i n assay. 3.  Immunoadsorbent DEAE-purified  Preparation  CAMAL-1 o r BLV-1 MAbs f o r immunoadsorbent  p r e p a r a t i o n were d i a l y z e d i n 0.05M b o r a t e b u f f e r , pH 8.0. Washed Sepharose 4B-CL beads were resuspended i n c o l d dH^O and stirred  i n a s m a l l beaker i n s i d e an i c e bucket.  CNBr i n  78  dimethylformamide (250 mg/ml) was added s l o w l y dropwise (33 mg/ml beads) over 1 min t o t h e beads. 10.5  The pH was k e p t between  and 11.0 u s i n g 4M NaOH over 30 min.  The a c t i v a t e d beads  were washed q u i c k l y on a g l a s s f u n n e l w i t h 250 ml c o l d dH^O f o l l o w e d by 500 ml c o l d 0.05M b o r a t e b u f f e r .  The a c t i v a t e d  beads were added 1:1 v/v t o t h e p u r i f i e d MAb (5.0 mg/ml) i n b o r a t e b u f f e r and t h e r e s u l t i n g s l u r r y was rocked o v e r n i g h t a t o 4 C.  The s u p e r n a t a n t was c o l l e c t e d t h e next day and t h e  coupled beads were washed i n lOmM T r i s b u f f e r , pH 8.0, b e f o r e o b e i n g resuspended  i n 0.16M ethanolamine  b l o c k any remaining  active sites.  overnight at 4 C to  F i n a l l y , t h e beads were  washed w e l l i n lOmM T r i s b u f f e r and s t o r e d a t 4°C i n PBS w i t h 0.02% NaN„. The 0D„„„ o f t h e MAb s o l u t i o n b e f o r e and a f t e r 3 280 c o u p l i n g were compared.  I n each case, between 92 and 93% o f t h e  o r i g i n a l MAb was determined  B.  Polyclonal Antiserum  t o have bound t o t h e beads.  (Rabbit) Antibodies s p e c i f i c f o r CAMAL was p r e p a r e d  female r a b b i t s hyperimmunized subcutaneously  from New Zealand White  w i t h P A G E - p u r i f i e d CAMAL  (30 yg CAMAL i n 50% complete Freund's a d j u v a n t ) .  R a b b i t s were  immunized a t monthly i n t e r v a l s and t h e i r b l o o d c o l l e c t e d by m a r g i n a l ear v e n i p u n c t u r e 2 weeks f o l l o w i n g each immunization. removed from t h e c l o t t e d b l o o d and assayed r e a c t i v i t y w i t h CAMAL (coated a t 1 ug/ml). was  raised i n a t o t a l of 6 rabbits.  Serum was  i n t h e ELISA f o r R a b b i t anti-CAMAL s e r a  A l l s e r a showed  analogous  s p e c i f i c i t y f o r CAMAL i n ELISA and i n FACS a n a l y s i s ; t h e s e d a t a were  79  p r e s e n t e d i n a Ph.D. t h e s i s by A . J . Malcolm  (501). Sera was s t o r e d a t  o -70 C i n a l i q u o t s u n t i l  used.  Normal r a b b i t serum (NRS) was p r e p a r e d by b l e e d i n g t h e same New Zealand White female r a b b i t s p r i o r t o t h e i r immunization w i t h CAMAL. T h i s serum was s t o r e d i n a s i m i l a r manner p r i o r t o u s e . R a b b i t a n t i - n o r m a l human serum (used as a p o s i t i v e c o n t r o l ) was p r e p a r e d by hyperimmunization with  o f female New Zealand White r a b b i t s  a membrane p r e p a r a t i o n d e r i v e d from s o n i c a t i o n o f p o o l e d normal  human p e r i p h e r a l b l o o d l e u c o c y t e s e m u l s i f i e d i n 50% complete adjuvant.  The r e s u l t i n g s e r a showed s t r o n g r e a c t i v i t y i n  fluorescence-activated c e l l  sorter  l e u c o c y t e s from a l l i n d i v i d u a l s C.  Freund's  (FACS) a n a l y s i s w i t h BM and PB  tested.  Secondary A n t i b o d i e s For  FACS a n a l y s i s u s i n g r a b b i t a n t i s e r a as t h e p r i m a r y  goat a n t i - r a b b i t IgG, F C a b ) ^  fragment,  label,  conjugated t o f l u o r e s c e i n  i s o t h i o c y a n a t e (FITC) was u t i l i z e d as t h e secondary l a b e l ( C a p p e l ) . T h i s and a l l p r i m a r y a n t i s e r a were r o u t i n e l y c e n t r i f u g e d a t 20,000 rpm f o r 5 min i n m i c r o f u g e tubes t o remove aggregates p r i o r to c e l l For  immediately  labeling.  i n d i r e c t immunoperoxidase s t u d i e s , u s i n g murine monoclonal  a n t i b o d i e s as t h e p r i m a r y l a b e l , r a b b i t anti-mouse  I g conjugated t o  h o r s e r a d i s h p e r o x i d a s e (HRP) was u t i l i z e d as t h e secondary (Cedarlane).  label  When r a b b i t anti-CAMAL serum was employed as t h e  p r i m a r y l a b e l , swine a n t i - r a b b i t I g - HRP secondary a n t i b o d y .  (Cedarlane) was used as t h e  80  Antigens 1.  CAMAL P r i o r t o t h e a v a i l a b i l i t y o f t h e CAMAL-1 MAb, CAMAL was  p u r i f i e d by Robert C. Shipman on p r e p a r a t i v e p o l y c r y l a m i d e g e l s f o r purposes o f r a b b i t immunization and ELISA.  Membrane  e x t r a c t s from p o o l e d PBL o f ANLL p a t i e n t s w i t h h i g h leukemic c e l l counts were passed over an immunoadsorbent column t o which r a b b i t a n t i - n o r m a l human a n t i b o d i e s were c o u p l e d .  The m a t e r i a l  which d i d n o t b i n d t o t h e column was e l e c t r o p h o r e s e d on 7.5% p o l y a c r y l a m i d e g e l s and t h e 68 KD p r o t e i n  band c o r r e s p o n d i n g t o  CAMAL was c u t o u t and e l u t e d from t h e g e l .  T h i s m a t e r i a l was  used t o c o a t ELISA p l a t e s and f o r immunization  purposes.  A f t e r t h e CAMAL-1 MAb became a v a i l a b l e , CAMAL was t h e r e a f t e r p u r i f i e d i n a one-step manner by e l u t i o n o f bound m a t e r i a l from a CAMAL-1 a f f i n i t y column over which  crude  e x t r a c t s from m y e l o i d leukemic c e l l s had been p a s s e d .  This  CAMAL p r e p a r a t i o n was used f o r ELISA, PAGE, and immunization o f r a b b i t s and was found t o have t h e same c h a r a c t e r i s t i c s as t h e p r e v i o u s l y p r e p a r e d CAMAL. In o r d e r t o study the p o s s i b l e e f f e c t o f a d d i t i o n o f CAMAL on m y e l o i d p r o g e n i t o r c e l l s  in vitro,  i t was important t h a t  p u r i f i e d CAMAL f o r t h e s e s t u d i e s be p r e p a r e d somewhat differently.  F o r these s t u d i e s t h e o r i g i n a l e x t r a c t which was  passed over t h e CAMAL-1 column c o n s i s t e d o f t h e s o l u b l e r e s u l t i n g from f r e e z e - t h a w i n g PBL o f c h r o n i c leukemia p a t i e n t s .  fraction  granulocytic  The p r o t e i n - c o n t a i n i n g f r a c t i o n s e l u t e d  from  the CAMAL-1 column were immediately p o o l e d and n e u t r a l i z e d w i t h  81  0.1 H NaOH, f o l l o w e d  by d i a l y s i s  i n PBS.  n e u t r a l i z a t i o n was c a r r i e d o u t i n o r d e r  This rapid  to maintain the antigen  i n a s t a t e r e s e m b l i n g i t s normal p h y s i o l o g i c a l one i f p o s s i b l e . Following  dialysis,  t h e CAMAL p r e p a r a t i o n was s t e r i l i z e d by  passage through a 0.45 u m i l l i p o r e f i l t e r and an a l i q u o t was reserved Biorad 2.  f o r determination  of p r o t e i n concentration  assay.  Negative c o n t r o l p r o t e i n antigens  progenitor purification  f o r use i n the myeloid  c e l l s t u d i e s were p r e p a r e d by a f f i n i t y i n t h e same manner as d e s c r i b e d  monoclonal a n t i b o d i e s , p u r i f i e d  Polyacrylamide Gel Electrophoresis  f o r CAMAL.  (PAGE) t o examine  e l u t e d from CAMAL-1 immunoadsorbent columns a f t e r passage  o f human plasma, c o n d i t i o n e d leukemia p a t i e n t s ' c e l l s . and  Murine  f o r these s t u d i e s .  Standard PAGE p r o c e d u r e s (502,503) were u t i l i z e d materials  chromatography  on a r a b b i t anti-mouse I g  immunoadsorbent column, were u t i l i z e d  E.  by t h e  sample m a t e r i a l s ,  were e l e c t r o p h o r e s e d  media, and s o l u b l e e x t r a c t s o f m y e l o i d  Ten p e r c e n t  reducing  g e l s were p r e p a r e d  i n c l u d i n g m o l e c u l a r weight s t a n d a r d s a t 50 V f o r 5 - 6 hours and s i l v e r  (Sigma),  stained  (504).  F.  Patient  Samples  P e r i p h e r a l blood  o r bone marrow samples were o b t a i n e d  Department o f Hematology, C e l l Laboratories,  from t h e  S e p a r a t o r and Immunotransplant  Vancouver G e n e r a l H o s p i t a l  (for a l l studies  described),  the T e r r y Fox L a b o r a t o r y , Vancouver, B.C. ( f o r t h e FACS s o r t i n g  82  s t u d i e s ) , U n i v e r s i t y C o l l e g e H o s p i t a l and Dr. M.  Greaves, ICRF,  L i n c o l n ' s Inn F i e l d s , London ( f o r many of the samples d e s c r i b e d i n Chapter  III).  I n a d d i t i o n , Dr. N. Buskard p r o v i d e d a number of  samples from m y e l o i d  leukemics  (through the A d u l t O u t p a t i e n t  Department and C r y o l a b o r a t o r y , Vancouver G e n e r a l H o s p i t a l ) and normals (through the Canadian Red  C r o s s , Vancouver) which were used  i n the experiments d e s c r i b e d i n Chapter  VI.  Diagnoses were e s t a b l i s h e d u s i n g r o u t i n e c r i t e r i a morphological  examination,  cytochemistry,  including  cytogenetics, colony  a s s a y s , s u r f a c e Ig and E - r o s e t t e t e s t s where a p p l i c a b l e . a d d i t i o n , i n d i r e c t immunofluorescence t e s t s f o r c e l l  In  surface antigens  were c a r r i e d out on samples o b t a i n e d from ICRF. A l l samples u t i l i z e d  i n t h e s e s t u d i e s were o b t a i n e d  p a t i e n t s t h a t had g i v e n t h e i r  G.  Cell  rpm  consent.  Preparations  Leucocytes 1,000  from  and  were p r e p a r e d  from BM  samples by s e d i m e n t a t i o n  at  c o l l e c t i o n of the b u f f y c o a t , o r by r o u t i n e  F i c o l l - H y p a q u e s e p a r a t i o n procedures  (505,506).  P e r i p h e r a l blood  l e u c o c y t e s (PBL) were o b t a i n e d from h e p a r i n i z e d samples u s i n g Plasmagel  ( L a b o r a t o i r e Roger B e l l o n , N e u i l l y , F r a n c e ) o r by  Ficoll-Hypaque technique. immunoperoxidase and  utilized.  removed by l y s i s f o r 3 - 4 necessary.  F o r a l l s t u d i e s performed u s i n g  f o r the m y e l o i d  Hypaque s e p a r a t i o n was  the  progenitor c e l l  Contaminating  assays,  Ficoll-  e r y t h r o c y t e s were  min w i t h 37°C T r i s - N H ^ C l a t pH  7.2,  if  83  H.  F l u o r e s c e n c e - A c t i v a t e d C e l l S o r t e r (FACS) S t u d i e s C e l l l a b e l i n g , FACS a n a l y s i s , and FACS s o r t i n g t e c h n i q u e s were  performed  as o u t l i n e d below.  1.  Cell Labeling L e u c o c y t e s p r e p a r e d from BM  o r PB samples were washed t w i c e  6 i n s t e r i l e PBS w i t h 5% FCS. resuspended 1:10  i n 0.2  i n PBS/5%FCS).  1 x 10  c e l l s were then  ml of t h e p r i m a r y a n t i b o d y s o l u t i o n  (diluted  A l l samples were l a b e l e d s e p a r a t e l y w i t h 3  p r i m a r y a n t i b o d i e s : ( 1 ) the p o s i t i v e c o n t r o l , r a b b i t a n t i - n o r m a l human serum; (2) the n e g a t i v e c o n t r o l , normal (3) r a b b i t anti-CAMAL serum. 1.5 of  r a b b i t serum; and  C e l l s were i n c u b a t e d on i c e f o r  h r , washed 3 times i n PBS/5%FCS, and resuspended  i n 0.2  secondary a n t i b o d y ( f l u o r e s c e i n a t e d goat a n t i - r a b b i t  d i l u t e d 1:20 ice,  i n PBS/5%FCS).  A f t e r another 1.5  ml  IgG,  h r i n c u b a t i o n on  c e l l s were washed once i n PBS/5%FCS then c e n t r i f u g e d a t  1,000  rpm  f o r 5 min  through 100% FCS.  Samples were  resuspended  i n 0.5 ml PBS/5%FCS p r i o r t o FACS a n a l y s i s . 2.  FACS A n a l y s i s o f L a b e l e d C e l l A B e c t o n - D i c k i n s o n FACS IV was  Samples u t i l i z e d f o r these s t u d i e s .  R o u t i n e o p e r a t i o n i n c l u d e d argon l a s e r s e t t i n g o f 400 mW, nm,  488  FITC f i l t e r and s t a n d a r d i z a t i o n w i t h g l u t a r a l d e h y d e - f i x e d  c h i c k e n r e d b l o o d c e l l s and f l u o r e s c e n t  monodispersed  carboxymethylated m i c r o s p h e r e s  m + 0.02  (d = 1.75  SD).  For a  g i v e n sample, 25,000 c e l l s were a n a l y z e d and r e c o r d e d f o r r e a c t i v i t y w i t h each of the p o s i t i v e , n e g a t i v e and sera.  anti-CAMAL  84  3.  FACS C e l l S o r t i n g Technique A f t e r a sample had been a n a l y z e d  as s i g n i f i c a n t l y p o s i t i v e  w i t h t h e r a b b i t anti-CAMAL serum, t h i s sample was s o r t e d u s i n g t h e FACS IV.  Two s o r t windows were determined, one c o n t a i n i n g  t h a t 25% o f t h e sample showing minimal r e l a t i v e f l u o r e s c e n c e and the o t h e r c o n t a i n i n g t h a t 25% o f t h e sample showing t h e h i g h e s t r e l a t i v e fluorescence  (see F i g u r e 2.1).  p o p u l a t i o n s were c o l l e c t e d i n s e p a r a t e PBS/5%FCS.  The two  cell  tubes c o n t a i n i n g 1 ml  The c e l l s between these two windows, r e p r e s e n t i n g  t h e remaining  50% o f t h e sample c e l l p o p u l a t i o n , were d i s c a r d e d  into the r e s e r v o i r f l a s k .  The head d r i v e frequency  was s e t a t  36 KHz, and 2,000 V were a p p l i e d a c r o s s t h e e l e c t r o s t a t i c deflection plates.  The Eput counters  recorded  t h e number o f  c e l l s c o l l e c t e d i n t h e r i g h t and l e f t d e f l e c t i o n tubes,  and t h e  FACS was r u n a t 5 d r o p l e t s p e r d e f l e c t i o n p u l s e w i t h t h e a b o r t mode i n o p e r a t i o n ; t h e d r o p l e t d e l a y was s e t a t 14 drops. C o o l i n g water (2°C) was c i r c u l a t e d around t h e c o l l e c t i o n and sample tubes.  Immediately f o l l o w i n g a s o r t r u n , 5,000  cells  from each o f t h e c o l l e c t e d p o p u l a t i o n s were examined w i t h r e s p e c t t o t h e i r r e l a t i v e f l u o r e s c e n c e , i n o r d e r t o determine the a c c u r a c y  o f the s o r t .  based on s o r t i n g accuracy standardized accuracy). the c e l l  The expected accuracy  was > 95%,  determined p r i o r t o each r u n u s i n g  green and r e d f l u o r e s c e n t beads ( g e n e r a l l y 97% S o r t i n g a c c u r a c i e s were found t o be comparable f o r  samples c o l l e c t e d ; t h e "low" p o p u l a t i o n showed 96.7 +  0.7% (SEM) accuracy (SEM) a c c u r a c y ,  and t h e " h i g h " p o p u l a t i o n showed 90.8 + 0.9%  t h e d i f f e r e n c e found t o be a t t r i b u t a b l e t o some  85  F i g u r e 2.1. C e l l p o p u l a t i o n s c o l l e c t e d by the f l u o r e s c e n c e - a c t i v a t e d cell s o r t i n g (FACS) t e c h n i q u e , u s i n g 3-dimensional parameters. (a) Schematic diagram of the 3 dimensions v i s u a l i z e d i n (b) and ( c ) . x a x i s , l i g h t s c a t t e r ( p r o p o r t i o n a l to c e l l s i z e ) ; y a x i s , r e l a t i v e f l u o r e s c e n c e i n t e n s i t y ; z a x i s , c e l l number. (b) T o t a l c e l l p o p u l a t i o n (25,000 c e l l s ) a n a l y z e d w i t h r a b b i t anti-CAMAL serum l a b e l , p r i o r to c e l l s o r t i n g . (c) C e l l p o p u l a t i o n s c o l l e c t e d f o l l o w i n g c e l l s o r t i n g , showing 2 s e p a r a t e p o p u l a t i o n s of lowest and h i g h e s t (25% each) f l u o r e s c i n g c e l l s .  87  cell  damage r e s u l t i n g i n d e b r i s which f e l l  outside  the  o r i g i n a l l y d e f i n e d s o r t window f o r the " h i g h " p o p u l a t i o n . c e l l s were then washed once i n PBS FCS  and  the sample was  S c i e n t i f i c ) a t 800 The  rpm  to remove p r o t e i n from the  into a cytocentrifuge  f o r 8 min  to obtain s l i d e  preparations.  The  immunoperoxidase t e c h n i q u e has  e x t e n s i v e l y on t i s s u e s e c t i o n s  We  p r e v i o u s l y been u t i l i z e d  been more l i m i t e d  adapted the i n d i r e c t immunoperoxidase procedure to  study CAMAL a n t i g e n e x p r e s s i o n preparations  Preparations  ( p a r a f f i n embedded or f r o z e n ) ; however  i n s t a i n i n g s i n g l e c e l l s has  (467,507-511).  and  l i g h t microscopy.  I n d i r e c t Immunoperoxidase S t a i n i n g of S i n g l e C e l l  i t s use  (John's  s l i d e s were s t a i n e d r o u t i n e l y w i t h Wright's s t a i n  examined by  I.  loaded  The  i n or on s i n g l e c e l l  of bone marrow or PBL.  cytospin  T h i s t e s t i s simple,  u s e f u l as a d i a g n o s t i c probe f o r the p r e s e n c e of CAMAL.  rapid  and  Processed  c e l l s were washed a t l e a s t 3 times i n serum-free medium (RPMI), resuspended a t 2 x 1 0 funnels  for slide  A f t e r 30 min  6  c e l l s / m l , and  immersion i n methanol-2% H„0„ the s l i d e s were 2 2 sprayed a g a i n  (CAMAL-1 o r c o n t r o l BLV-1  MAb  c o n t r o l r a b b i t serum a t 1:400 circle,  incubated  secondary a n t i b o d y at  1:200  incubated  (3 drops) i n t o c y t o s p i n  preparation.  sprayed, f l o o d e d , and  cell  loaded  i n PBS  (5 min).  d i l u t i o n i n PBS)  w i t h 1:25  was  a p p l i e d to  a t 20°C, and washed i n PBS.  (anti-mouse Ig-HRP a t 1:100  f o r 30 min  antibody  a t 10 yg/ml, r a b b i t anti-CAMAL or  f o r 30 min  d i l u t i o n i n PBS  Primary  The  or a n t i - r a b b i t Ig-HRP  normal human serum) was  a t 20°C and washed.  the  applied,  S l i d e s were developed f o r  88  10 min PBS,  i n diaminobenzidine  (10 mg)  and H^O^  (100 y l ) i n 50  washed, and c o u n t e r s t a i n e d w i t h e i t h e r 2% methyl  ml  green,  hematoxylin o r W r i g h t ' s . S l i d e s were examined by l i g h t microscopy.  S l i d e s were  scanned  c o m p l e t e l y t o examine a l l c e l l s and percentage p o s i t i v e counts were determined  a t XI,000 m a g n i f i c a t i o n .  r e a c t i v i t y was each s l i d e .  In cases where p o s i t i v e  > 10% of a sample, a t l e a s t 400  c e l l s were s c o r e d on  When samples showed lower r e a c t i v i t y , between 1,000  3,000 c e l l s were examined f o r each s l i d e  ( h i g h e s t numbers of  were s c o r e d f o r samples w i t h extremely low p e r c e n t a g e  counts as t h e i r presence was  t e s t s were i n i t i a l l y  cells  reactivity).  Mature and n u c l e a t e d e r y t h r o c y t e s were n o t i n c l u d e d i n the positive cell  and  too v a r i a b l e .  percentage A l l slide  c a r r i e d out a t l e a s t t w i c e on s e p a r a t e o c c a s i o n s  and examined b l i n d l y and  i n d e p e n d e n t l y by two  r e s e a r c h e r s (Drs. J .  Levy and P. Logan) t o ensure a c c u r a c y and r e p r o d u c i b i l i t y .  After  one  y e a r , t e s t s were o n l y d u p l i c a t e d when s l i d e p r e p a r a t i o n s were s u b o p t i m a l , as a means of v e r i f y i n g  J.  results.  B l i n d Study P r o t o c o l A b l i n d study, now  ending i t s t h i r d y e a r , was  between the Department o f M i c r o b i o l o g y , U.B.C.  established  and the Department of  P a t h o l o g y and D i v i s i o n of Hematopathology, Vancouver G e n e r a l Hospital.  T h i s study was  u s i n g CAMAL-1 and i n d i r e c t 1.  designed t o monitor ANLL p a t i e n t s ' BM  cells  immunoperoxidase.  P a t i e n t Group A l l p a t i e n t s a t t e n d i n g the Hematology Department o f Vancouver  G e n e r a l H o s p i t a l (as i n - o r o u t - p a t i e n t s ) and who  had BM  aspirations  89  performed were i n c l u d e d  i n t h i s study.  samples examined d u r i n g  t h i s study.  There were 701 p a t i e n t  Thirty-seven  percent  o f these  samples were from 86 d i f f e r e n t ANLL p a t i e n t s w h i l e 63% were from any other  i n d i v i d u a l s attending  General H o s p i t a l .  t h e Hematology Department o f Vancouver  T h i s l a t t e r group i n c l u d e d p a t i e n t s w i t h any o t h e r  type o f leukemia, h e m a t o l o g i c malignancy o r d y s c r a s i a , and normals. T h i s study r e p o r t s t h e r e s u l t s from 34 o f t h e 86 d i f f e r e n t ANLL p a t i e n t s examined d u r i n g t h i s time.  These 34 p a t i e n t s  represent  e v e r y ANLL p a t i e n t f o r whom bone marrow samples were o b t a i n e d diagnosis  and post-chemotherapy ( w i t h i n 1.0 + 0.5 months).  remaining (n = 52) ANLL p a t i e n t s were e i t h e r i n i t i a l l y l a t e r stages i n t h e i r d i s e a s e  Eighty percent at  diagnosis  study p e r i o d . achieved  CR.  The  evaluated  at  (n = 35), had i n s u f f i c i e n t amounts o f  bone marrow a s p i r a t e d t o enable s l i d e p r e p a r a t i o n o n l y seen a t d i a g n o s i s  both at  (n = 11) o r were  (n = 6 ) .  o f a l l ANLL p a t i e n t s t h a t we examined  (n = 40) a c h i e v e d  complete r e m i s s i o n  In the presently reported Differences  initially  (CR) d u r i n g t h e  group o f 34 p a t i e n t s , 92%  (80% v e r s u s 92%) were due t o f a i l u r e o f 5/6  remaining p a t i e n t s , examined i n i t i a l l y  at diagnosis,  to a t t a i n  CR.  No f o l l o w up samples were r e c e i v e d from t h e s e p a t i e n t s . The median p a t i e n t age was 48.4 years f o r t h e t o t a l ANLL group (n = 86) and 49.3 years f o r t h e group r e p o r t e d 2.  herein  (n = 34).  A n a l y s i s Groups S l i d e s made from p a t i e n t samples were coded by  successive  numbering so t h a t no p a t i e n t i n f o r m a t i o n would be a v a i l a b l e t o t h e principal investigator. at  diagnosis  The ANLL p a t i e n t s who had been examined both  and post-chemotherapy were d i v i d e d i n t o 2 groups.  90  Groups 1 and 2 were compared by t h e p r o d u c t - l i m i t method (Kaplan-Meier e s t i m a t e ) u s i n g l o g - r a n k  s t a t i s t i c s t o determine  d i f f e r e n c e s i n s u r v i v a l time p r i o r t o r e l a p s e . 1.  ANLL p a t i e n t s a t i n i t i a l p r e s e n t a t i o n whose CAMAL BM v a l u e s decreased  2.  ANLL p a t i e n t s a t i n i t i a l p r e s e n t a t i o n whose CAMAL BM values  3.  post-chemotherapy.  i n c r e a s e d o r s t a y e d t h e same post-chemotherapy.  S t a t i s t i c a l Analysis We have d e f i n e d CAMAL BM v a l u e s which had n o t changed  s i g n i f i c a n t l y p r e - and post-chemotherapy as b e i n g < 5.0%.  those which changed  T h i s f i g u r e was chosen from r e s u l t s o f an a n a l y s i s done on  20 s l i d e s , each read b l i n d l y 5 times i n a randomized manner, which were s e l e c t e d from a p o p u l a t i o n o f 700 s l i d e s so t h a t the complete range o f t r u e p r o p o r t i o n s o f r e c o r d e d represented.  CAMAL BM v a l u e s would be  I n no i n s t a n c e d i d t h e observed CAMAL BM v a l u e o f a  g i v e n s l i d e d i f f e r by > 4.5%, hence t h e f i g u r e 5.0% was chosen t o i n d i c a t e a s i g n i f i c a n t change. residuals and  The a b s o l u t e magnitude o f t h e  ( d i f f e r e n c e s between t h e f i v e r e c o r d i n g s  f o r t h e same s l i d e  t h e mean o f these r e c o r d i n g s ) never exceeded + 2.4%. No d i v i s i o n o f t h e p a t i e n t group was performed f o r a n a l y s i s o f  the data by t h e Cox p r o p o r t i o n a l hazards model which examined t h e f o l l o w i n g c o v a r i a t e s as they r e l a t e d t o d i s e a s e - f r e e s u r v i v a l time prior to relapse: BM v a l u e  sex, age, i n i t i a l  CAMAL BM v a l u e , change i n CAMAL  (from d i a g n o s i s t o post-chemotherapy).  91  K. 1.  M y e l o i d P r o g e n i t o r C e l l Assay General P r o t o c o l A system f o r s t u d y i n g t h e growth o f m y e l o i d p r o g e n i t o r s  ( s p e c i f i c a l l y c o l o n y - f o r m i n g u n i t s i n c u l t u r e o r CFU-c) was e s t a b l i s h e d i n Dr. J . Levy's l a b o r a t o r y a f t e r examining t h r e e such systems from d i f f e r e n t l a b o r a t o r i e s .  The systems i n i t i a l l y  examined  were those o f Dr. C. Eaves ( T e r r y Fox L a b o r a t o r y , Vancouver, B.C.), Dr. I . B e r n s t e i n (Fred H u t c h i n s o n Cancer I n s t i t u t e , Washington) and Dr. H. Messner Ontario).  ( O n t a r i o Cancer  Seattle,  I n s t i t u t e , Toronto,  While each o f t h e s e systems has i t s advantages and  d i s a d v a n t a g e s , t h e system based on Dr. H. Messner's p r o t o c o l was chosen f o r these s t u d i e s f o r t h e f o l l o w i n g r e a s o n s : (1) t h e u s e o f human plasma a l l o w s the c u l t u r e system t o be r e s t r i c t e d t o human o r even autologous m a t e r i a l s  (no FCS i s u s e d ) ; (2) human plasma  i n c r e a s e s t h e v i s c o s i t y o f t h e system and makes i t much e a s i e r t o a c c u r a t e l y determine c o l o n y numbers than m e t h y l c e l l u l o s e systems u s i n g FCS; (3) c o l o n i e s a r e e a s i l y p l u c k e d from m e t h y l c e l l u l o s e , when d e s i r e d , compared t o agar-bound c o l o n i e s ;  (4) human plasma promotes  the growth o f w e l l - d e f i n e d megakaryocyte c o l o n i e s i n v i t r o ; and (5) human plasma c o n t a i n s p r o t e i n t h a t adsorbs s p e c i f i c a l l y t o CAMAL-1 i n a f f i n i t y chromatography, a l l o w i n g study o f t h e e f f e c t o f i t s d e p l e t i o n from t h e system. The c o l o n y - p l u c k i n g and immunoperoxidase  s t a i n i n g o f t h e CGL  sample d e s c r i b e d i n Chapter IV was c a r r i e d o u t on c o l o n i e s o b t a i n e d from Dr. C. Eaves ( T e r r y Fox L a b o r a t o r y , Vancouver, B.C.).  Dr. Eaves  a l s o k i n d l y p r o v i d e d h e r l a b o r a t o r y ' s e x p e r t i s e i n t h i s system over a  92  p e r i o d o f 6 months i n o r d e r f o r t h i s i n v e s t i g a t o r t o l e a r n d e t a i l s of the  i n v i t r o c o l o n y assay. The  b a s i c components o f t h e m y e l o i d p r o g e n i t o r  outlined i n detail.  In general,  assay w i l l be  t o prepare d u p l i c a t e c u l t u r e s , the  f o l l o w i n g components were added t o 15 ml s t e r i l e t e s t 1.4 ml 2% m e t h y l c e l l u l o s e  ( c o n t a i n i n g 5 x 10  _5  tubes:  M  2-mercaptoethanol) 0.9 ml human plasma 0.3 ml c o n d i t i o n e d  medium 6  0.3 ml c e l l s  ( a t a p p r o x i m a t e l y 1 - 2 x 10  cells/ml) i n  I s c o v e ' s medium The  m i x t u r e was g e n t l y v o r t e x e d and 1.0 ml was d e l i v e r e d  d u p l i c a t e 35 mm 3 cc syringe. x 15 mm)  t i s s u e culture dishes  and  (Lux) w i t h an 18 ga n e e d l e and  The d u p l i c a t e c u l t u r e s were p l a c e d  p e t r i d i s h w i t h a 35 mm d i s h c o n t a i n i n g  for humidification.  