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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 Marie Logan D.V.M., The U n i v e r s i t y of Guelph, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1987 © P a t r i c i a Marie Logan, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada Department V6T 1Y3 DE-6(3/81) ABSTRACT Human acute nonlymphoblastic or myelogenous leukemia (ANLL or AML) i s a malignant disease of the bone marrow involving hemopoietic (blood-forming) c e l l s of the myeloid lineage. ANLL i s a complex neoplastic disease, whose fundamental nature i s only p a r t i a l l y understood despite intensive research. The disease i s complicated by i t s apparent heterogeneity i n terms of the degree of d i f f e r e n t i a t i o n of hemopoietic stem c e l l involvement i n d i f f e r e n t patients and the c e l l u l a r expression of immunologically defined surface markers. The presence of a common antigen i n myelogenous leukemia (CAMAL) has been previously i d e n t i f i e d . This th e s i s examines the expression of the CAMAL marker i n or on bone marrow (BM) and peripheral blood (PB) c e l l s using a monoclonal antibody-based i n d i r e c t immunoperoxidase s l i d e t e s t . Increased numbers of CAMAL-positive c e l l s were found i n or on BM and PB of myeloid leukemia patients (with acute or chronic forms of the disease) compared with those found i n normals or most lymphoid malignancies. Results presented herein have demonstrated that f l u c t u a t i o n s i n CAMAL BM values (% p o s i t i v e c e l l s ) c o r r e l a t e d with s u r v i v a l time p r i o r to relapse. In a b l i n d study, ANLL patients Whose CAMAL BM values decreased post-chemotherapy had s i g n i f i c a n t l y (p < 0.025) longer f i r s t remission times (x = 19.2 months) than patients with increasing or s t a t i c CAMAL BM values (x = 6.8 months). CAMAL BM values were often observed to increase during remission, p r i o r to relapse, suggesting the presence of r e s i d u a l s u b c l i n i c a l disease. Addition of excess p u r i f i e d leukemia-derived CAMAL to an i n v i t r o myeloid progenitor c e l l assay caused profound i n h i b i t i o n of normal CFU-c growth but had no i n h i b i t o r y e f f e c t on CFU-c growth from myeloid leukemia i i i p a tients i n a c t i v e disease states. Depletion of CAHAL from normal plasma and conditioned media (sources of numerous hemopoietic growth regulatory factors) 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 not myeloid leukemic, CFU-c growth. These r e s u l t s indicated that myeloid leukemic c e l l s possessed apparent differences i n responsiveness to CAMAL-mediated hemopoietic regulation compared to normal c e l l s . Lack of responsiveness to i n h i b i t i o n by leukemia-derived CAMAL may f a c i l i t a t e dominance of the malignant clone over normal c e l l s . i v TABLE OF CONTENTS P a g e CHAPTER I. INTRODUCTION 1 I. Hemopoiesis 1 A. Introduction 1 B. Assays f o r hemopoietic stem c e l l s 4 1. In vivo assays 4 2. In v i t r o assays . . . T 8 C. Regulation of hemopoiesis 15 1. The hemopoietic colony-stimulating factors 16 a. Murine Colony-Stimulating Factors 16 M u l t i p o t e n t i a l Colony-Stimulating Factor (Multi-CSF) 16 Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) .' 23 Macrophage Colony-Stimulating Factor (M-CSF) 24 Granulocyte Colony-Stimulating Factor (G-CSF) 24 Other Murine Regulatory Molecules with GM-CSF A c t i v i t y 25 Megakaryocyte Colony Stimulation 26 Eosinophil D i f f e r e n t i a t i o n Factor 26 Erythroid Colony-Stimulating Factors 26 Lymphocyte Growth Factors 27 i . T C e l l Growth Factor (TCGF) or Inter l e u k i n 2 (IL-2) 27 i i . B C e l l Stimulating Factors (BSF) . . . . 27 b. Human Colony-Stimulating Factors 28 Pluripotent Colony-Stimulating Factor 28 GM-CSF (CSF-a) 29 G-CSF (CSFfl)) 29 M-CSF (Human Urinary Colony-Stimulating Factor 30 Erythropoietin (epo) 30 Erythroid Potentiating A c t i v i t y (EPA) 31 Leukemic B l a s t Growth Factor (LBGF) 31 T C e l l Growth Factor (TCGF) or Interleukin 2 (IL-2) 31 B C e l l Stimulating Factors (BSF) 32 c. D i s t i n c t Membrane Receptors f o r Colony-Stimulating Factors 32 d. Other Functions of the Colony-Stimulating Factors 33 2. Nonspecific Substances with Hemopoietic Growth Promoting A c t i v i t y 34 V Page 3. Negative Granulopoietic Regulators 35 L a c t o f e r r i n 36 A c i d i c I s o f e r r i t i n s . 40 Interferons 41 T r a n s f e r r i n .. 42 E-Type Prostaglandins 43 Leukemia-Associated I n h i b i t o r 43 Tumor Necrosis Factor 44 Granulocytic Chalone 45 Other Reported I n h i b i t o r s of Myelopoiesis . . . . . 45 4. Microenvironmental Influences on Hemopoiesis . . . . 47 I I . THE HUMAN MYELOID LEUKEMIAS 50 A. Chronic Granulocytic Leukemia 50 C l i n i c a l Course and Pathogenesis 50 Stem C e l l O r i g i n 51 Chromosomal Changes and t h e i r Possible S i g n i f i c a n c e . . 52 Relationship Between CGL C e l l s and Growth Regulators . . 54 B. Acute Nonlymphoblastic Leukemia 56 C l i n i c a l Course and Pathogenesis 56 Stem C e l l O r i g i n 58 Chromosomal Changes and Their Possible S i g n i f i c a n c e . . 58 Relationship Between ANLL C e l l s and Growth Regulators 60 C. D i f f e r e n t i a t i o n Induction i n Myeloid Leukemia 61 I I I . Human Myeloid Leukemia-Associated Antigens . . 63 IV. Thesis Objectives 72 CHAPTER I I . MATERIALS AND METHODS 75 I. Detailed Procedures 75 A. Monoclonal Antibodies 75 1. Production 75 2. P u r i f i c a t i o n 77 3. Immunoadsorbent preparation 77 B. Po l y c l o n a l (Rabbit) Antibodies 78 C. Secondary Antibodies 79 v i Page D. Antigens 80 1. CAMAL 80 2. Negative control p r o t e i n antigens 81 E. Polyacrylamide Gel Electrophoresis (PAGE) 81 F. Patient Samples ' 81 G. C e l l Preparations 82 H. Fluorescence-Activated C e l l Sorter (FACS) Studies . . . . 83 1. C e l l Labeling 83 2. FACS Analysis of Labeled C e l l Samples 83 3. FACS C e l l Sorting Technique 84 I. I ndirect Immunoperoxidase Staining of Single C e l l Preparations 87 J. B l i n d Study Protocol 88 1. Patient Group 88 2. Analysis Groups 89 3. S t a t i s t i c a l Analysis 90 K. Myeloid Progenitor C e l l Assay 91 1. General Protocol 91 2. Preparation of Conditioned Medium 92 a. Placental Conditioned Medium (PCM) 93 b. Leucocyte Conditioned Medium (PHA-LCM) 93 3. Preparation of 2% Stock Methylcellulose 94 4. Preparation of Human Plasma 94 5. Preparation of C e l l Samples 96 6. Plucking and Staining of Colonies 96 7. Preparation of Human Plasma f o r CAMAL Depletion Studies 97 8. Preparation of Conditioned Medium f o r CAMAL Depletion Studies 97 9. CAMAL Addition Studies 98 L. S t a t i s t i c a l Analyses 98 CHAPTER I I I . EVALUATION AND DIAGNOSTIC IMPLICATIONS OF A RAPID SLIDE TEST FOR CAMAL 100 I. Introduction 100 I I . Results 102 A. Comparative Immunoperoxidase R e a c t i v i t y Between Monoclonal and Rabbit Antibodies 102 v i i Page B. CAMAL-1 Immunoperoxidase S l i d e Test 105 G. Ap p l i c a t i o n of the Indirect Immunoperoxidase S l i d e Test to the Study of Other Myeloid C e l l Markers 122 CHAPTER IV. CAMAL EXPRESSION IN LEUKEMIA 126 I. Introduction 126 I I . Results 126 A. Morphology of CAMAL-positive C e l l s . 126 1. FACS Sorting Studies 127 2. Immunoperoxidase Studies 136 a. Lack of C o r r e l a t i o n Between BM Bla s t C e l l Numbers and CAMAL BM Value 136 b. Lack of Co r r e l a t i o n Between Regenerative or Ap l a s t i c BM and CAMAL BM Value 136 c. The Morphology of CAMAL-positive C e l l s by Immunoperoxidase 138 B. ANLL Remission Pathology 146 C. The Presence of CAMAL i n Plasma 150 D. CAMAL Adsorption Studies 152 CHAPTER V. SIGNIFICANCE OF CAMAL AS A PROGNOSTIC MARKER FOR REMISSION IN ACUTE N0NLYMPH0BLASTIC LEUKEMIA 163 I. Introduction 163 I I . Results 165 A. Relationship Between Survival Time P r i o r to Relapse and Change i n CAMAL BM Values 166 B. C o r r e l a t i o n Between Decreasing CAMAL BM Values Post-chemotherapy and Longer Remission Times 166 C. CAMAL BM Values and Simultaneous BM Morphology 173 D. Supportive Data from Other ANLL Patients 173 E. Relationship Between F i r s t Remission Length and CAMAL BM Value at Diagnosis 174 F. Increasing CAMAL BM Values and Relapse 174 v i i i Page CHAPTER VI. THE POSSIBLE ROLE OF CAMAL IN MYELOPOIESIS 182 I. Introduction 182 I I . Results 182 A. CAMAL-1 P o s i t i v e Colonies i n CGL 182 B. Presence of CAMAL i n Conditioned Medium 183 C. CAMAL Depletion Studies 189 1. E f f e c t of CAMAL Depletion on Normal Bone Marrow . . . 190 2. E f f e c t of CAMAL Depletion on Normal Peripheral Blood 194 3. E f f e c t of CAMAL Depletion on ALL Bone Marrow . . . . 194 4. E f f e c t of CAMAL Depletion on Chronic Granulocytic Leukemia Peripheral Blood 197 5. E f f e c t of CAMAL Depletion on ANLL 197 D. CAMAL Addition Studies 199 1. E f f e c t of CAMAL Addition on Normal Myeloid Colony Growth 203 2. E f f e c t of CAMAL Addition on Myeloid Leukemia Patients' Colony Growth 205 E. Evidence that Normal and Leukemia-derived CAMAL May Not Be the Same 210 CHAPTER VII. DISCUSSION 213 I. Discussion 213 A. Evaluation and Diagnostic Implications of a Rapid S l i d e Test f o r CAMAL 213 B. CAMAL Expression i n Leukemia 217 C. S i g n i f i c a n c e of CAMAL as a Prognostic Marker f o r Remission i n ANLL 219 D. The Possible Role of CAMAL i n Myelopoiesis 224 I I . Summary and Conclusions 232 Appendix 234 References 239 i x LIST OF TABLES Page I The murine hemopoietic colony-stimulating factors 17-19 II The human hemopoietic colony-stimulating factors 20-22 II I Negative granulopoietic regulators 38 IV Comparison of myelogenous leukemia c e l l r e a c t i v i t y with rabbit anti-CAMAL and CAMAL-1 using immunoperoxidase . . . . 106 V Summary of immunoperoxidase s t a i n i n g r e s u l t s of c e l l s from nonlymphoblastic leukemia patients labeled with CAMAL-1 114 VI Summary of immunoperoxidase s t a i n i n g r e s u l t s of c e l l s from patients with chronic granulocytic leukemia 116 VII Summary of immunoperoxidase s t a i n i n g r e s u l t s of c e l l s from patients with preleukemia/MDS including RAEBIT . . . . 117 VIII Summary of immunoperoxidase s t a i n i n g r e s u l t s of c e l l s from patients with lymphoid malignancies or normals 119 IX FACS s o r t i n g r e s u l t s from two newly diagnosed ANLL patients' peripheral blood samples 128 X FACS s o r t i n g r e s u l t s from three ANLL remission patients' peripheral blood samples 131 XI FACS s o r t i n g r e s u l t s f o r CGL patients' p e r i p h e r a l blood c e l l s 133 XII Lack of c o r r e l a t i o n between the number of b l a s t c e l l s present i n bone marrow and the CAMAL BM value 137 XIII CAMAL BM values i n regenerating bone marrows 139 XIV CAMAL BM values i n non-regenerating bone marrows 140 XV Detection of CAMAL i n plasma by immunoaffinity chromatography 151 XVI Examples of ANLL patients whose CAMAL BM values decreased post-chemotherapy 168 XVII Examples of ANLL patients whose CAMAL BM values increased or remained the same post-chemotherapy 169 XVIII ANLL patients with increasing CAMAL BM values during remission 176 X Page XIX E f f e c t of CAMAL depletion on normal bone marrow 191 XX E f f e c t of CAMAL depletion on normal peripheral blood . . . . 195 XXI E f f e c t of CAMAL depletion on acute lymphoblastic leukemia (ALL) bone marrow 196 XXII E f f e c t of CAMAL depletion on chronic granulocytic leukemia peripheral blood . . . 198 XXIII E f f e c t of CAMAL depletion on ANLL 200 XXIV Summary of CAMAL depletion on myeloid colony growth 201 XXV E f f e c t of CAMAL addition on normal myeloid colony growth . . 204 XXVI E f f e c t of CAMAL addition on myeloid leukemic colony growth 207 XXVII E f f e c t of CAMAL addition on normal peripheral blood CAMAL depleted cultures 211 x i LIST OF FIGURES Page 1.1 Schematic diagram representing hemopoiesis 3 1.2 Interactions between the negative granulopoietic regulators . . 37 2.1 C e l l populations c o l l e c t e d by the fluorescence-activated c e l l s o r t i n g technique 85-86 2.2 Dose response curve f o r conditioned medium (PHA-LCM) on normal peripheral blood CFU-c 95 3.1 Comparative immunoperoxidase r e a c t i v i t y between rabbit anti-CAMAL serum and CAMAL-1 monoclonal antibody on ANLL bone marrow c e l l s 103-104 3.2 Immunoperoxidase s i n g l e c e l l s l i d e t e s t with CAMAL-1 107-111 3.3 ALL remission bone marrow c e l l s labeled by CAMAL-1 monoclonal antibody 121 3.4 Immunoperoxidase s i n g l e c e l l s l i d e t e s t with other myeloid-specific monoclonal antibodies 124-125 4.1 Peripheral blood c e l l s from an ANLL patient at diagnosis sorted on the basis of strong r e a c t i v i t y with rab b i t anti-CAMAL serum 130 4.2 Peripheral blood c e l l s from two CGL patients sorted on the basis of strong r e a c t i v i t y with rab b i t anti-CAMAL serum . . 134-135 4.3 Morphology of CAMAL-l-positive myeloid c e l l s i n ANLL remission patients 142-143 4.4 Lymphocytes from an ANLL remission patients' peripheral blood p o s i t i v e l y labeled by CAMAL-1 144 4.5 Diffuse granular s t a i n i n g of CGL peripheral blood c e l l s by CAMAL-1 145 4.6 FACS so r t i n g study of an ANLL post-bone marrow transplant patient's p e r i p h e r a l blood c e l l s 148-149 4.7 E l u t i o n p r o f i l e of material from normal human plasma bound by a CAMAL-1 immunoadsorbent column 153 4.8a ELISA r e a c t i v i t y of plasma-derived p u r i f i e d CAMAL with CAMAL-1 monoclonal antibody 154 4.8b Polyacrylamide gel electrophoresis of CAMAL from human plasma p u r i f i e d by a f f i n i t y chromatography 154b x i i Page 4.9 Immunoperoxidase s t a i n i n g of an ANLL post-bone marrow transplant patient's peripheral blood c e l l s , labeled with CAMAL-1, one month p r i o r to relapse 155 4.10 Normal peri p h e r a l blood c e l l s , incubated with increasing amounts of CAMAL and labeled by immunoperoxidase 157-160 4.11 Morphology of CAMAL-1 re a c t i v e c e l l s from a normal perip h e r a l blood sample a f t e r incubation with or without CAMAL 161-162 5.1 Kaplan-Meier s u r v i v a l curve showing s u r v i v a l time p r i o r to relapse f o r ANLL patients i n b l i n d study 170 5.2 CAMAL BM values over time f o r two ANLL patients with s i g n i f i c a n t l y decreased values post-chemotherapy 171 5.3 CAMAL BM values over time f o r three ANLL patients whose values increased or were unchanged following treatment . . . . 172 5.4 CAMAL BM values f o r one ANLL patient over the course of h i s disease 177 5.5 CAMAL-1 immunoperoxidase s l i d e t e s t r e s u l t s of peripheral blood c e l l s from an ANLL remission patient showing increasing r e a c t i v i t y 178-179 5.6 CAMAL-1 immunoperoxidase s l i d e t e s t r e s u l t s of peripheral blood c e l l s from an ANLL remission patient showing decreasing r e a c t i v i t y 181 6.1 C e l l s from CGL peripheral blood CFU-c labeled with CAMAL-1 by immunoperoxidase 184-185 6.2 E l u t i o n p r o f i l e of material bound to a CAMAL-1 immunoadsorbent column from p l a c e n t a l conditioned medium . . . 187 6.3 Binding of material from two sources of conditioned media to CAMAL-1 and negative control immunoaffinity columns . . . . 188 6.4 Normal marrow CFU-c from co n t r o l and CAMAL depleted cultures 193 6.5 Summary of the e f f e c t of CAMAL depletion on CFU-c from normals and myeloid leukemics 202 6.6 I n h i b i t i o n of normal marrow CFU-c by addition of p u r i f i e d leukemia-derived CAMAL 206 6.7 E f f e c t of addi t i o n of p u r i f i e d leukemia-derived CAMAL on CFU-c from ANLL patients 208 x i i i Page 6.8 Lack of i n h i b i t i o n of CGL peripheral blood CFU-c by addition of p u r i f i e d leukemia-derived CAMAL 209 x i v ACKNOWLEDGEMENTS The author i s g r a t e f u l f o r the post-doctoral fellowship support given throughout t h i s project by the Medical Research Council of Canada. Special thanks i s also due Dr. J u l i a Levy f o r her guidance, wisdom, and encouragement. A v a i l a b i l i t y of patient samples used throughout these studies depended l a r g e l y on the enthusiasm and cooperation received from collaborators at various i n s t i t u t i o n s . Acknowledgement of t h i s assistance goes to the following: The Department of Hematology, C e l l Separator Unit, and Dr. J . Denegri and the Immunotransplant Laboratory (Vancouver General Hospital) and Drs. A.C. and C. Eaves (Terry Fox Laboratory) f o r ki n d l y providing patient samples; Dr. Martin (St. Paul's Hospital) f o r providing the placenta f o r the preparation of conditioned media; Dr. Noel Buskard (Vancouver General Hospital and Canadian Red Cross) f o r h i s continued support and f o r providing samples from patients and normals; The Department of Pathology and D i v i s i o n of Hematopathology (Vancouver General Hospital) and, i n p a r t i c u l a r , Dr. Sheldon Naiman, f o r h i s expertise i n confirming s l i d e evaluations and f o r the many other ways i n which he provided support and cooperation f o r t h i s project; Professor Peter Isaacson and Keith M i l l e r (Morbid Anatomy, Un i v e r s i t y College, London), Dr. David Swirsky (Cambridge), Dr. Ray Powles (Royal Marsden H o s p i t a l , London), Dr. Dorothy Crawford (Department of Hematology, School of Medicine, U n i v e r s i t y College, London), Dr. Melvyn Greaves and Lynn Altass (ICRF, London), and Dr. A l i s o n Buchan (Department of Physiology, UBC, Vancouver) f o r t h e i r cooperation and advice; Dr. Ian McDonald f o r assistance i n obtaining numerous peripheral blood samples from normal volunters; The author wishes also to thank the following i n d i v i d u a l s f o r providing t r a i n i n g or t e c h n i c a l assistance: Dr. Hans Messner and h i s laboratory s t a f f and researchers, with s p e c i a l thanks to Nazir Jamal (Ontario Cancer I n s t i t u t e , Toronto, Ontario), and Dr. C. Eaves and her s t a f f (Terry Fox Laboratory) 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. Marshal Kadin (Hematopathology, U n i v e r s i t y of Washington) and Dr. Hugh Freeman (Gastroenterology, Department of Medicine, UBC, Vancouver) f o r providing laboratory space and equipment f o r carrying out i n i t i a l immunoperoxidase s t a i n i n g procedures; X V Dr. Dagmar Kalousek (Terry Fox Laboratory, Vancouver) f o r her cytogenetic analyses; Dan Zechini f o r h i s expertise and commitment i n helping to perform the FACS analyses and c e l l s o r t i n g procedures; Stephen Whitney, f o r h i s t e c h n i c a l help i n carrying out the immunoperoxidase s l i d e t e s t procedures; Other members of t h i s p roject, who have helped me by t h e i r support, advice and cooperation, including Dr. Andrew Malcolm, Dr. Robert Shipman, and Joan Shellard. Research support was also provided by the National Cancer I n s t i t u t e of Canada and the Medical Research Council of Canada. x v i LIST OF ABBREVIATIONS Abbreviations below are l i s t e d i n the order i n which they appear i n the t h e s i s . ANLL acute nonlymphoblastic leukemia CAMAL common antigen i n myelogenous (acute) leukemia BM bone marrow PB periph e r a l blood CAMAL BM value % CAMAL-1 p o s i t i v e c e l l s NK natural k i l l e r NC natural cytotoxic CFU-S colony-forming unit-spleen CFU-c colony-forming u n i t - c u l t u r e BFU-E burst-forming u n i t , erythroid CFU-E colony-forming u n i t , erythroid CFU-mega colony-forming u n i t , megakaryocyte CFU-GM colony-forming u n i t , granulocyte-macrophage CFU-eos colony-forming u n i t , eosinophil CFU-mast colony-forming u n i t , mast c e l l CFU-TL colony-forming u n i t , T lymphocyte CFU-BL colony-forming u n i t , B lymphocyte CFU-GEMM mixed colonies containing granulocytes, macrophages, megakaryocytes and erythroid CGL chronic granulocytic leukemia Ph 1 P h i l a d e l p h i a chromosome G-6-PD glucose-6-phosphate dehydrogenase CSF colony-stimulating f a c t o r IL Interleukin PSF p e r s i s t i n g c e l l stimulating f a c t o r HCGF hemopoietic c e l l growth fa c t o r BPA burst promoting a c t i v i t y MCGF mast c e l l growth f a c t o r SAF stem c e l l a c t i v a t i n g f a c t o r MG1 macrophage-granulocyte inducer CSA colony-stimulating a c t i v i t y DF d i f f e r e n t i a t i o n f a c t o r BSF B c e l l stimulatory f a c t o r BCGF B c e l l growth fa c t o r BGDF 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 BCDF 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 TRF T c e l l r eplacing f a c t o r GM granulocyte-macrophage G granulocyte M macrophage KD k i l o d a l t o n SCM spleen conditioned medium epo erythropoietin NIF-T neutrophil i n h i b i t o r y f a c t o r , derived from T c e l l s TCGF T c e l l growth f a c t o r GM-EA granulomonopoietic enhancing a c t i v i t y IFN i n t e r f e r o n xvii LF lactoferrin TF transferrin TNF tumor necrosis factor AIF acidic isoferritin LIA leukemia-associated inhibitory activity PGE prostaglandin E LAI leukemia-associated inhibitor CIL colony-inhibiting lymphokine PMN polymorphonuclear leucocyte MHC major histocompatability complex LAA leukemia-associated antigens FAB French-American-British ALL acute lymphoblastic leukemia CD cluster determinant FAL 3-a-fucosyl-N-acetyllactosamine LFA leucocyte function antigen CGL MBC/LBC chronic granulocytic leukemia, myeloid blast crisis/lymphoid blast crisis ELISA enzyme linked immunosorbent assay FACS fluorescence-activated cell sorter MAb monoclonal antibody CAMAL-1 monoclonal antibody specific for CAMAL PAGE polyacrylamide gel electrophoresis BLV bovine leukosis virus PEG polyethylene glycol DEAE diethyl amino ethyl Ig immunoglobulin FIT C fluorescein isothiocyanate HRP horseradish peroxidase FCS fetal calf serum SEM standard error of the mean CR complete remission PCM placental conditioned medium • PHA-LCM phytohemagglutin-lymphocyte conditioned medium MC methylcellulose BMT bone marrow transplant MDS myelodysplastic syndrome RAEBIT refractory anemia with excess blasts in transformation CLL chronic lymphocytic leukemia rbc erythrocytes PBS phosphate buffered saline LDH lactic dehydrogenase L acute leukemia R remission (R) relapse D dead T (in Fig 5 .4 ) bone marrow transplant CM conditioned medium 1 CHAPTER I INTRODUCTION I. HEMOPOIESIS A. Introduction Hemopoiesis r e f e r s to the formation and development of blood c e l l s . The control of t h i s process involves an i n t r i c a t e l y balanced set of in t e r a c t i o n s between various c e l l s and growth regulatory factors whose ultimate r o l e i s the maintenance of a homeostatic s i t u a t i o n within the i n d i v i d u a l . The changing requirements (increased or decreased demands) f o r any p a r t i c u l a r type of blood c e l l may thus be met i n any number of s i t u a t i o n s including normal c e l l death or consumption/loss r e s u l t i n g from i n f e c t i o n , inflammation, hemorrhage or other. Under normal conditions, the 9 production rate f o r the n e u t r o p h i l i c leucocyte alone i s 1.6 x 10 cells/kg/day i n the human (1). This huge number r e l a t e s d i r e c t l y to the f a c t that the h a l f - l i f e of mature c i r c u l a t i n g neutrophils i s only 6 - 7 hours (2,3). There are two broad groups of hemopoietic c e l l populations. These are: 1. myeloid c e l l s , including granulocytic, monocytic, megakaryocytic, and erythroid c e l l s , 2. lymphoid c e l l s , including B and T lymphocytes. Some c e l l s which reside i n various organs and tissues (non-circulating) were also o r i g i n a l l y derived from hemopoietic 2 precursors; these c e l l s include the f i x e d t i s s u e mast c e l l s and progeny of the monocytic s e r i e s , i e . Kupffer c e l l s ( l i v e r ) , Langerhans c e l l s ( s k i n ) , osteoclasts (bone), m i c r o g l i a (brain) and a l v e o l a r macrophages (lung). Natural k i l l e r (NK), natural cytotoxic (NC), non-B non-T (nul 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 found i n lymphoid t i s s u e s are l i k e l y the progeny of hemopoietic c e l l s as w e l l . From the yolk sac blood islands of the embryo, hemopoietic stem c e l l s migrate to the f e t a l l i v e r where they undergo rapid expansion and d i f f e r e n t i a t i o n . A f t e r b i r t h , hemopoiesis occurs i n the marrow of a l l bones. The demands f o r hemopoiesis decreases with maturity. As a r e s u l t , hemopoiesis i n adults takes place i n more r e s t r i c t e d locations ( p r i m a r i l y within the medullary space of f l a t bones and the epiphyses of long bones), although i n s i t u a t i o n s of extreme need, extramedullary hemopoiesis may occur i n the spleen, l i v e r and lymph nodes. The majority of c i r c u l a t i n g blood c e l l s represent the mature or end stages of d i f f e r e n t i a t i o n of hemopoietic c e l l s . I n i t i a l l y these c e l l s are thought to have ar i s e n from precursors within the bone marrow known as stem c e l l s . The stem c e l l concept i s c e n t r a l to our understanding of hemopoiesis. Stem c e l l s have the capacity to p r o l i f e r a t e extensively and to s e l f - r e p l i c a t e as well as to 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 f o r maturation are d i f f e r e n t , hence some are more "committed" than others to development along a p a r t i c u l a r pathway. A schematic representation of t h i s concept i s shown i n Figure 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 are r e f e r r e d to as pluripotent stem c e l l s since they are capable of producing stem c e l l progeny of 3 Figure 1.1. Schematic diagram representing hemopoiesis. Modified from references 4 , 5 . CFU-S I = colony-forming u n i t — spleen which form b l a s t c e l l colonies CFU-S II = colony-forming u n i t — spleen which form mixed colonies CFU-c = colony-forming u n i t (culture) BFU-E = burst-forming u n i t , e r y t h r o i d CFU-E = colony-forming u n i t , e r y t h r o i d CFU-mega = colony-forming u n i t , megakaryocyte CFU-GM = colony-forming u n i t , granulocyte-macrophage CFU-eos = colony-forming u n i t , eosinophil CFU-mast = colony-forming u n i t , mast c e l l 4 both the myeloid and lymphoid lineages. These i n turn give r i s e to more d i f f e r e n t i a t e d progenitor c e l l s within each lineage. As d i f f e r e n t i a t i o n along the developmental pathway occurs, there i s a concomitant decrease i n the capacity f o r self-renewal u n t i l t h i s capacity i s l o s t and fun c t i o n a l end-stage c e l l s are produced. This scheme of hemopoiesis i s thought to be pyramidal i n nature, involving an a m p l i f i c a t i o n of c e l l numbers at successive stages of d i f f e r e n t i a t i o n . B. ASSAYS FOR HEMOPOIETIC STEM CELLS 1. IN VIVO ASSAYS The hemopoietic stem c e l l concept as outlined i n Figure 1.1 has ar i s e n from both speculation and experimentation. Speculation that the mature fun 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 bloodstream arose from the d i f f e r e n t i a t i o n of p r i m i t i v e ancestral c e l l s located i n the bone marrow (BM) came o r i g i n a l l y from pathologists such as Maximow, Downey and Osgood (6-8). The groundwork f o r the s c i e n t i f i c i n v e s t i g a t i o n of hemopoiesis was l a i d i n 1961 by T i l l and McCulloch (9) when they described the spleen colony-forming 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 infused intravenously with BM c e l l s and, within 7 - 1 4 days, v i s i b l e d i s c r e t e colonies of x hemopoietic c e l l s were observed i n the spleens of these mice. The c e l l s presumed to be responsible f o r the production of these colonies were named "colony-forming u n i t — spleen" or CFU-S. That the CFU-S were c l o n a l i n o r i g i n (derived from a s i n g l e c e l l ) was demonstrated by the use of donor bone marrow with 5 radiation-induced gross chromosomal markers (10,11). In these in v e s t i g a t i o n s , a l l of the progeny of a s i n g l e CFU-S contained the same genetic marker, proving that they originated from a si n g l e clone. When the c e l l s within i n d i v i d u a l colonies were examined microscopically, i t was found that some CFU-S formed multilineage colonies c o n s i s t i n g of erythroid, megakaryocytic and granulocytic c e l l s ( i n d i c a t i n g that the karyotypic markers arose i n a very p r i m i t i v e stem c e l l ) while others formed only granulocytic or erythroid progeny ( i n d i c a t i n g that the markers originated i n less p r i m i t i v e c e l l s ) . The self-renewal capacity of CFU-S was shown by retransplantation studies (12). Even s i n g l e CFU-S co n s i s t i n g morphologically of erythroid progeny could, upon retransplantation, form colonies containing progeny of s i n g l e or mixed lineage. The f a c t that the t o t a l hemopoietic c e l l population could be reconstituted from the progeny of a s i n g l e CFU-S (more l i k e l y a subset of CFU-S) was strong i n d i c a t i o n f o r the p l u r i - or m u l t i p o t e n t i a l i t y of t h i s p r i m i t i v e stem c e l l (13,14). While some of the i n vivo CFU-S studies have strongly suggested that at l e a s t some CFU-S may be p l u r i p o t e n t i a l , there has always been much controversy concerning t h i s i n t e r p r e t a t i o n and many investigators b e l i e v e that CFU-S are myelopoietic stem c e l l s without lymphopoietic p o t e n t i a l . This uncertainty i s r e f l e c t e d i n Figure 1.1 by a dotted l i n e from the pl u r i p o t e n t to lymphoid stem c e l l . I t i s u n l i k e l y that the standard CFU-S assay alone w i l l ever resolve t h i s point. The f a c t that t h i s 6 assay involves a r e l a t i v e l y short duration and one which occurs i n a s p l e n i c microenvironment may help to explain the lack of observed lymphoid c e l l s within spleen colonies. The absence of a thymic microenvironment could c e r t a i n l y explain the lack of T-lymphocytes i n these colonies. While B-lymphocytes themselves have not been reported i n splenic colonies, c e l l s capable of forming B c e l l progeny have been (15). Other investigators have disagreed with these findings (16). I t has also been shown that most of the stem c e l l s i n j e c t e d into 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 rather than the spleen (5). Most l i k e l y a very 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 while most CFU-S are r e s t r i c t e d to myelopoiesis, at l e a s t under the conditions imposed by the routine spleen colony assay. Evidence supporting t h i s p o s s i b i l i t y came from studies of radiation-induced chromosomal markers i n r a t s , where the the same marker was sometimes found to be present i n both r e c i p i e n t erythroid spleen colonies and donor peri p h e r a l blood lymphocytes, i n d i c a t i n g that both c e l l types were the progeny of the same donor stem c e l l (17).* Convincing evidence e x i s t s i n d i c a t i n g that there i s a good deal of heterogeneity within CFU-S populations (18-20). This i s easy to imagine i f one views the e n t i r e process of hemopoiesis as a multitude 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 d i f f e r e n t i a t i o n s . The e a r l i e s t (most primitive^ or undifferentiated) c e l l s would have the greatest capacity f o r self-renewal and would produce greater numbers of progeny with l i m i t e d degrees of commitment along a s p e c i f i e d lineage and 7 hence increased p r o l i f e r a t i v e capacity. C e l l s further along the hemopoietic pathway would gradually lose t h e i r a b i l i t y to self-generate as they gained greater degrees of d i f f e r e n t i a t i o n u n t i l becoming mature f u n c t i o n a l blood c e l l s . The developmental pathway from stem c e l l to progenitor c e l l to morphologically recognizable immature to mature blood c e l l (as shown i n Figure 1.1) would thus occur i n a gradual ser i e s of "steps". This i s u s e f u l to consider when examining Figure 1.1 as well as the l i t e r a t u r e pertaining to hemopoietic colony assays. In vivo studies using the g e n e t i c a l l y anemic (stem c e l l v d e f i c i e n t ) mouse s t r a i n W/W , which have very low numbers of CFU-S, showed that t h i s defect could be cured by r e c o n s t i t u t i o n of these mice with normal syngeneic marrow containing radiation-induced markers or with marrow from another mutant s t r a i n , S l / S l , which had normal hemopoietic stem c e l l s but an abnormal "microenvironment" (21,22). These studies were paramount i n our understanding of the stem c e l l concept i t s e l f as well as some of the factors that influence stem c e l l r e gulation. Another i n vivo system, the d i f f u s i o n chamber culture, has been developed i n order to investigate the e f f e c t of long-range humoral factors on hemopoiesis (23-28). In t h i s system, mice (or other species) are implanted i n t r a p e r i t o n e a l l y with d i f f u s i o n chambers containing hemopoietic c e l l s i n medium with serum or plasma i n a plasma-clot or semi-solid agar support. The chambers allow free d i f f u s i o n of humoral factors to the ins i d e without permitting t r a n s f e r of c e l l s . Normal and 8 malignant hemopoietic c e l l s from t i s s u e c u l t u r e , bone marrow, periph e r a l blood, spleen, yolk sac, and f e t a l l i v e r have been cultured i n t h i s manner. A f t e r 7 - 1 4 days of growth, d i f f u s i o n chambers are removed and the c e l l s i n s i d e examined f o r colony (CFU-d) formation, enumeration, or subculture into i n v i t r o progenitor c e l l assay systems (the l a t t e r assay w i l l be discussed i n the following s e c t i o n ) . S i m i l a r i t i e s and differences i n responsiveness to a number of substances a f f e c t i n g hemopoietic c e l l s have been observed between bone marrow i n s i t u or i n v i t r o and within d i f f u s i o n chamber cultures (29-34). Moreover i t i s thought that d i f f u s i o n chamber granulocytic progenitors are 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 progenitors forming granulocytic colonies i n v i t r o (35). While these differences between d i f f u s i o n chamber cultures and other i n vivo or i n v i t r o hemopoietic stem c e l l assays c l e a r l y e x i s t , the d i f f u s i o n chamber culture technique i s s t i l l regarded by many to be the best system i n which to study the e f f e c t of long-range soluble factors on hemopoiesis i n vivo. The i n vivo assays f o r hemopoietic stem c e l l s l a i d the groundwork f o r our understanding of the complex nature of hemopoiesis. More recently, i n v i t r o studies have been developed which have r a p i d l y expanded our conceptual view of hemopoiesis as outlined i n Figure 1.1. 2. IN VITRO ASSAYS In 1966, two groups of inv e s t i g a t o r s developed an i n v i t r o technique capable of growing murine hemopoietic colonies (34,35). This methodology was r a p i d l y extended to the study of 9 human hemopoietic colonies i n v i t r o (36). The technique involves the growth of d i s c r e t e colonies, derived from i n d i v i d u a l hemopoietic precursor c e l l s , i n a semi-solid support medium (agar, methylcellulose or plasma clot-based) containing growth and serum f a c t o r s . The v i s c o s i t y of the support medium maintains c e l l s derived from an i n d i v i d u a l clonogenic precursor i n close proximity, allowing separate colonies to be enumerated, and analyzed. In 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 multilineage-associated progenitors i n the mouse and human have been developed (17-34). Colony-forming progenitor c e l l s have been given the general name CFU-c (colony-forming u n i t - c u l t u r e ) and include erythroid and granulocytic progenitors (Figure 1.1). Large erythroid progenitors or the colonies derived from them have been termed BFU-E (burst-forming u n i t s — ery t h r o i d ) ; t h e i r smaller, more mature progenitors have been termed CFU-E (colony-forming u n i t — e r y t h r o i d ) . Other myeloid progenitors have been named CFU-mega; CFU-GM, CFU-eos or CFU-mast to describe t h e i r p a r t i c u l a r lineage commitment (see Figure 1.1). While the terminology j u s t described i s i n common use, d i f f e r e n t terms have been proposed by various l a b o r a t o r i e s . The culture of T and B-lymphoid-committed progenitors (CFU-TL, CFU-BL) has also been developed (42,43,49,51) but most investigators b e l i e v e the majority of c e l l s forming these lymphoid colonies i n v i t r o are more mature than the myeloid 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 colonies v a r i o u s l y composed of granulocytes, macrophages, megakaryocytes, 10 and erythroid c e l l s (CFU-GEMM). Mixed colonies containing mast c e l l s , eosinophils, and lymphoid c e l l s (usually T-lymphocyte) have also been reported (53-58). These mixed colonies may contain CFU-S as well as m u l t i p o t e n t i a l and single-lineage progenitors of a v a r i e t y of commitment pathways (44,45,59,60). Extremely 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 c e l l s with b l a s t c e l l morphology have also been grown i n v i t r o (61-63); these colonies contain a large number of CFU-S as well 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 progenitor c e l l s . Evidence f o r the existence of a p l u r i p o t e n t i a l (with both myeloid and lymphoid p o t e n t i a l ) stem c e l l i n the mouse has already been discussed i n the previous section. In the human, evidence to support t h i s concept has come from studies involving neoplasms of hematopoietic c e l l s . In 90% of patients with chronic granulocytic leukemia (CGL), BM c e l l s contain a c h a r a c t e r i s t i c gross chromosomal t r a n s l o c a t i o n t(9;22) r e f e r r e d to as the Phi l a d e l p h i a (Ph 1) chromosome (66,67). The Ph 1 chromosome has been demonstrated i n nucleated erythroid c e l l s , granulocytes, monocytes, megakaryocytes and probably B-lymphocytes (68-71) i n d i c a t i n g that the karyotypic abnormality arose i n a stem c e l l common to both myeloid and lymphoid c e l l s . Further evidence f o r the c l o n a l i t y of t h i s disorder as well 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 studies. Glucose-6-phosphate dehydrogenase (G-6-PD) i s a polymorphic i n t r a c e l l u l a r enzyme with two isoenzyme types. The locus f o r G-6-PD i s on the X chromosome and since one X chromosome undergoes random i n a c t i v a t i o n during embryogenesis i n females, 11 i n d i v i d u a l c e l l s from G-6-PD heterozygous females w i l l produce one or the other G-6-PD isoenzyme but not both. The t o t a l c e l l population i n such a G-6-PD heterozygote w i l l , therefore, reveal a 50:50 isoenzyme synthesis p r o f i l e under normal circumstances. Fialkow et a l . have demonstrated that female G-6-PD heterozygotes with CGL (and some with acute nonlymphoblastic leukemia) had a si n g l e isoenzyme type i n many of t h e i r blood c e l l s (72-74). In chronic granulocytic leukemia, these c e l l s were shown to include myeloid and lymphoid (B) c e l l s . In acute myelogenous leukemia, a si n g l e G-6-PD isotype was found i n both leukemic leucocytes and erythrocytes. I n t e r e s t i n g l y , while CGL has been shown to 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 , the massive expansion of hemopoietic c e l l s occurs p r i m a r i l y within the granulocyte-monocyte lineage. Further evidence f o r the existence of a common stem c e l l f o r both the myeloid and lymphoid lineages i n the human came from G-6-PD analysis of such c e l l s from mixed colonies (75). In addition to the evidence already presented f o r m u l t i p o t e n t i a l colonies i n v i t r o , there i s indisputable evidence f o r the c l o n a l i t y of colonies: (a) micromanipulation and time-lapse cinematography techniques have shown that m u l t i p o t e n t i a l and s i n g l e lineage colonies of a v a r i e t y of types can be grown from s i n g l e c e l l s (46,76,77). (b) granulocyte-macrophage and erythroid colonies from G-6-PD heterozygotes were shown to possess only a si n g l e isoenzyme type (78,79). 