Cultures  into  i n s i d e a l a r g e (150 5 ml s t e r i l e  dH^O  were i n c u b a t e d a t 37°C, 100% h u m i d i t y  5% CO^ f o r 14 days a t which time they were read w i t h an  i n v e r t e d microscope.  Colonies  containing  > 20 c e l l s were s c o r e d .  two o c c a s i o n s d u r i n g  these s t u d i e s , counts were v e r i f i e d b l i n d l y by  another r e s e a r c h e r .  I n a d d i t i o n , counts were a l s o done b l i n d l y a t  i n t e r v a l s t o v e r i f y r e s u l t s by t h i s  investigator.  D e t a i l e d d e s c r i p t i o n s o f t h e components i n v o l v e d progenitor 2.  c e l l assay  Preparation  i n the myeloid  follow.  o f C o n d i t i o n e d Medium  C o n d i t i o n e d medium was used i n t h e m y e l o i d p r o g e n i t o r  cell  as a source of c o l o n y s t i m u l a t i n g a c t i v i t y f o r growth o f m y e l o i d progenitors  On  in vitro.  assay  93  a.  P l a c e n t a l C o n d i t i o n e d Medium  (PCM)  One e n t i r e p l a c e n t a was o b t a i n e d d u r i n g a scheduled C a e s a r i a n - s e c t i o n a t S t . P a u l ' s H o s p i t a l , Vancouver, B.C. p l a c e n t a was immediately  The  p l a c e d i n t o s t e r i l e PBS and t r a n s p o r t e d t o  the U n i v e r s i t y o f B r i t i s h  Columbia.  The p l a c e n t a was washed f r e e of  contaminating b l o o d w i t h s t e r i l e PBS, t h e t i s s u e was t e a s e d a p a r t and f o r c e d through a s t e r i l e s c r e e n t o o b t a i n a s i n g l e c e l l  suspension.  P l a c e n t a l c e l l s were washed 2 times i n RPMI and resuspended (each 1.0 ml volume o f packed c e l l s  i n RPMI  i n 20 mis) w i t h 5% FCS.  The  c e l l s were i n c u b a t e d a t 37°C i n 5% CO^ f o r 7 days, then p e l l e t e d at  2,000 rpm f o r 10 min and t h e supernatant was h a r v e s t e d .  a l i q u o t s of p o o l e d supernatant were s t o r e d a t -70°C u n t i l b.  ( p l a c e n t a l c o n d i t i o n e d medium o r PCM)  used.  Leucocyte C o n d i t i o n e d Medium  (PHA-LCM)  H e p a r i n i z e d p e r i p h e r a l b l o o d from normal i n d i v i d u a l s sedimented  Three ml  was  a t 37°C f o r 40 - 50 min and the l e u c o c y t e - r i c h plasma  was h a r v e s t e d .  The l e u c o c y t e s were then p e l l e t e d a t 400 g f o r 10 min  and resuspended  i n I s c o v e ' s medium w i t h 10% FCS and 1%  phytohemagglutinin  (PHA) a t a c o n c e n t r a t i o n of 1 x 10  F i f t y ml volumes were i n c u b a t e d  a t 37°C and 5% C 0  2  6  cells/ml.  f o r 7 days  a f t e r which the s u p e r n a t a n t s were h a r v e s t e d and s t o r e d i n a l i q u o t s a t o -70 C u n t i l  needed.  To ensure potency titrated  of the PCM and PHA-LCM p r e p a r a t i o n s , they were  i n t h e m y e l o i d p r o g e n i t o r c e l l assay u s i n g normal p e r i p h e r a l  b l o o d l e u c o c y t e s from a v o l u n t e e r whose c e l l s had p r e v i o u s l y been demonstrated t o be c a p a b l e of c o l o n y f o r m a t i o n i n t h a t assay.  Both  the PCM and PHA-LCM p r e p a r a t i o n s were found t o f u n c t i o n o p t i m a l l y as  94  s t i m u l a t o r s of m y e l o i d of  10%.  Figure 3.  c o l o n y f o r m a t i o n when used a t a c o n c e n t r a t i o n  A dose response  curve f o r one b a t c h of PHA-LCM i s shown i n  2.2.  P r e p a r a t i o n of 2% Stock M e t h y l c e l l u l o s e Twenty grams of m e t h y l c e l l u l o s e (MC)  2 l i t r e flask.  T h i s was  constant s t i r r i n g .  MC  autoclaved i n a  d i s s o l v e d i n 500 ml s t e r i l e hot dH^O  with  Another 500 ml of 2 X Iscove's medium was  a f t e r the s o l u t i o n had s o l u t i o n was  powder was  c o o l e d t o 20°C ( a p p r o x i m a t e l y  s t i r r e d o v e r n i g h t a t 4°C.  s o l u t i o n were p r e p a r e d  and  o  3 h r ) and  A l i q u o t s (100 ml)  this  of the  2%  s t o r e d a t 20°C f o r 7 days when they  were examined f o r c o n t a m i n a t i o n a l i q u o t s were s t o r e d a t -20  added  and d i s c a r d e d i f n e c e s s a r y .  C u n t i l needed.  P r i o r to use  (24  Sterile hr)  -5 an a l i q u o t was  thawed, 5 x 10  mixed by v i g o r o u s shaking. remaining  s t o c k MC  s o l u t i o n was  used f o r e x p e r i m e n t a t i o n ,  performed u s i n g normal PBL  4.  The  d i s c a r d e d w i t h i n 1 month of thawing.  m e t h y l c e l l u l o s e was  MC  M 2-mercaptoethanol was  added  and  used o r the  B e f o r e any b a t c h of a titration  was  t o determine the o p t i m a l c o n c e n t r a t i o n of  t o be used i n the system. P r e p a r a t i o n of Human Plasma  Samples of f a s t i n g human plasma p r e p a r e d from h e p a r i n i z e d p e r i p h e r a l b l o o d samples ( c o l l e c t e d from normal v o l u n t e e r s ) were t e s t e d f o r t h e i r growth promoting c a p a c i t y i n the m y e l o i d  progenitor o  c e l l assay.  A p p r o p r i a t e samples were s t o r e d i n a l i q u o t s a t -70  u n t i l used.  F o r s t a n d a r d i z a t i o n purposes r e q u i r e d i n the experiments  o u t i n e d i n Chapter  V I , plasma from a s i n g l e v o l u n t e e r was  q u a n t i t y (500-600 ml whole b l o o d donation) over 8 months.  T h i s plasma was  on 3 s e p a r a t e  C  obtained i n occasions  always of h i g h q u a l i t y w i t h r e g a r d to  95  PERCENT  CONDITIONED  MEDIUM ( P H A - L C M )  F i g u r e 2 . 2 . Dose response c u r v e f o r c o n d i t i o n e d medium (PHA-LCM) on normal p e r i p h e r a l b l o o d C F U - c .  96  growth promoting a b i l i t y i n the m y e l o i d Leucocytes  progenitor c e l l  assay.  from the same b l o o d sample were always used t o p r e p a r e  PHA-LCM a t the same time, t o f u r t h e r s t a n d a r d i z e t h e system.  Prior  to use i n t h e assay, plasma samples were thawed, c e n t r i f u g e d t o remove any f i b r i n c l o t s t h a t may have formed and f i l t e r e d through a 0.45 ym m i l l i p o r e membrane t o reduce background p r e c i p i t a t e s . 5.  P r e p a r a t i o n o f C e l l Samples Bone marrow o r p e r i p h e r a l b l o o d specimens were c o l l e c t e d i n  p r e s e r v a t i v e - f r e e h e p a r i n i z e d tubes.  Mononuclear c e l l - e n r i c h e d  p r e p a r a t i o n s were o b t a i n e d by l a y e r i n g samples over a F i c o l l - H y p a q u e g r a d i e n t as p r e v i o u s l y d e s c r i b e d .  Leucocytes  were washed t w i c e i n  I s c o v e ' s medium and resuspended a t 1 x 10^ c e l l s / m l . c e l l s were p l a t e d a t lower ( f o r CGL samples) o r h i g h e r  Occasionally, ( f o r ANLL  samples) c o n c e n t r a t i o n s i n o r d e r t o o b t a i n a p p r o p r i a t e numbers of colonies f o r assaying. To prepare non-adherent c e l l p o p u l a t i o n s f o r some experiments, adherent c e l l s were removed from mononuclear c e l l - e n r i c h e d o p o p u l a t i o n s by i n c u b a t i o n (37 C, 5% CO^) o v e r n i g h t a t 1 x 1 0 c e l l s / m l i n Iscove's medium w i t h 5% FCS i n t i s s u e c u l t u r e f l a s k s . 6  6.  P l u c k i n g and S t a i n i n g o f C o l o n i e s On some o c c a s i o n s , c o l o n i e s were p l u c k e d  i n d i v i d u a l l y from the  m e t h y l c e l l u l o s e ( w h i l e b e i n g examined through an i n v e r t e d microscope) u s i n g a drawn-out c a p i l l a r y tube and a t i n y rubber b u l b t o a s p i r a t e cells.  These a s p i r a t e d c e l l s were p l a c e d i n s i d e p r e v i o u s l y etched  circles  on g l a s s microscope s l i d e s and d i s p e r s e d w i t h i n the c i r c l e  u s i n g a j e t of f o r c e d a i r from a pump. stained with a modified Papanicolaou  A f t e r a i r - d r y i n g , c e l l s were  technique  o r Wright's s t a i n and  97  examined by  l i g h t microscopy.  s t a i n i n g of the CGL used was  immunoperoxidase  c o l o n i e s d e s c r i b e d i n Chapter  V I , the  the same as t h a t a l r e a d y d e s c r i b e d h e r e i n except  necessary  t o use an i n i t i a l  o r d e r to adequately 7.  F o r the i n d i r e c t  technique t h a t i t was  immersion i n 10% H 0^ - methanol i n 2 2  b l o c k endogenous p e r o x i d a s e .  P r e p a r a t i o n of Human Plasma f o r CAMAL D e p l e t i o n S t u d i e s Normal human plasma was  u s i n g a CAMAL-1 column.  d e p l e t e d of CAMAL by  Between 10 and  i n t h i s manner a t any g i v e n time. utilized,  plasma was  fractions.  passed  immunoadsorbence  20 ml of plasma was  A 6.5  ml a f f i n i t y column  s t e r i l i z e d by passage through  OD  23o a 0.45  m i l l i p o r e f i l t e r b e f o r e b e i n g s t o r e d i n a l i q u o t s a t -70°C. t h o r o u g h l y washing the column w i t h PBS showed n e g a t i v e OD was  _  280  was  over the column and c o l l e c t e d i n 1 ml  Those c e n t r a l f r a c t i o n s h a v i n g e q u i v a l e n t  r e a d i n g s were p o o l e d and  depleted  until  N HC1.  After  the unbound f i l t r a t e  r e a d i n g s , the m a t e r i a l adsorbed  e l u t e d i n 1 ml f r a c t i o n s w i t h 0.1  u  t o the column  Protein-containing  f r a c t i o n s were p o o l e d and s t o r e d . To prepare a p p r o p r i a t e c o n t r o l plasma f o r these batches  of plasma o b t a i n e d from the same normal v o l u n t e e r a t the same  time, were passed  i n an i d e n t i c a l manner over a 6.5  ( n e g a t i v e c o n t r o l MAb) h a v i n g OD  280  a f f i n i t y column.  ml  Those c e n t r a l  BLV-1 fractions  r e a d i n g s e q u i v a l e n t to the plasma b e f o r e column  passage were p o o l e d , 8.  experiments,  f i l t e r - s t e r i l i z e d and  stored.  P r e p a r a t i o n of C o n d i t i o n e d Medium f o r CAMAL D e p l e t i o n S t u d i e s C o n d i t i o n e d medium (PCM  and PHA-LCM) was  passage over a CAMAL-1 i m m u n o a f f i n i t y t h a t j u s t d e s c r i b e d f o r plasma.  d e p l e t e d of CAMAL by  column i n the same manner as  Appropriate control conditioned  98  media was a l s o p r e p a r e d by passage over a BLV-1 i m m u n o a f f i n i t y column.  Conditioned  medium so p r e p a r e d was f i l t e r - s t e r i l i z e d and o  s t o r e d i n a l i q u o t s a t -70 C u n t i l 9.  CAMAL A d d i t i o n Preparation  Studies  o f p u r i f i e d CAMAL o r n e g a t i v e  been d e s c r i b e d p r e v i o u s l y . i n phosphate b u f f e r e d had  used.  c o n t r o l p r o t e i n s have  These p r o t e i n s were m a i n t a i n e d a t pH 7.2  s a l i n e a t 4°C u n t i l u t i l i z e d .  been f i l t e r s t e r i l i z e d  The s o l u t i o n s  and B i o r a d p r o t e i n assays performed on an  a l i q u o t of the f i l t e r e d s o l u t i o n ( s ) . For these s t u d i e s , e q u i v a l e n t or n e g a t i v e  amounts ( 0 - 4 0 ug/ml) o f CAMAL  c o n t r o l p r o t e i n s were added t o r o u t i n e c e l l c u l t u r e s  p r i o r to p l a t i n g i n the progenitor  c e l l assay.  T o t a l volumes o f  p l a t i n g m i x t u r e were m a i n t a i n e d a t r o u t i n e volume ( e q u i v a l e n t m l / p l a t e ) by r e p l a c i n g p o r t i o n s  of the c e l l s o l u t i o n  medium) w i t h PBS w i t h o r w i t h o u t CAMAL o r n e g a t i v e The  preparations  t o 1.0  (Iscove's  control proteins.  were p l a t e d and c u l t u r e d i n a r o u t i n e manner.  Three  s e p a r a t e l y p r e p a r e d CAMAL p r o t e i n samples were u t i l i z e d  i n these  studies.  Each sample  Each sample came from a CGL p a t i e n t ' s c e l l s .  was t e s t e d b o t h on normal and m y e l o i d leukemic c e l l s n o n - s p e c i f i c t o x i c i t y of i n d i v i d u a l preparations Three s e p a r a t e l y p r e p a r e d n e g a t i v e similarly  L.  t o ensure t h a t  was n o t o c c u r r i n g .  c o n t r o l p r o t e i n samples were  utilized.  Statistical  Analyses  Two-sided Student's t - t e s t a n a l y s e s  were performed t o compare  the d a t a w i t h i n Chapter I I I and w i t h i n Chapter V I .  Comparisons were  made, between m y e l o i d leukemics and normals ( o r o t h e r  hematologic  99  m a l i g n a n c i e s ) , o f t h e average p e r c e n t CAMAL-1 p o s i t i v e c e l l s and PB.  i n BM  The r e s u l t s o f CFU-c growth i n c o n t r o l and CAMAL d e p l e t e d  c u l t u r e s were a l s o compared i n m y e l o i d the Students' two-sided Statistics  leukemics  and normals u s i n g  t-test.  utilized  f o r t h e d a t a p r e s e n t e d i n Chapter V i n c l u d e d  the Cox s u r v i v a l a n a l y s i s model i n t h e BMDP2L computer program t o determine  i f any o f p a t i e n t sex, age, CAMAL BM v a l u e a t d i a g n o s i s o r  change i n CAMAL BM v a l u e post-chemotherapy were s i g n i f i c a n t  factors  i n t h e p r e d i c t i o n o f which ANLL p a t i e n t s would have l o n g e r r e m i s s i o n times.  The Kaplan-Meier  e s t i m a t e w i t h l o g rank s t a t i s t i c a l  analysis  (512) was used t o compare s u r v i v a l time p r i o r t o r e l a p s e f o r ANLL p a t i e n t s w i t h (a) d e c r e a s i n g o r (b) i n c r e a s i n g / u n c h a n g i n g CAMAL BM v a l u e s post-chemotherapy.  100  CHAPTER I I I  EVALUATION AND DIAGNOSTIC IMPLICATIONS OF A RAPID SLIDE TEST FOR CAMAL  I.  INTRODUCTION The  s p e c i f i c i d e n t i f i c a t i o n and c l a s s i f i c a t i o n o f human  leukemias u s i n g immunologic c r i t e r i a , p a r t i c u l a r l y examination o f c e l l  serological  s u r f a c e a n t i g e n i c markers, has gained  acceptance over t h e l a s t decade.  widespread  C e l l s u r f a c e marker phenotyping has  i c o n t r i b u t e d s i g n i f i c a n t l y t o our u n d e r s t a n d i n g o f t h e n a t u r e o f human acute leukemias,  i n c l u d i n g t h e apparent  c e l l u l a r o r i g i n of p a r t i c u l a r  h e m a t o l o g i c m a l i g n a n c i e s and t h e r e l a t i o n s h i p o f c e r t a i n c e l l s u r f a c e phenotypes t o c l i n i c a l  prognosis.  e x t e n s i v e l y and reviewed  T h i s s u b j e c t has been i n v e s t i g a t e d  (469-472).  Chapter I o f t h i s t h e s i s o u t l i n e d a n t i g e n s o f s i g n i f i c a n c e i n ANLL i n p a r t i c u l a r .  T h i s o u t l i n e emphasized t h e problems o f  a n t i g e n i c h e t e r o g e n e i t y i n ANLL which c o m p l i c a t e d i a g n o s i s and p o s s i b l y treatment o f t h i s d i s e a s e .  A l t h o u g h t h e r e i s much  t h a t l e u k e m i a - a s s o c i a t e d a n t i g e n s , unique  evidence  o r n o t , e x i s t i n ANLL,  t h e r e has been l i m i t e d major success a t a c t u a l i s o l a t i o n and c h a r a c t e r i z a t i o n o f such a n t i g e n s  (452-464).  We have p r e v i o u s l y r e p o r t e d on r a b b i t h e t e r o a n t i s e r a r a i s e d t o p u r i f i e d CAMAL a n t i g e n d e r i v e d from s o n i c a t i o n o f p o o l e d p e r i p h e r a l b l o o d l e u c o c y t e s from ANLL p a t i e n t s w i t h h i g h leukemic c e l l (513,514).  counts  We have shown by ELISA, i n d i r e c t immunoflourescence  the f l u o r e s c e n c e - a c t i v a t e d c e l l  in  s o r t e r and by i m m u n o p r e c i p i t a t i o n  101  t h a t t h i s anti-CAMAL serum r e a c t s s t r o n g l y w i t h PBL p a t i e n t s w i t h ANLL (acute phase o r r e m i s s i o n ) or CGL  and BM  cells  from  and n o t a t  d e t e c t a b l e l e v e l s w i t h c e l l s from normal i n d i v i d u a l s o r most p a t i e n t s with lymphoproliferative disorders. More r e c e n t l y , we  r e p o r t e d the development of a monoclonal  antibody, CAMAL-1, d e r i v e d from a f u s i o n of NS-1  c e l l s and  s p l e n o c y t e s from mice immunized w i t h the same p u r i f i e d (515).  While  initial  soon became apparent  Balb/c  antigen  r e s u l t s u s i n g FACS a n a l y s i s were p r o m i s i n g , i t t h a t t h i s MAb  d i d not r e a c t , i n many i n s t a n c e s ,  w i t h ANLL p a t i e n t s ' c e l l s i n s u r f a c e f l u o r e s c e n c e assays.  This  MAb,  however, r e a c t e d v e r y s t r o n g l y w i t h s o n i c a t e s of ANLL c e l l s i n the ELISA and was  shown t o be c a p a b l e of p u r i f y i n g CAMAL from such  s o n i c a t e s u s i n g a f f i n i t y chromatography. MAb  t o study the presence  S i n c e we wished t o use  of CAMAL on leukemic  s i m p l e and r a p i d method of doing t h i s u s i n g an immunoperoxidase procedure  crude  on s i n g l e c e l l  this  c e l l s , we developed  a  indirect  slide preparations.  T h i s c h a p t e r d e s c r i b e s the c u m u l a t i v e r e s u l t s from more than t h r e e years of i n v e s t i g a t i o n u s i n g the immunoperoxidase procedure  which was  d e t a i l e d i n Chapter  [and normal bone marrow t r a n s p l a n t (BMT)  II.  We  examined a l l p a t i e n t s  donors] who  b l o o d and/or bone marrow a s p i r a t i o n s performed  staining  had p e r i p h e r a l  through the Department  of Hematology, Vancouver G e n e r a l H o s p i t a l as w e l l as some normal  PB  from v o l u n t e e r s a t the U n i v e r s i t y of B r i t i s h Columbia.  two  y e a r s , t h e s e s t u d i e s have been performed  F o r over  b l i n d l y ; t h e s e r e s u l t s were  comparable t o those gathered b e f o r e the b l i n d study o u t l i n e d i n Chapter MAb  II.  The d a t a p r e s e n t e d here show c l e a r l y t h a t the CAMAL-1  r e a c t s s p e c i f i c a l l y w i t h PB o r BM  c e l l s from p a t i e n t s w i t h  102  n o n l y m p h o b l a s t i c leukemia and s i g n i f i c a n t l y greater  t h a t most of t h e s e i n d i v i d u a l s have  numbers of CAMAL-positive c e l l s than do  o r p a t i e n t s w i t h lymphoid m a l i g n a n c i e s . been shown t o be  of g r e a t  s i g n i f i c a n c e f o r the  myeloid-associated antigens, using CAMAL-1) performed p o o r l y  This  s l i d e t e s t has study of  normals also  other  o t h e r MAb's, some of which  (like  i n i n d i r e c t immunofluorescence c e l l  surface  a n t i g e n assays.  RESULTS A.  Comparative Immunoperoxidase R e a c t i v i t y Between Monoclonal  and  Rabbit Antibodies Studies using have shown t h a t the  the r a b b i t a n t i b o d y w i t h i n d i r e c t immunoperoxidase l e v e l of d e t e c t i o n  of CAMAL-positive c e l l s  samples from p a t i e n t s w i t h ANLL i s comparable to r e s u l t s previously  by us u s i n g  Another study (515) FACS a n a l y s i s o f t e n no)  a s i m i l a r a n t i s e r u m and  and  i n d i c a t e d t h a t t h i s MAb  r e a c t i v i t y w i t h ANLL or CGL  antiserum.  Results  t h e s e f i n d i n g s and  from the  and  tested.  c e l l s than d i d the  rabbit  CAMAL-1 has  (and  in reactivity result  is illustrated  i n Figure  3.1  a,b  where  been observed i n a l a r g e number of be  BM  antibody  T h i s g e n e r a l d i f f e r e n c e between the  These d i s t i n c t i o n s may  the  (by CAMAL-1) a n t i g e n  same ANLL p a t i e n t were l a b e l e d w i t h r a b b i t  CAMAL-1 r e s p e c t i v e l y .  a n t i b o d y and  lower  i n p r e f e r e n t i a l membrane (by  r a b b i t antibody) versus i n t r a c e l l u l a r  c e l l s from the  CAMAL-1 i n  showed, i n g e n e r a l ,  i n d i c a t e t h a t the d i f f e r e n c e s  This point  (514).  i n d i r e c t immunoperoxidase assay support  from an o f t e n s t r i k i n g d i f f e r e n c e  recognition.  described  FACS a n a l y s i s  subsequent i n v e s t i g a t i o n s u s i n g  in  rabbit  samples  of importance i n d i s t i n g u i s h i n g  103  Figure 3.1. Comparative immunoperoxidase r e a c t i v i t y between r a b b i t anti-CAMAL serum and CAMAL-1 m o n o c l o n a l a n t i b o d y on ANLL bone marrow cells. (a) r a b b i t anti-CAMAL serum l a b e l e d , showing p r e f e r e n t i a l membrane staining; (b) CAMAL-1 MAb l a b e l e d , showing s t r o n g i n t e r n a l s t a i n i n g .  a  105  those c e l l s t h a t s y n t h e s i z e o r i n t e r n a l i z e t h i s a n t i g e n from t h a t may o n l y adsorb membrane. determinant It  those  s m a l l amounts o f s o l u b l e CAMAL on t h e i r plasma  C e l l s u r f a c e a n t i g e n m o d i f i c a t i o n may e f f e c t i v e l y mask t h e u n i q u e l y r e c o g n i z e d by t h e CAMAL-1 MAb.  s h o u l d be noted t h a t t h e same a f f i n i t y - p u r i f i e d  p r e p a r a t i o n was used  CAMAL  t o produce b o t h o f these a n t i s e r a and t h a t they  c o m p e t i t i v e l y i n h i b i t each o t h e r i n t h e ELISA when t e s t e d a g a i n s t p u r i f i e d CAMAL (Dr. Robert  Shipman, p e r s o n a l communication).  A comparison o f t h e a c t u a l percentage myelogenous leukemia  r e a c t i v i t i e s of  samples w i t h b o t h t h e r a b b i t a n t i b o d y and  CAMAL-1 u s i n g immunoperoxidase i s shown i n T a b l e IV.  While  r e a c t i v i t i e s w i t h these samples a r e o f t e n h i g h e r w i t h t h e r a b b i t a n t i b o d y , t h e r e a r e a l s o a h i g h e r number o f samples t h a t show background a r t i f a c t u a l s t a i n i n g w i t h t h e n e g a t i v e c o n t r o l serum than w i t h t h e MAbs. of  rabbit  The ease o f s t a n d a r d i z a t i o n , v i r t u a l l a c k  a r t i f a c t u a l s t a i n i n g , and acceptance by o t h e r i n v e s t i g a t o r s i n t h e  f i e l d were t h e major f a c t o r s i n v o l v e d i n c h o o s i n g CAMAL-1 f o r t h e study p r e s e n t e d B.  here.  CAMAL-1 Immunoperoxidase S l i d e T e s t The photomicrographs  shown i n F i g u r e 3.2 a-h i l l u s t r a t e many o f  the advantages o f t h e i n d i r e c t immunoperoxidase s i n g l e c e l l technique  ( w i t h monoclonal a n t i b o d y l a b e l i n g ) , as w e l l as a number of  t y p i c a l s t a i n i n g p a t t e r n s found normals. the t e s t .  staining  i n v a r i o u s p a t i e n t groups and  F i g u r e 3.2 a,b i l l u s t r a t e a number o f p o i n t s important i n BM c e l l s from an ANLL p a t i e n t a t d i a g n o s i s a r e shown,  counterstained with hematoxylin photomicrographs  and Wright's  respectively.  show a d i r e c t comparison between r e s u l t s  w i t h t h e s e two c o u n t e r s t a i n s on t h e same l a b e l e d c e l l  These obtained  sample.  Figure  106  Table  IV. Comparison o f myelogenous leukemia c e l l r e a c t i v i t y w i t h anti-CAMAL and CAMAL-1 u s i n g immunoperoxidase  Diagnosis  Sample  Percentage r e a c t i v i t y CAMAL-1 R a b b i t anti-CAMAL  ANLL, i n i t i a l presentation  BM PB  31 2  49 12  ANLL, i n i t i a l presentation  BM  4  95  PB  0  90  ANLL r e m i s s i o n (3 months p r i o r to relapse) ANLL, r e l a p s e  BM *  29  82  ANLL, r e l a p s e  BM  13  21  CGL, c h r o n i c phase  PB  23  43  CGL, c h r o n i c phase  PB  19  45  * Illustrated  i n F i g u r e 3.1 a,b.  A p p r o x i m a t e l y 25,000 c e l l s were a n a l y z e d  i n each case, w i t h each antiserum.  107  F i g u r e 3.2. Immunoperoxidase s i n g l e c e l l s l i d e t e s t w i t h CAMAL-1. BM c e l l s from a newly diagnosed ANLL p a t i e n t c o u n t e r s t a i n e d w i t h (a) hematoxylin and (b) Wright's s t a i n s ; ( c ) same c e l l sample l a b e l e d w i t h n e g a t i v e c o n t r o l MAb; (d) ANLL r e m i s s i o n BM sample, c o u n t e r s t a i n e d w i t h methyl green; (e) c h r o n i c phase CGL p e r i p h e r a l b l o o d c e l l s ; ( f ) BM c e l l s from a p a t i e n t w i t h preleukemia; (g) normal BM; (h) normal p e r i p h e r a l blood c e l l s .  108  109  110  112  3.2 b i n d i c a t e s t h a t , r e g a r d l e s s preparation  of the q u a l i t y of the s l i d e  i t s e l f , p o s i t i v e c e l l s are exceptionally w e l l  delineated.  The extreme v a r i a b i l i t y  (cytoplasmic,  perinuclear, nuclear,  i n antigenic d i s t r i b u t i o n and membrane) i n p o s i t i v e c e l l s  i s emphasized i n these two photomicrographs.  Figure  3.2 c  i l l u s t r a t e s t h e same c e l l sample l a b e l e d w i t h t h e n e g a t i v e monoclonal a n t i b o d y . performed. cells;  I n a l l cases a p p r o p r i a t e  negative  control  c o n t r o l s were  As i n F i g u r e 3.2 c they showed no p o s i t i v e l y s t a i n i n g  c o n s e q u e n t l y , f u r t h e r photomicrographs o f such  negative  c o n t r o l s w i l l n o t be p r e s e n t e d h e r e . I n F i g u r e 3.2 d, a BM sample from an ANLL r e m i s s i o n l a b e l e d w i t h CAMAL-1 and c o u n t e r s t a i n e d A very  patient  w i t h methyl green i s shown.  s i g n i f i c a n t number o f CAMAL-1 p o s i t i v e c e l l s have been  c o n s i s t e n t l y observed i n samples from ANLL r e m i s s i o n  patients,  i n d i c a t i n g t h a t we may be d e t e c t i n g a marker o f an u n d e r l y i n g a b n o r m a l i t y which i s s t i l l morphologically  occuring  i n t h e s e p a t i e n t s , even though no  i d e n t i f i a b l e malignant c e l l s are present.  I n F i g u r e 3.2 e, a t y p i c a l s t a i n i n g p a t t e r n i s i l l u s t r a t e d when PBL  from a CGL ( c h r o n i c phase) p a t i e n t were l a b e l e d w i t h CAMAL-1.  Immature and mature g r a n u l o c y t e s  comprised 95% o f t h e s e  CAMAL-1  r e a c t i v e c e l l s , one o f which demonstrates a n t i g e n - r i c h membrane blebbing.  The a b i l i t y t o d i r e c t l y determine t h e morphology o f many  p o s i t i v e l y s t a i n i n g c e l l s i s a major advantage o f t h i s t e s t and has confirmed observations  with fluorescence-activated  c e l l s t h a t a r e CAMAL-positive i n c l u d e many n o n b l a s t i n Chapter I V ) .  c e l l s o r t i n g that cells  (discussed  113  F i g u r e 3.2 f shows a s i n g l e i n t e n s e l y p o s i t i v e mononuclear from t h e BM o f a preleukemic  p a t i e n t l a b e l e d w i t h CAMAL-1.  cell  Granular  c y t o p l a s m i c s t a i n i n g , an i n t e n s e p e r i n u c l e a r r i n g and prominent clumped i n t r a n u c l e a r a n t i g e n d i s t r i b u t i o n a r e e v i d e n t i n t h i s  cell.  Many samples, a f t e r e x t e n s i v e p r o c e s s i n g , s t a i n l e s s r o u t i n e l y w i t h any c o u n t e r s t a i n ; however, t h e p o s i t i v e c e l l s a r e remarkable  i n their  s t a i n i n g p a t t e r n and stand out b o l d l y as seen i n t h i s photomicrograph. Approximately  75% o f t h e 31 normal BM samples t e s t e d have  r e v e a l e d a v e r y s m a l l number o f CAMAL-1 p o s i t i v e c e l l s %),  one o f which i s i l l u s t r a t e d  i n F i g u r e 3.2 g.  (average  0.6  T h i s was t h e case  whether F i c o l l - H y p a q u e o r b u f f y c o a t - s e p a r a t e d samples were examined.  P r e v i o u s methods o f c e l l a n a l y s i s u s i n g t h e FACS d i d n o t  d e t e c t t h i s v e r y low number o f CAMAL-expressing c e l l s i n normal BM samples.  F i g u r e 3.2 h i l l u s t r a t e s t h a t normal PBL l a b e l e d w i t h  CAMAL-1 show no ( o r < 0.1%) p o s i t i v e l y - r e a c t i n g  cells.  A l a r g e number o f p a t i e n t s ' c e l l s have now been examined i n t h i s way u s i n g CAMAL-1, VIII.  and t h e r e s u l t s a r e summarized i n T a b l e s V -  These r e s u l t s demonstrate t h a t s i g n i f i c a n t numbers o f  CAMAL-expressing c e l l s a r e p r e s e n t i n BM and PB o f m y e l o i d  leukemia  p a t i e n t s and i l l u s t r a t e a simple and r a p i d d i a g n o s t i c s l i d e t e s t i n which t h e CAMAL-1 MAb may be employed t o d e t e c t t h e presence  of t h i s  antigen. T a b l e V shows a summary o f r e s u l t s o b t a i n e d when BM and PBL from p a t i e n t s w i t h ANLL i n v a r i o u s s t a g e s o f t h e i r d i s e a s e were t e s t e d w i t h CAMAL-1 i n t h e i n d i r e c t immunoperoxidase s l i d e t e s t .  I t can be  seen t h a t a s i g n i f i c a n t number o f CAMAL-1 p o s i t i v e c e l l s a r e p r e s e n t  114  T a b l e V.  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s  acute n o n l y m p h o b l a s t i c  Diagnosis  from  leukemia p a t i e n t s l a b e l e d w i t h CAMAL-1  Sample  Number tested  P e r c e n t CAMAL-positive cells + SEM and range  ANLL, a t diagnosis  BM PB  75 50  14.3 + 2,.1 (0. 1 4.4 + 1..8 (0  ANLL, p o s t chemotherapy  BM PB  29 24  18.7 + 4,.9 (0 12.1 + 4,,7 (0  ANLL, r e m i s s i o n  BM PB  120 104  13.6 + 1,.7 (0. 1 - 100.0) - 80.0) 6.5 + 1,.3 (0  ANLL, r e l a p s e  BM PB  39 31  14.4 + 2 .4 (0. 2 - 80.0) - 100.0) 8.6 + 3,.9 (0  80.0) 80.0)  - 100.0) - 80.0)  115  i n BM o r PBL o f most ANLL p a t i e n t s , whether i n acute phase o f t h e i r d i s e a s e , post-chemotherapy o r d u r i n g r e m i s s i o n . been determined  Furthermore, i t has  (Chapter V) t h a t many p a t i e n t s showing low numbers of  p o s i t i v e c e l l s a t d i a g n o s i s subsequently  d i s p l a y very high values  post-chemotherapy and d u r i n g r e m i s s i o n .  As mentioned p r e v i o u s l y ,  these r e s u l t s have been gathered  over t h e p a s t 3 1/2 y e a r s and  t h e r e f o r e i n c l u d e s l i d e examinations  performed d u r i n g t h e b l i n d  study  d e s c r i b e d i n Chapter V as w e l l as those p r e v i o u s l y performed. A l l s l i d e s were examined b l i n d l y throughout  and a comparison of r e s u l t s  o b t a i n e d b e f o r e and a f t e r t h e b l i n d study i n d i c a t e d t h a t r e s u l t s were, i n most c a s e s , v e r y s i m i l a r i n terms o f average % p o s i t i v e c e l l s i n v a r i o u s p a t i e n t groups.  F o r example, average v a l u e s f o r  ANLL p a t i e n t BM samples a t d i a g n o s i s were found 14.3%  a f t e r the b l i n d  t o be 13.6 b e f o r e and  study.  T a b l e VI summarizes r e s u l t s o b t a i n e d when CGL p a t i e n t BM and PB c e l l s were examined.  Very h i g h numbers o f CAMAL-positive c e l l s a r e  p r e s e n t i n CGL samples i n c h r o n i c o r a c c e l e r a t e d phases o f t h e disease.  We were i n t e r e s t e d i n d e t e r m i n i n g  i f CAMAL was  still  p r e s e n t i n o r on c e l l s from CGL p a t i e n t s when they e n t e r e d an acute b l a s t c r i s i s s t a t e and i f t h e r e might be a d i f f e r e n c e i n a n t i g e n e x p r e s s i o n depending on t h e n a t u r e o f t h e b l a s t i c (myeloid vs lymphoid).  A l l p a t i e n t s i n myeloid  transformation  b l a s t c r i s i s (CGL  MBC) showed v e r y s i g n i f i c a n t numbers o f CAMAL-positive c e l l s i n t h e i r BM and PB; two p a t i e n t s i n lymphoid had  comparatively  blast crisis  (CGL LBC),  however,  few p o s i t i v e PBL.  BM and PB c e l l s from p a t i e n t s w i t h m y e l o d y s p l a s t i c syndromes (preleukemia/MDS i n c l u d i n g r e f r a c t o r y anemia w i t h excess b l a s t s i n  116  Table VI.  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s  from p a t i e n t s , w i t h c h r o n i c g r a n u l o c y t i c  Diagnosis  Sample  Number tested  CGL, c h r o n i c phase  BM PB  26 40  CGL, a c c e l e r a t e d phase  BM PB  3 4  CGL MBC *  BM PB  13 5  CGL LBC **  PB  2  * MBC = m y e l o i d b l a s t c r i s i s ** LBC = lymphoid b l a s t c r i s i s  leukemia  P e r c e n t CAMAL-positive + SEM and range  22.4 23.0  + +  cells  5.1 ( 0.8 - 100,.0) 4.2 ( 0 - 100,.0)  24.0 + 5.5 +  9.2 (12.0 2.9 ( 1.8 -  42,.0) 14,.0)  + +  6.7 ( 3.0 8.7 (10.0 -  80..0) 50,.0)  23.8 24.6  0.3 + 0.2  ( 0.1 -  0..5)  117  t r a n s f o r m a t i o n o r RAEBIT) were found t o have h i g h numbers of CAMAL-positive (Table V I I ) .  cells,  comparable t o those found i n m y e l o i d  S i n c e the CAMAL a n t i g e n i s common t o a l l forms of  m y e l o i d leukemia and s i n c e t h e s e end, t h i s was  leukemics  not an unexpected  d i s o r d e r s o f t e n progress to that finding.  T a b l e V I I I shows r e s u l t s o b t a i n e d w i t h ALL, p a t i e n t s as w e l l as normals.  CLL and  With the e x c e p t i o n of c e r t a i n  r e m i s s i o n p a t i e n t s , a l l o f t h e s e samples showed v e r y low numbers of CAMAL-positive ranges of p o s i t i v i t y ,  lymphoma ALL  average  c e l l s i n t h e i r BM o r PB and v e r y narrow  i n d i c a t i n g a d r a m a t i c d i f f e r e n c e between these  samples and the m y e l o i d leukemics o r m y e l o d y s p l a s t i c s . d i f f e r e n c e s i n average numbers of CAMAL-positive  These  c e l l s were found t o  be h i g h l y s i g n i f i c a n t i n the two-sided Student's t t e s t , w i t h a l l p v a l u e s < 0.001.  I t was  i n t e r e s t i n g t o n o t e t h a t the  CAMAL-positive  c e l l s found i n a p p r o x i m a t e l y h a l f o f the newly diagnosed  ALL  p a t i e n t s , w h i l e c o n s t i t u t i n g a v e r y s m a l l f r a c t i o n o f the t o t a l  cell  p o p u l a t i o n when compared t o m y e l o i d leukemics, were n e v e r t h e l e s s o f t e n p r e s e n t a t l e v e l s s l i g h t l y h i g h e r than those found i n normal  BM  o r PB samples.  The s i g n i f i c a n c e of t h i s o b s e r v a t i o n , i f any,  remains  u n c l e a r but may  be r e l a t e d t o some b a s i c a b n o r m a l i t y i n hemopoiesis.  Some p r e v i o u s r e s u l t s from FACS a n a l y s i s w i t h r a b b i t anti-CAMAL serum showed an o c c a s i o n a l ALL p a t i e n t w i t h h i g h numbers of BM  o r PB c e l l s  (514).  CAMAL-positive  R e c e n t l y t h e r e has been much e v i d e n c e  p u b l i s h e d t o support f i n d i n g s such as t h i s ; a number of i n v e s t i g a t o r s have found simultaneous e x p r e s s i o n o f m y e l o i d and lymphoid on leukemic c e l l s  (516-527).  antigens  Lineage i n f i d e l i t y i n some leukemia  p a t i e n t s ' c e l l s a p p a r e n t l y does e x i s t and t h i s d i s c o v e r y has h e l p e d  118  Table VII.  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s  from p a t i e n t s w i t h preleukemia/MDS i n c l u d i n g  Sample  Number tested  Preleukemia/ MDS  BM PB  25 19  RAEBIT  BM PB  6 7  Diagnosis  * MDS = m y e l o d y s p l a s t i c syndrome RAEBIT = r e f r a c t o r y anemia w i t h excess b l a s t s  RAEBIT*  P e r c e n t CAMAL-positive cells + SEM and range  11.5 + 2.3 (0.3 - 50.0) 7.7 + 5.3 (0.1 - 100.0) 14.0 + 2.9 (7.2 - 24.0) 15.5 + 11.1 (0.3 - 80.0)  i n transformation  119  Table V I I I .  Summary o f immunoperoxidase s t a i n i n g r e s u l t s o f c e l l s  from p a t i e n t s w i t h lymphoid  Diagnosis  m a l i g n a n c i e s o r normals  Sample  Number tested  ALL, a t diagnosis  BM PB  41 19  1.2 + 0.3 +  ALL,  BM PB  11 8  12.4 + 5.2 +  CLL *  BM PB  8 22  1.1 + 0.2 +  0.5 (0 0.1 (0  -  3.8) 1.5)  Lymphoma  BM PB  77 8  2.3 + 1.6 +  0.2 (0 0.7 (0  -  9.4) 5.5)  Normal  BM PB  31 38  remission  * CLL = c h r o n i c lymphocytic  leukemia  P e r c e n t CAMAL-positive cells + SEM and range  0.3 (0 0.1 (0  -  7.5) 2.0)  4.8 (0.1 - 50.0) 2.2 (0.4 - 18.0)  0.6 + 0.1 (0 2.5) <0.1 + <0.1 (0 - <0.1)  120  t o widen our u n d e r s t a n d i n g hemopoiesis.  of b o t h  leukemic  Greaves e t a l . suggested  most l i k e l y r e p r e s e n t s a n o r m a l l y  c e l l origins  and  t h a t such l i n e a g e p r o m i s c u i t y  t r a n s i e n t stage of gene e x p r e s s i o n  i n m u l t i - o r b i p o t e n t i a l p r e c u r s o r s which has been " a r r e s t e d " o r f r o z e n i n the acute The and PB  leukemic  h a l f of the ALL  samples examined showed l e v e l s of CAMAL-positive  Nearly h a l f  41 newly diagnosed  f a c t o n l y 2/41  had  similar situation.  (6/11 ALL  BM  had  ALL  r e m i s s i o n BM  samples had  H a l f (4/8)  not an  < 4% p o s i t i v e ) .  > 4 % positive.  5 % CAMAL-1 p o s i t i v e c e l l s 19 newly diagnosed  i n ANLL samples was  (5/11) of the ALL  CAMAL-1 p o s i t i v e c e l l s of  (528).  o b s e r v a t i o n t h a t approximately  comparable t o those found result.  state  3.3 10.5  of the ALL  (4/8 had  < 0.6  PB samples had  t h i s p o i n t i n an ALL  % positive.  None of the ALL  cells  expected samples had  > 8 %  In comparison, none  The p e r i p h e r a l b l o o d r e v e a l e d a r e m i s s i o n PB samples had %).  >  In comparison, none of  over 2 % p o s i t i v e c e l l s .  The  i n the s t r o n g l y  r e m i s s i o n samples were of the m y e l o i d  illustrates  BM  > 8 % positive cells; in  g r e a t m a j o r i t y of CAMAL-1 r e a c t i v e c e l l s found p o s i t i v e ALL  remission  r e m i s s i o n BM  lineage.  Figure  sample t h a t  was  r e m i s s i o n samples w i t h h i g h numbers  of p o s i t i v e c e l l s were o b t a i n e d from the ALL  p a t i e n t s examined i n  T a b l e V I I I a t d i a g n o s i s , t h e r e f o r e i t i s n o t p o s s i b l e t o determine i f any o r a l l of these p a t i e n t s had h i g h v a l u e s a t d i a g n o s i s as w e l l as during remission.  T h i s , however, i s v e r y u n l i k e l y c o n s i d e r i n g the  r e s u l t s from the newly diagnosed  ALL  group.  Another e x p l a n a t i o n f o r  these r e s u l t s i s more p r o b a b l e , based on the f o l l o w i n g d a t a . study p u b l i s h e d i n 1985  In a  (529), which demonstrated the p r e s e n c e of  CAMAL-positive c e l l s p r i o r t o r e l a p s e i n PB of acute  leukemia  121  Figure 3.3. ALL remission bone marrow c e l l s labeled by CAMAL-1 monoclonal antibody. Immature and mature myeloid c e l l s are p o s i t i v e l y labeled.  122  p a t i e n t s who  had  undergone BMT,  one  of the p a t i e n t s who  r e l a p s e a f t e r t r a n s p l a n t a t i o n (and who 3 months p r i o r ) was have found, i n two  o t h e r ALL (25 and  s i g n a l e d by  relapse.  We  (Chapter V) ALL  remission 30%)  S i m i l a r l y , we  p a t i e n t s , t h a t h i g h numbers of  were p r e s e n t  i n t h e i r BM  one month  p a t h o l o g i c a l mechanism i s o c c u r r i n g which  the CAMAL marker and which may  know t h i s t o be and  present  I t i s p o s s i b l e , t h e r e f o r e , t h a t i n some ALL  p a t i e n t s an u n d e r l y i n g be  50% p o s i t i v e c e l l s  o r i g i n a l l y diagnosed as an ALL.  CAMAL-positive c e l l s p r i o r to relapse.  had  died i n  be r e l a t e d t o  may  imminent  the case w i t h some ANLL p a t i e n t s  o n l y f u r t h e r i n v e s t i g a t i o n s w i t h g r e a t e r numbers of  p a t i e n t s can determine i f the same h o l d s t r u e f o r any p a t i e n t s i n  this  group.  C.  A p p l i c a t i o n of the I n d i r e c t Immunoperoxidase S l i d e T e s t t o Study of Other M y e l o i d  the  C e l l Markers  A group of f o u r i n v e s t i g a t o r s i n Dr.  J u l i a Levy's  laboratory  took p a r t i n the Second I n t e r n a t i o n a l Workshop on Human Leukocyte D i f f e r e n t i a t i o n A n t i g e n s to determine the e f f i c a c y of a p p l y i n g  the  i n d i r e c t immunoperoxidase s l i d e t e s t d e s c r i b e d h e r e i n to the study of other  antigens  (476).  on m a l i g n a n t m y e l o i d c e l l s u s i n g  T h i s study showed t h a t the s l i d e t e s t was  such i n v e s t i g a t i o n s and using  a p a n e l of MAbs  surface  could provide  immunofluorescence a s s a y s , such as  a n t i g e n i c d i s t r i b u t i o n and cells.  information  the morphology of  extremely u s e f u l i n not e a s i l y  intracellular  antigen-expressing  Comparisons of immunoperoxidase methods w i t h FACS a n a l y s i s  have shown c l o s e c o r r e l a t i o n s f o r the d e t e c t i o n of c e l l antigens  obtained  (511,530).  Of  s p e c i a l i n t e r e s t , and  surface  r e l a t e d to t h i s t h e s i s ,  123  was t h e d i s c o v e r y t h a t 10/14 MAbs which ( l i k e CAMAL-1) showed no o r minimal r e a c t i v i t y w i t h ANLL c e l l cell  l i n e s o r p a t i e n t samples u s i n g FACS  s u r f a c e a n t i g e n i c a n a l y s i s , d i d s t a i n s i g n i f i c a n t numbers o f  c e l l s i n a t l e a s t some o f these immunoperoxidase.  samples u s i n g  indirect  F i g u r e 3.4 a,b,c i l l u s t r a t e s some r e s u l t s from t h e  a p p l i c a t i o n o f t h i s t e s t t o o t h e r m y e l o i d - s p e c i f i c MAbs.  I t can  c l e a r l y be seen t h a t t h e s l i d e t e s t a l l o w s e f f e c t i v e v i s u a l i z a t i o n of the a n t i g e n s  f o r which these MAbs a r e s p e c i f i c .  immunoperoxidase s t a i n i n g o f HL60 c e l l s  F i g u r e 3.4 c shows  (a human p r o m y e l o c y t i c  cell  l i n e ) w i t h MAb 110, which was one o f a group o f MAbs t h a t were n o t supposed t o r e a c t s t r o n g l y w i t h ANLL c e l l  lines.  r e a c t i v i t y w i t h HL60 and MAb 110; furthermore p o s i t i v e s t a i n i n g w i t h immunoperoxidase.  We found 20.0%  t h i s MAb  showed i n t e n s e  Over t h e l a s t few y e a r s ,  t h e r e has been i n c r e a s e d i n t e r e s t i n t h e use o f t h i s t e c h n i q u e f o r studying antigens technique  on s i n g l e c e l l p r e p a r a t i o n s .  We b e l i e v e t h a t t h e  o f f e r s s i g n i f i c a n t advantages over t h e r o u t i n e  immunofluorescence assays t h a t have become so p o p u l a r ,  surface  and we have  shown t h a t i t can be v e r y u s e f u l f o r t h e study o f a n t i g e n s u s i n g a v a r i e t y o f MAbs which do n o t show s i g n i f i c a n t r e a c t i v i t y i n FACS analysis.  124  Figure 3.4. Immunoperoxidase s i n g l e c e l l s l i d e t e s t w i t h o t h e r m y e l o i d - s p e c i f i c monoclonal a n t i b o d i e s . (a) CGI c h r o n i c phase p e r i p h e r a l b l o o d c e l l s l a b e l e d w i t h MAb70, (b) HL-60 c e l l s l a b e l e d w i t h MAb30, (c) HL-60 c e l l s l a b e l e d w i t h M A b l l O .  a  126  CHAPTER IV  CAMAL EXPRESSION IN LEUKEMIA  I.  INTRODUCTION I t has been demonstrated t h a t t h e CAMAL a n t i g e n i s p r e s e n t i n o r on a s i g n i f i c a n t number o f BM and PB c e l l s nonlymphoblastic w i t h lymphoid  leukemia  in cells disorders  unusual  a n t i g e n i n t h a t i t has been found t o be p r e s e n t  (ANLL, CGL, p r e l e u k e m i a / m y e l o d y s p l a s t i c i n patients' cells  syndrome).  In  i n a l l stages of the disease  i n a l l FAB subgroups. The  cells  d i s c o v e r y t h a t CAMAL was p r e s e n t i n ANLL r e m i s s i o n p a t i e n t s '  i m p l i e d t h a t we were n o t s t u d y i n g an a n t i g e n r e s t r i c t e d t o t h e  malignant of  CAMAL appears t o be a v e r y  from p a t i e n t s w i t h many v a r i e t i e s o f m y e l o p r o l i f e r a t i v e  ANLL, CAMAL i s found and  compared t o normals o r most i n d i v i d u a l s  malignancies.  leukemia-associated  of p a t i e n t s with  b l a s t c e l l s themselves.  the c e l l s  We wished t o determine t h e n a t u r e  e x p r e s s i n g t h i s a n t i g e n i n v a r i o u s s t a g e s o f leukemia i n  o r d e r t o h e l p c l a r i f y our f i n d i n g s and f u r t h e r our u n d e r s t a n d i n g o f t h i s a n t i g e n . Such i n v e s t i g a t i o n s w i l l be d e s c r i b e d i n t h i s  II.  chapter.  RESULTS A.  Morphology o f CAMAL-positive Two approaches were taken  of had  CAMAL-positive c e l l s .  Cells  i n o r d e r t o determine t h e morphology  The f i r s t  involved c o l l e c t i n g c e l l s  that  shown s t r o n g p o s i t i v e f l u o r e s c e n c e when l a b e l e d w i t h t h e r a b b i t  anti-CAMAL serum and a n a l y z e d i n t h e f l u o r e s c e n c e - a c t i v a t e d c e l l  127  sorter.  The  II.  second approach i n v o l v e d u s i n g the CAMAL-1 MAb  The  d e t a i l s of t h i s p r o c e d u r e have been d e s c r i b e d i n Chapter  immunoperoxidase s t u d i e s ; these procedures 1.  in indirect  have a l s o been d e s c r i b e d .  FACS S o r t i n g S t u d i e s Three m y e l o i d  leukemic  p a t i e n t groups were examined i n t h i s  manner w i t h the r a b b i t anti-CAMAL serum (newly diagnosed r e m i s s i o n , and CGL).  In each i n s t a n c e , two  ANLL, ANLL  c e l l p o p u l a t i o n s were  c o l l e c t e d : t h a t 25% of the t o t a l l a b e l e d c e l l p o p u l a t i o n showing the (1) lowest  and  (2) h i g h e s t r e l a t i v e f l u o r e s c e n c e i n t e n s i t y w i t h  anti-CAMAL serum. collected.  The  remaining  50% of the sample p o p u l a t i o n was  determine the morphology of the c e l l s . were v e r i f i e d by Dr.  counts performed to  These d i f f e r e n t i a l  counts  Sheldon Naiman, a s e n i o r h e m a t o l o g i s t  Vancouver G e n e r a l H o s p i t a l . by F i c o l l - H y p a q u e  A l l samples a n a l y z e d  and  at  s e p a r a t i o n (and these a r e , t h e r e f o r e ,  T a b l e X, where c e l l s were p r e p a r e d and  203,  u s i n g the plasmagel s e p a r a t i o n  therefore contained a l l leucocyte  T a b l e IX shows r e s u l t s from two who  the  s o r t e d were  e n r i c h e d f o r mononuclear c e l l s ) w i t h the e x c e p t i o n of p a t i e n t  technique  not  These c e l l p o p u l a t i o n s were t r a n s f e r r e d to g l a s s s l i d e s ,  s t a i n e d w i t h Wright's and d i f f e r e n t i a l c e l l  prepared  the  types.  newly diagnosed  ANLL p a t i e n t s  showed s i g n i f i c a n t numbers of p o s i t i v e l y f l u o r e s c i n g c e l l s when  l a b e l e d w i t h the anti-CAMAL serum. the malignant  c e l l s were p r e s e n t  p o p u l a t i o n although, present  In both c a s e s , the m a j o r i t y of  i n the h i g h e s t f l u o r e s c i n g  as the FACS a n a l y s i s shows, almost a l l c e l l s  i n the t o t a l p o p u l a t i o n d i d l a b e l p o s i t i v e l y w i t h  anti-CAMAL serum.  Erythrocytes  ( r b c ) and  the  lymphocytes a l s o l a b e l e d  p o s i t i v e l y , and w h i l e most were d e t e c t e d i n the lowest f l u o r e s c i n g  128  T a b l e IX.  FACS s o r t i n g r e s u l t s from two newly diagnosed patients*  1)  p e r i p h e r a l b l o o d samples  FACS a n a l y s i s o f p e r i p h e r a l b l o o d  Antiserum  label  Differential cell  C e l l types  Blast Promyelocyte Myelocyte Metamyelocyte Staff Neutrophil Lymphocyte Monocyte rbc  cells  Number o f c e l l s f l u o r e s c i n g * P a t i e n t 101 P a t i e n t 102  a n t i - n o r m a l human normal r a b b i t serum anti-CAMAL  2)  ANLL  21,050 5,687 20,154  counts f o r s o r t e d  (anti-CAMAL r e a c t i v e )  P e r c e n t o f each c e l l P a t i e n t 101 LOW HIGH  23  62 3 12  23,846 5,744 21,874  73  occ. 22 5 occ.  samples  type p r e s e n t P a t i e n t 102 LOW HIGH  6 0 2 1 1 1 49 2 38  * a p p r o x i m a t e l y 25,000 c e l l s were a n a l y z e d i n each case ** p a t i e n t 102 had acute p r o m y e l o c y t i c leukemia  14 54 2 6 3 9 8 4  129  population, as w e l l .  i t was c l e a r t h a t CAMAL was d e t e c t e d on t h e i r  Moreover, s i n c e these two c e l l types  of those  listed  i n the myeloid  m a j o r i t y would f a l l  membranes  a r e s m a l l e r than any  s e r i e s , i t might be expected t h a t t h e  i n t h e lower f l u o r e s c i n g p o p u l a t i o n .  The  photomicrograph shown i n F i g u r e 4.1 d e p i c t s t h e c e l l s p r e s e n t  i n the  h i g h l y f l u o r e s c e n t p o p u l a t i o n from p a t i e n t 101 ( T a b l e I X ) , showing the predominance o f b l a s t c e l l s . present  The s t r i k i n g number o f lymphocytes  i n t h i s p o p u l a t i o n v e r i f i e d t h e p r e s e n c e o f CAMAL on t h e  membrane o f non-myeloid  cells.  Three ANLL r e m i s s i o n p a t i e n t s , whose c e l l s showed p o s i t i v e f l u o r e s c e n c e w i t h t h e anti-CAMAL serum were a l s o a n a l y z e d i n t h i s manner.  T a b l e X shows these r e s u l t s .  and s o r t e d  I t should be  emphasized t h a t ANLL r e m i s s i o n c r i t e r i a d e f i n e s t h a t no b l a s t are present  i n t h e p e r i p h e r a l b l o o d and l e s s than 5% b l a s t s  ( c o n s i d e r e d t o be w i t h i n normal range) a r e p r e s e n t of these p a t i e n t s .  populations.  i n t h e bone marrow  I t was o f s p e c i a l i n t e r e s t , t h e r e f o r e , t o  determine t h e c e l l types  processed  cells  found t o express t h e CAMAL a n t i g e n i n these  Samples from p a t i e n t s 201 and 202,.Table X, were  by F i c o l l - H y p a q u e  c e l l - e n r i c h e d sample.  s e p a r a t i o n , p r o v i d i n g a mononuclear  I n b o t h o f these  samples, p o s i t i v e l y  f l u o r e s c i n g c e l l s c o n s i s t e d m a i n l y o f lymphocytes. sample from p a t i e n t 203, p r o c e s s e d  Even i n t h e  by plasmagel s e p a r a t i o n t o o b t a i n  a t o t a l l e u c o c y t e p o p u l a t i o n , a l a r g e p e r c e n t a g e o f lymphocytes were were p r e s e n t , Neutrophils  even i n t h e h i g h l y f l u o r e s c e n t c e l l  comprised t h e m a j o r i t y o f c e l l s , however, i n t h i s  p o p u l a t i o n i n d i c a t i n g t h a t more CAMAL was p r e s e n t these  population.  c e l l s than on lymphocytes.  on t h e s u r f a c e of  130  Figure 4.1. P e r i p h e r a l b l o o d c e l l s from an ANLL p a t i e n t a t d i a g n o s i s s o r t e d on the b a s i s of s t r o n g r e a c t i v i t y w i t h r a b b i t anti-CAMAL serum. A p p r o x i m a t e l y 3/4 of t h e s e c e l l s were b l a s t s a l t h o u g h numerous c e l l s w i t h lymphoid morphology were p r e s e n t . The c e l l s i l l u s t r a t e d were from p a t i e n t 101 ( T a b l e I X ) .  131  T a b l e X.  FACS s o r t i n g r e s u l t s from t h r e e ANLL  remission  1)  patients*  p e r i p h e r a l blood  FACS a n a l y s i s o f p e r i p h e r a l b l o o d  Antiserum  label  2)  Differential cell  Cell  types  * approximately  cells  Number o f c e l l s f l u o r e s c i n g * P a t i e n t 101 P a t i e n t 202 P a t i e n t 203  a n t i - n o r m a l human normal r a b b i t serum anti-CAMAL  Myelocyte Metamyelocyte Staff Neutrophil Eosinophil Lymphocyte Monocyte rbc  samples  20,347 3,151 20,956  counts f o r s o r t e d  23,536 403 9,333  (anti-CAMAL r e a c t i v e )  24,644 89 22,734  samples  P e r c e n t o f each c e l l type p r e s e n t P a t i e n t 203 P a t i e n t 201 P a t i e n t 202 LOW HIGH LOW HIGH LOW HIGH  97 3  3 occ. 92 5  2 83 1 14  10 1 80 7 2  25,000 c e l l s were a n a l y z e d i n each case  2 4 3 5  1 5 55  78 1 7  28 1 8  132  Two  CGL  T a b l e XI.  p a t i e n t samples were examined and  In b o t h samples h i g h l y f l u o r e s c e n t p o p u l a t i o n s were  g r e a t l y e n r i c h e d f o r immature m y e l o i d metamyelocytes.  Again,  F i g u r e 4.2  cells,  a,b  c e l l s had  c e l l populations  the CAMAL a n t i g e n on  and  4.2  and 4.3,  we  Two  25%  hoped to  a s p e c i f i c p a t t e r n of r e a c t i v i t y w i t h  anti-CAMAL serum f o r any g i v e n c e l l seem t o be the case.  their  302.  (lowest 25% and h i g h e s t  f l u o r e s c i n g ) f o r each p a t i e n t i n T a b l e s 4.1, determine i f t h e r e was  i n this population  i l l u s t r a t e c e l l s c o l l e c t e d i n the h i g h l y  f l u o r e s c e n t p o p u l a t i o n from p a t i e n t s 301 By c o l l e c t i n g two  in particular,  lymphocytes were p r e s e n t  i n d i c a t i n g t h a t not o n l y m y e l o i d surface.  r e s u l t s are shown i n  the  t y p e . In g e n e r a l t h i s d i d not  p o i n t s , however, were c l e a r from the  comparison. a.  Lymphoid c e l l s were p r e s e n t  p o p u l a t i o n s of the m y e l o i d were from ANLL o r CGL  i n both h i g h and  low f l u o r e s c i n g  leukemia samples examined, whether they  patients.  T h i s was  most remarkable i n the ANLL  r e m i s s i o n p a t i e n t p e r i p h e r a l b l o o d samples, where they comprised most of b.  the p o s i t i v e mononuclear c e l l The  population.  r e l a t i v e s i z e of the c e l l  ( e r y t h r o c y t e and  type, f o r example, s m a l l  lymphocyte) o r l a r g e (most of the m y e l o i d  i n c l u d i n g n e u t r o p h i l and promyelocyte) was determining  the major  f a c t o r f o r d i f f e r e n c e s seen i n t h e i r numbers i n low  high populations. cases.  probably  series  However, t h i s c o u l d not be the o n l y f a c t o r i n a l l  F o r i n s t a n c e , p a t i e n t 101,  T a b l e IX, had  23% b l a s t s p r e s e n t  i n the low p o p u l a t i o n , a l l of which would have been l a r g e r than 22%  and  lymphocytes p r e s e n t  i n the h i g h p o p u l a t i o n .  