12 (c) mixed cultures of male and female c e l l s were shown by karyotypic analysis to form e i t h e r "male" or "female" colonies (80,81). The evidence that progenitor c e l l s which form colonies i n v i t r o are indeed the progeny of stem c e l l s observed i n the i n vivo assays i s as follows. (a) I n d i v i d u a l CFU-S form spleen colonies containing 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 of murine BM c e l l s can i s o l a t e populations that contain stem c e l l s but no progenitor c e l l s . This f r a c t i o n could, however, form spleen colonies containing progenitor c e l l s (82). (c) I t i s possible to generate murine progenitor c e l l s i n fra c t i o n a t e d f e t a l l i v e r suspension cultures that contain stem c e l l s but no progenitor c e l l s (83). Murine clonogenic hemopoietic precursor c e l l s (stem and progenitor c e l l s ) are present at a frequency of about 1.5% i n BM, the r i c h e s t source of such c e l l s (54). Spleen and peripheral blood (PB) also contain colony-forming c e l l s , a l b e i t at much lower frequencies. Numerous c e l l f r a c t i o n a t i o n techniques have been employed to separate and characterize stem and progenitor c e l l populations and subsets thereof. These techniques have included adherence to glass beads (84), density gradient separation (84-89), v e l o c i t y sedimentation (90-95), electrophoresis (96), fluorescence-activated c e l l s o r t i n g (97) and mitogen-binding (19,20,83,98). Separation and cha r a c t e r i z a t i o n of CFU-S subsets, progenitor c e l l s and CFU-E 13 have been possible u t i l i z i n g these techniques. Evidence that the clonogenic progenitor c e l l s i d e n t i f i e d i n v i t r o are, i n fa c t , the c e l l s responsible f o r the production of mature, fu n c t i o n a l end-stage blood c e l l s comes from a number of studies. (a) granulocyte-macrophage and erythroid colony-forming c e l l s are i n active c e l l c ycle i n vivo (54). (b) the frequency and p r o l i f e r a t i v e capacity of granulocyte-macrophage and erythroid colony-forming c e l l s i s s u f f i c i e n t to account f o r the required number of erythroid c e l l s and polymorphonuclear leucocytes i n vivo under steady-state conditions. (c) during states of p h y s i o l o g i c a l disturbance ( f o r example, act i v e regeneration of granulocytes and monocytes following i r r a d i a t i o n or other cytotoxic events, and following hypertransfusion) colony l e v e l s f l u c t u a t e i n corresponding fashion. The i n v i t r o clonogenic progenitor c e l l assay j u s t described has enormously increased our understanding of hemopoiesis and i t s regulatory f a c t o r s . The vast majority of colonies formed i n the semi-solid culture technique are derived from progenitors with r e l a t i v e l y advanced lineage commitment (fo r example, CFU-GM, CFU-E, GM cluster-forming c e l l s ) . CFU-GEMM (mixed) colony-forming c e l l s are c e r t a i n l y detectable and these c e l l s are l i k e l y to be the i n v i t r o counterpart of a CFU-S subset (CFU-S-II) (54,61,98-100). The c e l l thought to 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 v i t r o 14 i n the mouse (63,64) and i n human u m b i l i c a l cord blood (65). With the development of the long term bone marrow culture technique, i t became evident that p l u r i p o t e n t stem c e l l s that could form CFU-S could be maintained and were capable of r e c o n s t i t u t i n g the e n t i r e hemopoietic system i n i r r a d i a t e d mice (101-105). The basis f o r the long term maintenance of p r i m i t i v e hemopoietic c e l l s lay i n the formation i n culture of a stromal adherent c e l l layer (106,107). This stromal layer contained (pre-)adipocytes, f i b r o b l a s t - l i k e endothelial c e l l s , osteoblasts, reticulum and mesenchymal c e l l s and macrophages with associated hemopoietic c e l l s of a l l degrees of d i f f e r e n t i a t i o n . P r o l i f e r a t i o n of stem c e l l s i n the adherent layer could be maintained f o r up to months under c o n t r o l l e d conditions. The applications of the continuous bone-marrow culture technique have been reviewed (108) and include: (a) i n v e s t i g a t i o n of the e f f e c t of chemotherapeutic drugs and i r r a d i a t i o n on stromal c e l l exposure p r i o r to bone marrow transplantation (b) the study of hormonal influences ( c o r t i c o s t e r o i d s , peptides, growth factors) on hemopoiesis (c) the carcinogenic e f f e c t of viruses, i r r a d i a t i o n and chemical carcinogens on hemopoiesis (d) i n v e s t i g a t i o n of the influence of genetics on hemopoiesis (e) the establishment of factor-dependent and -independent hemopoietic c e l l l i n e s . Evidence supporting the concept of hemopoiesis as outlined i n Figure 1.1 has been presented. At t h i s point i t i s 15 appropriate to describe the nature of the regulatory molecules or factors that influence t h i s process. C. REGULATION OF HEMOPOIESIS During normal hemopoiesis, i n t e r a c t i o n s occur between stimulatory and i n h i b i t o r y molecules, the c e l l s that produce them and t h e i r target c e l l s , so that p h y s i o l o g i c a l l y appropriate numbers of blood c e l l s are produced. This complex i n t e r p l a y i s often disrupted during a c t i v e leukemic states, r e s u l t i n g i n the suppression of normal hemopoiesis. At present, there i s no t o t a l l y s a t i s f a c t o r y explanation f o r the complete suppression of normal myeloid progenitor c e l l s that occurs i n acute nonlymphoblastic leukemia (ANLL). This suppression takes place i n s p i t e of the f a c t that leukemic c e l l s have longer c e l l cycle times than do normal c e l l s . Simple explanations inv o l v i n g the "crowding out" of normal myeloid progenitors by the leukemic clone cannot answer the fundamental question of how the leukemic c e l l s gained a competitive advantage i n the f i r s t place. This i s an act i v e area of research at the present time. In order to appreciate the s i g n i f i c a n c e of the many factors regulating hemopoiesis, i t i s necessary to consider the roles played by 1) the hemopoietic colony-stimulating f a c t o r s , 2) other nonspecific molecules including serum components, 3) negative regulators, and 4) microenvironmental influences. While some factors regulating lymphopoiesis w i l l be described f o r the sake of completeness, i t i s not within the scope of the objectives of t h i s t h e s i s to cover t h i s aspect of hemopoiesis i n d e t a i l . Consequently, the emphasis w i l l f a l l on regulation of myelopoiesis. 16 1. THE HEMOPOIETIC COLONY-STIMULATING FACTORS The term "colony-stimulating f a c t o r " or CSF derives 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 of regulatory molecules shown to cause p r o l i f e r a t i o n of hemopoietic c e l l s r e s u l t i n g i n the formation of recognizable colonies i n v i t r o . Once i t had been recognized that a number of CSFs existed with a c t i v i t y on d i f f e r e n t progenitor c e l l lineages, terminology arose designed to i d e n t i f y these various CSFs. Often a p r e f i x or s u f f i x i s used to indic a t e which hemopoietic c e l l lineage(s) i s stimulated by the CSF (Tables I and I I ) . A l l of the well characterized CSFs are glycoproteins with reported molecular weights ranging from 15 - 70 KD. They are b i o l o g i c a l l y a c t i v e -11 -13 at extremely low m o l a r i t i e s (10 to 10 M). A l l are produced by a v a r i e t y of c e l l types and have functions other than stimulating the p r o l i f e r a t i o n of hemopoietic precursor c e l l s . These other functions include target c e l l s u r v i v a l , i n v i t r o induction of d i f f e r e n t i a t i o n (commitment to a p a r t i c u l a r c e l l lineage) and stimulation of a v a r i e t y of functions i n mature end stage c e l l s . a) MURINE COLONY-STIMULATING FACTORS M u l t i p o t e n t i a l Colony-Stimulating Factor (Multi-CSF) Multi-CSF i s produced by mitogen-stimulated T - c e l l s , cloned T c e l l l i n e s , c e r t a i n T lymphomas and hybridomas as well as by the murine myelomonocytic leukemia c e l l l i n e , WEHI-3B. The most common source f o r t h i s f a c t o r had been conditioned medium from pokeweed mitogen-stimulated mouse splenic lymphocytes (SCM) or Table 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 C o l o n y - s t i m u l a t i n g A l t e r n a t e P r o g e n i t o r Molecular P u r i f i e d Cloned A c t i v i t y on References f a c t o r nomenclature t a r g e t c e l l ( s ) weight human c e l l s M u l t i - C S F IL-3.PSF.HCGF, CFU-S,blast (stem) 23 KD \/ y/ X" 109-118 BPA,MCGF,SAF c e l l s . m u l t i p o t e n t stem cells,GM,G,M, eosinophil,mast °cell.megakaryocyte and e r y t h r o i d pro-g e n i t o r s GM-CSF MGI-1GM.CSA- GM,G,M p r o g e n i t o r s , 23 KD </ \/ * 119-123 GM i n i t i a l stages of e r y t h r o i d p r e c u r s o r s , f e t a l e o s i n o p h i l p r o g e n i t o r s * M-CSF CSF-1,MGI-1M, M ( p r i m a r i l y ) pro- 60-70 KD y/ X X 123-129 CSA-M . g e n i t o r s , some GM, (dimer) G p r o g e n i t o r s 28-35 KD (mono-mer) G-CSF MGI-1G.DF G ( p r i m a r i l y ) pro- 25 KD X (G) 123,130-g e n i t o r s , M only 133 at h i g h concentra-t i o n s , weak a c t i v i t y on i n i t i a l stages of 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 e c u r s o r s Table 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 (cont'd) C o l o n y - s t i m u l a t i n g A l t e r n a t e f a c t o r nomenclature P r o g e n i t o r t a r g e t c e l l ( s ) M o lecular weight P u r i f i e d Cloned A c t i v i t y on References human c e l l s 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 e o s i n o p h i l p r e c u r s o r s 46 KD ND 134 E r y t h r o p o i e t i n epo CFU-E 40-45 KD p a r t i a l 135 T c e l l growth f a c t o r TCGF, IL-2 a c t i v a t e d T c e l l s 21-30 KD 136-141 B c e l l s t i m u l a t o r y a)IL-4,BCGF-l, f a c t o r s (BSF) BSF p i b)BGDF,BCGF I I c)BCDF,TRF a c t i v a t e d B c e l l s ( p r o l i f e r -a t i o n ) 11-22 KD 20 KD (recombinant IL-4) a c t i v a t e d B c e l l s ( p r o l i f e r -a t i o n and Ig s e c r e t i o n ) induces d i f f e r e n -t i a t i o n ( I g produc-t i o n ) i n a c t i v a t e d , p r o l i f e r a t i n g B c e l l s 40-55 KD p a r t i a l ND p a r t i a l X X X V 142-150 Neuroleukin i n d u c t i o n of I g s e c r e t i o n i n B c e l l s 56 KD 151,152 !—1 T a b l e I . T h e m u r i n e 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 ) * r e c o m b i n a n t m u r i n e G M - C S F a t v e r y 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 t h e p r o l i f e r a t i o n o f e o s i n o p h i l , m e g a k a r y o c y t a n d e r y t h r o i d p r e c u r s o r s . A b b r e v i a t i o n s : '•. 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 ; H C G F , 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 ; M C G F , mast c e l l g r o w t h f a c t o r ; S A F , s tem 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 ; B C G F , B c e l l g r o w t h f a c t o r ; B G D F , 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 , granulocyte; M, macrophage. Table 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 l o n y - s t i m u l a t i n g A l t e r n a t e P r o g e n i t o r Molecular P u r i f i e d Cloned A c t i v i t y on References f a c t o r nomenclature t a r g e t c e l l ( s ) weight murine c e l l s IL-3 M u l t i - C S F B l a s t c e l l s , m u l t i -l i n e a g e myeloid c o l o n i e s , e r y t h r o i d , G, M, GM, e o s i n o p h i l , b a s o p h i l , megakaryo-cyte p r o g e n i t o r s 14-28 KD ND P l u r i p o i e t i n GM-CSF G-CSF M-CSF P l u r i p o t e n t CSF CSF<*, GM-CSF-<x, NIF-T Leukemic b l a s t growth f a c t o r CSFyg CSF-1, human u r i n e CSF LBGF 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 p r o g e n i t o r s 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 ** GM progeni t o r s ( p r i m a r i l y G) 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 B l a s t c e l l s 18 KD 14-35 KD 18-22 KD 35-45 KD, 18-26 KD horaodimers y y y y y y y ( d i f f e r e n t i a -t i o n o f WEHI-3B c e l l s ) 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 164-166 30 KD p a r t i a l ND 162,167 t o O Table 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 (cont'd) C o l o n y - s t i m u l a t i n g A l t e r n a t e f a c t o r nomenclature P r o g e n i t o r t a r g e t c e l l ( s ) M olecular P u r i f i e d Cloned A c t i v i t y on References weight murine c e l l s E r y t h r o p o i e t i n 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 T c e l l growth f a c t o r epo EPA TCGF, IL-2 B c e l l s t i m u l a t o r y a)BSF 1,BCGF-f a c t o r s (BSF) I b)BGDF,BCGF-I I c)BCDF,TRF Neu r o l e u k i n CFU-E BFU-E, CFU-E a c t i v a t e d T c e l l s a c t i v a t e d B c e l l s ( p r o l i f e r a t i o n ) a c t i v a t e d B c e l l s ( p r o l i f e r a t i o n and I g s e c r e t i o n ) I g s e c r e t i o n i n a c t i v a t e d , p r o l i -f e r a t i n g B c e l l s i n d u c t i o n o f I g s e c r e t i o n i n B c e l l s 39 KD 28 KD 15 KD y y y y 150-200 KD p a r t i a l * 60-80 KD (reported) > 60 KD p a r t i a l X 35 KD p a r t i a l X 56 KD y/ • (murine) (CFU-E) (BFU-E,CFU-E) y 168-170 171,172 136, 173-176 177-181 151,152 * Yang Y-C, C i a r l e t t a AB, Temple PA, Chung MP, Kovacic S, W i t e k - G i a n n o t t i J S , Leary AC, K r i z R, Donahue RE, Wong GG, C l a r k SC: Human IL-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 growth f a c t o r r e l a t e d to murine I L - 3 . C e l l 47:3, 1986. ** recombinant human GM-CSF at high 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, Tsuchiya M, Asano S, K a z i r o Y, Yamazaki T, Yamamoto 0, H i r a t a Y, Kubota N, Oheda M, Nomura H, Ono M: Mo 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 of cDNA f o r human granulocyte c o l o n y - s t i m u l a t i n g f a c t o r . Nature 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 f a c t o r s (cont'd) A b b r e v i a t i o n s : CSF, colony-stimulating factor; NIF-T, neutrophil inhibitory factor - derived from T cells; IL, interleukin; BCGF, B c e l l growth factor; BCDF, B c e l l differentiation factor; BGDF, B c e l l growth and differentiation factor; TRF, T c e l l replacing factor; GM, granulocyte-macrophage; G, granulocyte; M, macrophage. 23 WEHI-3B c e l l s u n t i l the a v a i l a b i l t i y of the cloned recombinant molecule (115). Multi-CSF stimulates the i n v i t r o formation of b l a s t c e l l , granulocyte (CFU-G), macrophage (CFU-M), granulocyte-macrophage (CFU-GM), eosinophil (CFU-eos), mast c e l l (CFU-mast), megakaryocyte (CFU-mega), erythroid (BFU-E and CFU-E), and mixed (CFU-GEMM) colonies from murine BM. Addition of erythropoietin (epo) enhances the formation and s i z e of erythroid colonies i n the presence of multi-CSF (100). Of great i n t e r e s t was the discovery that leukemia-derived (WEHI-3B) multi-CSF was d i f f e r e n t than multi-CSF from SCM. A si n g l e amino acid s u b s t i t u t i o n (114,115) appeared to 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 differences between these multi-CSFs has also been observed, r e l a t i n g to decreased f u n c t i o n a l a c t i v i t y of the leukemia-derived f a c t o r with regard to stimulation of erythroid and mast c e l l p r o l i f e r a t i o n (182). Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) GM-CSF may be derived from medium conditioned by murine lung (most commonly), heart, spleen, s a l i v a r y glands, thigh muscle, bone shaft, kidney, thymus or br a i n as well as from concanavalin A-stimulated T19.1 hybridoma c e l l s (119-121,183,184). As i t s name implies, GM-CSF stimulates the p r o l i f e r a t i o n of CFU-G, CFU-M, or CFU-GM. In f e t a l l i v e r c e l l c ultures, GM-CSF has also been shown to be capable of stimulating the production of eosinophil colonies. The 24 proportion of each developing colony type i s r e l a t e d to the concentration of GM-CSF used i n v i t r o . When bone marrow c e l l s were cultured with high concentrations of GM-CSF, extremely large (> 5000 c e l l s ) pure granulocytic colonies developed (92); at low concentrations of GM-CSF, only macrophage colonies developed (185,186). The number of CFU-GM colonies developing declined (as expected) with decreasing GM-CSF concentrations. At very high concentrations, recombinant GM-CSF has been shown to stimulate p r o l i f e r a t i o n of eosinophil, megakaryocyte and erythroid precursors as well (187-190). Macrophage Colony-Stimulating Factor (M-CSF) M-CSF has been characterized from three sources: L - c e l l conditioned medium (124), yolk sac conditioned medium (125) and pregnant mouse uterine extract (191). M-CSF p r i m a r i l y stimulates the p r o l i f e r a t i o n of macrophage colonies from bone marrow although i t can also stimulate a small number of G-containing colonies i n c u l t u r e . Synergism between M-CSF and hemopoietin 1 (IL-1) has been shown to r e s u l t i n the formation of very large macrophage colonies, by a mechanism of induction of M-CSF receptors on p r i m i t i v e hemopoietic c e l l s (192). Granulocyte Colony-Stimulating Factor (G-CSF) G-CSF, l i k e GM-CSF, has been derived from lung-conditioned medium from endotoxin-treated mice (132). G-CSF has also been derived from medium conditioned by thymus, heart, muscle, s a l i v a r y gland, bone shaft, kidney, spleen, and normal 25 pe r i t o n e a l c e l l s (131). This CSF has the capacity to stimulate the growth of small pure G colonies from murine bone marrow and at high concentrations, a small number of GM and occasionally M colonies form (133). G-CSF, unl i k e the previously described murine CSFs, can also stimulate the p r o l i f e r a t i o n of human G colonies i n v i t r o . Other Murine Regulatory Molecules with GM-CSF A c t i v i t y There have been reports of other murine CSFs with a c t i v i t y on GM progenitor c e l l s i n v i t r o . However, u n l i k e the previously described CSFs, none of these putative CSFs has been p u r i f i e d to homogeneity. As a r e s u l t t h e i r precise b i o l o g i c a l s i g n i f i c a n c e i s unclear. Three such factors w i l l be mentioned: (1) Conditioned medium from a cloned embryonic c e l l l i n e contained a 65 - 70 KD molecule which stimulated granulocyte and macrophage colony development (193). (2) Post-endotoxin mouse serum contains a molecule s i m i l a r to M-CSF but of lower (< 23 KD) molecular weight. I t i s not cl e a r i f t h i s serum a c t u a l l y contains GM-CSF or an aberrant M-CSF i n addition to G-CSF. (3) A molecule of molecular weight 65 KD, present i n inflammatory exudate c e l l conditioned medium, reportedly stimulated the p r o l i f e r a t i o n of CFU-GM and G colonies (194). Again, i t i s d i f f i c u l t to determine i f t h i s molecule represents contamination of the preparation with other known CSF molecules. 26 Megakaryocyte Colony Stimulation Murine multi-CSF appears to be the primary stimulus f o r the formation of CFU-Mega. Two other molecules have also been described with a c t i v i t y on murine megakaryocyte progenitors. A megakaryocyte enhancing f a c t o r has been i d e n t i f i e d i n medium conditioned by WEHI-3B, lung, bone shaft, 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), but i t i s unclear 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 of some other known mediator with multi-CSF. Human urine from patients with a p l a s t i c anemia or i d i o p a t h i c thrombocytopenic purpura has been shown to contain a MEG-CSF with a c t i v i t y on mouse megakaryocyte progenitors (196). Eosinophil D i f f e r e n t i a t i o n Factor A novel lymphokine was recently described which appears to be a regulatory molecule f o r eosinophil production (134). This eosinophil 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 clones r e a c t i v e to a l i o - and p a r a s i t e antigens derived from p a r a s i t i z e d mice, stimulated eosinophil d i f f e r e n t i a t i o n i n l i q u i d (but not semi-solid) bone marrow cultures. Presumably, EDF acts on committed bone marrow precursors to stimulate the production of normal, f u n c t i o n a l eosinophils. Erythroid Colony-Stimulating Factors Anemic mouse serum contains a molecule thought to be erythropoetin (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). This f a c t o r stimulates the p r o l i f e r a t i o n of l a t e r 27 stages of erythroid colony formation (CFU-E). Erythroid progenitors are stimulated by multi-CSF ( i n which case, often r e f e r r e d to as "burst promoting a c t i v i t y " or BPA), and to some extent by GM-CSF and G-CSF acting with erythropoietin. Recombinant murine GM-CSF by i t s e l f , at very high concentrations, stimulates p r o l i f e r a t i o n of e r y t h r o id precursors. Lymphocyte Growth Factors While i t i s not part of the objectives of t h i s t hesis to in v e s t i g a t e the lymphoid arm of the hemopoietic pathway i n d e t a i l , lymphocyte growth factors w i l l be b r i e f l y mentioned f o r the sake of presenting a complete overview of hemopoiesis. ( i ) T - C e l l Growth Factor (TCGF) or I n t e r l e u k i n 2 (IL-2) TCGF or IL-2 has been derived from murine T-lymphoma or hybridoma c e l l l i n e s and spleen c e l l conditioned media. This molecule stimulates p r o l i f e r a t i o n of antigen- or mitogen-primed T lymphocytes. ( i i ) B C e l l Stimulating Factors (BSF) 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 rs have been i d e n t i f i e d (Table I) although not completely characterized as yet. I n t e r l e u k i n 4 (IL-4), a putative B c e l l growth f a c t o r (BCGF), has been molecularly cloned and demonstrated to have p r o l i f e r a t i v e and immunoglobulin-secretion inductive e f f e c t s on activated B c e l l s (150). Table I l i s t s reported B c e l l growth and d i f f e r e n t i a t i o n factors which have been p a r t i a l l y p u r i f i e d and characterized. 28 Neuroleukin, produced by lec t i n - s t i m u l a t e d T c e l l s , has also been shown to induce immunoglobulin secretion i n cultured peripheral blood mononuclear c e l l s . b) HUMAN COLONY-STIMULATING FACTORS Interleukin-3 (IL-3) IL-3 or multi-CSF has very recently 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 to human GM-CSF, which i s believed to have ar i s e n from a common ancestral gene. Both are produced by activated T lymphocytes and the genes f o r both are t i g h t l y linked (9 kilobases apart) on 3 chromosome 5. IL-3 appears to react with more p r i m i t i v e progenitors or stem c e l l s than does GM-CSF and gives r i s e to greater numbers of b l a s t c e l l colonies with higher self-generative p o t e n t i a l than does GM-CSF. E s s e n t i a l l y a l l myeloid progenitors and stem c e l l s are apparently stimulated by IL-3 (Table I I ) . The marked degree of heterogeneity i n molecular weight of IL-3 i s due to i t s large content of carbohydrate, a s i t u a t i o n s i m i l a r to GM-CSF. Pluripotent Colony-Stimulating Factor Human pluri p o t e n t CSF ( p l u r i p o i e t i n ) , derived from medium conditioned by human bladder c e l l cancer l i n e 5637 and p u r i f i e d to homogeneity, has been reported to be capable of stimulating the p r o l i f e r a t i o n of m u l t i p o t e n t i a l stem c e l l s , GM and erythroid progenitors (153). This CSF appears to possess the combined 29 properties of human GM-CSF and G-CSF, discussed next. Whether or not p l u r i p o i e t i n i s the same as IL-3 has not been reported. GM-CSF (CSFa) Two CSFs with GM colony-stimulating 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 of human sources: conditioned media from per i p h e r a l blood (197), lung (198), and placenta (154,199) as well as from c e l l l i n e s (200-202) and a number of organ cultures (203). GM and G-CSF have been separated from each other, molecularly cloned, and shown to stimulate d i s t i n c t CFU-GM subsets (154). GM-CSF stimulates GM colony formation, peaking at 14 days, with a predominance of M colonies at that time (155,161,204). Eosinophil colony formation i s also stimulated d i r e c t l y by GM-CSF. In combination with erythropoietin, GM-CSF has been shown to stimulate m u l t i p o t e n t i a l and erythroid colony formation, p o s s i b l y by an i n d i r e c t mechanism. Recently, recombinant human GM-CSF has been shown to stimulate the p r o l i f e r a t i o n of b l a s t c e l l colonies from patients with acute myeloblastic leukemia (162) and induce macrophage production of IL-1, TNF-a, IL-2 receptors and mRNA for 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 stimulates G colony formation but can also stimulate some GM and occasional M colonies peaking at 7 days i n v i t r o . I t has been demonstrated that human G-CSF has 30 biochemical and b i o l o g i c a l properties comparable to murine G-CSF. Recently a f a c t o r with granulomonopoietic enhancing a c t i v i t y (GM-EA) has been i d e n t i f i e d (205). This f a c t o r had no colony-stimulating a c t i v i t y by i t s e l f but, when added to human bone marrow cultures containing a source of GM colony-stimulating a c t i v i t y , GM-EA caused an enhancement of GM colony numbers at both day 7 and day 14 incubation. GM-EA was shown to be produced by human monocyte-derived l i p i d - c o n t a i n i n g c e l l s . M-CSF (Human Urinary Colony-Stimulating Factor) Although of human o r i g i n , M-CSF has no strong d i r e c t stimulatory e f f e c t on the p r o l i f e r a t i o n of human colonies. However, on mouse bone marrow, t h i s molecule has e f f e c t s s i m i l a r to murine M-CSF, causing s e l e c t i v e p r o l i f e r a t i o n of macrophage colony formation (164). Monocytes, f i b r o b l a s t s and endothelial c e l l s are a l l sources of M-CSF. Human M-CSF i s thought to be involved i n the s u r v i v a l and a c t i v a t i o n of monocytes and macrophages. Erythr o p o i e t i n (epo) Human urine has been u t i l i z e d as a source f o r erythr o p o i e t i n (168,206). P u r i f i e d epo stimulates mature erythroid colony (CFU-E) formation. As mentioned previously, p l u r i p o i e t i n or epo i n combination with CSFa w i l l support the 31 s u r v i v a l and p r o l i f e r a t i o n of multilineage and early erythroid (BFU-E) colonies. Erythroid Potentiating A c t i v i t y (EPA) Erythroid-potentiating a c t i v i t y stimulates both early (BFU-E) and l a t e (CFU-E) erythroid colony formation i n humans and mice but has no e f f e c t on other myeloid colony growth (171,172). EPA has been p u r i f i e d from medium conditioned by the human T-lymphoblast c e l l l i n e Mo, inf e c t e d with the human T - c e l l lymphotropic v i r u s HTLV-II. EPA i s also produced by most other HTLV-II infe c t e d mature T-lymphoblast l i n e s and by some monocyte c e l l l i n e s . Leukemic Bl a s t Growth Factor (LBGF) Recently, a hemopoietic growth f a c t o r termed leukemic b l a s t growth fa c t o r or LBGF was p a r t i a l l y p u r i f i e d from a human bladder carcinoma c e l l l i n e HTB9 (167). LBGF was shown to cause stimulation of b l a s t c e l l growth from acute myeloblastic leukemia patients' p e r i p h e r a l blood. LBGF a c t i v i t y could be separated from GM-CSF a c t i v i t y , i n d i c a t i n g that LBGF may be a novel, d i s t i n c t hemopoietic growth f a c t o r . I t s r e l a t i o n s h i p or i d e n t i t y with IL-3 has not been described. T C e l l Growth Factor (TCGF) or Interleukin-2 (IL-2) As i n the mouse, human IL-2 has been derived from antigen-or mitogen-stimulated T lymphocytes and from primed T c e l l l i n e s or lymphomas (Jurkat). Human IL-2 w i l l not cause p r o l i f e r a t i o n 32 of unprimed T lymphocytes, acting only once these c e l l s have been "primed" by antigen, mitogen, or other a c t i v a t i n g agents. B C e l l Stimulating Factors (BSF) A s i m i l a r s i t u a t i o n with regard to B c e l l stimulatory 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 ors e x i s t s i n humans as that i n the murine system. Factors causing 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 of activated B c e l l s have been described and p a r t i a l l y characterized (Table I I ) . S i m i l a r l y , neuroleukin, capable of inducing immunoglobulin secretion 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 of activated B c e l l s has also been reported to be an e f f e c t of an interferon-alpha (IFNa.) (207). A c) DISTINCT MEMBRANE RECEPTORS FOR COLONY-STIMULATING FACTORS Membrane receptors f o r CSFs have been i d e n t i f i e d i n the mouse. The murine CSFs that regulate granulocyte-macrophage production have d i s t i n c t , coexpressed membrane receptors on t h e i r target c e l l s . This coincides with the observation that the CSFs appear to have evolved separately (not from a common ancestral gene), based on d i s s i m i l a r i t i e s of amino acid sequence analyses of CSFs within a species. Most murine granulocyte-macrophage (GM) c e l l s simultaneously express receptors f o r three or four CSFs (multi-CSF, GM-CSF, M-CSF and G-CSF). Each of these CSFs presumably has receptors on c e l l s of other hemopoietic lineages as evidenced by t h e i r a b i l i t y to stimulate p r o l i f e r a t i o n i n these c e l l s (see Table I ) . While no apparent 33 d i r e c t competition f o r receptor binding e x i s t s between these CSFs, the binding of a p a r t i c u l a r CSF often r e s u l t s i n the down-modulation and a c t i v a t i o n of receptors f o r other CSF types (208): 1) Receptor binding of multi-CSF resulted i n down-modulation of receptors f o r a l l other CSFs with GM a c t i v i t y ; 2) Receptor binding of GM-CSF caused down-modulation of receptors f o r G-CSF and M-CSF (but not multi-CSF); 3) GM-CSF receptors were down-modulated by high concentrations of M-CSF; and 4) M-CSF receptors were down-modulated by high concentrations of G-CSF. Of great i n t e r e s t was the discovery that the 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 to the product of the c-fms oncogene (209). d) OTHER FUNCTIONS OF THE COLONY-STIMULATING FACTORS As mentioned previously, i n addition to t h e i r p r o l i f e r a t i v e a c t i v i t y on hemopoietic precursor c e l l s , the myeloid CSF's have other demonstrated functions. These include: 1) maintenance of the i n v i t r o s u r v i v a l of hemopoietic c e l l s , from progenitor c e l l l e v e l up to mature post-mitotic polymorphonuclear leucocytes (210-213). 2) an a b i l i t y to i r r e v e r s i b l y commit m u l t i - or 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 d i f f e r e n t i a t i o n pathway (214,215). 3) stimulation of various functions i n mature (end) c e l l s . 34 The kinds of fun c t i o n a l a c t i v i t i e s stimulated by CSFs have been reported to include increased antibody-dependent neutrophil and eosinophil-mediated c y t o t o x i c i t y and increased phagocytosis by neutrophils and macrophages. I t has been demonstrated that GM-CSF and G-CSF l e v e l s increase dramatically following exposure to b a c t e r i a l products (227-229) and return to normal l e v e l s following elimination of the b a c t e r i a (or t h e i r products). In s i t u a t i o n s of acute b a c t e r i a l i n f e c t i o n of a non-immune host, such rapid increases i n serum l e v e l s of factors known to stimulate phagocytosis and 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 stages. 2. NONSPECIFIC SUBSTANCES WITH HEMOPOIETIC GROWTH PROMOTING  ACTIVITY In addition to the hemopoietic colony-stimulating factors already discussed, there are a number of molecules which possess poorly characterized growth promoting a c t i v i t y f o r hemopoietic c e l l s . These substances are of nonspecific nature, exerting t h e i r a c t i v i t y by i n d i r e c t means. Some of these molecules are mentioned here to i l l u s t r a t e further the complex nature of the influences on hemopoietic regulation. Phorbol esters and l i t h i u m s a l t s were shown to induce increased l e v e l s of GM-CSFs i n d i r e c t l y through a c t i v i t y on macrophages or other mononuclear c e l l s (230-233). There are a large number of serum components with reported growth promoting a c t i v i t i e s f o r cultured c e l l s . Indeed serum contains over f i v e 35 hundred d i f f e r e n t proteins whose functions are, i n general, poorly characterized at best. Serum components with known growth promoting a c t i v i t i e s include t r a n s f e r r i n , albumin and numerous hormones (the 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 , thyroid hormones, testosterone, 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 the hemopoietic colony stimulating factors has been discussed, but many of the other serum components l i s t e d can play a major r o l e i n hemopoietic regulation i n i n v i t r o hemopoietic c e l l assays. D i f f e r e n t batches of f e t a l c a l f serum or plasma have been demonstrated to contain markedly d i f f e r e n t amounts of thyroid hormones, gl u c o c o r t i c o i d s , testosterone, e s t r a d i o l and i n s u l i n (234). A p a r t i c u l a r serum batch can profoundly e f f e c t the a c t i v i t y of known CSFs. I t has been observed that with c e r t a i n serum batches, only M (or G) colonies developed (or at l e a s t predominated) using 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 to be the only e f f e c t i v e method to c o n s i s t e n t l y demonstrate an e f f e c t by a given serum component on colony formation i n v i t r o (234). These points can complicate i n t e r p r e t a t i o n s of reported e f f e c t s of p a r t i c u l a r substances on hemopoietic c e l l stimulation or i n h i b i t i o n and should always be considered i n such assays. 3. 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 regulation of many hormone systems. C o r t i s o l , an 36 adrenal s t e r o i d , acts as a negative feedback regulator i n the hypothalamic-pituitary-adrenocortical axis to l i m i t the production of p i t u i t a r y adrenocorticotropin (ACTH). In granulopoiesis, negative regulators have been studied extensively and conclusive evidence e x i s t s f o r t h e i r r o l e both i n v i t r o and i n vivo. While there have been many reports of granulopoietic i n h i b i t o r s (236-238, reviews), there are fewer i n h i b i t o r y molecules that have been investigated thoroughly (239). Furthermore, i t i s apparent from reviewing reports of the well characterized negative regulators of granulopoiesis that there are a myriad of i n t e r a c t i o n s occurring between these molecules and the c e l l s that produce them. A c l e a r p i c t u r e of the end r e s u l t i n terms of i n h i b i t i o n of granulopoiesis w i l l r e l y on an understanding of these i n t e r a c t i o n s . Figure 1.2 was created to emphasize the enormous complexity of i n t e r p l a y between c e l l s and i n h i b i t o r y factors i n granulopoiesis ( s p e c i f i c a l l y , i n the production of the neutrophil/monocyte/macrophage lineage). The negative regulators that w i l l be discussed are l i s t e d i n Table I I I . L a c t o f e r r i n L a c t o f e r r i n (LF), an iron-binding glycoprotein, has a number of important p h y s i o l o g i c a l r o l e s . In i t s iron-depleted form, LF has 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 properties. LF augments neutrophil adhesiveness and production of hydroxyl r a d i c a l s . I t i s synthesized i n immature granulocytic c e l l s and stored i n the secondary granules of mature c e l l s (325). 37 i Opolymorphonuclear leucocyte Figure 1.2. I n t e r a c t i o n s between the 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 . T8+, T4+, T lymphocytes bearing T8 or T4 c e l l surface antigens; 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 ; alFN, f l F N , i n t e r f e r o n a or y; TNF, tumor n e c r o s i s f a c t o r ; GM-CSF, granulocyte-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, colony-forming u n i t , granulocyte-macrophage; AIF, a c i d i c i s o f e r r i t i n ; LIA, leukemia-associated 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 , non-phagocytic, non-adherent, F c + bone marrow c e l l i n normals and myeloid leukemics b = LIA = leukemia-associated i n h i b i t o r y a c t i v i t y c = negative 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 responsiveness to negative r e g u l a t i o n by the f a c t o r i n myeloid leukemia p a t i e n t s versus 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 -» = negative (suppressive or i n h i b i t o r y ) s i g n a l 38 Table I I I . Negative granulopoietic regulators Regulator (abbreviation), Alternate Name Molecular Weight References 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) 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) Interferons (IFN-a, IFN-B, IFN-y) T r a n s f e r r i n (TF) E-type prostaglandins (PGEi,PGE2) Leukemia-associated i n h i b i t o r (LAI) Tumor necrosis f a c t o r (TNF), LuKII Granulocytic chalone Other: a) C o l o n y - i n h i b i t i n g lymphokine (CIL) b) negative regulator produced by normal adherent BM c e l l s c) unspecified c e l l products from leukemics or a p l a s t i c anemics 85-100 KD 19- 21 KD 15- 40 KD 80 KD 350 Native: > 500 KD Active: 150-170 KD Native: 40-45 KD SDS-PAGE: 17-18 KD components 4 peptides of 20-30 amino acids each 85 KD ND ND 240-259 260-272 273-284 239,255,285 286-300 301-303 304-310 311-314 315 316 317-324 ND = not described 39 There are numerous reports concerning the r o l e of LF i n the i n h i b i t i o n of granulocyte/macrophage colony forming c e l l s (CFU-GM) (Table I I I ) . As Figure 1.2 i l l u s t r a t e s , the cumulative e f f e c t of LF i n t h i s regard i s the r e s u l t of many in t e r a c t i o n s with other regulatory molecules and with i t s target c e l l population, a l l of which may vary i n amount at any given time. LF has been shown to e f f e c t the following: 1) binding to s p e c i f i c receptors on HLA-DR-positive human monocytes (246,253) and Ia (I-A and I-E/C) antigen-positive murine macrophages (258), i n h i b i t i n g the release of GM-CSF from these c e l l s (248,258). 2) i n h i b i t i o n of production and/or secretion of a monokine(s) from human monocytes which causes other c e l l types (T lymphocytes, f i b r o b l a s t s , endothelial c e l l s ) to release GM-CSF (250,256,257). 