the  These lymphocytes  133  Table XI.  FACS s o r t i n g r e s u l t s f o r CGL p a t i e n t s ' peripheral blood  1)  FACS a n a l y s i s o f p e r i p h e r a l b l o o d  Antiserum  label  Differential cell  C e l l types  Blast Promyelocyte Myelocyte Metamyelocyte Staff Neutrophil Eosinophil Basophil Lymphocyte Monocyte Normoblast L a t e normoblast  cells  Number o f c e l l s f l u o r e s c i n g * P a t i e n t 301 P a t i e n t 302  a n t i - n o r m a l human normal r a b b i t serum anti-CAMAL  2)  cells  19,330 698 11,164  counts o f s o r t e d  16,025 1,095 12,206  (anti-CAMAL r e a c t i v e )  samples  P e r c e n t o f each c e l l type p r e s e n t P a t i e n t 301 P a t i e n t 302 LOW HIGH LOW HIGH  1 5 24 36  32  14 15 42 18 2  1 1 35 22 7 5  9  7  2 21  1 10 17 39 10 7 1 1 8 3 1 2  * a p p r o x i m a t e l y 25,000 c e l l s were a n a l y z e d i n each case ** I l l u s t r a t e d i n F i g u r e 4 . 2 a ( p a t i e n t 301) and b ( p a t i e n t 302)  134  Figure 4.2. P e r i p h e r a l b l o o d c e l l s from two CGL p a t i e n t s s o r t e d on t h e b a s i s of s t r o n g r e a c t i v i t y w i t h r a b b i t anti-CAMAL serum. Predominant c e l l types from p a t i e n t s (a) 301 and (b) 302 (from T a b l e X I ) were immature m y e l o i d c e l l s .  135  b  136  must have had a g r e a t e r c o n c e n t r a t i o n o f membrane bound CAMAL did  than  t h e b l a s t s i n t h e low p o p u l a t i o n .  2.  Immunoperoxidase  Studies  a.  Lack o f C o r r e l a t i o n Between BM B l a s t C e l l Numbers and CAMAL BM Value We have shown t h a t many c e l l s b e s i d e s b l a s t s c o n t a i n t h e CAMAL  marker; t h i s was most obvious Even i n newly diagnosed  i n t h e case o f ANLL r e m i s s i o n  samples.  ANLL p a t i e n t s , no c o r r e l a t i o n seemed t o e x i s t  between t h e number o f b l a s t s and t h e number o f CAMAL-1 p o s i t i v e (CAMAL BM v a l u e ) .  Indeed, a number o f ANLL p a t i e n t s a t i n i t i a l  p r e s e n t a t i o n showed v e r y low CAMAL BM v a l u e s t h e r e were many malignant of  cells  c e l l s present.  (Chapter V) even though  T a b l e X I I shows t h i s  c o r r e l a t i o n between t h e number o f b l a s t c e l l s p r e s e n t  lack  i n ANLL  p a t i e n t s ' BM and t h e i r CAMAL BM v a l u e u s i n g immunoperoxidase.  For  example, w h i l e p a t i e n t sample 1302 showed v e r y s i m i l a r % b l a s t s and % < CAMAL-positive c e l l s 1089  (9 and 7%), sample 1150 (0 and 35%) and sample  (59 and 1.3%) showed t h a t , i n g e n e r a l , no such c o r r e l a t i o n  e x i s t e d and CAMAL'BM v a l u e s might be much h i g h e r o r much lower than % N  marrow b l a s t s .  Even i n t h e same p a t i e n t , no c o n s i s t e n t p o s i t i v e o r  n e g a t i v e c o r r e l a t i o n was found  to exist  T a b l e X I I ) u s i n g t h e immunoperoxidase b.  (see p a t i e n t s a, b, c and d,  assay.  Lack o f C o r r e l a t i o n Between R e g e n e r a t i v e  o r A p l a s t i c BM and  CAMAL BM V a l u e I t has been s p e c u l a t e d t h a t CAMAL e x p r e s s i o n i n c e l l s may simply be a marker o f r e g e n e r a t i n g BM, p a r t i c u l a r l y because o f t h e r e m i s s i o n p a t i e n t data.  T h i s has c l e a r l y been shown n o t t o be t h e case;  Tables  X I I I and XIV summarize CAMAL BM r e s u l t s from 26 r e g e n e r a t i n g and 15  Table X I I .  Lack o f c o r r e l a t i o n between t h e number o f b l a s t  cells  p r e s e n t i n bone marrow and t h e CAMAL BM v a l u e  P a t i e n t Code number  1302 1311 1021 1125 1089a 1557a 1577 1060 1524 1053b 1425b 1031c 1150c 1194c 1015 1372d 1406d  a ) b ) c )  % BM b l a s t s  9 32 23 occ. 59 18 20 1 36 53 15 2 0 13 1 55 1  CAMAL BM v a l u e (% p o s i t i v e c e l l s )  7.0 5.5 10.0 75.0 1.3 2.0 100.0 8.6 3.0 6.9 31.0 5.4 35.0 20.0 62.5 0.8 20.0  r e f e r t o samples examined f o r t h e same 4 p a t i e n t s (a,b,c,d) a t d i f f e r e n t times  138  non-regenerating were found  BM samples.  F o r these T a b l e s , o n l y samples t h a t  t o be i n a c t i v e s t a t e s o f r e g e n e r a t i o n  t o t a l l y non-regenerative included.  ( T a b l e X I I I ) and  o r a p l a s t i c s t a t e s ( T a b l e XIV) were  The e x p r e s s i o n o f CAMAL by c e l l s , t h e r e f o r e , appeared t o  have no c o r r e l a t i o n , e i t h e r p o s i t i v e o r n e g a t i v e , w i t h  regeneration  or l a c k thereof. c.  The Morphology o f CAMAL-positive C e l l s by Immunoperoxidase As we (510) and o t h e r s  method o f d e t e c t i n g c e l l u l a r  (530) have r e p o r t e d , t h e immunoperoxidase antigens  allows s t r a i g h t f o r w a r d  microscopic v i s u a l i z a t i o n of antigen-positive c e l l s provides  and, i n a d d i t i o n ,  i n f o r m a t i o n r e g a r d i n g t h e d i s t r i b u t i o n o f a n t i g e n on o r  w i t h i n i n d i v i d u a l c e l l s . The morphology o f r a b b i t  anti-CAMAL-positive  c e l l s has been d i s c u s s e d u s i n g t h e FACS method o f c e l l  collection.  Immunoperoxidase l a b e l i n g w i t h r a b b i t anti-CAMAL serum has v e r i f i e d these r e s u l t s .  Fewer c e l l s  w i t h CAMAL-1 MAb.  s t a i n p o s i t i v e l y when c e l l s a r e l a b e l e d  As mentioned p r e v i o u s l y , t h i s may be o f some  importance i n i d e n t i f y i n g c e l l s cells  t h a t produce CAMAL i n comparison t o  t h a t may adsorb ( p o s s i b l y v i a s p e c i f i c r e c e p t o r s ) CAMAL on  their cell  surface.  The m a j o r i t y o f CAMAL-positive c e l l s  (by immunoperoxidase  l a b e l i n g ) i n ANLL p a t i e n t s BM a t d i a g n o s i s o r r e l a p s e i n c l u d e malignant  b l a s t c e l l s and v e r y immature m y e l o i d  illustrated  i n Chapter I I I ( F i g u r e 3.2 a,b).  This i s not at a l l  s u r p r i s i n g s i n c e , i n many i n s t a n c e s , these c e l l s extremely  c e l l s , as a l r e a d y  are present at  high r e l a t i v e proportions.  I n ANLL r e m i s s i o n p a t i e n t BM, CAMAL-expressing c e l l s myeloid  include  c e l l s a t a l l m o r p h o l o g i c a l l y - i d e n t i f i a b l e stages o f m a t u r a t i o n  139  Table X I I I .  Patient  CAMAL BM v a l u e s  i n r e g e n e r a t i n g bone marrows  Status  CAMAL BM v a l u e (% p o s i t i v e c e l l s )  501  ANLL r e m i s s i o n w i t h good regeneration  502  ANLL r e m i s s i o n , r e g e n e r a t i n g bone marrow  16.5  503  ANLL r e m i s s i o n , t r i l i n e a r regeneration occurring  13.7  504  ANLL r e m i s s i o n w i t h a c t i v e granulocytic regeneration  2.5  505  Lymphoma post-BMT w i t h e x c e l l e n t t r i l i n e a r regeneration  1.5  Summary:  3.6  F o r 26 p a t i e n t s w i t h r e g e n e r a t i n g BM (23 ANLL, 1 ALL, 1 lymphoma and 1 post-BMT lymphoma) t h e mean CAMAL BM v a l u e was 11.4%.  140  T a b l e XIV.  Patient  CAMAL BM v a l u e s i n n o n - r e g e n e r a t i n g  Status  bone marrows  CAMAL BM v a l u e (% p o s i t i v e c e l l s )  35.0  601  ANLL; h y p o c e l l u l a r w i t h absence o f m y e l o i d and e r y t h r o i d p r e c u r s o r s  602  ANLL post-chemotherapy;  aplasia  0.1  603  ANLL post-chemotherapy;  aplasia  35.5  604  B i p h e n o t y p i c (ALL and ANLL) leukemia post-chemotherapy; a p l a s i a  6.0  605  ANLL post-chemotherapy;  0.2  Summary:  aplasia  F o r 15 p a t i e n t s (12 ANLL, 1 b i p h e n o t y p i c [ALL/ANLL], 1 ALL and 1 MDS) w i t h n o n - r e g e n e r a t i v e bone marrows, t h e mean CAMAL BM v a l u e was 12.6%.  141  of m a t u r a t i o n , leucocytes.  from b l a s t s t o end stage  polymorphonuclear  P o s i t i v e c e l l s from t h e g r a n u l o c y t e  ( F i g u r e 4.3 a , b ) ,  monocyte, megakaryocyte ( F i g u r e 4.3 c) and e r y t h r o i d l i n e a g e s have a l l been i d e n t i f i e d i n ANLL r e m i s s i o n p a t i e n t BM.  In general,  fewer  c e l l s i n the p e r i p h e r a l blood of remission p a t i e n t s s t a i n positively.  These i n c l u d e g r a n u l o c y t e s  and monocytes p r i m a r i l y , b u t  i n a number o f cases have i n c l u d e d a v e r y h i g h number o f lymphocytes ( F i g u r e 4.4).  The p o s s i b l e s i g n i f i c a n c e o f t h i s w i l l be d i s c u s s e d .  Observed d i f f e r e n c e s i n CAMAL d i s t r i b u t i o n w i t h i n c e l l s have been profound.  As photomicrographs i n Chapter I I I , F i g u r e 3.2, and i n  F i g u r e 4.3 i l l u s t r a t e , most o f t h i s p r o t e i n i s l o c a t e d i n t r a c e l l u l a r l y and has been i d e n t i f i e d i n a number o f c y t o p l a s m i c s t a i n i n g p a t t e r n s from d i f f u s e t o s t r o n g l y g r a n u l a r . p e r i n u c l e a r s t a i n i n g of myeloid frequency The  Intense  c e l l s has been observed w i t h  great  ( F i g u r e s 3.1 b, 3.2 a,b,d and 4.3 a , b ) . s i g n i f i c a n c e , i f any, o f apparent a n t i g e n shedding has n o t  been determined  ( F i g u r e s 3.2 e, 4.3 a ) .  I t has been commonly  observed i n c h r o n i c stage CGL p e r i p h e r a l b l o o d p r e p a r a t i o n s . a v e r y f r e q u e n t p a t t e r n o f CGL PB l a b e l i n g i s i l l u s t r a t e d  Indeed,  i n Figure  4.5 where a h e a v i l y g r a n u l a r s t a i n i n g o f PB l e u c o c y t e s i s seen. many cases  t h i s granular  as w i t h i n , t h e s e  cells.  l a b e l has been i d e n t i f i e d o u t s i d e , as w e l l CGL (BM o r PB) samples r o u t i n e l y c o n t a i n  g r e a t e r percentages o f CAMAL-positive c e l l s than any o t h e r leukemia group examined. myeloid  maturation  myeloid  CGL c e l l s a t v i r t u a l l y a l l s t a g e s o f  l a b e l p o s i t i v e l y with  CAMAL-1.  T h i s g e n e r a l p a t t e r n o f r e a c t i v i t y w i t h a l l stages w i t h i n the myeloid  In  lineage holds  t r u e i n BM samples from  of c e l l s  142  Figure 4.3. Morphology of CAMAL-1 p o s i t i v e m y e l o i d c e l l s i n ANLL remission patients. (a) - (c) i l l u s t r a t e p o s i t i v e l y l a b e l e d BM c e l l s from 3 r e m i s s i o n p a t i e n t s i l l u s t r a t i n g (a) and (b) p o s i t i v e g r a n u l o c y t e s and (c) megakaryocytes.  143  144  F i g u r e 4.4. Lymphocytes from an ANLL r e m i s s i o n p a t i e n t ' s p e r i p h e r a l p o s i t i v e l y l a b e l e d by CAMAL-1.  blood  145  F i g u r e 4 . 5 . D i f f u s e g r a n u l a r s t a i n i n g o f CGL p e r i p h e r a l b l o o d c e l l s by CAMAL-1. T h i s type of l a b e l i n g i s h i g h l y c h a r a c t e r i s t i c of CGL PB cells.  146  preleukemi.es/ m y e l o d y s p l a s t i c s , 3.3)  remission patients (Figure  and normals, even though r e l a t i v e percentages of  CAMAL-l-reactive c e l l s may myeloid  B.  some ALL  o f t e n be v e r y s m a l l i n comparison w i t h  leukemics.  ANLL Remission P a t h o l o g y In o r d e r to i n v e s t i g a t e the p o s s i b i l i t y t h a t  e l e v a t e d numbers of CAMAL-1 r e a c t i v e c e l l s may u n d e r l y i n g pathology PBL,  we  chose to study  c e l l s possessed clinical  present  significantly  represent  i n a p p a r e n t l y normal r e m i s s i o n BM  or  an ANLL r e m i s s i o n p a t i e n t whose m a l i g n a n t  a c h a r a c t e r i s t i c k a r y o t y p e (47,XY,+6).  diagnosis  an  (ANLL FAB  M5),  At  initial  c y t o g e n e t i c e v a l u a t i o n of a d i r e c t  bone marrow p r e p a r a t i o n w i t h G-banding performed by Dr. Dagmar Kalousek ( T e r r y Fox L a b o r a t o r y , p r e s e n c e of two morphological  cell  lines  Vancouver, B.C.)  (46,XY/47,XY,+6).  the 23 examined.  A t the same time, immunoperoxidase l a b e l i n g of  t e s t s 74% b l a s t s were p r e s e n t  r e l a p s e was  normal, but  abnormal metaphase (48,XY,+6,+8) i n  showed 10% r e a c t i v i t y w i t h CAMAL-1.  hyperdiploidy  the  Four months l a t e r  e v a l u a t i o n of t h i s p a t i e n t ' s BM was  c y t o g e n e t i c a n a l y s i s r e v e a l e d one  revealed  Three weeks f o l l o w i n g  i n the BM,  (48,XY,+6,+8), and  BM  these  24/25 metaphases showed  a clinical  d i a g n o s i s of ANLL i n  made.  Another ANLL (FAB M4) secondary t o p r e v i o u s  p a t i e n t , whose leukemia may  have been  chemotherapy f o r o v a r i a n carcinoma,  i n v e s t i g a t e d c y t o g e n e t i c a l l y a t p r e s e n t a t i o n f o r ANLL.  was  G-banding  chromosome a n a l y s i s r e v e a l e d a unique h y p o d i p l o i d k a r y o t y p e i n a l l metaphases s t u d i e d  [44,XX,-4,-17,-21,+MAR,7q~,t(5q;llp;13q),  147  t(9q;19q)]. phase.  An i d e n t i c a l twin BM t r a n s p l a n t was performed i n acute  The p a t i e n t ' s c y t o g e n e t i c  analyses  2 and 4 weeks  p o s t - t r a n s p l a n t were normal (46,XX) i n d i c a t i n g t h a t h e r m a l i g n a n t c e l l s had a p p a r e n t l y  been s u c c e s s f u l l y e r a d i c a t e d .  However, a t 4  weeks p o s t - t r a n s p l a n t we found t h a t 100% o f h e r PBL r e a c t e d w i t h t h e r a b b i t anti-CAMAL serum i n FACS a n a l y s i s .  When t h e s e l a b e l e d  were s o r t e d t o o b t a i n t h a t 25% o f t h e p o p u l a t i o n r e l a t i v e f l u o r e s c e n c e , we d i s c o v e r e d routine morphological  criteria.  cells  showing t h e h i g h e s t  t h a t these c e l l s were normal by  F i g u r e 4.6 a,b shows t h a t  typical  c e l l s s o r t e d o u t i n t h i s manner i n c l u d e d b o t h mature n e u t r o p h i l s and lymphocytes. blood  There were no m a l i g n a n t c e l l s d e t e c t a b l e  o r BM a t t h i s time.  i n e i t h e r her  Four months l a t e r , t h i s p a t i e n t s u f f e r e d  c l i n i c a l ANLL r e l a p s e w i t h t h e emergence once a g a i n o f h e r o r i g i n a l hypodiploid  c e l l c l o n e , which had undergone minor e v o l u t i o n .  observation  t h a t h e r normal c e l l s were p o s i t i v e f o r membrane CAMAL  p o s t - t r a n s p l a n t c o u l d imply transplanted expression The  adsorption  c e l l s and/or i n f o r m a t i o n  by t h e i d e n t i c a l t w i n donor  The  o f s o l u b l e CAMAL by exchange r e s u l t i n g i n CAMAL cells.  r e s u l t s from t h e two ANLL p a t i e n t s s t u d i e d  using  immunoperoxidase and CAMAL-1 o r FACS a n a l y s i s and r a b b i t anti-CAMAL, when c o r r e l a t e d w i t h m o r p h o l o g i c a l two  important r e l a t e d f i n d i n g s : 1) m o r p h o l o g i c a l  r e m i s s i o n may be i n c a p a b l e all be  and c y t o g e n e t i c  cases,  analyses,  evaluation of  o f a c c u r a t e l y gauging r e m i s s i o n  and 2) u n d e r l y i n g  revealed  state i n  seminal changes i n ANLL may i n v o l v e ( o r  i d e n t i f i e d by) c l o n e ( s ) o f c e l l s t h a t a r e normal b o t h  morphologically  and c y t o g e n e t i c a l l y and t h a t i n c l u d e t e r m i n a l l y  differentiating  lines.  148  F i g u r e 4.6. FACS s o r t i n g study of an ANLL post-bone marrow t r a n s p l a n t patient's p e r i p h e r a l blood c e l l s . Cells labeling strongly with r a b b i t anti-CAMAL serum 4 months p r i o r t o leukemic r e l a p s e i n c l u d e d m o r p h o l o g i c a l l y (and k a r y o t y p i c a l l y ) normal mature (a) n e u t r o p h i l s and lymphocytes; (b) h i g h e r m a g n i f i c a t i o n of same.  a  150  C.  The Presence o f CAMAL i n Plasma I t was p o s s i b l e t h a t t h e p r e s e n c e o f CAMAL-positive c e l l s i n  ANLL r e m i s s i o n p a t i e n t s , p a r t i c u l a r l y lymphoid c e l l s ,  r e s u l t e d from  a d s o r p t i o n o f s o l u b l e CAMAL which had been produced and s e c r e t e d by other c e l l s .  Were t h i s t o be t h e case, i t would be r e a s o n a b l e t o  assume t h a t s o l u b l e CAMAL might be p r e s e n t i n t h e plasma o f t h e s e individuals.  To examine t h i s p o s s i b i l i t y , we looked f o r CAMAL i n the  plasma o f a number o f m y e l o i d leukemia p a t i e n t s as w e l l as normals u s i n g a f f i n i t y chromatography as d e s c r i b e d i n Chapter I I .  Affinity  chromatography e x t r a c t i o n o f CAMAL from plasma was performed a f t e r numerous u n s u c c e s s f u l attempts a t q u a n t i t a t i n g serum o r plasma CAMAL l e v e l s u s i n g ELISA and radio-immunoassay. T a b l e XV shows r o u g h l y e s t i m a t e d plasma l e v e l s o f CAMAL i n m y e l o i d leukemics and normals.  CAMAL p r o t e i n c o n c e n t r a t i o n s were  e s t i m a t e d by B i o r a d p r o t e i n a s s a y o f e l u t e d m a t e r i a l bound t o a CAMAL-1 immunoadsorbent column.  column a f t e r plasma had been p a s s e d o v e r the  There a r e obvious problems w i t h t h i s method o f e s t i m a t i n g  plasma CAMAL c o n c e n t r a t i o n s . These i n c l u d e t h e immunological cross-reactivity  o f CAMAL-1 w i t h human serum albumin (Shipman and  S h e l l a r d , p e r s o n e l communication) and t h e f a c t t h a t t h i s method would, a t b e s t , o n l y e s t i m a t e p r o t e i n c o n c e n t r a t i o n .  With t h i s i n  mind, t h e f o l l o w i n g e s t i m a t e s a r e r e p o r t e d , b u t i t i s conceded t h a t t h e s e e s t i m a t e s may be q u a n t i t a t i v e l y q u i t e i n a c c u r a t e .  Normal  6) plasma l e v e l s were 21 ug/ml on average; l e v e l s i n m y e l o i d o r p r e l e u k e m i c s (n = 4) averaged 52 ug/ml.  I t i s not possible to  claim with c e r t a i n t y that these l e v e l s represent s i g n i f i c a n t d i f f e r e n c e s between plasma CAMAL l e v e l s i n normals and m y e l o i d  (n =  151  T a b l e XV.  D e t e c t i o n o f CAMAL i n plasma by immunoaffinity  Diagnosis  Estimated  chromatography  Plasma CAMAL L e v e l s (yg/ml)  CGL CGL  50.0 66.0  Preleukemia  47.0  ANLL r e m i s s i o n  44.7  Normal Normal Normal Normal Normal Normal  19.0 25.9 15.0 13.5 26.7 26.5  152  leukemics  a l t h o u g h t h i s may be t h e case.  The e l u t e d CAMAL ( F i g u r e  4.7) r e a c t e d t o t h e same e x t e n t i n ELISA w i t h CAMAL-1 MAb, whether p u r i f i e d from normals o r m y e l o i d  leukemics  same m o b i l i t y i n PAGE, r u n n i n g as expected  D.  CAMAL A d s o r p t i o n  ( F i g u r e 4.8a) and had t h e a t 68 KD ( F i g u r e 4.8b).  Studies  I t has been demonstrated t h a t p e r i p h e r a l b l o o d can, on o c c a s i o n , c o n t a i n s i g n i f i c a n t l y h i g h numbers o f CAMAL-positive c e l l s during c l i n i c a l  r e m i s s i o n i n ANLL.  from a c u t e leukemia  even  Moreover, i n a b l i n d study o f PBL  p a t i e n t s who had undergone BMT (529), those ANLL  p a t i e n t s who r e l a p s e d showed i n c r e a s e d numbers o f CAMAL-1 p o s i t i v e cells  ( u s i n g immunoperoxidase) up t o 3 months p r i o r t o r e l a p s e  ( F i g u r e 4.9).  We wished t o determine i f normal PBL would become  CAMAL-1 r e a c t i v e i f i n c u b a t e d i n t h e presence  o f excess  CAMAL,  p a r t i c u l a r l y s i n c e t h e r e was t h e p o s s i b i l i t y t h a t m y e l o i d  leukemic  plasma might c o n t a i n h i g h e r l e v e l s o f CAMAL than normals. Whole h e p a r i n i z e d p e r i p h e r a l b l o o d samples from two normal i n d i v i d u a l s were i n c u b a t e d o v e r n i g h t w i t h i n c r e a s i n g c o n c e n t r a t i o n s of  s o l u b l e CAMAL d e r i v e d from m y e l o i d  leukemia  cell  extracts (or  e q u i v a l e n t amounts o f i r r e l e v a n t p r o t e i n o r PBS) as o u t l i n e d i n Chapter  II.  F o l l o w i n g t h i s i n c u b a t i o n , PBL were i s o l a t e d from  these  samples by F i c o l l - H y p a q u e s e p a r a t i o n and s l i d e p r e p a r a t i o n s were made.  The i n d i r e c t immunoperoxidase assay was then c a r r i e d out on  these s l i d e s u s i n g CAMAL-1 o r an i r r e l e v a n t MAb. In a l l o f t h r e e normal samples t e s t e d i n t h i s manner, CAMAL-1 r e a c t i v e (membrane p o s i t i v e ) c e l l s were i d e n t i f i e d on s l i d e s o f c e l l s which had been p r e v i o u s l y i n c u b a t e d w i t h > 12.5 yg/ml CAMAL.  No  153  Figure 4.7. Elution profile of material from normal human plasma bound by a CAMAL-1 immunoadsorbent column. • O  • CAMAL-1 column eluate O negative control MAb column eluate  - LOG  OF D I L U T I O N  Figure 4.8a. ELISA reactivity of plasma-derived affinity purified CAMAL with CAMAL-1 monoclonal antibody. O 9  O normal plasma 0 ANLL remission plasma  154b  1  2  3  4  5  6  7  8  F i g u r e 4.8b. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c p r o f i l e o f CAMAL p u r i f i e d by a f f i n i t y chromatography from human plasma. Lanes 1,8: 66 KD m o l e c u l a r weight s t a n d a r d s Lanes 2,7: 62/66 KD d o u b l e t o f CAMAL, p u r i f i e d by a f f i n i t y chromatography, from m y e l o i d leukemia c e l l l y s a t e s (62/66 KD d o u b l e t was c o n s i s t e n t l y p u r i f i e d by one CAMAL-1 column). Lanes 3,4: CAMAL p u r i f i e d by a f f i n i t y chromatography from two normal human plasma samples Lanes 5,6: CAMAL p u r i f i e d by a f f i n i t y chromatography from two m y e l o i d leukemia plasma samples.  155  Figure 4.9. Immunoperoxidase staining of an ANLL post-bone marrow transplant p a t i e n t ' s peripheral blood c e l l s , labeled with CAMAL-1, one month p r i o r to relapse.  156  p o s i t i v e l y l a b e l e d c e l l s were seen i n s l i d e s l a b e l e d w i t h t h e i r r e l e v a n t MAb o r i n s l i d e s c o n t a i n i n g c e l l s p r e - i n c u b a t e d w i t h < 5 yg/ml CAMAL.  F i g u r e 4.10 a - f i l l u s t r a t e s c e l l s from one normal PB  sample, i n c u b a t e d w i t h i n c r e a s i n g amounts o f CAMAL and l a b e l e d w i t h CAMAL-1 o r t h e n e g a t i v e c o n t r o l MAb i n t h e immunoperoxidase showing obvious  l a b e l i n g of c e l l s  ( p r i m a r i l y lymphoid  membranes) w i t h 12.5 and 50.0 yg/ml excess CAMAL.  assay,  and e r y t h r o i d  F i g u r e 4.11 a,b  (from another normal PB sample) i l l u s t r a t e s an even more s t r i k i n g d i f f e r e n c e , w i t h r b c , lymphocytes, l a b e l i n g w i t h CAMAL-1.  monocytes and g r a n u l o c y t e s  These r e s u l t s i n d i c a t e t h a t , a t a c e r t a i n  c o n c e n t r a t i o n o f excess CAMAL (> 12.5 ug/ml), normal PBL can become CAMAL-positive. of CAMAL-positive  I t i s p o s s i b l e , t h e r e f o r e , t h a t t h e i n c r e a s e d number PBL ( e s p e c i a l l y lymphoid  c e l l s ) d e t e c t e d i n ANLL  r e m i s s i o n (Chapter V) o r BMT p a t i e n t s p r i o r t o r e l a p s e may be due t o a s i m i l a r a d s o r p t i o n , r e s u l t i n g from i n c r e a s e d amounts o f s o l u b l e CAMAL i n t h e plasma o f these i n d i v i d u a l s a t such  times.  157  Figure 4.10. Normal p e r i p h e r a l b l o o d c e l l s , i n c u b a t e d w i t h amounts of CAMAL and l a b e l e d by immunoperoxidase.  a.  0 yg/ml CAMAL, l a b e l e d w i t h CAMAL-1.  increasing  158  b.  5 yg/ml CAMAL, l a b e l e d w i t h CAMAL-1.  c.  12.5 yg/ml CAMAL, l a b e l e d w i t h CAMAL-1.  1 i  • d.  e.  %  •  +  12.5 yg/ml CAMAL, l a b e l e d w i t h n e g a t i v e c o n t r o l  50 yg/ml CAMAL, l a b e l e d w i t h n e g a t i v e c o n t r o l  MAb.  MAb.  _  «  •  160  f.  50 ug/ml CAMAL, l a b e l e d w i t h CAMAL-1.  161  Figure 4.11. Morphology of CAMAL-1 r e a c t i v e c e l l s from a normal p e r i p h e r a l b l o o d sample a f t e r i n c u b a t i o n w i t h o r w i t h o u t CAMAL. (a) C e l l s i n c u b a t e d w i t h 50 ug/ml a f f i n i t y p u r i f i e d CAMAL and l a b e l e d w i t h CAMAL-1, i l l u s t r a t i n g p o s i t i v e l y s t a i n i n g m y e l o i d ( n e u t r o p h i l s , monocytes, e r y t h r o c y t e s ) and lymphoid c e l l s ; (b) the same c e l l sample i n c u b a t e d w i t h 0 ug/ml CAMAL and l a b e l e d w i t h CAMAL-1, i l l u s t r a t i n g no p o s i t i v e l y l a b e l e d c e l l s .  a  162  >  *  • ^ ^ £4 ^  • b  t  163  CHAPTER V  SIGNIFICANCE OF CAMAL AS A PROGNOSTIC MARKER FOR REMISSION IN ACUTE NONLYMPHOBLASTIC LEUKEMIA  I.  INTRODUCTION Studies of myeloid  l i n e a g e - a s s o c i a t e d a n t i g e n s have c o n t r i b u t e d  s i g n i f i c a n t l y t o our p r e s e n t u n d e r s t a n d i n g types i n v o l v e d i n acute n o n l y m p h o b l a s t i c  of the complexity  leukemia.  of c e l l  As mentioned  p r e v i o u s l y , t h e attempt t o c o r r e l a t e F r e n c h - A m e r i c a n - B r i t i s h (FAB) group c l a s s i f i c a t i o n s o f ANLL w i t h s p e c i f i c c e l l  s u r f a c e phenotypes  (472-476) has met w i t h l i m i t e d s u c c e s s , w i t h t h e e x c e p t i o n o f c e r t a i n d e f i n e d a s s o c i a t i o n s (M4/M5 w i t h CD14 a n t i g e n , f o r example). a n t i g e n i c h e t e r o g e n e i t y c o m p l i c a t e s d i a g n o s i s and treatment  Marked o f ANLL,  p a r t i c u l a r l y i n areas c u r r e n t l y o f widespread i n t e r e s t , namely d i a g n o s i s o f r e s i d u a l d i s e a s e and immuno-purging o f bone marrow f o r autologous  bone marrow t r a n s p l a n t a t i o n purposes.  ANLL without bone marrow t r a n s p l a n t a t i o n (BMT) g e n e r a l l y has a poor l o n g term p r o g n o s i s .  Leukemic r e l a p s e c o n t i n u e s t o be a s e r i o u s  problem i n t r e a t e d ANLL and a t p r e s e n t t h e r e i s l i m i t e d u s e f u l i n f o r m a t i o n a v a i l a b l e c o n c e r n i n g p r o g n o s t i c i n d i c a t o r s i n ANLL. With r e g a r d t o c h r o n i c g r a n u l o c y t i c leukemia  (CGL),  s t u d i e s have  i n d i c a t e d t h a t i t might be p o s s i b l e t o i d e n t i f y c e r t a i n p o t e n t i a l c a n d i d a t e s f o r s u c c e s s f u l BMT i n t h e younger CGL p a t i e n t group by examining a number o f c l i n i c a l  parameters i n c l u d i n g serum  lactic  dehydrogenase (LDH) a c t i v i t y ,  sex, s p l e e n s i z e , h e m a t o c r i t ,  platelet  164  count, c i r c u l a t i n g n u c l e a t e d basophils plus eosinophils Longer r e m i s s i o n s  r b c s , as w e l l as % marrow b l a s t s , (531).  