3) i n h i b i t i o n of the production and/or release by human monocytes of two other i n h i b i t o r y molecules, a c i d i c i s o f e r r i t i n s (251) and prostaglandin E (247). Figure 1.2 shows that the e f f e c t of LF i s not the same i n myeloid leukemia patients as that seen i n normals. This d i f f e r e n c e i s manifest i n two ways. In the f i r s t place, polymorphonuclear leucocytes (PMN's) from leukemia patients have lower l e v e l s of endogenous LF than normals, and (perhaps of greater s i g n i f i c a n c e ) t h e i r PMN's contain low or undetectable l e v e l s of the a c t i v e form of LF (240,245,254,326,327). Secondly the LF-mediated suppression of GM-CSF from monocytes of leukemia patients i s decreased compared to that of normals 40 (253,328,329). While part of the explanation f o r t h i s may reside i n the decreased number of PMN's present i n acute myeloid leukemics i n the act i v e state of t h e i r disease, t h i s f a c t o r alone cannot explain the s i t u a t i o n of decreased responsiveness and/or amount of LF, e s p e c i a l l y i n the case of chronic myeloid leukemics. As Figure 1.2 points out (* = di f f e r e n c e i n e f f e c t of i n h i b i t o r s i n myeloid leukemics compared to normals), t h i s s i t u a t i o n i s but the f i r s t of many such s i t u a t i o n s i n which the e f f e c t s seen by a negative regulator of normal myelopoiesis are d i f f e r e n t i n the case of leukemics. The cumulative e f f e c t s of these differences may be of tantamount importance i n the explanation of how the leukemic clone gains a foothold over normal myeloid progenitor c e l l s , enabling the malignant c e l l s to a t t a i n a small but d i s t i n c t growth advantage over t h e i r normal counterparts. A c i d i c I s o f e r r i t i n s The i s o f e r r i t i n s are comprised of two types of subunits, H (molecular weight 21 KD) which i s associated with heart t i s s u e and L (molecular weight 19 KD) which i s associated with l i v e r . The a c i d i c i s o f e r r i t i n s (AIF) are composed mainly of H subunits and are produced by HLA-DR-positive monocytes/macrophages (268,330). Target c e l l s are s i m i l a r l y r e s t r i c t e d by Class II antigenic expression. AIF have been shown to cause a suppression of i n v i t r o CFU-GM colony formation i n the mouse and of CFU-GEMM, BFU-E and CFU-GM colony formation i n humans (266,270). Both interferon-alpha and prostaglandin E can 41 augment the e f f e c t of AIF while both LF and interferon-gamma can i n h i b i t i t s e f f e c t (Figure 1.2). As with LF, there i s a di f f e r e n c e between normals and myeloid leukemics with respect to the quantity of and s e n s i t i v i t y to AIF. AIF l e v e l s are higher i n leukemia patients* c e l l s (261,268) but, since there i s a de f i c i e n c y of Ia antigens on the myeloid progenitor c e l l s of leukemia p a t i e n t s , they lack s e n s i t i v i t y to the i n h i b i t o r y e f f e c t s of AIF. A study of a population of r a p i d l y p r o l i f e r a t i n g myeloid leukemic b l a s t c e l l s i n v i t r o also demonstrated t h e i r lack of s e n s i t i v i t y to i n h i b i t i o n by AIF (263). Interferons Interferons (IFN) are comprised of a group of glycoproteins which are produced and secreted by c e l l s i n response to i n f e c t i o n with viruses and other s t i m u l i . There are three types of interferons — IFN-beta ( f i b r o b l a s t IFN) which i s produced by f i b r o b l a s t s , IFN-gamma (immune IFN) which i s produced by T lymphocytes, and IFN-alpha (leucocyte IFN), of leucocyte o r i g i n . I t i s c l e a r that IFNs play an i n h i b i t o r y r o l e i n myelopoiesis (Table I I I ) . Colony formation by m u l t i p o t e n t i a l (CFU-GEMM) and erythroid (BFU-E) progenitors i s suppressed by a l l three IFN's to the same degree (239). IFN-gamma i s most e f f e c t i v e i n the suppression of CFU-GM (IFN-y > IFN-o > IFN-B). The i n h i b i t o r y e f f e c t on colony formation i s d i r e c t (not a r e s u l t of action upon an intermediary c e l l type or i t s products). I t has been shown that IFN-alpha blocks 42 d i f f e r e n t i a t i o n beyond the myelocyte l e v e l (277,280). As Figure 1.2 i l l u s t r a t e s , there are a number of in t e r a c t i o n s that occur between IFNs and other negative regulatory molecules. IFN-alpha and IFN-gamma synergize to suppress colony formation i n v i t r o at concentrations of each that have no e f f e c t whatsoever were each to be used alone (310). The production and/or release of 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 to be no dif f e r e n c e between the s e n s i t i v i t i e s of myeloid progenitor c e l l s of leukemia patients or normals with respect to the IFN's, with the exception that prostaglandin E suppresses IFN-alpha-induced expression of Ia antigens, a s i t u a t i o n that could e f f e c t the actions of other negative regulators known to be so r e s t r i c t e d (LF, AIF, TF). Tr a n s f e r r i n T r a n s f e r r i n (TF), l i k e LF, i s an iron-binding glycoprotein, plays 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 released by a population of T8-positive T lymphocytes and suppresses the release of GM-CSF from le c t i n - s t i m u l a t e d T4-positive, Class II antigen-positive c e l l s (285). In normals, TF i s released from Class II antigen-positive T8 c e l l s . However, i n leukemia patients, TF i s released from Class I I antigen-negative T8 c e l l s and the TF-mediated i n h i b i t o r y a c t i v i t y derived from these c e l l s i s increased (239). 43 E-Type Prostaglandins E-type prostaglandins (PGE^ and PGE^) are produced by human blood monocytes and murine pe r i t o n e a l macrophages (288). PGEs act by s e l e c t i v e i n h i b i t i o n of Ia antigen-positive monocyte-macrophage progenitors (267,299). Their i n h i b i t o r y e f f e c t i s augmented by t h e i r a b i l i t y to stimulate Ia antigen expression on myeloid progenitor c e l l s , thereby increasing the responsiveness of these c e l l s to negative regulation by AIF and the PGEs themselves (239). I n t e r e s t i n g l y , PGEs can cause a 30 -70% enhancement of BFU-E production 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 to the expression of Class II antigens on the BFU-E progenitors (331). Monocyte/macrophage-derived CSF stimulates the production of PGE. LF can i n h i b i t PGE production. These i n t e r a c t i o n s are summarized i n Figure 1.2 and further serve to i l l u s t r a t e the complex nature of the stimulatory and i n h i b i t o r y influences of the regulators of myelopoiesis. Again, an abnormal responsiveness to the e f f e c t of PGE has been i d e n t i f i e d i n myeloid leukemia patients* progenitor c e l l s (293-296). Decreased s e n s i t i v i t y to PGE-mediated i n h i b i t i o n , r e l a t e d to an i n s u f f i c i e n c y of Class II antigens on the progenitor c e l l s , was observed compared to normal controls. Leukemia-Associated I n h i b i t o r Leukemia-associated i n h i b i t o r (LAI) has been reported to reduce the number of normal CFU-c i n S-phase but has no e f f e c t on clonogenic c e l l s from acute or chronic myeloid leukemics 44 (301) . The i n h i b i t o r y a c t i v i t y of t h i s molecule was shown to be present i n a subunit (150-170 KD) of i t s native form (> 500 KD) (302) . LAI i s produced by c e l l s present i n both normals and myeloid leukemics i n d i c a t i n g that i t may play a regulatory r o l e i n normal myelopoiesis. The phenotype of LAI-producing c e l l s , the same i n both normal and leukemic states (303), i s a large, hon-phagocytic, non-adherent, Fc-receptor p o s i t i v e bone marrow c e l l . Increased production of such an i n h i b i t o r , capable of reducing the p r o l i f e r a t i v e rate of normal granulopoietic progenitors, could help to explain the growth advantage of malignant myeloid c e l l s over normals. Tumor Necrosis Factor Tumor necrosis f a c t o r (TNF) was named over a decade ago to describe a serum substance induced by endotoxin that was capable 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 that time, TNF has been p u r i f i e d from rab b i t and mouse serum (332) and from human c e l l l i n e s (305,306). Human TNF has been cloned (305,333) and i t i s now apparent that a molecule (or, as i n the case of the int e r f e r o n s , a family of re l a t e d molecules) i d e n t i c a l to that described as TNF i s produced by human monocytes (334) and natural k i l l e r c e l l s (308) as well as by myeloid (eg. HL-60) and lymphoblastoid (eg. LuKII) c e l l l i n e s . Human TNF has been shown to cause s i g n i f i c a n t (50 - 98%) i n h i b i t i o n of CFU-GM as well as i n h i b i t i o n of BFU-E and CFU-GEMM (310). Unlike the previously described negative regulators of myelopoiesis, TNF causes more i n h i b i t i o n of CFU-c (cl u s t e r s < 40 45 c e l l s ) i n non-remission ANLL patients than i n those i n remission or normals (310). Granulocytic Chalone The concept of granulocytic "chalones" dates back over two decades. Chalones are described as t i s s u e - s p e c i f i c , species-nonspecific negative feedback regulator substances that are produced by mature c e l l s within the same lineage as that i n which t h e i r e f f e c t s are manifest i n immature c e l l s (335). The granulocytic chalone has been reported to be a polypeptide of 20 - 30 amino acids (311,312). The existence of the granulocytic chalone as o r i g i n a l l y defined has been questioned (314), although many inve s t i g a t i o n s have concluded that mature polymorphonuclear leucocytes (neutrophils) contain negative feedback regulatory substances. These molecules have been reported to suppress the p r o l i f e r a t i o n of granulocytic precursor c e l l s . In l i g h t of the inve s t i g a t i o n s concerning the release of l a c t o f e r r i n (LF) from these c e l l s , i t i s very po s s i b l e that t h i s substance was responsible f o r the i n h i b i t o r y e f f e c t s observed by soluble products derived from polymorphonuclear leucocytes, although i t i s d i f f i c u l t to re c o n c i l e the reported differences i n molecular weight (LF = 85 - 100 KD, chalone = 25 amino acids) of these substances. Other Reported I n h i b i t o r s of Myelopoiesis A number of other le s s well characterized i n h i b i t o r s of granulopoiesis have been reported (315-324). Whether or not 46 these substances are d i f f e r e n t from those already investigated or have relevance i n vivo has yet to be proven. a) Co l o n y - i n h i b i t i n g lymphokine Col o n y - i n h i b i t i n g lymphokine (CIL) i s a substance of reported molecular weight 85 KD which i s synthesized and released from a human hybridoma c e l l l i n e (6TM), a fusion between PHA-stimulated normal lymphocytes and a mutant Jurkat T lymphoma c e l l l i n e (315). A c o r r e l a t i o n between expression of Class II antigens and s e n s i t i v i t y to CIL was found i n human c e l l l i n e s and bone marrow progenitors (336). CIL caused a massive i n h i b i t i o n of CFU-GM as well as CFU-GEMM, BFU-E and CFU-E. This very i n t e r e s t i n g lymphokine may prove to have s i g n i f i c a n c e i n the r e gulation of normal and/or abnormal myelopoiesis. Recent evidence indicates that CIL s p e c i f i c a l l y recognizes a molecular structure common to both human and murine Ia molecules (336) and i s capable of blocking stimulator c e l l recognition i n mixed lymphocyte reactions (MLR). The generation of immune responses and regulation of hemopoiesis must be linked i n a great many ways; CIL may prove to be one of the regulatory molecules involved i n t h i s i n t e r a c t i o n . b) An undefined negative regulatory 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 normal progenitor c e l l s to go i n and out of c e l l c y c l e . This negative con t r o l on the p r o l i f e r a t i v e capacity of normal blood progenitors had no e f f e c t on CGL progenitors of marrow or blood o r i g i n , which remained continuously i n cycle i n the presence of 47 the normal marrow adherent c e l l s (316). This appears to be another case i n which normal 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 the control of leukemic progenitor c e l l s , c) Unspecified c e l l products from patients with hemopoietic disorders There have been a number of reports which have indicated that myeloid leukemic c e l l s can suppress the p r o l i f e r a t i o n of co-cultured normal CFU-GM i n v i t r o (317-319,321,323,324). S i m i l a r l y , the co-culture of marrow or blood c e l l s from a p l a s t i c anemics with normals has been shown to cause i n h i b i t i o n of normal CFU-c and erythroid colony production (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 of the factors involved i n t h i s suppression of normal hemopoiesis awaits further study and may prove to include some of the negative regulators previously described. 4. MICROENVIRONMENTAL INFLUENCES ON HEMOPOIESIS The r o l e of s p e c i f i c t i s s u e microenvironments i n the regulation of hemopoiesis i s c l e a r l y established, although i n most cases, not p r e c i s e l y characterized. In the case of lymphopoiesis, microenvironmental regulation appears to be an absolute requirement f o r the complete development of both T and B lymphocytes. Pre-T c e l l s from the bone marrow migrate to the thymus and become "educated" within t h i s organ to become mature a n t i g e n - s p e c i f i c , MHC-restricted 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 nurse c e l l s within the cortex are thought to be responsible f o r t h i s regulation 48 through the production 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 within secondary lymphoid organs, notably the spleen and lymph nodes. Microenvironmental influences on myelopoiesis have also been established. Most evidence has come from the growth of CFU-S i n i r r a d i a t e d r e c i p i e n t mice. CFU-S developing i n the spleens of such r e c i p i e n t s were predominantly eryth r o i d , while CFU developing i n the BM of the same mice were composed p r i m a r i l y of granulocytic c e l l s (5,337). This observation implied that " e r y t h r o p o i e t i c " and "granulopoietic" niches existed within d i f f e r e n t hemopoietic organs. When bone shafts were grafted into the spleen, developing colonies displayed both granulocytic ( i n the marrow cavity) and erythroid ( i n adjacent s p l e n i c tissue) lineage r e s t r i c t i o n . Analysis of s p l e n i c colonies has, however, indicated that 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 within most colonies. Moreover, the requirement f o r microenvironmental c e l l influences are not always absolute, as i t has been established that a s i n g l e colony-forming c e l l i n the absence of other c e l l s can develop into a mixed colony i n v i t r o containing various progenitor c e l l p o t e n t i a l i t i e s within i t s own m i l i e u ( 5 ) . The Dexter long-term bone marrow culture system has demonstrated that lineage commitment by m u l t i p o t e n t i a l stem c e l l s does occur i n the presence of adherent stromal c e l l l ayers. The p r e c i s e mechanisms c o n t r o l l i n g such commitment as well as stem c e l l s u r v i v a l i n t h i s system are not well 49 characterized at present and may require the establishment of cloned stromal c e l l l i n e s i n order to define the influences of c e l l u l a r matrices and/or humoral regulators within the microenvironment. The c l a s s i c a l example pertaining to the importance of the bone marrow stromal microenvironment i n the regulation of hemopoiesis comes from the study of S l / S l mice, which possess d a genetically-determined macrocytic anemia t r a i t . S l / S l mice have normal stem c e l l s , as evidenced by the a b i l i t y of t h e i r bone marrow to 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 . Transplantation of mouse d stem c e l l s from normal BM f a i l e d to cure the S l / S l defect, i n d i c a t i n g that i n Sl/Sl** mice, the hemopoietic de f i c i e n c y r e l a t e d to an abnormal stromal microenvironment, poorly conducive to the s u r v i v a l and p r o l i f e r a t i o n of hemopoietic stem c e l l s (22). Furthermore, i n long-term "Dexter" marrow cultures, S l / S l adherent stromal c e l l s f a i l e d to support hemopoiesis d but S l / S l hemopoietic stem c e l l s p r o l i f e r a t e d normally when grown on normal BM stromal c e l l s (338). F i n a l l y , Z i p o r i proposed that, 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 p o s i t i o n within or nearby the marrow stromal c e l l population would determine i t s fate. I t was demonstrated that exogenous crude or pure sources of CSF had decreased a c t i v i t y i n the presence of increasing numbers of adherent murine marrow stromal 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 with increased s e l f renewal progenitor CFU-c maintenance. Obviously both microenvironmental c e l l s within hemopoietic 50 tissues and humoral factors play important r o l e s i n the basic regulation of hemopoiesis, both l i k e l y operating at a l l c e l l l e v e l s (stem to progenitor c e l l populations). THE HUMAN MYELOID LEUKEMIAS There are two major forms of human myeloid leukemia based on t h e i r c l i n i c a l nature — chronic granulocytic leukemia (CGL) and acute nonlymphoblastic leukemia (ANLL). Both are c l o n a l malignancies o r i g i n a t i n g i n hemopoietic stem c e l l s . Such c e l l s are l i k e l y optimal targets f o r neoplastic transformation due to t h e i r normally extensive p o t e n t i a l f o r p r o l i f e r a t i o n . A. CHRONIC GRANULOCYTIC LEUKEMIA C l i n i c a l Course and Pathogenesis Chronic granulocytic leukemia i s a myeloid c e l l malignancy which runs a chronic course of two to three years duration. CGL i s characterized by a massive expansion of myeloid progenitor c e l l populations, r e s u l t i n g i n excessive numbers of (predominantly) granulocytes at a l l stages of maturation which i n f i l t r a t e the c i r c u l a t i o n , the spleen and other tissues (340-342). Evidence shows that CGL may develop as a r e s u l t of r a d i a t i o n exposure, a notable increase i n the incidence of the disease being observed i n survivors of the atomic bomb attack on Hiroshima (343,344). However i n the majority of cases, 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 patient having undergone allogeneic bone marrow transplantation, relapse of the disease i n donor c e l l s (with a c q u i s i t i o n of the Ph' chromsome) was recently 51 reported (345) i n d i c a t i n g the p o s s i b i l i t y that a p e r s i s t i n g oncogenic agent or event could be involved i n some cases. Over 90% of CGL patients demonstrate the 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 the long arms of chromosomes 9 and 22, i n the leukemic clone (66,67). During progression of the disease, the Ph' chromosome-positive c e l l s become more numerous due to t h e i r growth advantage over normal c e l l s . Eventually greater than 99% of the d i v i d i n g c e l l s i n the bone marrow are Ph'-positive. Ultimately most patients enter an "accelerated" phase or an acute leukemia s i t u a t i o n (blast c r i s i s ) . In the b l a s t c r i s i s stage, approximately 65 - 75% of patients develop an acute myeloid-type leukemia often r e f r a c t o r y to treatment. The remaining 25 - 35% develop an acute lymphoblastic leukemia, often responsive to treatment with v i n c r i s t i n e and prednisone. Stem C e l l O r i g i n Karyotypic (Ph* chromosome) and enzymatic (G-6-PD) studies have provided conclusive evidence that CGL i s a c l o n a l disease of plu r i p o t e n t stem c e l l s . Indeed, as mentioned previously, t h i s disease has given credence to the existence of a common lymphoid-myeloid stem c e l l i n humans. The Ph' chromosome has been found i n granulocytes, monocytes, macrophages, nucleated erythroid c e l l s , megakaryocytes, basophils, eosinophils and t h e i r progenitors as well as B c e l l s (68-70,346,347). Sex chromosome mosaicism (46 XY Ph'/47 XXY) i n 2 CGL patients indicated the monoclonality of the disease (348,349). 