i n ANLL have been shown t o be a s s o c i a t e d w i t h  such f a c t o r s (many o f which a r e i n t e r r e l a t e d ) as low i n i t i a l c i r c u l a t i n g b l a s t count, low pretreatment and  LDH o r f i b r i n o g e n l e v e l s  r a p i d development o f complete r e m i s s i o n s t a t u s (532).  In v i t r o  assays f o r h e m o p o i e t i c p r o g e n i t o r c e l l s o r t h e i r a s s o c i a t e d r e g u l a t o r y f a c t o r s have been examined f o r t h e i r  prognostic  i m p l i c a t i o n s i n ANLL (533-537).  very  Unfortunately,  i n f o r m a t i o n has been w e l l c h a r a c t e r i z e d c o n c e r n i n g  little  factors  (cell  markers, serum p r o t e i n s , e t c . ) t h a t might s e r v e as common p r o g n o s t i c i n d i c a t o r s i n ANLL d u r i n g  remission.  Over a decade ago, i t was observed t h a t t h e n a t u r e colony-forming  of myeloid  c e l l growth i n v i t r o c o u l d a s s i s t i n t h e m o n i t o r i n g o f  i n d i v i d u a l ANLL p a t i e n t s i n terms o f p r e d i c t i n g t h e onset o f r e m i s s i o n o r r e l a p s e (538,539).  Baker e t a l . were a b l e t o p r e d i c t  r e l a p s e i n 21/26 ANLL r e m i s s i o n p a t i e n t s ' BM u s i n g murine heteroantiserum  s p e c i f i c f o r a myeloblast  antigen  (461).  In a  c o l l a b o r a t i v e b l i n d study u s i n g an i n d i r e c t immunoperoxidase  slide  t e s t and t h e CAMAL-1 MAb, we were a b l e t o d e t e c t i n c r e a s e d amounts o f CAMAL i n p e r i p h e r a l b l o o d or more p r i o r t o r e l a p s e Although present  l e u c o c y t e s o f BMT p a t i e n t s up t o 3 months (529).  i n ANLL b l a s t c e l l s , CAMAL i s n o t s t r i c t l y  b l a s t c e l l a n t i g e n and has been demonstrated i n o r on many c e l l w i t h i n t h e myeloid  l i n e a g e , as d e s c r i b e d i n Chapter IV.  a types  On o c c a s i o n  we have found d e t e c t a b l e l e v e l s o f CAMAL on t h e s u r f a c e o f lymphoid c e l l s i n ANLL r e m i s s i o n p a t i e n t s as w e l l (514, Ch.IV).  While CAMAL  165  appears  t o be expressed a t much h i g h e r l e v e l s i n PBL o r BM c e l l s o f  myelogenous leukemia p a t i e n t s , i t has been e s t a b l i s h e d (Chapter I I I ) t h a t CAMAL i s a l s o p r e s e n t i n normal BM c e l l s , a l b e i t a t s i g n i f i c a n t l y lower l e v e l s . approximately (510).  1% CAMAL-1 r e a c t i v e c e l l s by immunoperoxidase  The remarkable  high levels,  As d e s c r i b e d , normal BM c o n t a i n s assay  f i n d i n g t h a t CAMAL was p r e s e n t , o f t e n a t v e r y  i n BM c e l l s from ANLL r e m i s s i o n p a t i e n t s ,  l e d us t o  question the p o s s i b l e p r o g n o s t i c s i g n i f i c a n c e of i t s presence.  This  c h a p t e r d e s c r i b e s t h e r e s u l t s o b t a i n e d from a b l i n d study c a r r i e d out w i t h ANLL p a t i e n t s a t t e n d i n g t h e Department o f Hematology, Vancouver General H o s p i t a l .  We wished t o determine  of CAMAL-1 r e a c t i v e BM c e l l s  whether o r n o t t h e number  (CAMAL BM v a l u e ) r e f l e c t e d  clinical  p r o g n o s i s i n ANLL p a t i e n t s over t h e c o u r s e o f t h e i r d i s e a s e .  Using  the i n d i r e c t immunoperoxidase s l i d e t e s t , we examined BM c e l l s the CAMAL-1 MAb i n an attempt  t o determine  with  any c o r r e l a t i o n between  l e n g t h o f c l i n i c a l r e m i s s i o n and changes i n p a t i e n t s ' CAMAL BM v a l u e s f o l l o w i n g chemotherapy. change appears  Results presented here i n d i c a t e that t h i s  t o be a u s e f u l p r o g n o s t i c i n d i c a t o r f o r r e m i s s i o n i n  ANLL.  RESULTS D e t a i l s o f t h e b l i n d study p r o t o c o l have been g i v e n i n Chapter I I ; some p o i n t s w i l l be r e i t e r a t e d here f o r c l a r i t y .  Only d a t a  from  ANLL p a t i e n t s a r e p r e s e n t e d h e r e , a l t h o u g h t h e b l i n d study i n c l u d e d examination  o f over 700 samples from a l l p a t i e n t s (and BMT donors)  a t t e n d i n g t h e Department o f Hematology, Vancouver G e n e r a l All  s l i d e s were p r e p a r e d  Hospital.  and coded n u m e r i c a l l y so t h a t no p a t i e n t  166  i n f o r m a t i o n was a v a i l a b l e t o t h e s l i d e r e a d e r .  ANLL p a t i e n t s (n =  34) whose BM had been s e r i a l l y examined a t t h e a p p r o p r i a t e times were d i v i d e d i n t o two groups f o r a n a l y s i s by t h e p r o d u c t - l i m i t method (Kaplan-Meier e s t i m a t e ) : 1.  ANLL p a t i e n t s a t d i a g n o s i s whose CAMAL BM v a l u e s  decreased  post-chemotherapy ( w i t h i n 1 + 0 . 5 months), and 2.  ANLL p a t i e n t s a t d i a g n o s i s whose CAMAL BM v a l u e s  increased or  remained t h e same post-chemotherapy,  A.  R e l a t i o n s h i p Between S u r v i v a l Time P r i o r t o Relapse and Change i n CAMAL BM V a l u e s The e n t i r e group (n = 34) o f ANLL p a t i e n t s was examined,  the Cox s u r v i v a l a n a l y s i s model i n t h e BMDP2L b i o m e d i c a l program,  using  computer  i n o r d e r t o determine i f t h e r e was any s i g n i f i c a n t  v a r i a b l e ( s ) t h a t c o u l d be u s e f u l t o p r e d i c t which p a t i e n t s would have l o n g e r r e m i s s i o n times.  C o v a r i a t e s a n a l y s e d were p a t i e n t age, sex,  CAMAL BM v a l u e a t d i a g n o s i s and change i n CAMAL BM v a l u e post-chemotherapy.  The o n l y s i g n i f i c a n t  (p = 0.05) v a r i a b l e i n  p r e d i c t i n g p a t i e n t s w i t h l o n g e r r e m i s s i o n times was t h e change i n t h e CAMAL BM v a l u e post-chemotherapy.  B.  C o r r e l a t i o n Between D e c r e a s i n g  CAMAL BM V a l u e s Post-chemotherapy  and Longer R e m i s s i o n Times S i n c e t h e change i n CAMAL BM v a l u e  ( p r e - t o post-chemotherapy)  had been shown t o be s i g n i f i c a n t i n terms o f p r o g n o s i s ,  the t o t a l  ANLL group was d i v i d e d i n t o two subgroups based on t h i s parameter. Group 1 (n = 10) c o n s i s t e d o f ANLL p a t i e n t s whose CAMAL BM v a l u e s  16 7  decreased  s i g n i f i c a n t l y post-chemotherapy ( T a b l e XVI).  The average  r e m i s s i o n l e n g t h f o r t h i s group was 19.2 months, w i t h 50% o f these patients s t i l l remaining  in first  remission.  T h i s was compared t o t h e  ANLL p a t i e n t s (Group 2) whose CAMAL BM v a l u e s  either  i n c r e a s e d o r remained unchanged post-chemotherapy ( T a b l e X V I I ) .  The  average r e m i s s i o n l e n g t h f o r Group 2 was 6.8 months, w i t h o n l y 25% still  i n f i r s t remission.  A comparison o f t h e s u r v i v a l time p r i o r t o  r e l a p s e was made between t h e s e two groups u s i n g t h e Kaplan-Meier estimate with log-rank s t a t i s t i c a l a n a l y s i s . the s u r v i v a l curves  F i g u r e 5.1 i l l u s t r a t e s  f o r these two groups; a s i g n i f i c a n t d i f f e r e n c e (p  < 0.025) was found between them.  There was v e r y l i t t l e d i f f e r e n c e  between r e m i s s i o n l e n g t h s f o r p a t i e n t s whose v a l u e s i n c r e a s e d (x = 6.1 months) and those whose v a l u e s remained unchanged (x = 7.4 months) post-chemotherapy.  The improved p r o g n o s i s was c o r r e l a t e d  o n l y w i t h a d e c r e a s i n g CAMAL BM v a l u e . F i g u r e 5.2 i l l u s t r a t e s CAMAL BM v a l u e s p r e - and post-chemotherapy f o r two ANLL p a t i e n t s whose v a l u e s s i g n i f i c a n t l y f o l l o w i n g treatment.  dropped  These p a t t e r n s may be compared  w i t h those from t h r e e ANLL p a t i e n t s whose CAMAL BM v a l u e s o r were unchanged f o l l o w i n g chemotherapy ( F i g u r e 5.3).  increased  It is  apparent from these examples t h a t t h e a b s o l u t e p e r c e n t a g e i n c r e a s e (or  d e c r e a s e ) does n o t seem t o d e f i n e p r e c i s e l y how l o n g any  i n d i v i d u a l r e m i s s i o n w i l l be.  However, t h e r e l a t i v e change  (decrease  o r i n c r e a s e , +/-) post-chemotherapy i s d i r e c t l y c o r r e l a t e d w i t h average r e m i s s i o n  length.  T a b l e XVI.  ANLL p a t i e n t s whose CAMAL BM  v a l u e s decreased  Patient  101 102 103 104 105 106 108 107 109 110  post-chemotherapy (Group 1)  CAMAL BM v a l u e ( p e r c e n t p o s i t i v e c e l l s ) At d i a g n o s i s Post-chemotherapy  21.0 18.9 80.0 31.0 31.0 50.0 21.0 12.3 14.2 21.0  0.7 7.5 0.1 13.0 0.1 11.0 2.5 6.3 0.5 0.1  0 6.5 10 14 17 20 32 24.5 32 36  T o t a l number of p a t i e n t s = 10 Average r e m i s s i o n l e n g t h = 19.2 months  Present  s t a t u s o f a l l p a t i e n t s i n Group 1 n  1st remission 2nd r e m i s s i o n Relapse Dead  5 1 1 3  Remission length (months)  Percent 50 10 10 30  169  T a b l e XVII.  ANLL p a t i e n t s whose CAMAL BM v a l u e s  o r remained t h e same post-chemotherapy  Patient  201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224  increased  (Group 2)  CAMAL BM v a l u e ( p e r c e n t p o s i t i v e c e l l s ) At d i a g n o s i s Post-chemotherapy  0.5 80.0 0.1 5.0 2.5 0.5 4.5 2.5 11.0 1.5 5.5 3.0 5.5 4.2 8.0 1.4 1.8 2.2 0.1 0.8 0.1 2.2 0.7 1.3  7.0 100.0 25.0 20.0 4.0 2.9 12.3 45.5 6.5 62.5 4.9 6.3 1.5 6.0 6.5 0.5 2.5 0.7 21.0 20.0 2.2 16.5 13.7 3.5  0 0 1 1.0 2.5 3 3.5 3.5 3.5 5 5.5 6 7 7.0 8 8 8 8 10.0 13 14 14 16 16  T o t a l number o f p a t i e n t s = 24 Average r e m i s s i o n l e n g t h = 6.8 months Present  s t a t u s o f a l l p a t i e n t s i n Group 2 n  1st remission 2nd r e m i s s i o n Relapse Dead BMT  6 1 2 11 4  Remission length (months)  Percent 25 4 8 46 17  170  1.0 0.9 0.8 0.7-I  LU  (A < _ l Ul <  <2 >  g5 fc  o CC Q.  0.6 _l GROUP 1  0.5 0.4-  UJ  0.30.2  GROUP 2  0.1 0  "T  r~i  r  5  I  I  I  1  r—i—i—i—i—i—|—i—i—i—,—i—i—r—i—i—i——i—i—i—i—i—i—i—|—i  10  MONTHS  15  POST  20  25  30  35  CHEMOTHERAPY  F i g u r e 5.1. K a p l a n - M e i e r s u r v i v a l c u r v e showing s u r v i v a l time p r i o r t o r e l a p s e f o r ANLL p a t i e n t s i n b l i n d s t u d y . Group 1, ANLL p a t i e n t s (n = 10) whose CAMAL BM v a l u e s d e c r e a s e d s i g n i f i c a n t l y post-chemotherapy; Group 2, ANLL p a t i e n t s ( n = 2 4 ) whose CAMAL BM v a l u e s i n c r e a s e d o r remained unchanged f o l l o w i n g chemotherapy.  MONTHS  POST-CHEMOTHERAPY  F i g u r e 5.2. CAMAL BM v a l u e s over time f o r two ANLL p a t i e n t s w i t h s i g n i f i c a n t l y decreased v a l u e s post-chemotherapy. S u r v i v a l times p r i o r t o r e l a p s e were (a) 10 months and (b) s t i l l i n f i r s t remission. L, acute leukemia; R, r e m i s s i o n ;  ( R ) , r e l a p s e ; D, dead  172  F i g u r e 5.3. CAMAL BM v a l u e s over time f o r t h r e e ANLL p a t i e n t s whose v a l u e s i n c r e a s e d o r were unchanged f o l l o w i n g treatment. S u r v i v a l times p r i o r t o r e l a p s e were (a) 1.3 months, (b) 10 months and (c) 8 months. L, acute  leukemia; R, r e m i s s i o n ;  ( R ) , r e l a p s e ; D, dead  173  C.  CAMAL BM Values and Simultaneous M o r p h o l o g i c a l examination  BM Morphology  o f CAMAL-1 p o s i t i v e c e l l s a t d i a g n o s i s  r e v e a l e d t h a t , i n a l l ANLL p a t i e n t s examined, t h e predominant p o s i t i v e c e l l s were b l a s t c e l l s and o t h e r v e r y immature m y e l o i d as expected.  cells  However, when post-chemotherapy s l i d e s were examined,  t h e r e were d i f f e r e n c e s among t h e ANLL p a t i e n t s w i t h r e s p e c t t o t h e morphology o f CAMAL-1 p o s i t i v e c e l l s .  A l l ANLL p a t i e n t s whose CAMAL  BM v a l u e s i n c r e a s e d post-chemotherapy showed s i m i l a r k i n d s o f p o s i t i v e c e l l s t o those seen a t d i a g n o s i s ( p r i m a r i l y immature m y e l o i d cells,  including blasts).  I n g e n e r a l , t h i s was t h e case as w e l l f o r  p a t i e n t s whose CAMAL BM v a l u e s d i d n o t change post-chemotherapy, a l t h o u g h a few p a t i e n t s showed p o s i t i v e c e l l s a t a l l s t a g e s o f myeloid maturation.  However, i n those p a t i e n t s whose CAMAL BM v a l u e s  decreased post-chemotherapy, p o s i t i v e c e l l s r o u t i n e l y i n c l u d e d myeloid  c e l l s a t a l l l e v e l s of maturation,  of mature polymorphonuclear  D.  some w i t h a preponderance  leucocytes.  S u p p o r t i v e Data from Other ANLL P a t i e n t s The  c o r r e l a t i o n between c l i n i c a l p r o g n o s i s and r e l a t i v e CAMAL BM  v a l u e p r e - and post-chemotherapy was supported by r e s u l t s from ANLL r e l a p s e and BMT p a t i e n t s examined.  5/6 ANLL r e l a p s e p a t i e n t s whose  CAMAL BM v a l u e s i n c r e a s e d s i g n i f i c a n t l y a f t e r treatment a t t a i n 2nd r e m i s s i o n .  f a i l e d to  T h i s was compared w i t h 4/4 ANLL r e l a p s e  p a t i e n t s w i t h s i g n i f i c a n t l y decreased  v a l u e s post-chemotherapy who  d i d a c h i e v e 2nd r e m i s s i o n s . F i n a l l y t h e r e were 7 ANLL p a t i e n t s who underwent BMT and f o r whom CAMAL BM v a l u e s had been r e c o r d e d p r e - and post-BMT.  5/7 o f  174  t h e s e p a t i e n t s had remission  ( a t 6.5  post-BMT; one  decreased CAMAL BM - 34 months).  2/7  values  and  a l l are s t i l l  in  showed i n c r e a s e d CAMAL BM  of these p a t i e n t s r e l a p s e d a t 3 months, the  values  other  s u f f e r e d f o c a l u t e r i n e leukemic r e l a p s e a t 8 months.  E.  R e l a t i o n s h i p Between F i r s t R e m i s s i o n Length and at  CAMAL BM  Value  Diagnosis  I t was  i n t e r e s t i n g t o n o t e t h a t a l l p a t i e n t s i n Group 1  elevated  ( w e l l above normal) CAMAL BM  x = 30).  These were the p a t i e n t s who  s i g n i f i c a n t l y longer f i r s t  values  remissions.  (n = 24,  P a t i e n t s i n Group 2 who  x = 6).  T h i s l e d t o the  However, when a Cox  performd on a l l ANLL p a t i e n t s (n = 40)  CAMAL BM  be more  responsive  survival analysis  whose BM  to p r e d i c t i n g remission  value at diagnosis.  had,  speculation  was  was  examined a t  d i a g n o s i s , none of the f o l l o w i n g c o v a r i a t e s were found t o s i g n i f i c a n t with regard  10,  had much lower  t h a t c e l l s i n t h i s s t a t e of a n t i g e n e x p r e s s i o n may t o s u c c e s s f u l treatment.  (n =  f a r e d b e t t e r i n terms of  on average, s i g n i f i c a n t l y s h o r t e r r e m i s s i o n s pre-treatment values  pre-treatment  had  length:  A d e c r e a s e i n the CAMAL BM  be age,  sex,  or  value  post-chemotherapy appeared to be the o n l y s i g n i f i c a n t f a c t o r i n t h i s regard.  F.  I n c r e a s i n g CAMAL BM In a p r e v i o u s  values present  V a l u e s and  Relapse  i n v e s t i g a t i o n (529)  we  showed t h a t CAMAL  i n c r e a s e d p r i o r to r e l a p s e i n a BMT study a l s o i n d i c a t e d t h a t t h i s was  v a l u e s were monitored d u r i n g r e m i s s i o n .  group s t u d i e d .  PBL The  the case when CAMAL  BM  There were 9 p a t i e n t s whose  175  s e q u e n t i a l CAMAL BM d a t a showed an i n c r e a s e o c c u r r i n g d u r i n g remission.  F o r 4 o f t h e s e p a t i e n t s , we had o b t a i n e d samples w i t h i n  the few months immediately  p r i o r to r e l a p s e .  T a b l e XVIII  shows these  4 p a t i e n t s ' r e c o r d e d CAMAL BM v a l u e s over t h e c o u r s e o f t h e i r r e m i s s i o n . I n each case, t h e s e v a l u e s were observed t o 3 months p r i o r t o r e l a p s e .  t o i n c r e a s e 1.5  We found t h a t 78% (7/9) o f p a t i e n t s  who had i n c r e a s i n g r e m i s s i o n v a l u e s s u f f e r e d r e l a p s e w h i l e o n l y 33% (3/9) o f p a t i e n t s w i t h d e c r e a s i n g r e m i s s i o n v a l u e s d i d .  S i n c e no  samples were examined f o r these l a t t e r 3 p a t i e n t s f o r 5 - 8 p r i o r t o r e l a p s e , i t cannot be determined had  months  i f t h e i r CAMAL BM v a l u e s  increased during that period. We were a b l e t o o b t a i n v e r y r e g u l a r samples from one ANLL  p a t i e n t over t h e 19 month c o u r s e o f h i s d i s e a s e .  F i g u r e 5.4  i l l u s t r a t e s t h i s p a t i e n t ' s r e c o r d e d CAMAL BM v a l u e s over time. t o each o f h i s 3 r e l a p s e s , i t was observed increased s i g n i f i c a n t l y  Prior  t h a t h i s CAMAL BM v a l u e s  (5.0 - 20.0%, 0.8 - 12.0% and 33.5 -  75.0%).  I t may be t h a t r e m i s s i o n PB samples w i l l prove t o be as u s e f u l as BM i n r o u t i n e m o n i t o r i n g procedure u s i n g immunoperoxidase.  At  p r e s e n t , l a c k o f r e a s o n a b l y s e q u e n t i a l PB samples i n key p a t i e n t s has p r e v e n t e d us from making more d e f i n i t i v e statements  on PB.  C e r t a i n l y , t h e same t r e n d as t h a t seen w i t h BM samples has been i n d i c a t e d thus f a r .  The f o l l o w i n g s e r i e s of photomicrographs  i l l u s t r a t e s these p o i n t s .  clearly  F i g u r e 5.5 a, b, c shows immunoperoxidase  s l i d e t e s t r e s u l t s o f PB samples from an ANLL p a t i e n t w i t h v a l u e s (a) post-chemotherapy ( 0 . 2 % p o s i t i v e ) , r e m i s s i o n (now 8.6% p o s i t i v e ) , and ( c ) another d u r i n g r e m i s s i o n (32.0% p o s i t i v e ) .  increasing  (b) 2 weeks l a t e r , i n 2 weeks l a t e r ,  also  W i t h i n 2-1/2 months o f t h e l a s t  Table X V I I I .  Patient  ANLL p a t i e n t s w i t h i n c r e a s i n g CAMAL BM v a l u e s d u r i n g r e m i s s i o n  CAMAL BM v a l u e ( p e r c e n t p o s i t i v e c e l l s ) d u r i n g r e m i s s i o n Months a f t e r  301 302 303 304  1  2  8.5 3.5 8.6 -  20 32 5.4  (R) or ( ) = r e l a p s e  3  -  4  5.5 -  5  6  30 (80) 35  diagnosis  7  8  9  10  11  (R) 2.2  -  -  1.0  -  27.5  (20)  12  13  14  15  16  20  -  -  (R)  17  L  R(R)  R  (R)  T  (R)D  MONTHS  F i g u r e 5.4. CAMAL BM v a l u e s f o r one ANLL p a t i e n t o v e r t h e c o u r s e o f h i s disease. L, acute leukemia; R, r e m i s s i o n ; ( R ) , r e l a p s e ; T, bone marrow t r a n s p l a n t ; D, dead.  178  F i g u r e 5.5. CAMAL-1 immunoperoxidase s l i d e t e s t s r e s u l t s o f p e r i p h e r a l b l o o d c e l l s from an ANLL r e m i s s i o n p a t i e n t showing i n c r e a s i n g reactivity. (a) 0.2% p o s i t i v e , f o l l o w i n g chemotherapy; (b) 8.6% p o s i t i v e , 2 weeks f o l l o w i n g ( a ) , w h i l e i n c l i n i c a l r e m i s s i o n ; ( c ) 32.0% p o s i t i v e , another 2 weeks l a t e r , s t i l l i n r e m i s s i o n b u t 2-1/2 months p r i o r t o r e l a p s e .  a  179  180  t e s t , t h i s p a t i e n t was i n r e l a p s e .  T h i s may be compared t o another  ANLL p a t i e n t whose PB v a l u e s decreased  during remission.  a, b, c i l l u s t r a t e s immunoperoxidase PB v a l u e s post-chemotherapy, i n r e m i s s i o n (1.3% p o s i t i v e ) , (0.5% p o s i t i v e ) . over 24 months.  F i g u r e 5.6  (a) 1 month  (12.0% p o s i t i v e ) ,  and ( c ) 1-1/2 months l a t e r , s t i l l  (b) 2 months l a t e r during  T h i s p a t i e n t has remained i n f i r s t  remission  remission f o r  181  F i g u r e 5.6. CAMAL-1 immunoperoxidase s l i d e t e s t r e s u l t s o f p e r i p h e r a l b l o o d c e l l s from an ANLL r e m i s s i o n p a t i e n t showing d e c r e a s i n g reactivity. (a) 12.0% p o s i t i v e 4 weeks post-chemotherapy, d u r i n g c l i n i c a l r e m i s s i o n ; (b) 1.3% p o s i t i v e 2 months l a t e r ; ( c ) 0.5% p o s i t i v e , another 1-1/2 months l a t e r d u r i n g r e m i s s i o n which has now l a s t e d > 24 months.  a  182  CHAPTER VI  THE POSSIBLE ROLE OF CAMAL IN MYELOPOIESIS  I.  INTRODUCTION CAMAL has been shown t o be a s i g n i f i c a n t marker b o t h i n t h e p r e d i c t i o n o f r e l a p s e i n ANLL BMT p a t i e n t s (529) and i n t h e prognostic chapter.  assessment o f r e m i s s i o n The o b s e r v a t i o n  i n ANLL, as shown i n t h e p r e v i o u s  t h a t CAMAL BM ( o r PBL) v a l u e s  often  i n c r e a s e d p r i o r t o r e l a p s e i n ANLL p a t i e n t s , l e d t o t h e s p e c u l a t i o n t h a t CAMAL might be i n v o l v e d i n some way i n t h e onset o f abnormal myelopoiesis  which must o c c u r  i n ANLL p a t i e n t s a t some p o i n t  the c l i n i c a l d i a g n o s i s o f r e l a p s e can be made. i n d i c a t e that during remission  There i s e v i d e n c e t o  i n ANLL, a b n o r m a l i t i e s  in cellular  i n t e r a c t i o n s occur which may be r e l a t e d t o t h e e v e n t u a l of t h e leukemic c l o n e support  (536-538).  This chapter  before  re-emergence  o u t l i n e s evidence to  t h e p o s s i b l e r e g u l a t o r y r o l e o f CAMAL i n m y e l o p o i e s i s  i n c l u d i n g i t s i n h i b i t i o n o f normal m y e l o p o i e s i s .  Such i n h i b i t i o n  c o u l d c o n t r i b u t e t o t h e growth ( p r o l i f e r a t i v e ) advantage t h a t leukemic c e l l s p o s s e s s over normal  II.  cells.  RESULTS A.  CAMAL-1 P o s i t i v e C o l o n i e s In o r d e r  the c e l l s antigen,  i n CGL  t o determine whether o r n o t m y e l o i d p r o g e n i t o r s (and  i n t h e c o l o n i e s t h a t they formed) c o n t a i n e d i n d i v i d u a l c o l o n i e s were manually p l u c k e d  m e t h y l c e l l u l o s e c u l t u r e s , spread  t h e CAMAL  out o f  on s l i d e s and a i r - d r i e d .  The s l i d e s  183  were then s u b j e c t e d t o a m o d i f i e d  i n d i r e c t immunoperoxidase assay  u s i n g CAMAL-1, as o u t l i n e d i n Chapter I I .  Some d i f f i c u l t y  was  encountered w i t h t h i s p r o c e d u r e but c l e a r r e s u l t s were o b t a i n e d  in  the s t a i n i n g of s l i d e s from a p a t i e n t w i t h c h r o n i c g r a n u l o c y t i c leukemia.  Seventy-five percent  (18/24) of c o l o n i e s p l u c k e d  m e t h y l c e l l u l o s e showed p o s i t i v e s t a i n i n g w i t h the CAMAL-1 Twenty-five  percent  anti-CAMAL serum.  from MAb.  (6/24) of c o l o n i e s were p o s i t i v e w i t h the r a b b i t Furthermore, a l l c e l l s i n each of these  colonies  s t a i n e d p o s i t i v e l y , i n d i c a t i n g t h a t the a n t i g e n appeared to present  i n a l l c e l l s d e r i v e d from a common p r o g e n i t o r .  c o l o n y types No  i n c l u d e d g r a n u l o c y t e , macrophage and  B.  (Chapter  p r o c e d u r e s t h a t w i l l now  Presence of CAMAL i n C o n d i t i o n e d Conditioned  a,  patient.  the  This  t h e p r e v i o u s l y d e s c r i b e d f i n d i n g t h a t excess  CAMAL c o u l d adsorb t o normal PBL experimental  F i g u r e 6.1  c e l l s stained with  immunoperoxidase p r o c e d u r e on c o l o n i e s from the CGL p r e l i m i n a r y r e s u l t , and  Positive  erythroid bursts.  p o s i t i v e c o l o n i e s were found i n 2 normals t e s t e d .  b, c i l l u s t r a t e s t y p i c a l p o s i t i v e and n e g a t i v e  be  be  I V ) , prompted  the  described.  Medium  medium f o r i n v i t r o m y e l o i d  progenitor  studies  c o n t a i n s h e m o p o i e t i c growth f a c t o r s r e q u i r e d by these p r o g e n i t o r s f o r t h e i r p r o l i f e r a t i o n and f o r human c e l l s may  differentiation.  be p r e p a r e d  p l a c e n t a - c o n d i t i o n e d medium (PCM)  E f f e c t i v e c o n d i t i o n e d media  i n a number of ways i n c l u d i n g (1) (199), p e r i p h e r a l b l o o d  c o n d i t i o n e d medium of which p h y t o h e m a g g l u t i n i n - s t i m u l a t e d c o n d i t i o n e d medium i s most common (PHA-LCM) (197), organ c o n d i t i o n e d medium (198,203) and  leucocyte lymphocyte  (3) o t h e r human  (4) v a r i o u s human tumor  cell  184  Figure 6.1. C e l l s from CGL p e r i p h e r a l b l o o d CFU-c l a b e l e d w i t h r a b b i t anti-CAMAL by immunoperoxidase. (a) c e l l s from a g r a n u l o c y t e c o l o n y p o s i t i v e l y l a b e l e d ; (b) p o s i t i v e macrophage c o l o n y c e l l ; (c) c e l l from n e g a t i v e l y s t a i n e d megakaryocyte c o l o n y .  185  c  186  lines  (200-202).  D e t a i l s o f t h e p r e p a r a t i o n o f PCM and PHA-LCM used  i n t h e f o l l o w i n g s t u d i e s have been g i v e n i n Chapter I I . In o r d e r t o determine i f CAMAL was p r e s e n t (a s i t u a t i o n t h a t might be expected i f , indeed, r o l e i n myelopoiesis)  i n c o n d i t i o n e d medium CAMAL p l a y e d some  c o n d i t i o n e d media (CM) prepared  by t h r e e  d i f f e r e n t methods were passed over a CAMAL-1 immunoadsorbent R e s u l t s showed t h a t a l l 3 types and p e r i p h e r a l b l o o d  column.  o f c o n d i t i o n e d media (PCM, PHA-LCM  c e l l CM p r e p a r e d  by Dr. N. D e n e g r i ' s  laboratory)  c o n t a i n e d m a t e r i a l t h a t bound s p e c i f i c a l l y t o t h e CAMAL-1 column. F i g u r e 6.2 i l l u s t r a t e s t h a t when PCM was passed over a CAMAL-1 column, t h e e l u a t e c o n t a i n e d  d e t e c t a b l e amounts o f CAMAL b u t when t h e  same amount o f RPMI/5% FCS was passed over t h e same column, no d e t e c t a b l e m a t e r i a l had adsorbed.  The o n l y d i f f e r e n c e between  these  two p r e p a r a t i o n s was t h e c o n d i t i o n i n g o f t h e former by p l a c e n t a l cells.  To v e r i f y these  f i n d i n g s , another p r e p a r a t i o n o f PCM (PCM-2)  was passed over a CAMAL-1 immunoadsorbent results.  column w i t h  similar  A t t h e same time an e q u a l amount o f PCM-2 was passed over  an i r r e l e v a n t  ( n e g a t i v e c o n t r o l ) MAb column; F i g u r e 6.3 a shows t h a t  m a t e r i a l i n t h e PCM-2 bound s p e c i f i c a l l y t o t h e CAMAL-1 and n o t t o the n e g a t i v e  c o n t r o l column.  Another source  o f CM, PHA-LCM, was  prepared  and s u b j e c t e d t o immunoadsorbence w i t h t h e CAMAL-1 and  negative  c o n t r o l MAb columns.  present  i n t h i s source  and n o t t h e n e g a t i v e  F i g u r e 6.3 b i l l u s t r a t e s t h a t m a t e r i a l  o f CM a l s o bound s p e c i f i c a l l y t o t h e CAMAL-1,  c o n t r o l , column.  P r o t e i n assays were performed on t h e p o o l e d , n e u t r a l i z e d f r a c t i o n s c o l l e c t e d from t h e CAMAL-1 column u s i n g t h e B i o r a d p r o t e i n  187  0.10-  o •+  1  1  1  1  1  1  1  1  1  1  1  1  2  3  4  S  6  7  8  9  10  11  12  COLUMN  1  1  13  14  FRACTION  F i g u r e 6.2. E l u t i o n p r o f i l e o f m a t e r i a l bound t o a CAMAL-1 immunoadsorbent column from p l a c e n t a l c o n d i t i o n e d medium. • — • PCM r u n over CAMAL-1 column • • same volume o f RPMI/5% FCS r u n o v e r CAMAL-1 column.  Figure 6.3. Binding of material from two sources of conditioned media ( PCM: b, PHA-LCM) to CAMAL-1 • — • and negative control •—O immunoaffinity columns.  189  assay method w i t h BSA as t h e s t a n d a r d . determined  R e s u l t s from t h e s e assays  t h a t t h e f o l l o w i n g e s t i m a t e d amounts o f CAMAL were  adsorbed from t h e v a r i o u s s o u r c e s o f CM. Type o f CM  C.  CAMAL c o n c e n t r a t i o n  1. PCM-1  4.7 ug/ml  2. PCM-2  16.4 ug/ml  3. PHA-LCM  8.0 ug/ml  4. Denegri's CM  7.2 ug/ml  CAMAL D e p l e t i o n S t u d i e s S i n c e i t had been demonstrated  t h a t b o t h t h e human plasma  (Chapter IV) and t h e c o n d i t i o n e d medium ( j u s t d e s c r i b e d ) used i n v i t r o m y e l o i d c l o n o g e n i c p r o g e n i t o r assay appeared CAMAL, experiments were performed  i n the  to contain  t o determine t h e e f f e c t ,  i f any, o f  d e p l e t i o n o f CAMAL from t h e s e two sources on t h e growth o f m y e l o i d progenitors.  CAMAL was d e p l e t e d from b o t h plasma and CM by  immunoadsorbence u s i n g a CAMAL-1 column. d e p l e t i o n was assayed on normal and PB.  The e f f e c t o f t h i s  BM and PB, ALL BM, CGL PB and ANLL BM  A p p r o p r i a t e c o n t r o l s f o r t h e s e experiments  included  (1) t h e  use o f plasma and CM t h a t had n o t been t r e a t e d i n any way, and (2) the u s e o f plasma and CM t h a t had been s u b j e c t e d t o immunoadsorbence w i t h an i r r e l e v a n t MAb (BLV-1) column. c o n t r o l s , a number o f "system"  In a d d i t i o n t o these  c o n t r o l s were performed  internal  t o ensure  the e f f e c t s observed were n o t due t o t e c h n i c a l problems w i t h t h i s system.  These system c o n t r o l s i n c l u d e d t h e f o l l o w i n g :  that  190  1.  t h e use o f two s e p a r a t e l y p r e p a r e d CAMAL-1 columns f o r d e p l e t i o n which ensured t h a t t h e e f f e c t s seen were n o t due t o a problem w i t h a s i n g l e column  2.  t h e use o f two d i f f e r e n t types (PCM and PHA-LCM) and two  (PCM)  o r t h r e e (PHA-LCM) d i f f e r e n t batches o f c o n d i t i o n e d medium  to  ensure t h a t t h e e f f e c t s seen were o f a g e n e r a l n a t u r e and n o t due t o c o n d i t i o n s p a r t i c u l a r t o any g i v e n CM s o u r c e o r preparation 3.  t h e use o f plasma samples  from two d i f f e r e n t normal v o l u n t e e r s and  o f plasma c o l l e c t e d a t f o u r d i f f e r e n t times from t h e  same normal v o l u n t e e r t o a g a i n ensure t h a t t h e e f f e c t s seen were of a g e n e r a l n a t u r e . Normal PB was used i n t h e m y e l o i d p r o g e n i t o r assay t o examine the p o s s i b i l i t y o f a r t i f a c t s u s i n g t h e c o n t r o l s j u s t d e s c r i b e d .  Once  i t had been e s t a b l i s h e d t h a t t h e system d i d n o t have i n h e r e n t a r t i f a c t u a l problems, t h e experiments t h a t w i l l now be d e s c r i b e d were performed. individual  F o r a l l o f t h e s e experiments, plasma from t h e same normal ( t e s t e d f o r i t s h i g h q u a l i t y i n t h e assay) and PHA-LCM  p r e p a r e d from PHA-stimulated lymphocytes were u t i l i z e d 1.  from t h e same i n d i v i d u a l  i n o r d e r t o f u r t h e r s t a n d a r d i z e t h e system.  E f f e c t o f CAMAL D e p l e t i o n on Normal Bone Marrow Bone marrow samples  experiments.  A complete  from 10 normal BMT donors were used i n t h e s e list  a r e p r e s e n t e d i n t h e Appendix  o f t h e r e s u l t s from t h e s e experiments ( T a b l e XIX-A) f o l l o w i n g Chapter V I I .  F o r purposes o f c l a r i t y t h r e e examples o f t h e s e r e s u l t s and a summary a r e i n c l u d e d here ( T a b l e X I X ) .  T a b l e XIX.  Patient  Code  Number o f CFU-c per 10-> c e l l s  1  0 B C  268 + 255.5 + 210 +  2  0 B C  3*  0 B C  Average o f r e s u l t s Mean % B*s: Mean % C s :  Code:  E f f e c t o f CAMAL d e p l e t i o n on normal bone marrow  74 72 56  + + +  %  %  Inhibition  3 4.5 4  100 95.3 78.4  0 4.7 21.6  1 0 2  100 97.3 75.7  0 2.7 24.3  100 102.4 74.3  0 -2.4 25.7  127.5 + 12 131 + 8 95 + 7.5  from 10 samples :  96.8% 76.4%  0 = no t r e a t m e n t o f plasma o r CM B = plasma and CM passed o v e r n e g a t i v e (BLV-1) MAb C = plasma and CM passed o v e r CAMAL-1 column  * Nonadherent c e l l s were  plated  column  192  A l l o f 10 normal BM samples  t e s t e d showed a d e c r e a s e i n the  number o f CFU-c when CAMAL was d e p l e t e d from t h e plasma and CM. I n h i b i t i o n o f t h e s e normals ranged from 15.7 t o 32.2% (average 23.6%) of t h e u n t r e a t e d c o n t r o l s , o r 14.0 t o 27.6% (average 20.4%) o f t h e n e g a t i v e MAb  column-treated c o n t r o l s .  When a two-sided Student's  t - t e s t a n a l y s i s was performed t o compare t h e r e s u l t s o f t h e n e g a t i v e MAb  c o l u m n - t r e a t e d c o n t r o l s and t h e CAMAL-1 c o l u m n - t r e a t e d  (CAMAL-depleted)  CFU-c, t h e d i f f e r e n c e between t h e s e r e s u l t s was  found t o be h i g h l y s i g n i f i c a n t w i t h p < 0.001. The r e s u l t s shown f o r p a t i e n t 3, T a b l e XIX, u s i n g  non-adherent  c e l l s i n d i c a t e d t h a t t h e i n h i b i t o r y e f f e c t seen i n normal BM was n o t due t o an i n d i r e c t a c t i o n mediated through adherent c e l l s  (known t o  be r e s p o n s i b l e f o r p r o d u c t i o n o f v a r i o u s r e g u l a t o r y f a c t o r s ) .  The  s i g n i f i c a n t i n h i b i t i o n o f a p o p u l a t i o n o f normal m y e l o i d p r o g e n i t o r s by CAMAL d e p l e t i o n i n d i c a t e d t h a t t h i s p r o t e i n may be i n v o l v e d i n some r e g u l a t o r y manner i n normal m y e l o p o i e s i s .  The p r e s e n c e o f CAMAL  i n t h i s system appeared t o be a requirement f o r t h e growth o f a t l e a s t some normal m y e l o i d c l o n o g e n i c p r o g e n i t o r s .  Whether t h i s  i n h i b i t i o n observed w i t h CAMAL d e p l e t i o n was due t o i n h i b i t i o n o f a p a r t i c u l a r s u b p o p u l a t i o n o f CFU-c o r due t o a g e n e r a l i z e d  inhibition  has n o t been determined as y e t . Both t h e number and average s i z e o f marrow CFU-c grown under CAMAL-depleted cultures.  c o n d i t i o n s were o f t e n reduced i n comparison t o c o n t r o l  These e f f e c t s were apparent by 7-10 days and p e r s i s t e d f o r  a t l e a s t t h r e e weeks i n v i t r o .  F i g u r e 6.4 i l l u s t r a t e s t y p i c a l CFU-c  from (a) c o n t r o l and (b) CAMAL-depleted BM  sample.  c u l t u r e s o f t h e same normal  193  F i g u r e 6.4. Normal marrow CFU-c from ( a ) c o n t r o l and (b) CAMAL d e p l e t e d c u l t u r e s , showing a d e c r e a s e i n s i z e and number o f CFU-c.  194  2.  E f f e c t o f CAMAL D e p l e t i o n on Normal P e r i p h e r a l Blood T a b l e XX shows r e s u l t s from two o f t h e f o u r  performed  on normal PB samples.  experiments  As was t h e case w i t h t h e normal  CAMAL d e p l e t i o n caused a s i g n i f i c a n t  BM,  (p < 0.005) d e c r e a s e i n t h e  number o f CFU-c when compared t o c o n t r o l s .  A complete  data f i l e i s  p r e s e n t e d i n t h e Appendix ( T a b l e XX-A) f o r ^ r e v i e w i f d e s i r e d .  The  i n h i b i t i o n observed ranged from 26.3 t o 45.1% o f c o n t r o l v a l u e s . While t h i s may appear t o be o f g r e a t e r magnitude than t h a t  observed  f o r normal BM, t h e i n c r e a s e d i n h i b i t i o n was o n l y seen f o r two normal PB samples from t h e same i n d i v i d u a l were t e s t e d ) .  ( r o u t i n e and non-adherent  cells  I t was i n t e r e s t i n g , and may e v e n t u a l l y prove t o be  s i g n i f i c a n t , t h a t t h e s e two samples r e p r e s e n t e d a c o m p l e t e l y autologous s i t u a t i o n ; t h e c e l l s , plasma and c o n d i t i o n e d medium were all 3.  from t h e same i n d i v i d u a l i n t h i s case. E f f e c t o f CAMAL D e p l e t i o n on ALL Bone Marrow Two bone marrow samples from ALL p a t i e n t s were t e s t e d t o  determine formation. XXI.  t h e e f f e c t o f CAMAL d e p l e t i o n on t h e i r m y e l o i d c o l o n y The r e s u l t s from t h e s e experiments  a r e shown i n T a b l e  These r e s u l t s show v e r y s i m i l a r l e v e l s o f i n h i b i t i o n w i t h CAMAL  d e p l e t i o n t o those observed i n normals  (14.9 and 22.2 %  inhibition  compared t o n e g a t i v e c o l u m n - t r e a t e d c o n t r o l v a l u e s ) , i m p l y i n g t h a t s i m i l a r CAMAL r e g u l a t o r y c o n t r o l s may be i n e f f e c t f o r b o t h and a t l e a s t some ALL p a t i e n t s .  This i s a reasonable  normals  assumption  because, u n l i k e t h e s i t u a t i o n i n ANLL, normal m y e l o i d p r o g e n i t o r s a r e n o t suppressed i n ALL and t h e i r c o l o n y numbers i n v i t r o r e f l e c t t h e l e v e l of malignant c e l l  infiltration  itself  (391).  T a b l e XX.  Patient  E f f e c t o f CAMAL d e p l e t i o n on normal p e r i p h e r a l b l o o d  Code  Number o f CFU-c p e r 10-* c e l l s  %  % Inhibition  0 B C  34 +0 25 +4 17.5 + 2.  100 87.7 61.4  0 12. 38,  0 B C  22.5 + 1.5 21 +0 11.5 + 2.5  100 93.3 51.1  0 6. 48.  Average o f 4 normal p e r i p h e r a l b l o o d samples : Mean % B*s: Mean % C*s:  Code:  88.6 % 53.1 %  0 = no treatment o f plasma o r CM B = plasma and CM p a s s e d o v e r n e g a t i v e (BLV-1) MAb C = plasma and CM p a s s e d o v e r CAMAL-1 column  column  196  T a b l e XXI.  E f f e c t of CAMAL d e p l e t i o n on a c u t e  l y m p h o b l a s t i c leukemia (ALL) bone marrow  Patient  Code  Number of CFU-c p e r 10^ c e l l s  (remission)  0 B C  168 153 128  2 (not i n remission)  0 B C  121.5 + 3.5 111.5 + 1.5 84.5 + 4.5  Mean % B's: Mean % C*s:  Code:  ± 9 ± 4 + 5  %  Inhibition  100 91.1 76.2  0 8.9 23.8  100 91.8 69.6  0 8.2 30.4  91.5 % 72.9 %  0 = no treatment o f plasma o r CM B = plasma and CM passed over n e g a t i v e (BLV-1) MAb C = plasma and CM passed over CAMAL-1 column  column  197  4.  E f f e c t o f CAMAL D e p l e t i o n  on C h r o n i c  Granulocytic  Leukemia  P e r i p h e r a l Blood Having e s t a b l i s h e d t h a t a s i g n i f i c a n t i n h i b i t o r y e f f e c t was caused by d e p l e t i o n o f CAMAL from t h e m y e l o i d p r o g e n i t o r  assay i n  b o t h normals and ALL p a t i e n t s , a s e r i e s o f s i m i l a r experiments were performed u s i n g CGL PBL t o determine i f t h i s e f f e c t h e l d t r u e f o r m y e l o i d leukemics as w e l l . illustrated  Data from 2 o f 7 such experiments a r e  i n T a b l e XXII; a complete l i s t o f a l l e x p e r i m e n t a l  r e s u l t s are given  i n t h e Appendix ( T a b l e XXII-A).  I n no case was any  e f f e c t observed by CAMAL d e p l e t i o n i n these CGL samples, whether r o u t i n e o r non-adherent c e l l s were p l a t e d .  Both CFU-c numbers and  s i z e were i n d i s t i n g u i s h a b l e i n CAMAL d e p l e t e d control cultures.  c u l t u r e s from those i n  S t a s t i s t i c a l a n a l y s i s by Student's two-sided  t - t e s t v e r i f i e d t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e between these c u l t u r e s .  T h i s l a c k o f e f f e c t i n CAMAL d e p l e t e d  c u l t u r e s was  n o t due t o t h e u s e o f a d i f f e r e n t b a t c h o f p r e p a r e d plasma o r CM; t h i s p o s s i b i l i t y was t e s t e d a number o f times ( u s i n g t h e same plasma and  CM p r e p a r a t i o n s  normals).  shown t o have s i g n i f i c a n t i n h i b i t o r y e f f e c t s i n  Indeed, i t appeared t h a t m y e l o i d c l o n o g e n i c  were n o t i n f l u e n c e d i n any way by CAMAL d e p l e t i o n . demonstrated t h a t CGL PB c e l l s c o n t a i n s i g n i f i c a n t l y  progenitors  I t has been increased  amounts o f CAMAL compared t o normals (Chapter I I I ) , and y e t i t appeared t h a t CAMAL d e p l e t i o n had no e f f e c t on t h e s e c e l l s i n terms of t h e i r m y e l o i d p r o g e n i t o r 5.  c e l l s u r v i v a l and p r o l i f e r a t i o n .  E f f e c t o f CAMAL D e p l e t i o n  on ANLL  Four ANLL samples were examined t o determine i f t h e i r m y e l o i d progenitors  were u n r e s p o n s i v e t o CAMAL d e p l e t i o n as i n t h e case o f  198  T a b l e XXII.  E f f e c t o f CAMAL d e p l e t i o n on c h r o n i c  granulocytic  Patient  Code  0 B C 0 B C  Summary o f 7 CGL PB samples Mean % B's: Mean % C*s:  Code:  leukemia p e r i p h e r a l b l o o d  Number o f CFU-c p e r 5 x 10^ c e l l s  91 + 1 84 + 1 87 + 2.5 193 194 204  + 7 ± 11 + 14  %  % Inhibition  100 92.6 96  0 7.4 4  100 100.5 105.7  0 -0.5 -5.7  tested  102.2 107.7  0 = no treatment o f plasma o r CM B = plasma and CM passed over n e g a t i v e (BLV-1) MAb C = plasma and CM passed over CAMAL-1 column  column  199  the CGL samples t e s t e d . experiments  T a b l e XXIII shows r e s u l t s from two such  on r e c e n t l y diagnosed ANLL p a t i e n t s .  A g a i n no e f f e c t was  observed f o r any ANLL c u l t u r e s d e p l e t e d o f CAMAL, b o t h i n t h e two ANLL p a t i e n t s shown i n T a b l e XXIII and i n an ANLL r e m i s s i o n p a t i e n t tested  ( T a b l e XXIII-A,  c e l l s were p l a t e d .  Appendix), whether r o u t i n e o r non-adherent  There was no s i g n i f i c a n t d i f f e r e n c e between CAMAL  d e p l e t e d and c o n t r o l c u l t u r e s when a n a l y z e d by t h e Student's two-sided t - t e s t .  Here was another s i t u a t i o n i n which c e l l s  from  p a t i e n t s shown t o have h i g h r e l a t i v e CAMAL BM and PB v a l u e s compared t o normals were u n a f f e c t e d by t h e i n h i b i t o r y e f f e c t t h a t had been observed f o r CAMAL i n normals. T a b l e XXIV and F i g u r e 6.5 summarize t h e e f f e c t o f CAMAL d e p l e t i o n on CFU-c i n t h e sample groups d e s c r i b e d .  There was no  s i g n i f i c a n t d i f f e r e n c e between mean % CFU-c numbers i n n e g a t i v e column-treated c o n t r o l normals/ALL;  ("B") c u l t u r e s i n m y e l o i d leukemics v e r s u s  however, a s i g n i f i c a n t d i f f e r e n c e  (p < 0.025) was found  between mean % CFU-c i n CAMAL d e p l e t e d ("C") c u l t u r e s i n m y e l o i d leukemics v e r s u s normals/ALL.  These r e s u l t s i n d i c a t e e i t h e r  that  v e r y d i f f e r e n t CAMAL-mediated r e g u l a t o r y c o n t r o l s a r e i n o p e r a t i o n i n m y e l o p o i e s i s i n t h e s e two groups o r t h a t t h e c e l l s r e s p o n s i v e t o t h i s r e g u l a t i o n a r e n o t p r e s e n t i n t h e m y e l o i d leukemics D.  tested.  CAMAL A d d i t i o n S t u d i e s Once i t had been e s t a b l i s h e d t h a t CAMAL d e p l e t i o n i n t h e m y e l o i d  p r o g e n i t o r assay had no e f f e c t on m y e l o i d leukemics t e s t e d b u t caused s i g n i f i c a n t i n h i b i t i o n o f normals,  experiments were designed t o  determine t h e e f f e c t , i f any, o f a d d i t i o n o f excess CAMAL t o these cultures.  P r e l i m i n a r y experiments which showed t h a t CAMAL was  200  Table XXIII.  Patient  Code  E f f e c t o f CAMAL d e p l e t i o n on ANLL  Number o f CFU-c p e r 10-> c e l l s  1* (PB)  0 B C  63 70 69  2 (BM)  0 B C  35 +2 30 + 0.5 38.5 + 1.5  +2 +1 +9  %  % Inhibition  100 111 109.5  0 -11 - 9.5  100 85. 110  0 14.3 -10  Average o f 4 ANLL samples t e s t e d Mean % B's: Mean % C s :  Code:  99.2 % 108.2 %  0 = no treatment o f plasma o r CM B = plasma and CM passed over n e g a t i v e (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column  *The c o l o n y numbers shown f o r p a t i e n t 1 were from 4 x 10^ c e l l s ; no i n h i b i t o r y e f f e c t was found (but c o l o n y numbers were low) a t 10^ c e l l s plated.  201  T a b l e XXIV.  Sample  Summary o f CAMAL d e p l e t i o n  on m y e l o i d c o l o n y growth  Number  Mean % CFU-c i n B*  Mean % CFU-c number i n C*  Normal BM  10  96.8  76.4  20.4  Normal PB  4  88.6  53.1  35.5  ALL  2  91.5  72.9  18.6  CGL  7  102.2  107.7  -5.5  ANLL  4  99.2  108.2  -9.0  * = compared t o u n t r e a t e d c o n t r o l c u l t u r e s B = plasma and CM passed over n e g a t i v e (BLV-1) MAb C = plasma and CM passed over CAMAL-1 column  %  Difference (B - C)  column  Student's t - t e s t r e s u l t s : a) b)  B cultures: normal/ALL C cultures:  no s i g n i f i c a n t d i f f e r e n c e between m y e l o i d leukemics vs significant difference  (p < 0.025)  202  in K UJ  ~  CO  o S O  2  60-  40u. O o ce  20-  o m  o  <#  z  CO  u  z < Ui  S NORMALS 5 = 13  Figure 6 . 5 . normals  MYELOID LEUKEMICS x = 10  Summary o f t h e e f f e c t o f CAMAL d e p l e t i o n on CFU-c from and myeloid l e u k e m i c s .  0 = no treatment o f plasma o r c o n d i t i o n e d medium (CM) B = plasma and CM passed o v e r n e g a t i v e c o n t r o l MAb column C = plasma and CM passed o v e r CAMAL-1 column  203  p r e s e n t i n t h e plasma o f b o t h normals and m y e l o i d leukemics  (Chapter  IV) i n d i c a t e d t h a t t h e r e may be a d i f f e r e n c e i n t h e amount o f t h i s p r o t e i n i n t h e plasma o f these two groups, w i t h g r e a t e r amounts o f CAMAL b e i n g p r e s e n t i n t h e m y e l o i d  leukemic plasma.  I t was  s p e c u l a t e d t h a t t h i s might be o f some b i o l o g i c a l s i g n i f i c a n c e w i t h r e s p e c t t o leukemogenesis,  p a r t i c u l a r l y s i n c e a d d i t i o n o f excess  (between 5.0 and 12.5 yg/ml) CAMAL t o normal p e r i p h e r a l b l o o d had been shown t o r e s u l t i n a d s o r p t i o n o f t h e p r o t e i n t o normal l e u c o c y t e s (Chapter I V ) . t h e s e experiments  D e t a i l s of the t e c h n i c a l procedures f o r  a r e g i v e n i n Chapter I I .  Briefly,  excess  ( l e u k e m i a - d e r i v e d ) p u r i f i e d CAMAL ( o r PBS o r i r r e l e v a n t p r o t e i n ) was added t o r o u t i n e c u l t u r e s o f normal o r m y e l o i d leukemic c e l l s and CFU-c counts were 1.  performed.  E f f e c t o f CAMAL A d d i t i o n on Normal M y e l o i d Colony Growth Three normal samples were t e s t e d  t h e s e experiments.  T a b l e XXV shows t h e r e s u l t s o f a d d i t i o n o f excess  CAMAL t o t h e s e samples.  I n a l l cases shown h e r e , excess  (> 10 yg/ml) caused massive w i t h an average  (two BM and one PB sample) i n  CAMAL  i n h i b i t i o n o f normal m y e l o i d c o l o n i e s  i n h i b i t i o n o f 74.8 + 13.9 (SEM) % a t l e v e l s o f 20  yg/ml a d d i t i o n a l CAMAL.  No i n h i b i t i o n by up t o 40 yg/ml  a d d i t i o n a l i r r e l e v a n t p r o t e i n were observed normal PB sample t e s t e d .  i n one normal BM and one  I n h i b i t i o n appeared  t o o c c u r r a p i d l y once  l e v e l s o f a p p r o x i m a t e l y 15 yg/ml excess CAMAL were added. i n t e r e s t i s t h e p r e v i o u s demonstration  Of  o f CAMAL a d s o r p t i o n by normal  l e u c o c y t e s a t l e v e l s o f 12.5 yg/ml excess CAMAL a d d i t i o n .  These  and p r e v i o u s r e s u l t s i n d i c a t e d t h a t , w h i l e a c e r t a i n amount of CAMAL appeared  t o be r e q u i r e d f o r normal m y e l o p o i e s i s t o o c c u r , an  204  T a b l e XXV.  Sample  E f f e c t o f CAMAL a d d i t i o n on normal m y e l o i d  yg/ml excess protein  Number o f CFU-c p e r 1 0 c e l l s (BM), p e r 2 x 1 0 c e l l s (PB)  %  c o l o n y growth  %  inhibition  5  5  0  18.5 +  1.5  100  CAMAL  10 20 40  25 + 0.5 + 0.5 +  2 0.5 0.5  135.1 2.7 2.7  -35.1 97.3 97.3  NEG PROT  10 20 40  27 19 22  1 1 3  145.9 102.7 118.9  -45.9 - 2.7 -18.9  0 1 5 10 15 20  148 148.5 154.5 137 98.5 75  + 0 + 13.5 + 9.5 + 5 + 0.5 + 2  100 100.3 104.4 92.6 66.6 50.7  0 - 0.3 - 4.4 7.4 33.4 49.3  + + +  100 97.8 108.9 33.3 22.2  0 2.2 - 8.9 66.7 77.8  1 (BM)  2* (BM) CAMAL  3 (PB) CAMAL  0 1 10 15 20  N o n a d h e r e n t c e l l s were p l a t e d  22.5 22 24.5 7.5 5  + + +  +  0.5 1 5 1.5 0  0  205  e x c e s s i v e amount o f t h i s p r o t e i n ( o r p o s s i b l y , an a l t e r e d form o f CAMAL) was c a p a b l e o f c a u s i n g s i g n i f i c a n t shut-down o f normal myelopoiesis.  F i g u r e 6.6 i l l u s t r a t e s t h e r e s u l t s o b t a i n e d from one  normal BM ( p a t i e n t 2, T a b l e XXV) examined i n t h i s manner;  this  i n d i v i d u a l ' s CFU-c were i n h i b i t e d 49.3% o f c o n t r o l v a l u e s a t 20 yg/ml excess CAMAL. i n h i b i t i o n observed  T h i s , i n f a c t , was t h e lowest l e v e l o f i n t h e normals a t t h i s CAMAL c o n c e n t r a t i o n .  normal BM a c t u a l l y showed almost t o t a l i n h i b i t i o n  One  (97.3%) a t t h i s  concentration. 2.  E f f e c t o f CAMAL A d d i t i o n on M y e l o i d Leukemia P a t i e n t s '  Colony  Growth The  same e x p e r i m e n t a l p r o t o c o l was performed  leukemia p a t i e n t s .  on t h r e e m y e l o i d  Two o f t h e s e p a t i e n t s were i n a c t i v e stages o f  t h e i r d i s e a s e (one c h r o n i c phase CGL, one r e c e n t l y diagnosed  ANLL)  and one was an ANLL BMT p a t i e n t w i t h normal BM who had been i n r e m i s s i o n post-BMT f o r 17 months and had a normal CAMAL BM v a l u e (by i n d i r e c t immunoperoxidase). experiments.  T a b l e XXVI shows t h e r e s u l t s o f these  The ANLL BMT p a t i e n t showed s i g n i f i c a n t i n h i b i t i o n o f  m y e l o p o i e s i s a t l e v e l s o f 10 and 15 yg/ml excess CAMAL ( p a t i e n t 1, T a b l e XXVI and F i g u r e 6.7 a ) , s i m i l a r t o t h a t observed w i t h t h e normals.  No i n h i b i t o r y e f f e c t whatsoever on m y e l o p o i e s i s by a d d i t i o n  of up t o 20 yg/ml excess CAMAL, was seen i n t h e remaining  ANLL  ( F i g u r e 6 . 7 b , p a t i e n t 3, T a b l e XXVI) and t h e CGL p a t i e n t ( F i g u r e 6.8, p a t i e n t 2, T a b l e XXVI). used  i n b o t h t h e experiments  S i n c e t h e same CAMAL p r e p a r a t i o n was on normals and on t h e m y e l o i d  leukemics,  t h e r e was no p o s s i b i l i t y t h a t t h e p r e p a r a t i o n i t s e l f was o f a n o n s p e c i f i c t o x i c nature.  Furthermore,  the negative control protein  206  Ul  U  in  o  160 • 140 120 100  O  (J  80 60  CC Ul CD  s 3 2  40 20 0  0  1  5  /ug/ml C A M A L  Figure  6.6.  10  15  20  ADDED  I n h i b i t i o n o f normal marrow CFU-c by a d d i t i o n o f p u r i f i e d l e u k e m i a - d e r i v e d CAMAL a t l e v e l s o f 15 - 20 ug/ml.  207  T a b l e XXVI. E f f e c t o f CAMAL a d d i t i o n on m y e l o i d leukemic c o l o n y growth  Patient  vg/ml excess protein  Number o f CFU-c*  %  % inhibition  0  35.5 +  0.5  CAMAL  10 15  19.5 + 5.5 +  1.5 0.5  54.9 15.5  45.1 84.5  NEG PROT  10 15  31 33  4 1  87.3 93  12.7 7  0 1 5 10 15 20  195 192 198 201 211.5 196  + 5 + 13 + 11 + 6 + 1.5 + 8.5  100 98.5 101.5 103.5 108.5 100.5  0 1.5 1.5 3.5 8.5 0.5  +  2  100  1 (BM)  2** (PB) CAMAL  3 (PB)  0  15  + +  100  0  -  0  CAMAL  5 10 20  20.5 + 20 + 20 +  2.5 8 1  136.7 133.3 133.3  -36.7 -33.3 -33.3  NEG PROT  5 10 20  14.5 + 14.5 + 16.5 +  0.5 0.5 1.5  96.7 96.7 110  3.3 3.3 -10  * = patient 1 c e l l s plated at 10 c e l l s per dish p a t i e n t 2 c e l l s p l a t e d a t 7.5 x 10^ c e l l s p e r d i s h p a t i e n t 3 c e l l s p l a t e d a t 1.5 x 10-> c e l l s p e r d i s h 3  ** = nonadherent c e l l s were p l a t e d  208  rh  ill -h r h 2520-  NEG  /ug/ml PROTEIN ADDED  /ug/ml CAMAL ADDED  NEG  /ug/ml PROTEIN ADDED  fig/ml CAMAL ADDED  F i g u r e 6.7. E f f e c t o f a d d i t i o n o f p u r i f i e d l e u k e m i a - d e r i v e d CAMAL on CFU-c from ANLL p a t i e n t s . ( a ) ANLL bone marrow t r a n s p l a n t (17 months post-BMT) p a t i e n t ' s marrow CFU-c, showing i n h i b i t i o n a t l e v e l s o f 10 and 15 yg/ml excess CAMAL; (b) newly diagnosed ANLL p a t i e n t ' s PB CFU-c, showing no i n h i b i t i o n a t l e v e l s up t o 20 yg/ml excess CAMAL.  209  2 O  o u o CC UJ CO  S 3 2  jug/ml  NEG  /ug/iml CAMAL ADDED  PROTEIN ADDED  I  F i g u r e 6.8. Lack o f i n h i b i t i o n o f CGL p e r i p h e r a l b l o o d CFU-c by a d d i t i o n of p u r i f i e d l e u k e m i a - d e r i v e d CAMAL. No i n h i b i t i o n was observed a t l e v e l s up t o 20 ug/ml excess CAMAL.  210  added was p r e p a r e d i n an i d e n t i c a l manner t o t h e CAMAL p r o t e i n , u s i n g immunoadsorption, control  and no i n h i b i t o r y e f f e c t was observed f o r those  cultures.  The r e s u l t s from t h e m y e l o i d leukemia p a t i e n t s c l e a r l y  indicated  t h a t p a t i e n t s i n whom a c t i v e d i s e a s e , and t h e r e f o r e abnormal m y e l o p o i e s i s , were o c c u r r i n g were t o t a l l y u n a f f e c t e d i n terms o f t h e i r CFU-c growth i n t h e presence o f l e v e l s o f l e u k e m i a - d e r i v e d CAMAL t h a t were i n h i b i t o r y t o normal m y e l o p o i e s i s .  The i m p l i c a t i o n s  of these r e s u l t s a r e obvious w i t h r e g a r d t o a p o s s i b l e mechanism by which m y e l o i d leukemic c e l l s can cause t h e shut-down o f normal m y e l o i d p r o g e n i t o r s and thus a f f o r d themselves advantage  over normal  following  Chapter.  E.  cells.  