52 Glucose-6-phosphate dehydrogenase isoenzyme studies i n heterozygous females with CGL also indicated that the disease was of 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 stem c e l l . This has been discussed previously (72-74). Strong evidence supporting the p l u r i p o t e n t stem c e l l o r i g i n of CGL has come from inv e s t i g a t i o n s of the c e l l s involved i n b l a s t c r i s i s phase of the disease. While most CGL b l a s t c r i s i s patients* c e l l s have been shown to be of myeloid 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). Greaves and colleagues (352-354) have demonstrated using immunological membrane markers, that approximately one-third of CGL b l a s t c r i s i s patients examined possessed a lymphoid c e l l phenotype, most often of the common (c)ALL v a r i e t y , l e s s frequently of n u l l c e l l phenotype. Much less frequent have been reports of CGL b l a s t c r i s e s i n v o l v i n g c e l l s with T-lymphocyte phenotypes (355-357). A l l of these lymphoid b l a s t c e l l s were Ph' p o s i t i v e , i n d i c a t i n g that they arose from the same clone as that involved i n the chronic "granulocytic" phase of the disease. Chromosomal Changes and Their Possible S i g n i f i c a n c e Two known c e l l u l a r (proto-) oncogenes, c - s i s and c-abl, have been i d e n t i f i e d as being involved i n the r e c i p r o c a l t (9;22) t r a n s l o c a t i o n of the Ph' chromosome (358-361). Chromosome 22 i s the normal l o c a t i o n of c - s i s , the c e l l u l a r homologue of 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 fa c t o r (362). Chromosome 9 i s the normal l o c a t i o n of c-abl, the c e l l u l a r 53 homologue of the murine Abelson leukemia v i r u s oncogene. The Ph* chromosome involves r e c i p r o c a l t r a n s l o c a t i o n of both proto-oncogenes. Expression of c - s i s i s not generally detectable i n CGL, making i t l i k e l y that t h i s proto-oncogene plays a l e s s e r r o l e , i f any, i n the pathogenesis of CGL. However, the consistent 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 area on chromosome 22 near the lambda l i g h t chain immunoglobulin gene locus 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-abl gene (359,361,363,364). This abnormal gene product has been shown to have tyrosine kinase a c t i v i t y s i m i l a r to that of many known v i r a l transforming proteins (365) and u n l i k e that of i t s normal c e l l u l a r homologue. This aberrant expression of c-abl may be of s i g n i f i c a n c e i n the abnormal expression of hemopoiesis i n CGL. Considerable evidence e x i s t s which supports a multistep pathogenesis f o r CGL. In many cases, a c q u i s i t i o n of the Ph' chromosome appeared to be a secondary event i n the progression of the disease. A number of patients presenting with c l i n i c a l CGL that was Ph' negative were l a t e r shown to become Ph' p o s i t i v e (366-368). The presence of Ph' negative b l a s t c e l l c r i s i s following Ph' p o s i t i v e chronic phase and the discovery of Ph' negative B-lymphocytes derived from the same G-6-PD isoenzyme clone as the Ph' p o s i t i v e leukemia c e l l s provided further evidence that CGL had a multistep pathogenesis (369-371). Many investigators b e l i e v e that CGL, 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 stem c e l l , involves a succession of genetic changes (one of which i s a c q u i s i t i o n of the Ph' chromosome). These successive a l t e r a t i o n s , to genes that are l i k e l y c l o s e l y t i e d to regulation of the basic c e l l u l a r processes of 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 , 54 are thought to r e s u l t i n a progressive uncoupling of the normal signals 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 maturation (354-372). In l i g h t of t h i s view, the chronic phase of CGL may j u s t i f i a b l y be considered as a "premalignant" or at l e a s t "pre-acute leukemic" phase. The b l a s t c r i s i s stage of CGL i s associated with a d d i t i o n a l chromosomal changes i n over 75% of patients. These commonly include trisomy 8, isochromosome 17 or Ph' chromosome d u p l i c a t i o n (373,374). I f the genes aff e c t e d by these a d d i t i o n a l chromosomal changes were involved i n coupling p r o l i f e r a t i o n and maturation, the resultant progressive uncoupling of these processes could e f f e c t the evolution of a subclone with in 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 , including "maturation a r r e s t " . This w i l l be discussed further i n acute nonlymphoblastic leukemia. Relationship Between CGL C e l l s and Growth Regulators The frequency of i n v i t r o colony-forming c e l l s i n CGL i s extremely elevated — 100-fold f o r bone marrow and 1,000 -60,000-fold f o r peripheral blood i n untreated patients (375-377). That these colony-forming c e l l s belonged to the malignant clone was demonstrated by the presence of the Ph* chromosome within the d i v i d i n g c e l l s of most colonies (378-380). Long term bone marrow cultures of CGL c e l l s recently demonstrated that r e s i d u a l normal progenitors were indeed present and, i n most cases, e a s i l y detectable a f t e r 3 weeks i n culture (381). There i s uniform expansion of a l l myeloid progenitor compartments during the chronic phase of the disease. With the onset of b l a s t c r i s i s , major changes occur i n t h i s pattern of i n v i t r o growth, u n t i l a pattern s i m i l a r to that seen i n 55 acute nonlymphoblastic leukemia i s attained (to be discussed). Throughout the e n t i r e course of the disease, CGL colony-forming c e l l s r e t a i n an absolute dependency on CSF f o r growth. CFU-c from CGL chronic phase patients e x h i b i t normal morphology and degrees of maturation. There have been reports that CGL c e l l responsiveness to stimulation by CSF i s s l i g h t l y l e s s than normal (382,383). As already described, the responsiveness of myeloid leukemia c e l l s to many negative regulators (LF, AIF, TF, PGE, LAI, TNF) i s not the same as normal c e l l s . In most cases, a s i g n i f i c a n t l y decreased s e n s i t i v i t y to 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 with regard to the p r o l i f e r a t i v e advantage of the malignant clone over normal hemopoietic c e l l s . The s i g n i f i c a n c e of autostimulation of leukemia c e l l s by production of t h e i r own growth f a c t o r s has been an area of controversy. Supporting evidence includes the observation that multi-CSF dependent hemopoietic c e l l l i n e s became leukemogenic and acquired the a b i l i t y to secrete multi-CSF simultaneously (384-385). When one of these CSF-dependent c e l l l i n e s was transfected with a r e t r o v i r u s construct containing the gene f o r GM-CSF, subclones became leukemogenic at the same time as autonomously-producing GM-CSF (386). Further evidence showed that when chicken macrophages immortalized with v-myc were subsequently inf e c t e d with the v-mil 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 synthesis of avian M-CSF (387). T c e l l l i n e s transformed by HTLV became TCGF secretors and had a decreased dependency on exogenous TCGF (388). However, there are other cases whereby CSF-dependent c e l l l i n e s were infected with Abelson leukemia 56 i v i r u s , became leukemic and did not synthesize or express increased receptor numbers f o r CSF (389,390). There i s c e r t a i n l y evidence that CGL monocytic c e l l s can secrete t h e i r own growth factors (CSFs), and i n chronic phase, CSF l e v e l s are generally normal. 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 of the leukemic clone however, since both normal and CGL c e l l s are s i m i l a r l y responsive to, and absolutely require, exogenous CSF i n v i t r o . The f a c t that normal c e l l s have shorter c e l l c ycle times than leukemic c e l l s further complicates t h i s i n t e r p r e t a t i o n (391). Furthermore, i t i s l i k e l y that a large number of c e l l s other than normal or leukemic monocytic c e l l s can produce CSF within the body, and the cumulative influences of such production, although not characterized, make i t highly u n l i k e l y that autocrine stimulation of growth by myeloid leukemic c e l l s can be of c r i t i c a l importance i n the i n i t i a t i o n of 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 Pathogenesis Acute nonlymphoblastic leukemia (ANLL) or acute myelogenous leukemia (AML) i s a r a p i d l y f a t a l disease i f untreated. Patients e x h i b i t an increasing number of immature, poorly or non-differentiated b l a s t c e l l s of the granulocyte-monocyte lineage which eventually s p i l l over into the peripheral c i r c u l a t i o n . Maturation of c e l l s i n ANLL i s defective and, as a r e s u l t , e arly immature c e l l s do not d i f f e r e n t i a t e to any great extent and hence increase i n number. C l i n i c a l signs involve the associated decrease i n normal hemopoietic c e l l s , including anemia, thrombocytopenia and 57 mature granulocytopenia/monocytopenia. In approximately one-third of ANLL patients, a preleukemic or myelodysplastic state preceeds the development of acute leukemia (392). Chemotherapy treatment allows approximately 75% of adult ANLL patients to achieve complete remission. The duration of remission i s v a r i a b l e and leukemic relapse continues to be a serious problem i n ANLL. In most cases of ANLL no s p e c i f i c e t i o l o g i c a l agent(s) or event(s) have been i d e n t i f i e d , although r a d i a t i o n exposure and chemotherapeutic (a l k y l a t i n g ) agents are known to be capable of causing damage to genetic material which may be associated with the development of acute leukemia (393-398). ANLL i s an extremely heterogeneous disease i n terms of morphologically-defined subgroups and immunologically-defined c e l l u l a r markers. The French-American-British (FAB) co-operative group 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 follows (399-400): Ml: myeloblastic leukemia without maturation, M2: myeloblastic leukemia with maturation, M3: hypergranular promyelocytic leukemia, M4: myelomonocytic leukemia, M5: monocytic leukemia, and M6: erythroleukemia. While d i f f e r i n g responses to various chemotherapeutic agents have been reported between these subgroups, no s p e c i f i c differences i n pathogenesis have been established. 58 Stem C e l l O r i g i n Most, i f not a l l , cases of ANLL are thought to be monoclonal i n o r i g i n , based on chromosomal and G-6-PD studies. G-6-PD studies have indicated that, while ANLL i s a disease of stem c e l l o r i g i n , there appears to be a degree of heterogeneity present with regard to the l e v e l of stem c e l l involvement. In some, pos s i b l y most, ANLL patients the stem c e l l involved seems to be the granulocyte-monocyte precursor (401). Other patients have demonstrated leukemic o r i g i n to be i n more p r i m i t i v e (GM and erythroid) or more r e s t r i c t e d (monocyte only) progenitors (73,402,403). Chromosomal studies have also shown that, i n some ANLL patients, c l o n a l markers were present i n both er y t h r o i d precursors and the leukemic c e l l s (404). Recently, B lymphocyte involvement was also demonstrated i n some but not most ANLL patients by studies including i d e n t i f i c a t i o n of heavy chain immunoglobulin gene rearrangements and G-6-PD analysis (405,406). Whether t h i s heterogeneity r e f l e c t s true differences i n stem c e l l o r i g i n or a dif f e r e n c e i n r e s t r i c t i o n of d i f f e r e n t i a t i o n has not been established, nor have these differences been correlated with pathogenetic, prognostic or therapeutic differences between ANLL patients. These p o s s i b i l i t i e s await further i n v e s t i g a t i o n s . Chromosomal Changes and Their Possible S i g n i f i c a n c e Cytogenetic studies of ANLL c e l l s have indicated that with the advent of new procedures ( i n p a r t i c u l a r , incubation of marrow c e l l s f o r several days) a l l cases of ANLL may involve chromosomal changes. Commonly reported karyotypic abnormalities i n ANLL have included nonrandom translocations (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 or t o t a l trisomy of chromosome 1, loss of chromosome 7, 21, X or Y, and trisomy of chromosome 8 or 21 (reviewed i n 407). An acquired d e l e t i o n of the long arm of chromosome 5 (5q ) has also been shown to be associated with a number of hematologic dysplasias including ANLL (408). Of 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 recent discovery that the proto-oncogene c-fms (the c e l l u l a r homologue of v-fms, the transforming gene of the McDonough s t r a i n of the f e l i n e sarcoma virus) was located near the breakpoint(s) on chromosome 5 and was, i n f a c t , deleted from the 5q chromosome (409). The c-fms gene i s normally expressed i n hemopoietic c e l l s and has been shown to 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 to the M-CSF (CSF-1) receptor (209). In the 5q syndrome, hemopoietic c e l l s are hemizygous f o r c-fms. This suggests that a l t e r e d M-CSF receptor numbers may be capable of causing an abnormal response to normal growth factor-mediated 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 that i n other systems, a l t e r i n g the normal pattern of expression of proto-oncogenes can contribute to malignant transformation (410). In ANLL, the a m p l i f i c a t i o n of the c-myc gene (411,412) and the c-myb gene (413) have been reported. Abnormal expression of proto-oncogenes, whose normal r o l e i s thought to be i n the regulation of basic c e l l u l a r 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 processes, may well be part of leukemogenesis i n ANLL. Like CGL, ANLL i s presumed to be a disease of multi-stage nature (414). The f i r s t stage (which may also consist of multiple "steps") can be equated to the preleukemic/myelodysplastic s i t u a t i o n , which could include the chronic phase of CGL. Here, stem c e l l s are 60 affec t e d i n such a way that, although g e n e t i c a l l y abnormal, they are s t i l l capable of expressing some control over d i f f e r e n t i a t i o n programs. 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 differences i n c e l l s at some maturation l e v e l s would occur but s i m i l a r types of d i f f e r e n t i a t i o n programs would s t i l l be followed. In the second stage (again, p o s s i b l y multi-step i t s e l f ) , the disease advances as genetic controls 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 ogressively uncouple. The ensuing dysregulation of hemopoiesis now allows the expression of formerly "forbidden" programs. P r o l i f e r a t i o n continues but d i f f e r e n t i a t i o n does not follow, as the l i n k between these processes has been a l t e r e d or broken. A maturation " a r r e s t " occurs which now manifests c l i n i c a l l y as ANLL or CGL b l a s t c r i s i s . Relationship Between ANLL C e l l s and Growth Regulators ANLL c e l l s cultured 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 chronic phase CGL c e l l s i n the same system. Cloning e f f i c i e n c y and growth patterns vary considerably between ANLL patients. T y p i c a l growth behavior can range from no growth, small-large c l u s t e r (4 - 39 c e l l clones) formation only, to an abnormally high cluster:colony r a t i o (341,415,416) with c e l l s often revealing lack of normal maturation, or i n some systems, leukemic b l a s t c e l l morphology (417,418). There i s cytogenetic evidence that clones produced i n v i t r o by ANLL c e l l s are derived from the o r i g i n a l i n vivo leukemic clone (378,419). During complete remission, i n v i t r o colony growth patterns return to normal i n terms of both numbers and a b i l i t y to mature. In 61 long-term ANLL bone marrow cultures, i t was possible to demonstrate that most newly diagnosed ANLL patients s t i l l retained a pool of normal hemopoietic stem c e l l s which were apparent by 4 - 6 weeks incubation and had presumably been suppressed i n vivo (381). As i n the case of CGL, the growth of ANLL c e l l s i n v i t r o remains completely dependent on a source of CSF through the course of the disease. In general, ANLL and normal c e l l s appear to be s i m i l a r l y s e n s i t i v e to stimulation by CSF, although some inve s t i g a t o r s have reported increased (382) and decreased (420) responsiveness by ANLL c e l l s . Some i n t e r e s t i n g evidence concerning subnormal CSF l e v e l s i n the immediate v i c i n i t y of ANLL c e l l s i n the marrow has been reported. Non-existent or very low CSF production by adherent marrow c e l l s has been observed i n patients with le s s d i f f e r e n t i a t e d forms of ANLL (421). A correspondingly poor response to chemotherapy was also noted i n these patients, although i f complete remission was attained, normal l e v e l s of 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 the case of CGL, decreased responsiveness of ANLL c e l l s to i n h i b i t o r y molecules (LF,AIF,PGE,LAI) has been observed. Possibly a combination of decreased amounts of 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 lack of negative regulation are enough to i n i t i a t e or at l e a s t promote, the emergence of a clone of c e l l s possessing a growth advantage over normal c e l l populations. C. DIFFERENTIATION INDUCTION IN MYELOID LEUKEMIA Reversion of the malignant phenotype i n myeloid leukemia c e l l s by the use of various agents capable of inducing d i f f e r e n t i a t i o n has 62 been a subject of widespread research. This work has contributed to our understanding of some aspects of the o r i g i n and evolution of t h i s disease, and of the process of normal d i f f e r e n t i a t i o n , as well as defi n i n g p o s s i b i l i t i e s f o r novel therapeutic modalities. As previously discussed, acute myeloid leukemic c e l l s self-generate with l i t t l e , i f any, concomitant d i f f e r e n t i a t i o n . I t follows that 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 neoplastic c e l l population. Some time ago, Ichikawa observed that murine Ml myeloid leukemic c e l l cultures could be induced to d i f f e r e n t i a t e by the addition of some types of conditioned media (422). 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 to be s i m i l a r l y inducible by adding a source of CSF to cultured c e l l s (423,424). Furthermore, i t was discovered that, within the cloned Ml and WEHI-3B c e l l l i n e s , i n d i v i d u a l c e l l s could form clones with markedly d i f f e r e n t responsiveness to CSF-induced d i f f e r e n t i a t i o n . Subclones were developed from human (425,426) and murine (422,427-431) myeloid leukemia c e l l s that could be induced to d i f f e r e n t i a t e (D +) to mature macrophages or granulocytes by CSF. Other clones (D ) were established which were blocked i n t h e i r a b i l i t y to be induced to d i f f e r e n t i a t e . In these l a t t e r clones, s p e c i f i c chromosomal changes were often observed. Clonal differences i n d i f f e r e n t i a t i o n i n d u c i b i l i t y mediated by other compounds than CSF were also reported. Change i n c o n s t i t u t i v e p r o t e i n expression that apparently i n h i b i t e d normal induction 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 + c e l l s (432). These changes, often d i f f e r e n t f o r i n d i v i d u a l inducing compounds, were thought to be due to 63 c o n s t i t u t i v e expression of d i f f e r e n t s p e c i f i c gene expression pathways. Indeed, 2-dimensional gel e l e c t r o p h o r e t i c p r o f i l e s examining the expression of hundreds of proteins during normal and induced myeloid d i f f e r e n t i a t i o n suggested that there were multiple, p a r a l l e l , i n d i v i d u a l l y programmed gene expression pathways involved i n d i f f e r e n t i a t i o n (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 the CSF sources were shown to be G-CSF and (weakly) GM-CSF i n the mouse, GM-CSF (CSFa) and G-CSF (CSFB) i n humans (391). Other compounds with the a b i l i t y to induce d i f f e r e n t i a t i o n i n myeloid leukemia c e l l s include dimethyl sulphoxide (DMSO), s t e r o i d s , phorbol esters, butyrate and many cytotoxic drugs used as chemotherapeutic agents i n myeloid leukemia. Numerous investigations have been c a r r i e d out i n order to determine i f the d i f f e r e n t i a t i o n induced myeloid leukemia c e l l s were a c t u a l l y equivalent to "normal" c e l l s . While c e r t a i n c e l l membrane . antigenic markers of normal mature c e l l s have been i d e n t i f i e d on induced c e l l s (434), many studies have reported abnormal r e a c t i v i t i e s with a number of monoclonal antibodies known to react with normal mature c e l l s (435). Biochemical (436) and f u n c t i o n a l membrane markers (437) have also been reported to be a t y p i c a l or defective i n the induced c e l l s , i n d i c a t i n g that leukemic c e l l s which have been induced to d i f f e r e n t i a t e are not equivalent to t h e i r normal mature c e l l counterparts. I I I . HUMAN MYELOID LEUKEMIA-ASSOCIATED ANTIGENS There has been intense i n t e r e s t i n the study of human leukemia-associated antigens (LAA) or markers of leukemic c e l l s . 