a significant  growth  T h i s w i l l be d i s c u s s e d f u r t h e r i n t h e  E v i d e n c e t h a t Normal and Leukemia-derived  CAMAL May Not be t h e  Same S o l u b l e CAMAL, p u r i f i e d from leukemic c e l l s , was added t o normal PB CAMAL-depleted c e l l c u l t u r e s i n o r d e r t o determine treatment would " r e v e r s e " t h e apparent d e p l e t i o n on normal  CFU-c growth.  i f such  i n h i b i t o r y e f f e c t o f CAMAL  CAMAL, a t 10 yg/ml, was added t o  b o t h CAMAL d e p l e t e d and n e g a t i v e MAb column-treated c o n t r o l s ( T a b l e XXVII); t h i s amount was c a l c u l a t e d t o be a p p r o x i m a t e l y e q u a l t o t h e amount o f CAMAL d e p l e t e d from t h e plasma and c o n d i t i o n e d medium utilized  i n t h i s assay.  CAMAL d e p l e t i o n i t s e l f r e s u l t e d i n 14.7%  i n h i b i t i o n compared t o c o n t r o l c u l t u r e s , s i m i l a r t o t h a t p r e v i o u s l y reported.  As T a b l e XXVII demonstrates,  a d d i t i o n o f 10 ug/ml  l e u k e m i a - d e r i v e d CAMAL r e s u l t e d i n a p p r o x i m a t e l y 36% i n h i b i t i o n o f  211  T a b l e XXVII.  E f f e c t of CAMAL a d d i t i o n on normal p e r i p h e r a l b l o o d CAMAL d e p l e t e d  cultures  Code  Average number of CFU-c  B cultures  54.5 + 3.5  C cultures  (CAMAL d e p l e t e d )  %  %  Inhibition  100  0  46.5+3.5  85.3  14.7  A d d i t i o n of l e u k e m i a -  (B  34.5+4.0  63.3  36.7  d e r i v e d CAMAL (10 ug/ml)  (C  29.5+0.5  63.4  36.6  Code:  B = plasma and CM passed over n e g a t i v e (BLV-1) MAb C = plasma and CM p a s s e d over CAMAL-1 column  column  212  CFU-c i n b o t h "B" ( c o n t r o l ) and "C" (CAMAL d e p l e t e d ) c u l t u r e s .  These  d a t a imply t h e f o l l o w i n g : 1.  t h e same c o l o n i e s t h a t a r e s e n s i t i v e t o CAMAL (normal) - d e p l e t i o n a r e p r o b a b l y a l s o CAMAL (leukemic) - a d d i t i o n s e n s i t i v e , s i n c e t h e r e was no d i f f e r e n c e between t h e % i n h i b i t i o n o f CFU-c i n B v e r s u s C c u l t u r e s .  2.  t h e r e a r e more CAMAL (leukemic) - a d d i t i o n s e n s i t i v e CFU-c than CAMAL (normal) - d e p l e t i o n s e n s i t i v e CFU-c, s i n c e g r e a t e r  %  i n h i b i t i o n was observed i n t h e c u l t u r e s t o which l e u k e m i a - d e r i v e d CAMAL was added.  An a l t e r n a t e and, i n t h e  a u t h o r ' s o p i n i o n , more p r o b a b l e , e x p l a n a t i o n (normal) was n o t t o t a l l y d e p l e t e d 3.  i s t h a t CAMAL  from plasma and CM.  Leukemia-derived CAMAL may n o t be i d e n t i c a l t o normal CAMAL, s i n c e i t was n o t p o s s i b l e t o r e c o n s t i t u t e CAMAL d e p l e t i o n by CAMAL (leukemic) a d d i t i o n .  (normal)  213  CHAPTER V I I  DISCUSSION AND SUMMARY  I.  DISCUSSION A.  Evaluation  and D i a g n o s t i c  Implications  o f a Rapid S l i d e T e s t f o r  CAMAL The using  development o f an i n d i r e c t immunoperoxidase s l i d e t e s t ,  t h e CAMAL-1 MAb t o demonstrate t h e p r e s e n c e o f t h e CAMAL marker  i n o r on i n d i v i d u a l b l o o d o r bone marrow c e l l s , has r e s u l t e d i n a r a p i d , s i m p l e and i n e x p e n s i v e assay f o r t h e d e t e c t i o n o f CAMAL. an assay c o u l d laboratory  e a s i l y be t r a n s f e r r e d t o a r o u t i n e  clinical  Such  testing  s i n c e , i n i t s p r e s e n t form, i t r e q u i r e s no s p e c i a l i z e d  s k i l l s t o p e r f o r m beyond those a l r e a d y  i n u s e i n such a s e t t i n g .  We  have v e r i f i e d t h i s by i n s t r u c t i n g seven i n d i v i d u a l s a t d i f f e r e n t times and i n f o u r d i f f e r e n t l a b o r a t o r i e s i n t h e p r o c e d u r e o u t l i n e d herein.  Advantages o f t h i s type o f assay f o r t h e d e t e c t i o n o f  a n t i g e n i c markers i n c l u d e , 1.  i n a d d i t i o n t o those mentioned above:  i d e n t i f i c a t i o n of the i n t e r n a l d i s t r i b u t i o n perinuclear,  or nuclear) of the antigen  (cytoplasmic,  w i t h i n c e l l s as w e l l as  on t h e membrane 2.  a permanent r e c o r d  i n t h e form o f s t a i n e d , mounted s l i d e s ,  u s e f u l i n r e t r o s p e c t i v e a n a l y s e s and f o r t e a c h i n g 3.  very  purposes  t h e a b i l i t y t o s t o r e o r s t o c k p i l e s l i d e s i n an u n l a b e l e d  state  f o r up t o a t l e a s t 3 months ( f o r CAMAL) p r i o r t o p e r f o r m i n g t h e  214  assay,  a l l o w i n g more f l e x i b i l i t y w i t h r e g a r d t o a v a i l a b i l i t y o f  time and p e r s o n n e l 4.  t h e a b i l i t y t o d e t e c t as few as one p o s i t i v e c e l l among thousands  5.  d i r e c t determination The  complexity  discussed  (Chapter  o f t h e morphology o f a n t i g e n - p o s i t i v e c e l l s .  of a n t i g e n i c heterogeneity I).  i n ANLL has been  L i t e r a t u r e review has i n d i c a t e d t h a t s e v e r a l  putative leukemia-associated  antigens  (LAA) e x i s t on m y e l o i d  leukemia  cells.  S t u d i e s u t i l i z i n g a p a n e l o f absorbed h e t e r o a n t i s e r a d e f i n i n g  myeloid  LAA r e v e a l e d t h a t some LAA were common t o both ANLL and CGL  cells,  some were p r e s e n t on o n l y c e r t a i n i n d i v i d u a l ' s c e l l s ,  some malignant (464).  while  b l a s t s were u n r e a c t i v e w i t h any o f t h e s e r a examined  More r e c e n t s t u d i e s have i n d i c a t e d t h a t no i n d i v i d u a l  myeloid-specific  ( o r a s s o c i a t e d ) MAbs o r c y t o c h e m i c a l  r e c o g n i z e d t h e malignant (466-472).  markers  b l a s t c e l l s o f every ANLL p a t i e n t  Even w i t h i n i n d i v i d u a l p a t i e n t s , t h e r e was a c o n s i d e r a b l e  degree o f h e t e r o g e n e i t y w i t h r e s p e c t t o c e l l u l a r e x p r e s s i o n o f t h e markers examined.  D e f i n i t i v e evidence  i s lacking to implicate either  t r u e g e n e t i c h e t e r o g e n e i t y o r t h e e x i s t e n c e o f asynchronous  limited  d i f f e r e n t i a t i o n as t h e e x p l a n a t i o n f o r t h i s i n a l l cases o f ANLL, as mentioned p r e v i o u s l y . i n d i c a t e d t h a t leukemic of a combination expressed  A g r e a t d e a l o f s u p p o r t i v e evidence has c e l l phenotypes a r e l i k e l y t o be t h e r e s u l t  o f t h e e x p r e s s i o n o f normal genes ( n o t n e c e s s a r i l y  i n a normal q u a n t i t a t i v e manner) and abnormal, asynchronous  differentiation. The  r e s u l t s o f t h e CAMAL-1 i n d i r e c t immunoperoxidase t e s t have  e s t a b l i s h e d t h a t CAMAL i s p r e s e n t  i n a s i g n i f i c a n t l y i n c r e a s e d number  of BM o r p e r i p h e r a l b l o o d c e l l s from i n d i v i d u a l s w i t h myelogenous  215  leukemia  (ANLL, acute phase o r r e m i s s i o n and CGL) when compared t o  v e r y low numbers o f c e l l s e x p r e s s i n g CAMAL i n normal i n d i v i d u a l s o r most p a t i e n t s w i t h lymphoid m a l i g n a n c i e s . s t a n d p o i n t , CAMAL appears  From a d i a g n o s t i c  t o be a u s e f u l marker i n BM f o r t h e  d i s c r i m i n a t i o n o f ANLL from ALL a t d i a g n o s i s , a l t h o u g h l i k e a l l o t h e r m y e l o i d LAAs, t h i s i s n o t t h e case f o r every ANLL p a t i e n t .  As T a b l e  V showed, n o t a l l ANLL p a t i e n t s expressed s i g n i f i c a n t l y i n c r e a s e d numbers o f CAMAL-positive  c e l l a t d i a g n o s i s . The f a c t t h a t i n c r e a s e d  numbers o f CAMAL-positive  c e l l s were p r e s e n t i n BM and p e r i p h e r a l  b l o o d o f p r e l e u k e m i c , m y e l o d y s p l a s t i c and c h r o n i c g r a n u l o c y t i c leukemia p a t i e n t s i m p l i e d a common h e m o p o i e t i c  abnormality  r e l a t e d t o t h e l i k e l i h o o d o f development o f acute leukemia) i n these c o n d i t i o n s .  (possibly existing  T h i s was a l s o i n d i c a t e d by t h e "common" n a t u r e  of CAMAL e x p r e s s i o n i n a l l FAB subgroups o f ANLL.  I t further implied  t h a t i n c r e a s e d CAMAL e x p r e s s i o n was an e a r l y event i n t h e onset o f acute leukemia.  Were t h i s t o be t h e case, ANLL p a t i e n t s a t d i a g n o s i s  w i t h low numbers o f CAMAL-positive  c e l l s may be i n d i v i d u a l s w i t h more  advanced d i s e a s e , h a v i n g passed beyond t h e stage o f e x p r e s s i o n o f t h i s marker. later  T h i s i m p l i c a t i o n w i l l be d i s c u s s e d i n more d e t a i l i n a  section. There  i s no c l e a r e x p l a n a t i o n y e t f o r t h e o b s e r v a t i o n t h a t CAMAL  BM o r PB v a l u e s i n some p a t i e n t s w i t h lymphoid m a l i g n a n c i e s  (ALL a t  d i a g n o s i s o r lymphoma), w h i l e s i g n i f i c a n t l y lower than most myeloid leukemics, were n o n e t h e l e s s i n c r e a s e d somewhat above normal BM o r PB values (Table V I I I ) .  S i m i l a r l y , at t h i s point only speculation  surrounds t h e o b s e r v a t i o n t h a t h a l f o f t h e ALL r e m i s s i o n BM and PB samples examined demonstrated  numbers of CAMAL-expressing m y e l o i d  216  c e l l s comparable t o t h o s e found i n many ANLL p a t i e n t s .  As d i s c u s s e d  p r e v i o u s l y , two ALL r e m i s s i o n p a t i e n t s examined d u r i n g t h e b l i n d study showed h i g h numbers (25 and 30%) o f CAMAL-positive BM c e l l s one month p r i o r t o r e l a p s e and an ALL BMT p a t i e n t demonstrated a CAMAL PB v a l u e o f 50% t h r e e months p r i o r t o r e l a p s e ALL  (529).  A l a r g e r group o f  r e m i s s i o n p a t i e n t s needs t o be s e r i a l l y examined  d e f i n i t i v e remarks can be made c o n c e r n i n g  before  the p o s s i b i l i t y  that  i n c r e a s e d CAMAL e x p r e s s i o n may s i g n a l t h e onset o f r e l a p s e i n some ALL p a t i e n t s .  I t i s i n t e r e s t i n g t h a t t h e CAMAL-expressing  c e l l s i n ALL BM a r e d e f i n i t e l y o f m y e l o i d morphology. important a t t h i s p o i n t t o remember t h a t s t i m u l a t e d cell  l i n e s a r e a major source o f m y e l o p o i e t i c  factors  I t may be  lymphoid c e l l s o r  colony-stimulating  (GM-CSF, G-CSF, -» EPA) as w e l l as o t h e r  regulators of granulopoiesis  remission  important  (interferon-gamma, t r a n s f e r r i n ) and t h a t  t h e r e i s an enormously complex i n t e r p l a y between these r e g u l a t o r s , the c e l l s t h a t produce them and t h e i r t a r g e t c e l l s . this,  i t i s conceivable  In l i g h t of  t h a t d u r i n g t h e onset o f abnormal  lymphopoiesis o c c u r r i n g p r i o r t o r e l a p s e i n ALL, abnormal s t i m u l a t i o n of and i n t e r a c t i o n s between m y e l o i d r e g u l a t o r y f a c t o r s c o u l d and be "marked" by i n c r e a s e d CAMAL e x p r e s s i o n  by m y e l o i d c e l l s .  s p e c u l a t i o n i s o f f e r e d o n l y as food f o r thought; s p e c i f i c c o u l d be performed t o t e s t t h i s p o s s i b i l i t y .  Alternate  such as l i n e a g e i n f i d e l i t y p r e v i o u s l y d i s c u s s e d be  occur This  research  explanations,  (Chapter I I I ) c o u l d  invoked f o r a l i m i t e d number o f ALL p a t i e n t s a t d i a g n o s i s  expressing  b o t h CAMAL and ( p r i m a r i l y ) lymphoid a n t i g e n s ,  but t h i s  c o u l d c e r t a i n l y n o t e x p l a i n t h e s i t u a t i o n i n ALL r e m i s s i o n p a t i e n t s .  217  In c o n c l u s i o n , i t appears t h a t CAMAL i s a u s e f u l a d j u n c t i v e d i a g n o s t i c marker a t d i a g n o s i s f o r ANLL. determining  The main v a l u e o f  t h e number o f CAMAL-positive BM c e l l s a t d i a g n o s i s ,  however, appears t o be i n i t s changing e x p r e s s i o n and  post-chemotherapy  d u r i n g r e m i s s i o n , as w i l l be d i s c u s s e d i n a l a t e r s e c t i o n .  B.  CAMAL E x p r e s s i o n Cell  i n Leukemia  l a b e l i n g s t u d i e s w i t h r a b b i t anti-CAMAL serum ( i n t h e  fluorescence activated c e l l immunoperoxidase) allowed CAMAL-positive c e l l s cells  s o r t e r ) and CAMAL-1 MAb ( i n  i d e n t i f i c a t i o n o f t h e morphology o f  i n myeloid  i n c l u d e d myeloid  leukemic p a t i e n t s .  CAMAL-1 p o s i t i v e  c e l l s a t a l l l e v e l s of maturation,  from b l a s t s  (predominant a t d i a g n o s i s i n ANLL) t o end stage g r a n u l o c y t e s and monocytes.  I n ANLL r e m i s s i o n p e r i p h e r a l b l o o d samples, CAMAL-1  positive cells  i n c l u d e d mature m y e l o i d  c e l l s and, i n a number o f  samples w i t h h i g h numbers o f p o s i t i v e c e l l s were s t r o n g l y l a b e l e d as w e l l .  (up t o 80%), lymphocytes  I n g e n e r a l , r a b b i t anti-CAMAL serum  ( u s i n g i n d i r e c t immunofluorescence) l a b e l e d more numerous b u t s i m i l a r cell  types  i n myeloid  leukemia p a t i e n t s .  Many more c e l l s  lymphoid morphology were l a b e l e d by t h e h e t e r o a n t i s e r u m . probably  with This  r e l a t e s t o t h e p r e f e r e n t i a l r e c o g n i t i o n o f membrane bound  CAMAL by t h e r a b b i t anti-CAMAL serum, which presumably many more a n t i g e n i c d e t e r m i n a n t s d i s p l a y e d by c e l l  recognizes  s u r f a c e CAMAL than  does t h e CAMAL-1 MAb. The  q u e s t i o n remains as t o whether o r n o t t h e m o r p h o l o g i c a l l y  mature CAMAL-expressing c e l l s a r e a c t u a l l y d e r i v e d from t h e o r i g i n a l malignant clone.  G-6-PD and chromosomal a n a l y s e s  o f ANLL have  218  i n d i c a t e d t h a t i t i s a d i s e a s e of stem c e l l o r i g i n ; however the p r e c i s e nature  of the l e v e l of stem c e l l  e s t a b l i s h e d c a t e g o r i c a l l y and heterogeneity the GM  i n t h i s regard.  precursor  involvement has not been  i s complicated  by  apparent  While i t i s g e n e r a l l y b e l i e v e d t h a t  i s most commonly i n v o l v e d i n the stem o r i g i n of  ANLL, some i n v e s t i g a t o r s have c l e a r l y i m p l i c a t e d much more p r i m i t i v e cells  ( w i t h e r y t h r o i d and B lymphoid p o t e n t i a l i n a d d i t i o n t o GM)  occasionally, monocyte-restricted  precursors  or  (73,401-406).  There appeared to be no d i r e c t r e l a t i o n s h i p between number of m a l i g n a n t b l o o d c e l l s and number of CAMAL-positive c e l l s diagnosis.  at  Moreover the l a c k of c o r r e l a t i o n of CAMAL e x p r e s s i o n  e i t h e r a c t i v e l y r e g e n e r a t i n g o r a p l a s t i c BM  argues a g a i n s t a  with  cell  c y c l e dependent e x p r e s s i o n of t h i s marker, which might have e x p l a i n e d why  o n l y some of the m a l i g n a n t c e l l s were CAMAL-positive.  The  d e m o n s t r a t i o n of a s m a l l p r o p o r t i o n of normal CAMAL-positive BM i m p l i e d t h a t not a l l c e l l s , even i n m y e l o i d  leukemics,  cell  t h a t express  CAMAL were n e c e s s a r i l y d e r i v e d from the m a l i g n a n t c l o n e . The ANLL r e m i s s i o n p a t h o l o g y where i n one  case i t was  s t u d i e s o u t l i n e d i n Chapter  determined t h a t 100%  IV,  of p e r i p h e r a l b l o o d  c e l l s w i t h normal morphology and k a r y o t y p e l a b e l e d s t r o n g l y CAMAL positive,  i m p l i e d t h a t these CAMAL-expressing c e l l s were not  from the o r i g i n a l h y p o d i p l o i d leukemic c l o n e .  Since relapse  derived followed  w i t h i n f o u r months ( w i t h re-emergence of the o r i g i n a l c l o n e ) , i t i s obvious t h a t , i n f a c t , u n d e r l y i n g p a t h o l o g y nonetheless. normal PBL  The  was  indeed  still  present  r e s u l t s of the CAMAL a d s o r p t i o n s t u d i e s , wherein  became CAMAL-positive a f t e r i n c u b a t i o n w i t h excess CAMAL,  suggested t h a t i t was  p o s s i b l e f o r normal PBL,  i n c l u d i n g lymphocytes,  219  t o b i n d excess amounts o f s o l u b l e CAMAL on t h e i r membrane. labeled-CAMAL membrane b i n d i n g s t u d i e s i n d i c a t e d  I  non-saturable  l a b e l i n g o f normal and CGL c e l l s by p u r i f i e d CAMAL, o f s i m i l a r magnitude t o t h e t r a n s f e r r i n c o n t r o l .  I t should be f e a s i b l e t o  i s o l a t e s p e c i f i c CAMAL r e c e p t o r s , i f they e x i s t , from c e l l membrane e x t r a c t s by a f f i n i t y chromatography u s i n g a CAMAL column. conceivable,  and c o n s i s t e n t w i t h these r e s u l t s , t h a t  amounts o f CAMAL i n t h e plasma o f m y e l o i d  It is  increased  leukemics p r i o r t o r e l a p s e  (when i n c r e a s e d numbers o f CAMAL-expressing c e l l s have been demonstrated) can account f o r t h e i n c r e a s e d appearance o f p o s i t i v e c e l l s a t t h e s e times.  Furthermore, t h e ALL r e m i s s i o n samples showing  the p r e s e n c e o f p o s i t i v e m y e l o i d o t h e r than those the CAMAL marker.  c e l l s also indicated that  cells  i n v o l v e d i n t h e o r i g i n a l m a l i g n a n t c l o n e may express E v i d e n c e has been p r e s e n t e d  i n this thesis that  p o i n t s t o a r e l a t i o n s h i p between t h e i n c r e a s e d e x p r e s s i o n o f CAMAL and  C.  leukemic  pathology.  S i g n i f i c a n c e o f CAMAL as a P r o g n o s t i c Marker f o r Remission i n ANLL The marked a n t i g e n i c h e t e r o g e n e i t y ,  abnormalities  and u n c e r t a i n t y c o n c e r n i n g  v a r i a b l e chromosomal t h e l e v e l o f stem c e l l  involvement i n ANLL a r e r e f l e c t e d i n t h e l a c k o f u s e f u l common prognostic remission.  i n d i c a t o r s o r markers f o r t h i s d i s e a s e , e s p e c i a l l y I t i s c l e a r that routine monitoring  p r i m a r i l y of morphological fall  and c y t o c h e m i c a l  during  protocols (consisting  e v a l u a t i o n o f BM c e l l s )  f a r s h o r t o f i d e a l , p a r t i c u l a r l y i n t h e i r f a i l u r e t o adequately  d e t e c t r e s i d u a l leukemia i n a c l i n i c a l l y u s e f u l manner. U s u a l l y by  220  the time t h a t i t becomes obvious t h a t r e s i d u a l leukemia i s p r e s e n t , r e l a p s e i s w e l l e s t a b l i s h e d and w i t h i t , an extremely poor long term prognosis. pathology  The i n a b i l i t y t o a s c e r t a i n t h e p r e s e n c e o f u n d e r l y i n g complicates  the establishment  of r a t i o n a l  therapeutic  regimens as w e l l as t h e e v a l u a t i o n o f t h e i r e f f i c a c y a f t e r c o n s o l i d a t i o n chemotherapy.  initial  The i n a b i l i t y t o p r e d i c t which p a t i e n t s  w i l l have l o n g o r s h o r t r e m i s s i o n s  f u r t h e r increases the d i f f i c u l t y  o f p a t i e n t management d e c i s i o n s r e g a r d i n g t h e i m p l i c a t i o n o f n o v e l forms o f treatment i n s e l e c t e d p a t i e n t s . The  p r e l i m i n a r y r e s u l t s presented  from t h e b l i n d study i n  Chapter V o f f e r some grounds f o r optimism i n t h i s r e g a r d .  The  r e l a t i v e CAMAL BM v a l u e , b e f o r e and a f t e r c o n s o l i d a t i o n chemotherapy, does appear t o be o f p r o g n o s t i c  significance.  I n t h i s study,  a  c l e a r l y b e t t e r (p < 0.025) p r o g n o s t i c t r e n d was i n d i c a t e d i n t h e ANLL group whose CAMAL BM v a l u e s dropped s i g n i f i c a n t l y post-chemotherapy. F o r p a t i e n t s i n t h i s group, t h e average r e m i s s i o n l e n g t h was 2.8 times l o n g e r than those ANLL p a t i e n t s whose v a l u e s  increased or  remained t h e same post-chemotherapy. It values  i s v a l i d t o argue t h a t p a t i e n t s w i t h low (< 5%) c o u l d n o t show a s i g n i f i c a n t decrease  initial  post-treatment.  N o n e t h e l e s s , i t i s e q u a l l y v a l i d t o say t h a t p a t i e n t s w i t h  initial  v a l u e s o f < 100% ( r e a l l y , < 95%) c o u l d show e i t h e r an i n c r e a s e o r a decrease.  The d a t a has i n d i c a t e d t h a t t h i s was n o t t h e t r e n d seen.  I t has been c l e a r l y demonstrated t h a t t h e o n l y s i g n i f i c a n t v a r i a b l e i n p r e d i c t i n g i n c r e a s e d s u r v i v a l time p r i o r t o r e l a p s e was t h e change (decrease)  i n t h e CAMAL BM v a l u e p r e - t o post-chemotherapy.  g r e a t e r p r o p o r t i o n o f CAMAL-1 p o s i t i v e mature m y e l o i d  A much  c e l l s were  221  observed post-chemotherapy i n those p a t i e n t s whose v a l u e s  decreased,  perhaps i n d i c a t i n g t h a t the CAMAL-1 p o s i t i v e c e l l p o p u l a t i o n  at  d i a g n o s i s had undergone a g r e a t e r degree of d i f f e r e n t i a t i o n i n t h i s p a t i e n t group.  I n c r e a s i n g d i f f e r e n t i a t i o n i s i n v e r s e l y r e l a t e d to  p r o l i f e r a t i o n and c e l l s expressing Now  c o u l d , t h e r e f o r e , account f o r decreased  numbers of  CAMAL post-chemotherapy i n t h i s p a t i e n t group.  that three-quarters  of a d u l t ANLL p a t i e n t s a c h i e v e  complete  r e m i s s i o n f o l l o w i n g i n t e n s i v e chemotherapy, the problem of maintaining  these p a t i e n t s i n r e m i s s i o n s t a t e assumes much g r e a t e r  importance than ever b e f o r e .  Procedures such as maintenance  chemotherapy d u r i n g r e m i s s i o n t o p r e v e n t demonstrated to be  r e l a p s e have been  i n e f f e c t i v e i n t h i s r e g a r d , p o s s i b l y due  to  i n a b i l i t y to determine the times a t which such treatment might critical  our be  on an i n d i v i d u a l p a t i e n t b a s i s .  Very r e c e n t l y , the polymerase c h a i n r e a c t i o n (PCR) been u t i l i z e d  technique  has  i n the d e t e c t i o n of minimal r e s i d u a l d i s e a s e i n human 5  f o l l i c u l a r lymphoma (540).  As  little  as 1 i n 10  cells carrying  the t(14;18) t r a n s l o c a t i o n c h a r a c t e r i s t i c of 90% of these cases was  detectable using t h i s technique,  lymphoma  exceeding the d e t e c t i o n  l i m i t s of o t h e r r e p o r t e d methods ( f l o w cytometry or Southern b l o t a n a l y s i s f o r c l o n a l immunoglobulin gene rearrangements). o l i g o n u c l e o t i d e s f l a n k i n g the c r o s s o v e r s i t e s of the t r a n s l o c a t i o n a c t e d as p r i m e r s  f o r a PCR,  Synthetic  t(14;18)  a l l o w i n g DNA  sequences  f l a n k i n g the c r o s s o v e r s i t e s t o be amplied e x p o n e n t i a l l y , and be made e a s i l y d e t e c t a b l e .  T h i s e x c i t i n g new  however, be p r a c t i c a l i n d i s e a s e s  technique  thus t o  would not,  such as ANLL where even though  c e r t a i n nonrandom chromosomal a b n o r m a l i t i e s o c c u r ,  they are m u l t i p l e  222  and would r e q u i r e the p r e p a r a t i o n i n d i v i d u a l genetic  of d i f f e r e n t p r i m e r s f o r each  change on the b a s i s of DNA  sequencing.  Moreover,  many ANLL p a t i e n t s demonstrate no  chromosomal a b n o r m a l i t i e s  conventional  t e c h n i q u e would be r e a s o n a b l e f o r  techniques.  The  PCR  the m o n i t o r i n g of minimal r e s i d u e d i s e a s e ,CGL  i n Ph'  chromosome p o s i t i v e  p a t i e n t s r e c e i v i n g a l l o g e n e i c bone marrow t r a n s p l a n t s . I n ANLL, e a r l i e r  anti-myeloblast  i n v e s t i g a t i o n s by Baker and  remission Neither  colleagues  i n the BM  p a t i e n t s s e v e r a l months p r i o r t o r e l a p s e  the nature, of the r e a c t i v e c e l l s nor  were d e s c r i b e d  and  of many ANLL  (461,462).  the a n t i g e n ( s )  We  p r e v i o u s l y demonstrated the p r e s e n c e of CAMAL-positive PB p a t i e n t s up  In c o n t r a s t , 14/14 unrelated  acute leukemia BMT 13/15  p a t i e n t s dying  p a t i e n t s who  f o r the 12 month study p e r i o d , showed no CAMAL-1 r e a c t i v e c e l l s  cells  (by  in  (529).  of causes  remained i n  remission  s i g n i f i c a n t numbers of  immunoperoxidase).  In the 3 y e a r b l i n d study r e p o r t e d h e r e i n  (Chapter V ) , a more  complex s i t u a t i o n e x i s t e d than t h a t of the acute leukemia  of age  have  to s e v e r a l months p r i o r t o r e l a p s e  t o r e l a p s e , and  p a t i e n t s , s i n c e no  detected  i t i s p o s s i b l e t h a t these i n v e s t i g a t o r s were  i d e n t i f y i n g the CAMAL marker, o r some r e l a t e d a n t i g e n .  ANLL BMT  using  h e t e r o a n t i s e r u m i n immunofluorescence experiments,  demonstrated t h a t r e a c t i v e c e l l s were p r e s e n t  6/6  using  information  was  a v a i l a b l e on the p o s s i b l e e f f e c t s  ( o b v i o u s l y a much wider range was  treatment p r o t o c o l s on CAMAL e x p r e s s i o n . are l i m i t e d and p r e l i m i n a r y ,  BMT  examined) or d i f f e r e n t While the r e s u l t s p r e s e n t e d  t h e r e are c l e a r i n d i c a t i o n s t h a t  i n c r e a s i n g numbers of CAMAL-positive c e l l d u r i n g  r e m i s s i o n may  the o n s e t of r e l a p s e w i t h i n the f o l l o w i n g few months.  signal  Indeed, when  223  we o b t a i n e d  s u f f i c i e n t l y s e q u e n t i a l samples from an i n d i v i d u a l  p a t i e n t , i t was c l e a r t h a t t h i s p a t t e r n h e l d t r u e Table  ( F i g u r e 5.4 and  XVIII). Recent d a t a has a l s o i n d i c a t e d t h a t over 83% o f ANLL p a t i e n t s  g i v e n chemotherapy when t h e i r CAMAL BM v a l u e s were h i g h  (> 10%)  responded more s u c c e s s f u l l y t o chemotherapy (had l o n g e r  remission  t i m e s ) than those p a t i e n t s t r e a t e d when t h e i r v a l u e s were < 10%. The r e s u l t s from t h i s b l i n d study may prove t o have i m p l i c a t i o n s i n t h e following situations: 1.  determination  of " t r u e " remission  maintenance o f low o r d e c r e a s i n g to 2.  s t a t u s i n ANLL ( t h e CAMAL BM v a l u e s )  this,  determination  of optimal  times f o r p e r f o r m i n g BMT on ANLL  remission p a t i e n t s (while i n " t r u e " remission 3.  