64 These investigations have provided i n s i g h t into leucocyte d i f f e r e n t i a t i o n and the c e l l u l a r o r i g i n of leukemia as well as provided diagnostic reagents 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 from lymphoid leukemias. I n i t i a l l y , many studies focussed on the i d e n t i f i c a t i o n of antigenic markers that would be s p e c i f i c f o r (unique to) leukemic b l a s t s . I t was hoped that such markers could be u t i l i z e d both f o r i n i t i a l diagnosis and f o r detection of r e s i d u a l disease during remission and following bone marrow transplantation. Upon intensive i n v e s t i g a t i o n , putative leukemia-specific antigens have generally been found to be leukemia-associated antigens. These LAA have been shown to be present on normal myeloid c e l l s at c e r t a i n stages of d i f f e r e n t i a t i o n or during embryogenesis and, i n many instances, on non-hemopoietic t i s s u e s as well. E a r l y i nvestigations provided evidence f o r the existence of human leukemia-associated antigens. C e l l u l a r immune responses ( b l a s t formation) by remission lymphocytes i n the presence of i n a c t i v a t e d leukemic c e l l s from the same patients were reported (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 leukemic c e l l s but not to t h e i r remission leucocytes (440). Delayed type h y p e r s e n s i t i v i t y reactions by patients 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 antigens (441,442). Attempts by other investigators to reproduce some of these observations, u t i l i z i n g s i m i l a r methodologies have f a i l e d to substantiate some of these findings (443,444). Several examples of the production of autoantibodies reactive with p a t i e n t s ' own leukemic b l a s t s have been reported. In some instances, an increase i n these autoantibodies during remission was 65 reported (445,446). Antibody dependent c y t o t o x i c i t y f o r leukemic myeloblasts by patient sera has also been described (447,448). Immunoglobulins have been i d e n t i f i e d on the surface of leukemic c e l l s (449,450) and, a f t e r e l u t i o n from these c e l l s , were shown to have c y t o t o x i c i t y against leukemic c e l l s (450). A humoral response to LAA, separable from an a n t i - a l l o a n t i g e n response, was demonstrated i n many ANLL patients following immunotherapy with i r r a d i a t e d allogeneic leukemic b l a s t s (451). Characterization of the putative LAA recognized i n these studies has not been c a r r i e d out and t h e i r 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. Antisera to human leukemia c e l l s have been ra i s e d i n a number of non-human species (xeno-antisera) with the aim of de f i n i n g human LAA. In most cases, extensive absorption of such heteroantisera with normal hemopoietic c e l l s was necessary to render 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 . Heteroantisera i d e n t i f y i n g human myeloid LAA have been ra i s e d i n non-human primates (452-453), rabbits (453-456) and mice (457-458). By inducing tolerance i n mice to remission leucocytes, mice l a t e r immunized with leukemic c e l l s from the same patients produced a n t i s e r a s p e c i f i c f o r the leukemic c e l l type (ANLL vs ALL) (457-459). The same investigators also produced monkey anti-myeloblast serum with myeloid leukemic (ANLL and CGL b l a s t c r i s i s ) s p e c i f i c i t y , r e a c t i v e with a myeloblast antigen of molecular weight 75 - 80 KD and p i 7.8 shed i n v i t r o from the surface of leukemic myeloblasts (460). This compound was one of a seri e s of c h a r a c t e r i s t i c compounds shed from the surface of leukemic myeloblasts that 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 from compounds shed by other types of leukemic or nonleukemic c e l l s . 66 Their murine heteroantiserum has been shown to be capable of p r e d i c t i n g relapse i n ANLL remission patients (461,462). Other inv e s t i g a t o r s have reported simian anti-ANLL serum which, when absorbed against normal human blood c e l l s , recognized b l a s t c e l l s from an ANLL patient but not blood c e l l s from h i s normal i d e n t i c a l twin. This patient's leukemic b l a s t s , when used as stimulator c e l l s i n a mixed lymphocyte reaction, caused s i g n i f i c a n t stimulation of h i s normal twin's lymphocytes and h i s own remission lymphocytes (463). That several LAA existed on myeloid leukemic c e l l s was indicated by heteroantisera absorption studies with acute and chronic myeloid leukemia c e l l s (464). When i n d i v i d u a l ANLL or CGL patient's c e l l s were used f o r absorption, i t was apparent that some antigens detected by the sera were cross-reactive (ANLL and CGL), some were unique to c e r t a i n donors, while some myeloid leukemia patients' b l a s t s f a i l e d to react with any of a panel of simian a n t i s e r a to myeloid LAA. With the advent of monoclonal antibody technology i t became poss i b l e to i d e n t i f y i n d i v i d u a l antigenic markers of hemopoietic c e l l s with increasing ease. O r i g i n a l l y described by Kohler and M i l s t e i n (465), t h i s technique allowed the production of hybrid c e l l l i n e s , each producing homogeneous monoclonal antibodies dir e c t e d to a si n g l e antigenic determinant of a s i n g l e antigen. Such antibodies are capable of being produced i n v i t r o or i n vivo i n large q u a n t i t i e s . Much research e f f o r t has been spent on attempts to produce monoclonal antibodies that react s p e c i f i c a l l y with antigens on malignant c e l l s and not with normal c e l l u l a r antigens, or that d i s t i n g u i s h acute myeloid from acute lymphoid leukemia. The most f r u i t f u l r e s u l t s of these in v e s t i g a t i o n s have been the 67 ch a r a c t e r i z a t i o n of human leucocyte d i f f e r e n t i a t i o n antigens. The large number of reported myeloid-specific monoclonal antibodies (MAbs) emerging from these studies prompted the establishment of annual "International Workshops on Human Leukocyte D i f f e r e n t i a t i o n Antigens" i n 1983 (466-468). These workshops have enabled the standardization of nomenclature f o r leucocyte d i f f e r e n t i a t i o n antigens and allowed j o i n t analysis of hundreds of MAbs and t h e i r r e l a t i o n s h i p to each other, previously impossible to sort out by i n d i v i d u a l investigators (or groups) working independently. The number of " c l u s t e r determinant" (CD) groups or subgroups distinguished i n t h i s manner are now 50, including antigens i d e n t i f i e d on T and B c e l l s , myeloid c e l l s and activated c e l l s . Previous studies (reviewed 469-472) describing marker p r o f i l e s of human myeloid leukemia c e l l s have indicated that no i n d i v i d u a l MAbs or cytochemical markers recognized the malignant b l a s t c e l l s of every ANLL patient. Moreover, within the b l a s t c e l l population of i n d i v i d u a l ANLL patients, considerable heterogeneity also existed i n terms of these markers. I t i s not c l e a r i f t h i s s i t u a t i o n r e f l e c t s true genetic heterogeneity or 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 that the proportion of patient b l a s t c e l l s with s p e c i f i c markers u s u a l l y varies from below 15% to 90% (472). A number of investigarors have t r i e d to c o r r e l a t e FAB c l a s s i f i c a t i o n of ANLL with r e a c t i v i t y to defined myeloid antigens (472-476). While i t appears that most of these studies have not shown any cl e a r c o r r e l a t i o n between ANLL c e l l surface phenotypes and s p e c i f i c FAB classes, recent co-operative studies have described an association between c e r t a i n antigenic markers and FAB classes (CD14 antigen with 68 M4/M5) (477). Since markers of leukemic c e l l s appear to be normal gene products, i t i s us e f u l to discuss the better characterized molecules i n t h i s group. The "Leukocyte Common Antigen", formerly referred to as T200 or B220, i s now e n t i t l e d CD45. The CD45 antigen i s expressed at high density on the surface of hemopoietic c e l l s (leucocytes) (478). While not lineage s p e c i f i c , i t has been demonstrated to be c l o s e l y involved i n l i n e a g e - s p e c i f i c functions of lymphocytes, including T - c e l l mediated c y t o t o x i c i t y . The T200 gene encodes four forms of the same molecule by alternate s p l i c i n g (mol wt 220, 205, 190 and 180 KD) which appear to be d i f f e r e n t i a l l y expressed on subsets of T c e l l s , B c e l l s , monocytes and granulocytes (reviewed 468). A large panel of granulocyte-reactive MAbs have been investigated and found to i d e n t i f y a serie s of "granulocyte-associated" antigens. CD16 antigen, the Fc receptor on granulocytes, has been reported to range i n molecular weight from 54 - 62 KD, presumably due to microheterogeneity of the molecule (479). A 2.2 - 3.5 f o l d increase i n binding of anti-Fc MAbs was demonstrated a f t e r induced a c t i v a t i o n of neutrophils (480). A carbohydrate antigen, 3-a-fucosyl-N-acetyllactosamine (FAL) has also been i d e n t i f i e d i n asso c i a t i o n with a serie s of granulocyte membrane proteins of molecular weights 220, 180, 155, 130 and 98 KD) (479,481,482). FAL (or X-hapten) i s now e n t i t l e d CD15 by standardized nomenclature. FAL i s thought to be involved i n neutrophil functions such as margination, phagocytosis and resistance to b a c t e r i a l hydrolases (483). In addition to being strongly expressed by granulocytes, CD15 has also been i d e n t i f i e d on ANLL 69 patient c e l l s , e s p e c i a l l y M4 and M5 classes as well as on CGL c e l l s (484). Springer and Anderson (479) also i d e n t i f i e d three other 125 antigens i n I-labeled granulocyte lysates. These were molecules of 68, 65 (CAMAL) and 27 KD. "Monocyte-associated" antigens include CD14, strongly expressed on monocytes and found i n as s o c i a t i o n with (primarily) M4 and M5 stages of ANLL (486). MAbs s p e c i f i c f o r pl50/95, the l e a s t characterized member of the leucocyte function antigen family, are re a c t i v e with monocytes and were included i n the CD14 group (484). Most myeloid-associated antigens are expressed to varying degrees on both granulocytic and monocytic c e l l lineages. The leucocyte function antigen (LFA) family of heterodimers j u s t r e f e r r e d to also includes CD11 (C3bi receptor) and LFA-1. These c e l l surface glycoproteins share a common B subunit (mol wt 95 KD) noncovalently linked with d i f f e r e n t a subunits. 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 the LFA family. In f a c t , the s p e c i f i c antigen i d e n t i f i e d i s presumably the B subunit, since anti-CDwl8 MAbs can immunoprecipitate the (at least) 3 heterodimers within t h i s family — LFA-1, CD11/C3bi receptor and pl50/95. CD11, the C3bi receptor (mol wt 170/95 KD) i s expressed on granulocytes, monocytes and NK c e l l s (reviewed 486), and mediates the adherence and ingestion of C3bi-coated p a r t i c l e s (487). The CD11 antigen has been demonstrated on c e l l s from many cases of M4 and M5 ANLL, Ml, M2, and M3 to a l e s s e r degree, and CGL (488). LFA-1 (mol wt 180/95 KD) i s expressed on B and T c e l l s , NK c e l l s , monocytes and granulocytes. LFA-1 i s thought to represent an important adhesion glycoprotein that f a c i l i t a t e s leucocyte i n t e r a c t i o n v i a transmembrane s i g n a l l i n g 70 (489) . A myelomonocytic-associated g l y c o l i p i d (CD17), lactosylceramide i s expressed on most granulocytes, many monocytes, p l a t e l e t s and c e l l s (or c e l l l i n e s ) from ANLL and ALL patients (484). I t i s thought that oligosaccharides l i k e lactosylceramide, expressed i n normal and malignant hemopoietic c e l l populations, may be involved i n some way 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^ regulation (481-483). An increase i n lactosylceramide content was demonstrated to be associated with granulocyte d i f f e r e n t i a t i o n (490) . I n t e r e s t i n g l y , a study of the expression of 20 d i f f e r e n t g l y c o l i p i d s i n acute leukemia c e l l s indicated that i t was p o s s i b l e to c l a s s i f y even poorly d i f f e r e n t i a t e d acute leukemias as myeloid or lymphoid on the basis of t h e i r g l y c o l i p i d s and that the structure of c e l l surface g l y c o l i p i d s became more complex with increasing d i f f e r e n t i a t i o n (491). Antigens present 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 antigens, absent from most mature myeloid i c e l l s , are l i k e l y most c l o s e l y associated with c e l l s at p r i m i t i v e stages of d i f f e r e n t i a t i o n . The My-10 or CD34 antigen (mol wt 115 KD), l i k e many others i d e n t i f i e d by monoclonal antibodies, reacts with hemopoietic progenitor c e l l s (494). CD34 + progenitor c e l l s have been demonstrated to be capable of producing progeny of both myeloid and lymphoid (B and T) lineages, i n d i c a t i n g that the expression of t h i s antigen occurs 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 c e l l s . A recent study by Lowenberg and Bauman of the surface phenotypes of ANLL colony forming c e l l s (CFU) indicated that the CFU showed i n t e r p a t i e n t antigenic heterogeneity as well as differences between the leukemic c e l l populations i n expression of antigens i n 71 the same patient (496). Other studies have supported these findings (497) . Variable CFU expression (including absence) of antigens present i n the t o t a l leukemic c e l l population has important implications f o r the c l i n i c a l detection or purging of r e s i d u a l leukemic c e l l s . Surface marker studies are generally not necessary f o r the diagnosis and monitoring of chronic phase CGL, due to the c h a r a c t e r i s t i c overproduction of recognizable granulocytic c e l l s . The b l a s t c r i s i s phase (CGL BC) however, may be of lymphoid or myeloid type. With d i f f e r i n g treatments and prognoses, i t i s e s s e n t i a l to attempt to d i s t i n g u i s h between these two forms. Myeloid CGL BC c e l l s have surface marker phenotypes comparable to those i n ANLL. Lymphoid CGL-BC c e l l s have the same phenotype as that of ALL c e l l s . Immunophenotyping of CGL-BC has i d e n t i f i e d other le s s common variants (erythroid, mixed, megakaryocytic and u n d i f f e r e n t i a t e d (498) . From the numerous inv e s t i g a t i o n s regarding antigenic analysis of normal and leukemic myelopoiesis, there has emerged strong evidence that leukemic c e l l phenotypes probably r e s u l t from a combination of the expression of normal genes (not always i n normal quantitative fashion) and abnormal, asychronous d i f f e r e n t i a t i o n , including apparent maturation a r r e s t i n ANLL. The study of the expression of normal myeloid d i f f e r e n t i a t i o n antigens ( f o r example, loss of HLA-DR antigen and gain or loss of many others with increasing states of d i f f e r e n t i a t i o n ) has enabled important comparisons between malignant and normal gene expression i n hemopoiesis. 72 THESIS OBJECTIVES The existence of a common antigen i n (myelogenous) acute leukemia, (CAMAL), thought to be a c e l l surface p r o t e i n on BM and PB c e l l s of myeloid leukemia patients, had been previously established. Rabbit heteroantisera s p e c i f i c f o r the CAMAL antigen was demonstrated to react with myeloid leukemia c e l l membrane extracts i n the ELISA and with BM or PB c e l l s from myeloid leukemia patients (ANLL at diagnosis and during remission and CGL) i n fluorescence-activated c e l l s o r t e r (FACS) analysis but not at detectable l e v e l s with c e l l s from normals or most lymphoid leukemia patients (501,513,514). I d e n t i f i c a t i o n of the c e l l type(s) expressing t h i s marker, p a r t i c u l a r l y i n remission pa t i e n t s , had not been c a r r i e d out. A monoclonal antibody r e f e r r e d to as CAMAL-1 was subsequently developed which demonstrated s p e c i f i c i t y f o r polyacrylamide gel p u r i f i e d CAMAL i n ELISA (515). This MAb, however, showed l i m i t e d and inconsistent r e a c t i v i t y with BM, PB c e l l s and c e l l l i n e s from patients with nonlymphoblastic leukemia i n FACS analysis f o r c e l l membrane CAMAL (467,515). The following objectives of t h i s thesis project focus on es t a b l i s h i n g i n s i g h t into the nature of CAMAL expression by c e l l s and the po s s i b l e s i g n i f i c a n c e of such expression. 1. A simple rapid monoclonal antibody-based t e s t was required to detect the presence of the CAMAL marker i n BM and PB c e l l s . The nature of the dif 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 heteroantibodies needed to be established. 73 The p o s s i b l e presence of CAMAL i n normal hemopoietic c e l l s needed to be examined, and the i d e n t i f i c a t i o n of c e l l types expressing CAMAL was required. The p o s s i b l e s i g n i f i c a n c e of the frequent detection of CAMAL i n BM or PB of ANLL remission patients needed to be investigated. The question of i t s r e l a t i o n s h i p to c l i n i c a l prognosis needed to be answered. The author wished to examine the p o s s i b i l i t y that the CAMAL prot e i n might possess some fu n c t i o n a l a c t i v i t y r e l a t e d to hemopoietic c e l l growth, 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 . The i n v i t r o myeloid progenitor c e l l assay, a us e f u l system f o r t h i s evaluation, could be u t i l i z e d to investigate t h i s . Normal CAMAL could be s e l e c t i v e l y depleted from the system and leukemia-derived CAMAL could be added to the system, with subsequent evaluation of myeloid colony growth. 74 Footnotes 1. Recent evidence f o r the existence of 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 studies. R. Mulligan presented data (4th Terry Fox Cancer Conference, Stem C e l l s and Autologous Bone Marrow Transplantation, Vancouver, B.C., September 21-22, 1987) showing that i d e n t i c a l genomic in t e g r a t i o n patterns were observed i n lymphocytes (B and T c e l l s ) and macrophages when murine bone marrow, infected with a r e t r o v i r u s , was used to rec 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 indicated that i t was possible to i n f e c t p l u r i p o t e n t stem c e l l s without a f f e c t i n g t h e i r function and that very l i m i t e d numbers of clones gave r i s e to the majority of hemopoietic c e l l s at any given time, implying a sequential contribution by small numbers of marked stem c e l l clones. 2. Clark SC, Kamen R: The human hematopoietic colony-stimulating f a c t o r s . Science 236:1229, 1987, and Yang Y-C, C i a r l e t t a AB, Temple PA, Chung MP, Kovacic S, Witek-Giannotti JS, Leary AC, K r i z R, Donahue RE, Wong GG, Clark SC: Human IL-3 (multi-CSF): I d e n t i f i c a t i o n by expression cloning of a novel hematopoietic growth f a c t o r r e l a t e d to murine IL-3. C e l l 47:3, 1986. 3. B i o l o g i c a l properties of human inte r l e u k i n - 3 , presented by S. Clark at the 4th Terry Fox Cancer Conference, Stem C e l l s and Autologous Bone Marrow Transplantation, Vancouver, B.C., September 21-22, 1987. 