and, r e l a t e d  state)  development and e v a l u a t i o n o f n o v e l t h e r a p e u t i c  protocols  including: a.  e a r l y a d d i t i o n a l treatments f o r ANLL p a t i e n t s predictably shorter remission  lengths,  with  i n p a r t i c u l a r those  demonstrating a l a r g e i n c r e a s e i n CAMAL BM v a l u e post-chemotherapy b.  a d m i n i s t r a t i o n of r e m i s s i o n  chemotherapy t o i n d i v i d u a l ANLL  p a t i e n t s based on a s i g n i f i c a n t i n c r e a s e i n CAMAL BM v a l u e c.  a d m i n i s t r a t i o n o f a d d i t i o n a l chemotherapy post-BMT based on a s i g n i f i c a n t i n c r e a s e i n CAMAL BM v a l u e .  These a p p l i c a t i o n s a r e o n l y suggested here, based on p r e l i m i n a r y findings.  We a r e p l a n n i n g  a national c l i n i c a l t r i a l  to evaluate the  224  r e l a t i o n s h i p between changing CAMAL BM (and PB) v a l u e s  and c l i n i c a l  p r o g n o s i s i n a l a r g e group o f c l o s e l y monitored ANLL p a t i e n t s a t three  leukemia c e n t e r s .  these p r e l i m i n a r y  D.  This t r i a l  should  e s t a b l i s h whether o r n o t  findings are v a l i d .  The P o s s i b l e R o l e o f CAMAL i n M y e l o p o i e s i s I t has now been e s t a b l i s h e d t h a t CAMAL can a c t as a s i g n i f i c a n t  marker i n t h e p r e d i c t i o n o f r e m i s s i o n r e l a p s e i n ANLL p a t i e n t s . CAMAL i s simply  l e n g t h and i n t h e onset o f  The q u e s t i o n now s u r f a c e s  as t o whether  a c o n v e n i e n t a n t i g e n i c marker, a s s o c i a t e d  indirectly  w i t h m y e l o i d leukemogenesis, o r whether CAMAL may be i n v o l v e d i n a more d i r e c t b i o l o g i c a l manner. between w e l l and p o o r l y myelopoietic  Due t o t h e c o m p l e x i t y o f i n t e r a c t i o n s  c h a r a c t e r i z e d p o s i t i v e and n e g a t i v e  r e g u l a t o r s , t h e i r t a r g e t c e l l s o r m o l e c u l e s , and t h e  c e l l s p r o d u c i n g them, p r e c i s e answers t o t h i s q u e s t i o n become v e r y difficult  t o approach e x p e r i m e n t a l l y ,  certainty.  l e t alone r e s o l v e with  Some headway has now been made towards t h i s end and  f u t u r e e x p e r i m e n t a l approaches have become b e t t e r D e f i n i t i v e answers c o n c e r n i n g myelopoiesis  defined.  t h e p o s s i b l e r o l e o f CAMAL i n  await m o l e c u l a r a n a l y s i s o f t h i s p r o t e i n i n b o t h normals  and m y e l o i d leukemics.  F o r t h e moment, c e r t a i n i n t r i g u i n g r e s u l t s  have emerged. A number o f important f a c t o r s need t o be c o n s i d e r e d i n e x p e r i m e n t a l approaches aimed a t d e m o n s t r a t i n g e i t h e r s t i m u l a t i o n o r i n h i b i t i o n of hemopoietic colony derived  substance.  F i r s t of a l l ,  formation there  by any b i o l o g i c a l l y -  i s always a problem w i t h  i n t e r p r e t a t i o n o f t h e d e t e c t i o n and c h a r a c t e r i z a t i o n o f p u t a t i v e  225  regulatory factors using  an i n v i t r o t i s s u e c u l t u r e system.  systems a r e i n h e r e n t l y a r t i f i c i a l concentrations  i n terms o f i n v i v o  Such  cell  and o t h e r n a t u r a l m i c r o e n v i r o n m e n t a l i n f l u e n c e s .  However, t h e s i m p l i c i t y and c a p a c i t y t o s t a n d a r d i z e  such systems can  o f f e r advantages i m p o s s i b l e  t o d u p l i c a t e otherwise.  Furthermore t h e  i n v i t r o myeloid progenitor  c e l l assay has been demonstrated t o be a  u s e f u l and r e l e v a n t system f o r such i n v e s t i g a t i o n s . Other important c o n s i d e r a t i o n s putative regulatory  i n c l u d e the presence of a  f a c t o r i n v i v o a t l e v e l s s i m i l a r t o those  r e q u i r e d t o demonstrate e f f e c t s i n v i t r o , d e m o n s t r a t i o n of p h y s i o l o g i c a l l y r e l e v a n t i n v i v o f l u c t u a t i o n s o f t h e f a c t o r , and t h e a b i l i t y t o e f f e c t i v e l y p u r i f y t h e f a c t o r from r e l e v a n t CAMAL has been demonstrated t o be p r e s e n t plasma and i n BM and PB c e l l s .  j u s t i f y any statements c o n c e r n i n g  greater)  studies  i n v i v o , b o t h i n human  G i v e n t h a t methods used t o q u a n t i f y  CAMAL plasma l e v e l s a r e n o t c o n c l u s i v e ,  CAMAL a d s o r p t i o n  sources.  i t i s more d i f f i c u l t t o  relevant  i n vivo levels.  However,  i n d i c a t e d t h a t a p p r o x i m a t e l y 12 ug/ml ( o r  a f f i n i t y - p u r i f i e d CAMAL bound t o normal PB l e u c o c y t e s , and  t h i s l e v e l i s remarkably s i m i l a r t o those demonstrated t o cause i n h i b i t i o n of normal m y e l o i d c o l o n y  growth.  With r e g a r d  to relevant  p h y s i o l o g i c a l f l u c t u a t i o n i n v i v o , i t has been shown t h a t numbers o f CAMAL-positive c e l l s f l u c t u a t e i n v i v o , and t h a t t h i s f l u c t u a t i o n was s i g n i f i c a n t i n terms o f c l i n i c a l p r o g n o s i s . p u r i f i e d e f f e c t i v e l y by immunoadsorption. leukemic c e l l s p r o v i d e d S p e c i f i c antibodies others  F i n a l l y , CAMAL can be Soluble  extracts of  sufficient quantities f o r i n vitro  assays.  c o u p l e d t o a f f i n i t y columns have been u t i l i z e d by  t o s u c c e s s f u l l y p u r i f y b o t h M-CSF and IL-2 (128,175).  226  As d i s c u s s e d  i n Chapter I, serum ( o r plasma) c o n t a i n s  numerous  components w i t h known growth promoting a c t i v i t i e s f o r c u l t u r e d hemopoietic c e l l s .  The  selective, restrictive  ( o r absent) growth  and  p r o l i f e r a t i o n of some or a l l h e m o p o i e t i c c e l l s i n v i t r o , depending  on  the b a t c h of f e t a l c a l f serum or plasma u t i l i z e d , a r e t e s t i m o n y f o r the major r e g u l a t o r y r o l e t h a t some of t h e s e components can p l a y . has  been w e l l e s t a b l i s h e d t h a t the o n l y c o n s i s t e n t method f o r  d e m o n s t r a t i o n of any  p o s s i b l e e f f e c t ( s ) of a serum o r plasma  component i s the use  of s e l e c t i v e d e p l e t i o n  s e l e c t i v e l y depleted  (by a f f i n i t y chromatography) from the plasma  conditioned  observed.  Reconstitution  c e l l s were u n s u c c e s s f u l , albumin (up  When CAMAL  was  attempts u s i n g CAMAL d e r i v e d  was from leukemic  as were s i m i l a r attempts u s i n g human serum  to 40 yg/ml), a growth-promoting serum component w i t h  Depletion  some immunological c r o s s - r e a c t i v i t y w i t h  of CAMAL from the same normal human plasma  and  conditioned  medium f a i l e d t o i n h i b i t CFU-c development i n m y e l o i d  leukemics.  These r e s u l t s i m p l i e d t h a t , i n normal m y e l o p o i e s i s ,  be  i n v o l v e d i n some p o s i t i v e r e g u l a t o r y manner, but  exist.  CAMAL  that i n  m y e l o i d leukemia, such r e g u l a t i o n ( o r the c e l l s r e s p o n s i v e not  and  c e l l assay, a  o f t e n s i z e ) of normal CFU-c  s i m i l a r m o l e c u l a r weight and CAMAL.  (234).  medium used i n the m y e l o i d p r o g e n i t o r  d e c r e a s e i n the number (and  may  It  to i t ) do  F u t u r e s t u d i e s aimed a t c h a r a c t e r i z i n g t h i s e f f e c t i n  normals would i n c l u d e comparative m o r p h o l o g i c a l examination of c o l o n i e s from d e p l e t e d  and  a s p e c i f i c subpopulation(s)  c o n t r o l c u l t u r e s i n order of CFU-c was  s i z e of many c o l o n i e s from d e p l e t e d  affected.  t o determine i f The  decrease i n  marrow c u l t u r e s c o u l d r e l a t e t o  227  more r a p i d d i f f e r e n t i a t i o n  (and t h e r e f o r e , d e c r e a s e d p r o l i f e r a t i v e  capacity) w i t h i n these c o l o n i e s . myelopoiesis, two  I t i s p o s s i b l e t h a t , i n normal  CAMAL may be i n v o l v e d i n t h e t r a n s i t i o n between t h e s e  processes. One  very  important a s p e c t  concerning  the c h a r a c t e r i z a t i o n of  CAMAL which has n o t been c l e a r l y e s t a b l i s h e d , r e l a t e s t o t h e c e l l ( s ) responsible  f o r p r o d u c i n g i t . As these s t u d i e s a r e p a r t o f o t h e r  investigators' research  o b j e c t i v e s , experiments i n t o t h e n a t u r e of  c e l l s p r o d u c i n g CAMAL have n o t been attempted h e r e .  The p r e s e n c e o f  CAMAL i n human serum CAMAL and plasma i m p l i e d t h a t CAMAL i s r e l e a s e d from i t s c e l l ( s ) o f o r i g i n . and  leucocyte  conditioned  The p r e s e n c e o f CAMAL i n human p l a c e n t a l  media i m p l i e d t h a t t h e s e c e l l s  either  a c t i v e l y produce and s e c r e t e CAMAL o r r e l e a s e CAMAL from i n t r a c e l l u l a r stores. human p r o m y e l o c y t i c  Recently  cell  i t has been demonstrated t h a t t h e  l i n e HL-60 produces CAMAL-1 r e a c t i v e  m a t e r i a l when grown i n serum-free medium ( S h e l l a r d ,  personal  communication), i n d i c a t i n g t h a t m y e l o i d c e l l s a r e t h e p r o b a b l e source of CAMAL.  Whether m y e l o i d c e l l s o f a l l l e v e l s o f m a t u r a t i o n (known  t o c o n t a i n CAMAL) a c t u a l l y c o n t i n u e t o produce i t , o r merely and/or s e c r e t e same c e l l leukemics. was  store  i t , has n o t been e s t a b l i s h e d , n o r do we know i f t h e  type(s)  are responsible  f o r CAMAL p r o d u c t i o n  i n normals and  When t h e e f f e c t o f a d d i t i o n o f excess CAMAL t o c u l t u r e s  i n v e s t i g a t e d , leukemic (CGL) c e l l s were used as a c o n v e n i e n t  source f o r o b t a i n i n g testing.  s u f f i c i e n t amounts o f p u r i f i e d CAMAL f o r  I t was d i s c o v e r e d  t h a t a d d i t i o n o f > 10 - 15 v&/ml excess  l e u k e m i a - d e r i v e d CAMAL r e s u l t e d i n s i g n i f i c a n t i n h i b i t i o n o f normal CFU-c b u t t h e same p r e p a r a t i o n  had no e f f e c t on c o l o n y  growth i n  228  m y e l o i d leukemia p a t i e n t s w i t h a c t i v e d i s e a s e . d i d n o t appear t o be i n d i r e c t l y mediated  The i n h i b i t o r y  effect  through adherent a c c e s s o r y  c e l l s , a s i t u a t i o n s i m i l a r t o t h a t i n t h e p r e v i o u s CAMAL d e p l e t i o n studies.  While t h e s e r e s u l t s a t f i r s t  seemed d i s c r e p a n t w i t h t h e  p r e v i o u s s t u d i e s , t h e o b s e r v a t i o n t h a t l e u k e m i a - d e r i v e d CAMAL c o u l d n o t r e c o n s t i t u t e CAMAL d e p l e t e d normal  cultures indicated that there  might be a f u n c t i o n a l d i f f e r e n c e between CAMAL p r e s e n t i n normals and t h a t i n m y e l o i d leukemics. The e x i s t e n c e o f a h e m o p o i e t i c (multi-CSF) d e r i v e d from m a l i g n a n t c e l l s from i t s normal (114,115).  factor  (WEHI-3B) t h a t i s d i f f e r e n t  c o u n t e r p a r t has been documented p r e v i o u s l y  A s i n g l e amino a c i d d i f f e r e n c e between these two  m o l e c u l e s a p p a r e n t l y accounts f o r decreased f u n c t i o n a l a c t i v i t y of the WEHI-3B-derived m u l t i - C S F i n t h e s t i m u l a t i o n o f h e m o p o i e t i c proliferation  (182).  F u t u r e s t u d i e s u s i n g CAMAL d e r i v e d from  cell  normal  s o u r c e s a r e i n d i c a t e d , as i s d e t e r m i n a t i o n o f t h e p r i m a r y amino a c i d sequence o f CAMAL from normal  and leukemic s o u r c e s .  A number o f a d d i t i o n a l q u e s t i o n s immediately a r i s e , which r e l a t e to future research d i r e c t i o n s . 1.  What i s t h e t a r g e t c e l l ( s ) o r m o l e c u l e ( s ) f o r CAMAL?  2.  I s CAMAL from m y e l o i d leukemia p a t i e n t s " n o n - f u n c t i o n a l " by i t s e l f but capable of b l o c k i n g receptors f o r other required factors  ( f a c t o r s which normal  c e l l s r e q u i r e but myeloid  leukemics do n o t ) ? 3.  Does leukemic CAMAL b i n d some important r e g u l a t o r y m o l e c u l e i n c o n d i t i o n e d medium o r plasma, a d d i t i o n o f i n c r e a s e d CSF?  and can i t s e f f e c t be overcome by  229  4.  Does a d d i t i o n o f CAMAL t o PHA-stimulated  leucocyte cultures  cause any change i n p r o d u c t i o n o f CSF? 5.  Does l e u k e m i a - d e r i v e d  CAMAL need t o be p r e s e n t throughout t h e  c u l t u r e p e r i o d i n o r d e r t o e x e r t i t s e f f e c t , o r would p r e i n c u b a t i o n o r d e l a y e d a d d i t i o n a l s o be e f f e c t i v e ? 6.  Is a s i m i l a r molecule  p r e s e n t i n mice o r i s human CAMAL  f u n c t i o n a l i n t h e murine system, a l l o w i n g i n v i v o t e s t i n g ? 7.  What i s t h e m o l e c u l a r n a t u r e o f t h e mechanism by which CAMAL e x e r t s i t s i n h i b i t o r y e f f e c t on normal h e m o p o i e t i c  8.  Does CAMAL cause an i n c r e a s e o r decrease  cells?  i n the production  and/or r e l e a s e o f w e l l c h a r a c t e r i z e d n e g a t i v e r e g u l a t o r s ( L F , AIF, The  IFN, TF, PGE)? r e l a t i o n s h i p , i f any, o f leukemic  r e g u l a t o r s would be important similarity  leukemia  responsiveness PGE  to establish.  The most prominant  appears t o be t h e d i f f e r e n c e i n r e s p o n s i v e n e s s  r e g u l a t i o n by leukemic Myeloid  CAMAL t o d e f i n e d n e g a t i v e  and CAMAL.  to negative  c e l l s compared t o t h e i r normal c o u n t e r p a r t s .  p a t i e n t s ' c e l l s have been shown t o have  decreased  to the suppressive o r i n h i b i t o r y e f f e c t s of LF, AIF, I n t h e case o f A I F and PGE, t h i s i s r e l a t e d t o t h e i r  Class I I antigen target c e l l  specificity.  Since there i s a  d e f i c i e n c y o f C l a s s I I a n t i g e n s on m y e l o i d p r o g e n i t o r s o f leukemia p a t i e n t s , t h e s e r e g u l a t o r s have decreased patients. cells  e f f e c t s on c e l l s from these  Indeed, AIF i s p r e s e n t i n e l e v a t e d amounts i n leukemic  (261,268), a somewhat s i m i l a r s i t u a t i o n t o t h a t o f CAMAL.  possibility  o f C l a s s I I (HLA-DR) a n t i g e n - p o s i t i v e t a r g e t c e l l  specificity  o f CAMAL c o u l d be examined e x p e r i m e n t a l l y .  The  230  The v i r t u a l l y cells  total  s u p p r e s s i o n o f normal h e m o p o i e t i c p r o g e n i t o r  i n the f a c e of the emerging  more remarkable occurrence.  i n the absence  m a l i g n a n t c l o n e i n ANLL i s a l l the  of c o n c l u s i v e e x p l a n a t i o n s f o r i t s  Normal h e m o p o i e t i c c e l l s have p r o f o u n d r e g e n e r a t i v e  c a p a c i t i e s i n s i t u a t i o n s o f consumption c y c l e time of normal h e m o p o i e t i c c e l l s leukemic c e l l s .  And  or l o s s .  Moreover, the  cell  i s s h o r t e r than t h a t of  y e t the s t r i k i n g p u z z l e remains  c o n c e r n i n g the  a b i l i t y o f the m a l i g n a n t c l o n e t o g a i n a f o o t h o l d i n terms of increased s e l f  g e n e r a t i o n over normal  c u l t u r e s of b o t h CGL demonstrated  cells.  Long-term marrow  and newly diagnosed ANLL p a t i e n t s *  c e l l s have  t h a t normal h e m o p o i e t i c p r o g e n i t o r c e l l s a r e  p r e s e n t , even though  suppressed, i n v i v o  e s t a b l i s h e d t h a t m y e l o i d leukemia c e l l s c o m p l e t e l y dependent on CSF  (381).  I t has been  (ANLL and CGL)  f o r growth i n v i t r o .  remain  Large  i n r e s p o n s i v e n e s s t o CSF by m y e l o i d leukemia c e l l s normals have n o t been i n d i c a t e d .  still  differences  compared t o  P o s s i b l e mechanisms f o r c l o n a l  dominance by m y e l o i d leukemic c e l l s  include f a i l u r e  to i n t e r a c t  a p p r o p r i a t e l y w i t h s t r o m a l c e l l s , s t r o m a l c e l l d y s f u n c t i o n and/or abnormal CSF p r o d u c t i o n i n the immediate v i c i n i t y of the  leukemic  c l o n e . The most c o n v i n c i n g e x p l a n a t i o n s f o r the emergence and dominance of the leukemic c l o n e i n v o l v e the profound d e c r e a s e i n responsiveness to negative r e g u l a t i o n that these c e l l s e x h i b i t . has been d i s c u s s e d p r e v i o u s l y i n d e t a i l .  Such a mechanism p r o v i d e s  an i d e a l method f o r g a i n i n g a p r o l i f e r a t i v e advantage cells.  This  over  normal  E x p e r i m e n t a l r e s u l t s have i n d i c a t e d t h a t t h i s mechanism  may  be the manner i n which CAMAL p l a y s an o p e r a t i o n a l r o l e i n abnormal myelopoiesis.  I t i s apparent t h a t r e s i d u a l leukemic c e l l s may  exist  231  a t v e r y low o r u n d e t e c t a b l e l e v e l s i n ANLL d u r i n g r e m i s s i o n . apparent  It is  t h a t CAMAL-expressing c e l l s can a l s o e x i s t a t low l e v e l s i n  the same s i t u a t i o n .  The o b s e r v a t i o n t h a t i n c r e a s i n g numbers o f c e l l s  (whether p a r t o f t h e m a l i g n a n t  c l o n e o r n o t ) e x p r e s s i n g CAMAL appear  to h e r a l d t h e onset o f r e l a p s e may r e l a t e t o t h e s u p p r e s s i v e e f f e c t of i n c r e a s i n g amounts o f leukemic  CAMAL on normal h e m o p o i e t i c  cells.  T h i s event would, by i t s n a t u r e , be r e q u i r e d t o take p l a c e p r i o r t o the c l i n i c a l re-emergence o f t h e m a l i g n a n t  c l o n e , and t h i s does  indeed c o i n c i d e w i t h i n c r e a s i n g CAMAL e x p r e s s i o n .  The o b s e r v a t i o n  t h a t i n c r e a s i n g CAMAL BM v a l u e s post-chemotherapy c o r r e s p o n d t o s h o r t e r r e m i s s i o n l e n g t h s than do d e c r e a s i n g v a l u e s , i s a l s o i n accordance  with t h i s  interpretation.  Many q u e s t i o n s and s p e c u l a t i o n s c o n c e r n i n g t h e n a t u r e o f t h e CAMAL marker remain t o be examined.  I n a d d i t i o n t o those a l r e a d y  discussed, there i s a d i s t i n c t p o s s i b i l i t y that the e f f e c t s  observed  on i n v i t r o m y e l o p o i e s i s , which we have a s c r i b e d as b e i n g mediated by the p u r i f i e d 68 KD p r o t e i n , may be t h e r e s u l t o f some o t h e r c l o s e l y a s s o c i a t e d and c o - p u r i f i e d w i t h CAMAL. encoding  molecule,  U n t i l t h e gene  CAMAL i s i s o l a t e d and subsequent recombinant  protein i s  produced, t h i s p o s s i b i l i t y may be v e r y d i f f i c u l t t o examine i n detail.  The p o s s i b i l i t y t h a t normal and l e u k e m i a - d e r i v e d CAMAL may  not be i d e n t i c a l must a l s o be examined more c l o s e l y . P o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n s o f t h e p r o t e i n may o c c u r i n leukemic function.  c e l l s t h a t a l t e r CAMAL*s normal e x p r e s s i o n as w e l l as i t s Such p o s s i b i l i t i e s a r e now b e i n g addressed by o t h e r  investigators.  232  Summary and Conclusions The existence of an antigen (CAMAL) commonly associated with a l l forms of human myeloid leukemia had been previously established. This thesis describes a simple, convenient assay for the detection of CAMAL in or on BM or PB cells using a modified indirect immunoperoxidase single c e l l slide test with a CAMAL-specific MAb referred to as CAMAL-1. This assay established the specificity of CAMAL-1 and demonstrated the presence of significantly increased numbers of CAMAL-positive cells in myeloid leukemia patients compared with those found in normals or most individuals with lymphoid malignancies.  CAMAL was shown to be expressed primarily  intracellularly by numerous c e l l types (predominantly myeloid) in patients with preleukemia, CGL and ANLL (during active disease and often in remission as well).  Fluctuations in CAMAL BM value was  discovered to be a significant factor in the prediction of survival time prior to relapse in ANLL patients.  A decrease in the CAMAL BM  value post-chemotherapy was shown to be associated with significantly longer f i r s t remission length.  CAMAL BM values were often observed  to increase during remission, prior to relapse.  These results  indicated that i t may be possible to more accurately gauge remission status in ANLL patients by u t i l i z i n g the CAMAL-1 indirect immunoperoxidase test as a means of monitoring patients.  Novel  therapeutic approaches aimed at increasing disease-free survival based on detection of changes in CAMAL expression by cells appear to be possible.  In vitro myeloid progenitor cell studies indicated that  CAMAL may play, or be associated with, some regulatory role in hemopoiesis.  Depletion of CAMAL from normal plasma and conditioned  233  medium r e s u l t e d i n s i g n i f i c a n t i n h i b i t i o n o f normal had no e f f e c t on m y e l o i d leukemic c o l o n y growth.  CFU-c growth b u t  A d d i t i o n o f > 10  ug/ml l e u k e m i a - d e r i v e d excess CAMAL caused p r o f o u n d i n h i b i t i o n o f normal  CFU-c b u t had no e f f e c t on CFU-c growth from m y e l o i d  patients i n active disease states.  These r e s u l t s  d i f f e r e n c e s i n h e m o p o i e t i c r e g u l a t i o n by normal t h a t have n o t been p r e v i o u s l y examined.  leukemia  suggested  and leukemic  cells  A mechanism whereby m y e l o i d  leukemic c e l l s can cause t h e i n h i b i t i o n o f normal  hemopoiesis,  f a c i l i t a t i n g t h e i r own c l o n a l dominance, was suggested.  Quantitative  and p o s s i b l y q u a l i t a t i v e d i f f e r e n c e s i n CAMAL e x p r e s s i o n by leukemic c e l l s compared t o normals have been e s t a b l i s h e d which may r e l a t e t o leukemogenesis  and i n c r e a s e our u n d e r s t a n d i n g o f t h e p a t h o l o g y  u n d e r l y i n g t h i s complex h e m o p o i e t i c  malignancy.  234  APPENDIX A complete l i s t o f d a t a p e r t a i n i n g t o Chapter VI ( c , d, and e) i s p r e s e n t e d h e r e . The f o l l o w i n g T a b l e s i l l u s t r a t e a l l d a t a accumulated from which s e l e c t e d examples were chosen i n Chapter V I .  T a b l e XIX-A:  Patient  E f f e c t o f CAMAL d e p l e t i o n on normal bone marrow  Code  Number o f CFU-c per 1 0 cells  %  % Inhibition  5  + + +  3 4.5 4  100 95.3 78.4  0 4.7 21.6  74 72 56  + + +  1 0 2  100 97.3 75.7  0 2.7 24.3  0 B C  128 131 95  + + +  12 8 7.5  100 102.4 74.3  0 -2.4 25.7  4*  0 B C  170 163 121  + + +  2 16 17  100 95.6 71.2  0 4.4 28.8  5  0 B C  123 123.5 95.5  + + +  3 0.5 2.5  100 100.4 77.6  0 -0.4 22.4  6  0 B C  186.5 178 126.5  + + +  3.5 11 9.5  100 95.4 67.8  0 4.6 32.2  7  0 B C  70 69.5 59  + + +  7 7.5 5  100 99.3 84.3  0 0.7 15.7  8  0 B C  132.5 132 112  + + +  2.5 1 6  100 99.1 81  0 0.9 19.0  1  0 B C  2  0 B C  3*  268 255.5 210  235  Patient  Code  Number o f CFU-c per IO-* c e l l s  9  0 B C  135 121.4 102.4  + 10.1 + 9.5 + 7.8  10  0 B C  131.5 + 122 + 102 +  9.5 5.5 1.5  %  %  100 89.9 75.9  0 10.1 24.1  100 92.8 77.9  0 7.2 22.1  Average r e s u l t s from above experiments : Mean % B's: Mean % C s : Code:  96.8% 76.4%  0 = no treatment of plasma o r CM B = plasma and CM passed over n e g a t i v e (BLV-1) C = plasma and CM passed over CAMAL-1 column  *Nonadherent c e l l s were p l a t e d  i n t h e s e samples  Inhibition  column  T a b l e XX-A.  Patient  E f f e c t of CAMAL d e p l e t i o n on normal p e r i p h e r a l b l o o d  Code  Number of CFU-c per 10-> c e l l s  %  % Inhibition  1  0 B C  34 + 0 + 4 25 17.5 + 2.5  100 87 .7 61 .4  0 12.3 38.6  2*  0 B C  22.5 + 1.5 21 + 0 11.5 + 2.5  100 93 .3 51 .1  0 6.7 48.9  3*  0 B C  15.5 + 1.5 14 + 2 7 + 0  100 90 .3 45 .2  0 9.7 54.8  4  0 B C  26.5 + 3.5 22 + 0 14.5 + 1.5  100 83 54 .7  0 17 45.3  Average o f 4 normal p e r i p h e r a l b l o o d samples : Mean % B's: Mean % C s :  Code:  88.6 53.1  % %  0 = no t r e a t m e n t of plasma o r CM B = plasma and CM passed over n e g a t i v e (BLV-1) MAb C = plasma and CM passed o v e r CAMAL-1 column  *Nonadherent c e l l s were p l a t e d .  column  T a b l e XXII-A. E f f e c t o f CAMAL d e p l e t i o n on c h r o n i c g r a n u l o c y t i c leukemia p e r i p h e r a l b l o o d  Patient  Code  1  0 B C  91 84 87  + + +  2  0 B C  193 194 204  + + +  2*  0 B C  210 206 205  3  0 B C  24.5 31  3*  0 B C  22 29.5 28.5  4  0 B C  4*  0 B C  Summary o f 7 samples Mean % B's: Mean % C's: Code:  Number o f CFU-c p e r 5 x 10^ c e l l s  %  % Inhibition  100 92.6 96  0 7.4 4  7 11 14  100 100.5 105.7  0 - 0.5 - 5.7  + + +  2 1 5  100 98.1 97.6  0 1.9 2.4  +  1.5  100  0  +  3  126  -26  + + +  2 2.5 2.5  100 134.1 129.5  0 -34.1 -29.5  180 169 191  + + +  4 9.5 1.5  100 93.9 106.1  0 6.1 - 6.1  193 180 178  + + +  6.5 3 7  100 93.8 92.7  0 6.2 7.3  1 1 2.5  ND  tested :  102.2 % 107.7 %  0 = no treatment o f plasma o r CM B = plasma and CM passed over n e g a t i v e (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column  * = nonadherent c e l l s were p l a t e d ND = n o t determined  i n t h e s e experiments  T a b l e XXIII-A.  Patient  Code  E f f e c t of CAMAL d e p l e t i o n on ANLL  Number o f CFU-c per 1()5 c e l l s  1 (PB)  0 B C  63 70 69  2 (BM)  0 B C  3 (PB)  3*  %  %  Inhibition  100 111 109.5  0 -11 - 9.5  35 ± 2 30 + 0.5 38.5 + 1.5  100 85.7 110  0 14.3 -10  0 B C  ND 7.5 + 1.3 8.5 + 3.3  100 113.3  0 -13.3  0 B C  6.8 6.8  ± 2 + 1 ± 9  ND + 2.5 + 3.2  100 100  0 0  Summary o f samples t e s t e d : Mean % B's: Mean % C s : Code:  99.2 % 108.2 %  0 = no treatment o f plasma o r CM B = plasma and CM passed over n e g a t i v e (BLV-1) MAb C = plasma and CM passed over CAMAL-1 column  column  * = nonadherent c e l l s were p l a t e d ND = n o t determined P a t i e n t s 1 and 2 were r e c e n t l y diagnosed ANLLs; p a t i e n t 3 was an ANLL remission patient. P a t i e n t 3 experiments were performed i n q u a d r u p l i c a t e .  239  REFERENCES  1.  Boggs DR: The k i n e t i c s of n e u t r o p h i l i c l e u k o c y t e s i n h e a l t h and disease. 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