75 CHAPTER II MATERIALS AND METHODS I. DETAILED PROCEDURES A. Monoclonal Antibodies 1. Production The monoclonal antibody (MAb) s p e c i f i c f o r the CAMAL antigen was prepared by Dr. Andrew J. Malcolm i n Dr. J u l i a G. Levy's laboratory. This MAb, CAMAL-1 (subclass IgG-1), was prepared from a fusion of NS-1 myeloma c e l l s and Balb/c splenocytes from mice immunized with p u r i f i e d [by polyacrylamide gel electrophoresis (PAGE)] CAMAL antigen. The hybridoma procedure followed that described by Oi and Herzenberg (499) with modifications by previous researchers i n t h i s laboratory (500), and was the same as that now described f o r the preparation of appropriate negative control MAbs. The negative c o n t r o l MAbs were chosen from a ser i e s of MAbs produced which were s p e c i f i c f o r p24, the major s t r u c t u r a l p r o t e i n of the Bovine Leukosis Virus (BLV). Five to s i x week old female Balb/c mice were immunized subcutaneously with 30 ug of g e l - p u r i f i e d BLV antigen i n 50% complete Freund's adjuvant. Two weeks following a second immunization, sera from these mice were tested f o r r e a c t i v i t y i n ELISA with p u r i f i e d p24. One week l a t e r , a strongly-immune mouse was given 20 ug of p u r i f i e d p24 i n 0.1 ml s t e r i l e PBS intravenously on 3 consecutive days and 2 days l a t e r the spleen was removed f o r 76 fusion with log phase NS-1 c e l l s . A r a t i o of 5:1 splenocytes:NS-l c e l l s was used. These c e l l s were fused with polyethylene g l y c o l (PEG) and plated into 96 well t i s s u e culture plates at 1.6 x 10~* c e l l s / w e l l i n Dulbecco's modified minimal e s s e n t i a l medium with 20% FCS, hypoxanthine (2 yg/ml), aminopterin (0.18 yg/ml), thymidine (0.4 yg/ml) and treated 5 Balb/c thymocytes from 5 week old mice at 10 feeder c e l l s / w e l l . A f t e r incubation at 37°C and 10% C0 2 f o r 10-14 days, 0.1 ml culture supernatants were assayed f o r r e a c t i v i t y i n ELISA with p24 (coated at 1.0 yg/ml). Hybridomas that showed p o s i t i v e r e a c t i v i t y with p24 (and negative r e a c t i v i t y with a battery of i r r e l e v a n t antigens, including CAMAL) were cloned 3 times by l i m i t i n g d i l u t i o n , expanded and grown as as c i t e s i n pristane-treated Balb/c mice. Monoclonal antibodies from a p a r t i c u l a r anti-p24 hybridoma (BLV-1) were chosen as an appropriate control f o r CAMAL-1 studies f o r the following reasons: (1) t h i s MAb was the same subclass as CAMAL-1 (IgG-1); (2) i t reacted i n i n d i r e c t immunoperoxidase te s t s with PHA-stimulated lymphocytes from BLV-infected c a t t l e ; and (3) i t was capable of performing 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 preparation i n a f f i n i t y chromatography. Since these are a l l properties shared by CAMAL-1, and since an appropriate negative control MAb was c r i t i c a l f o r many of the experiments described i n Chapter VI, BLV-1 was chosen from 12 anti-p24 l i n e s f o r these purposes. 77 2. P u r i f i c a t i o n CAMAL-1 and BLV-1 were p u r i f i e d from t h e i r respective a s c i t e s f l u i d i n the same manner using diethylaminoethyl (DEAE) chromatography. Ascites was centrifuged f o r 10 min at 400 g to remove f i b r i n c l o t s . A 50% saturated (NH.KSO. s o l u t i o n 4 2 4 was added dropwise with constant s t i r r i n g to the as c i t e s f l u i d . This s a l t cut was kept overnight at 4°C and centrifuged at 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 of 0.03M T r i s b u f f e r (pH 7.4) equivalent to the o r i g i n a l volume of a s c i t e s f l u i d , and o dialyzed overnight at 4 C i n 2 changes of the same buffer. DEAE-Sephacel was poured into a chromatography column and eq 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 prepared a s c i t e s was passed over the DEAE-Sephacel and the beads were washed with b u f f e r u n t i l OD = 0. The p u r i f i e d antibody was eluted from the column using a continuous gradient (0.05-0.2M NaCl i n 0.03M T r i s ) . C o llected f r a c t i o n s were monitored f o r absorbance at 280 nm and those containing Ig were pooled, concentrated over PEG, dialyzed extensively i n PBS, and tested i n the ELISA f o r r e a c t i v i t y with CAMAL. Protein concentrations of the p u r i f i e d monoclonal antibodies were determined using the Biorad p r o t e i n assay. 3. Immunoadsorbent Preparation DEAE-purified CAMAL-1 or BLV-1 MAbs f o r immunoadsorbent preparation were dialyzed i n 0.05M borate buffer, pH 8.0. Washed Sepharose 4B-CL beads were resuspended i n cold dH^O and s t i r r e d i n a small beaker in s i d e an i c e bucket. CNBr i n 78 dimethylformamide (250 mg/ml) was added slowly dropwise (33 mg/ml beads) over 1 min to the beads. The pH was kept between 10.5 and 11.0 using 4M NaOH over 30 min. The activated beads were washed qui c k l y on a glass funnel with 250 ml cold dH^O followed by 500 ml cold 0.05M borate buffer. The activated beads were added 1:1 v/v to the p u r i f i e d MAb (5.0 mg/ml) i n borate b u f f e r and the r e s u l t i n g s l u r r y was rocked overnight at o 4 C. The supernatant was c o l l e c t e d the next day and the coupled beads were washed i n lOmM T r i s b u f f e r , pH 8.0, before o being resuspended i n 0.16M ethanolamine overnight at 4 C to block any remaining a c t i v e s i t e s . F i n a l l y , the beads were washed well i n lOmM T r i s b u f f e r and stored at 4°C i n PBS with 0.02% NaN„. The 0D„„„ of the MAb s o l u t i o n before and a f t e r 3 280 coupling were compared. In each case, between 92 and 93% of the o r i g i n a l MAb was determined to have bound to the beads. B. Polyclonal (Rabbit) Antibodies Antiserum s p e c i f i c f o r CAMAL was prepared from New Zealand White female rabbits hyperimmunized subcutaneously with PAGE-purified CAMAL (30 yg CAMAL i n 50% complete Freund's adjuvant). Rabbits were immunized at monthly i n t e r v a l s and t h e i r blood c o l l e c t e d by marginal ear venipuncture 2 weeks following each immunization. Serum was removed from the c l o t t e d blood and assayed i n the ELISA f o r r e a c t i v i t y with CAMAL (coated at 1 ug/ml). Rabbit anti-CAMAL sera was r a i s e d i n a t o t a l of 6 ra b b i t s . A l l sera showed analogous s p e c i f i c i t y f o r CAMAL i n ELISA and i n FACS anal y s i s ; these data were 79 presented i n a Ph.D. thesis by A.J. Malcolm (501). Sera was stored at o -70 C i n aliquots u n t i l used. Normal rabb i t serum (NRS) was prepared by bleeding the same New Zealand White female rabbits p r i o r to t h e i r immunization with CAMAL. This serum was stored i n a s i m i l a r manner p r i o r to use. Rabbit anti-normal human serum (used as a p o s i t i v e control) was prepared by hyperimmunization of female New Zealand White rabbits with a membrane preparation derived from sonication of pooled normal human peripheral blood leucocytes emulsified i n 50% complete Freund's adjuvant. The r e s u l t i n g sera showed strong r e a c t i v i t y i n fluorescence-activated c e l l s o r t e r (FACS) analysis with BM and PB leucocytes from a l l i n d i v i d u a l s tested. C. Secondary Antibodies For FACS analysis using rabb i t a n t i s e r a as the primary l a b e l , goat a n t i - r a b b i t IgG, FC a b ) ^ fragment, conjugated to f l u o r e s c e i n isothiocyanate (FITC) was u t i l i z e d as the secondary l a b e l (Cappel). This and a l l primary antisera were r o u t i n e l y centrifuged at 20,000 rpm f o r 5 min i n microfuge tubes to remove aggregates immediately p r i o r to c e l l l a b e l i n g . For i n d i r e c t immunoperoxidase studies, using murine monoclonal antibodies as the primary l a b e l , r a b b i t anti-mouse Ig conjugated to horseradish peroxidase (HRP) was u t i l i z e d as the secondary l a b e l (Cedarlane). When rabbi t anti-CAMAL serum was employed as the primary l a b e l , swine a n t i - r a b b i t Ig - HRP (Cedarlane) was used as the secondary antibody. 80 Antigens 1. CAMAL P r i o r to the a v a i l a b i l i t y of the CAMAL-1 MAb, CAMAL was p u r i f i e d by Robert C. Shipman on preparative polycrylamide gels f o r purposes of rabbit immunization and ELISA. Membrane extracts from pooled PBL of ANLL patients with high leukemic c e l l counts were passed over an immunoadsorbent column to which r a b b i t anti-normal human antibodies were coupled. The material which d i d not bind to the column was electrophoresed on 7.5% polyacrylamide gels and the 68 KD pro t e i n band corresponding to CAMAL was cut out and eluted from the g e l . This material was used to coat ELISA plates and f o r immunization purposes. A f t e r the CAMAL-1 MAb became a v a i l a b l e , CAMAL was thereafter p u r i f i e d i n a one-step manner by e l u t i o n of bound material from a CAMAL-1 a f f i n i t y column over which crude extracts from myeloid leukemic c e l l s had been passed. This CAMAL preparation was used f o r ELISA, PAGE, and immunization of rabbits and was found to have the same c h a r a c t e r i s t i c s as the previously prepared CAMAL. In order to study the possible e f f e c t of addition of CAMAL on myeloid progenitor c e l l s i n v i t r o , i t was important that p u r i f i e d CAMAL f o r these studies be prepared somewhat d i f f e r e n t l y . For these studies the o r i g i n a l extract which was passed over the CAMAL-1 column consisted of the soluble f r a c t i o n r e s u l t i n g from freeze-thawing PBL of chronic granulocytic leukemia patients. The protein-containing f r a c t i o n s eluted from the CAMAL-1 column were immediately pooled and ne u t r a l i z e d with 81 0.1 H NaOH, followed by d i a l y s i s i n PBS. This rapid n e u t r a l i z a t i o n was c a r r i e d out i n order to maintain the antigen i n a state resembling i t s normal p h y s i o l o g i c a l one i f po s s i b l e . Following d i a l y s i s , the CAMAL preparation 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 aliqu o t was reserved f o r determination of pro t e i n concentration by the Biorad assay. 2. Negative co n t r o l p r o t e i n antigens f o r use i n the myeloid progenitor c e l l studies were prepared by a f f i n i t y chromatography p u r i f i c a t i o n i n the same manner as described f o r CAMAL. Murine monoclonal antibodies, p u r i f i e d on a rabbi t anti-mouse Ig immunoadsorbent column, were u t i l i z e d f o r these studies. E. Polyacrylamide Gel Electrophoresis (PAGE) Standard PAGE procedures (502,503) were u t i l i z e d to examine materials eluted from CAMAL-1 immunoadsorbent columns a f t e r passage of human plasma, conditioned media, and soluble extracts of myeloid leukemia patients' c e l l s . Ten percent reducing gels were prepared and sample materials, including molecular weight standards (Sigma), were electrophoresed at 50 V f o r 5 - 6 hours and s i l v e r stained (504). F. Patient Samples Peripheral blood or bone marrow samples were obtained from the Department of Hematology, C e l l Separator and Immunotransplant Laboratories, Vancouver General Hospital ( f o r a l l studies described), the Terry Fox Laboratory, Vancouver, B.C. (f o r the FACS so r t i n g 82 s t u d i e s ) , U n i v e r s i t y College Hospital and Dr. M. Greaves, ICRF, Lincoln's Inn F i e l d s , London ( f o r many of the samples described i n Chapter I I I ) . In addition, Dr. N. Buskard provided a number of samples from myeloid leukemics (through the Adult Outpatient Department and Cryolaboratory, Vancouver General Hospital) and normals (through the Canadian Red Cross, Vancouver) which were used i n the experiments described i n Chapter VI. Diagnoses were established using routine c r i t e r i a i ncluding morphological examination, cytochemistry, cytogenetics, colony assays, surface Ig and E-rosette t e s t s where applicable. In addition, i n d i r e c t immunofluorescence tests f o r c e l l surface antigens were c a r r i e d out on samples obtained from ICRF. A l l samples u t i l i z e d i n these studies were obtained from patients that had given t h e i r consent. G. C e l l Preparations Leucocytes were prepared from BM samples by sedimentation at 1,000 rpm and c o l l e c t i o n of the buffy coat, or by routine Ficoll-Hypaque separation procedures (505,506). Peripheral blood leucocytes (PBL) were obtained from heparinized samples using Plasmagel (Laboratoire Roger Bellon, N e u i l l y , France) or by the Ficoll-Hypaque technique. For a l l studies performed using immunoperoxidase and f o r the myeloid progenitor c e l l assays, F i c o l l -Hypaque separation was u t i l i z e d . Contaminating erythrocytes were removed by l y s i s f o r 3 - 4 min with 37°C Tris-NH^Cl at pH 7.2, i f necessary. 83 H. Fluorescence-Activated C e l l Sorter (FACS) Studies C e l l l a b e l i n g , FACS ana l y s i s , and FACS so r t i n g techniques were performed as outlined below. 1. C e l l Labeling Leucocytes prepared from BM or PB samples were washed twice 6 i n s t e r i l e PBS with 5% FCS. 1 x 10 c e l l s were then resuspended i n 0.2 ml of the primary antibody s o l u t i o n ( d i l u t e d 1:10 i n PBS/5%FCS). A l l samples were labeled separately with 3 primary antibodies:(1) the p o s i t i v e c o n t r o l , r a b b i t anti-normal human serum; (2) the negative c o n t r o l , normal rab b i t serum; and (3) rab b i t anti-CAMAL serum. C e l l s were incubated on i c e f o r 1.5 hr, washed 3 times i n PBS/5%FCS, and resuspended i n 0.2 ml of secondary antibody (fluoresceinated goat a n t i - r a b b i t IgG, d i l u t e d 1:20 i n PBS/5%FCS). A f t e r another 1.5 hr incubation on i c e , c e l l s were washed once i n PBS/5%FCS then centrifuged at 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 to FACS an a l y s i s . 2. FACS Analysis of Labeled C e l l Samples A Becton-Dickinson FACS IV was u t i l i z e d f o r these studies. Routine operation included argon l a s e r s e t t i n g of 400 mW, 488 nm, FITC f i l t e r and standardization with glutaraldehyde-fixed chicken red blood c e l l s and fluorescent monodispersed carboxymethylated microspheres (d = 1.75 m + 0.02 SD). For a given sample, 25,000 c e l l s were analyzed and recorded f o r r e a c t i v i t y with each of the p o s i t i v e , negative and anti-CAMAL sera. 84 3. FACS C e l l Sorting Technique A f t e r a sample had been analyzed as s i g n i f i c a n t l y p o s i t i v e with the rabb i t anti-CAMAL serum, t h i s sample was sorted using the FACS IV. Two sort windows were determined, one containing that 25% of the sample showing minimal r e l a t i v e fluorescence and the other containing that 25% of the sample showing the highest r e l a t i v e fluorescence (see Figure 2.1). The two c e l l populations were c o l l e c t e d i n separate tubes containing 1 ml PBS/5%FCS. The c e l l s between these two windows, representing the remaining 50% of the sample c e l l population, were discarded into the r e s e r v o i r f l a s k . The head drive frequency was set at 36 KHz, and 2,000 V were applied across the e l e c t r o s t a t i c d e f l e c t i o n p l a t e s . The Eput counters recorded the number of c e l l s c o l l e c t e d i n the r i g h t and l e f t d e f l e c t i o n tubes, and the FACS was run at 5 droplets per d e f l e c t i o n pulse with the abort mode i n operation; the droplet delay was set at 14 drops. Cooling water (2°C) was c i r c u l a t e d around the c o l l e c t i o n and sample tubes. Immediately following a sort run, 5,000 c e l l s from each of the c o l l e c t e d populations were examined with respect to t h e i r r e l a t i v e fluorescence, i n order to determine the accuracy of the sort. The expected accuracy was > 95%, based on so r t i n g accuracy determined p r i o r to each run using standardized green and red fluorescent beads (generally 97% accuracy). Sorting accuracies were found to be comparable f o r the c e l l samples c o l l e c t e d ; the "low" population showed 96.7 + 0.7% (SEM) accuracy and the "high" population showed 90.8 + 0.9% (SEM) accuracy, the di f f e r e n c e found to be a t t r i b u t a b l e to some 85 Figure 2.1. C e l l populations c o l l e c t e d by the fluorescence-activated c e l l s o r t i n g (FACS) technique, using 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 axis, l i g h t s c a t t e r (proportional to c e l l s i z e ) ; y axis, r e l a t i v e fluorescence i n t e n s i t y ; z axis, c e l l number. (b) T o t a l c e l l population (25,000 c e l l s ) analyzed with rabbit 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 populations c o l l e c t e d following c e l l s o r t i n g , showing 2 separate populations of lowest and highest (25% each) f l u o r e s c i n g c e l l s . 87 c e l l damage r e s u l t i n g i n debris which f e l l outside the o r i g i n a l l y defined sort window f o r the "high" population. The c e l l s were then washed once i n PBS to remove p r o t e i n from the FCS and the sample was loaded into a cytocentrifuge (John's S c i e n t i f i c ) at 800 rpm f o r 8 min to obtain s l i d e preparations. The s l i d e s were stained r o u t i n e l y with Wright's s t a i n and examined by l i g h t microscopy. I. I n d irect Immunoperoxidase Staining of Single C e l l Preparations The immunoperoxidase technique has previously been u t i l i z e d extensively on t i s s u e sections ( p a r a f f i n embedded or frozen); however i t s use i n s t a i n i n g s i n g l e c e l l s has been more l i m i t e d (467,507-511). We adapted the i n d i r e c t immunoperoxidase procedure to study CAMAL antigen expression i n or on s i n g l e c e l l cytospin preparations of bone marrow or PBL. This t e s t i s simple, rapid and u s e f u l as a diagnostic probe f o r the presence of CAMAL. Processed c e l l s were washed at l e a s t 3 times i n serum-free medium (RPMI), resuspended at 2 x 10 6 c e l l s / m l , and loaded (3 drops) into cytospin funnels f o r s l i d e preparation. A f t e r 30 min immersion i n methanol-2% H„0„ the s l i d e s were 2 2 sprayed, flooded, and sprayed again i n PBS (5 min). Primary antibody (CAMAL-1 or control BLV-1 MAb at 10 yg/ml, rabb i t anti-CAMAL or control r a b b i t serum at 1:400 d i l u t i o n i n PBS) was applied to the c e l l c i r c l e , incubated f o r 30 min at 20°C, and washed i n PBS. The secondary antibody (anti-mouse Ig-HRP at 1:100 or a n t i - r a b b i t Ig-HRP at 1:200 d i l u t i o n i n PBS with 1:25 normal human serum) was applied, incubated f o r 30 min at 20°C and washed. Slide s were developed f o r 88 10 min i n diaminobenzidine (10 mg) and H^O^ (100 y l ) i n 50 ml PBS, washed, and counterstained with e i t h e r 2% methyl green, hematoxylin or Wright's. Slides were examined by l i g h t microscopy. Slides were scanned completely to examine a l l c e l l s and percentage p o s i t i v e counts were determined at XI,000 magnification. In cases where p o s i t i v e r e a c t i v i t y was > 10% of a sample, at l e a s t 400 c e l l s were scored on each s l i d e . When samples showed lower r e a c t i v i t y , between 1,000 and 3,000 c e l l s were examined f o r each s l i d e (highest numbers of c e l l s were scored f o r samples with extremely low percentage r e a c t i v i t y ) . Mature and nucleated erythrocytes were not included i n the percentage p o s i t i v e c e l l counts as t h e i r presence was too v a r i a b l e . A l l s l i d e t e s t s were i n i t i a l l y c a r r i e d out at l e a s t twice on separate occasions and examined b l i n d l y and independently by two researchers (Drs. J. Levy and P. Logan) to ensure accuracy and r e p r o d u c i b i l i t y . A f t e r one year, t e s t s were only duplicated when s l i d e preparations were suboptimal, as a means of v e r i f y i n g r e s u l t s . J. B l i n d Study Protocol A b l i n d study, now ending i t s t h i r d year, was established between the Department of Microbiology, U.B.C. and the Department of Pathology and D i v i s i o n of Hematopathology, Vancouver General Hos p i t a l . This study was designed to monitor ANLL patients' BM c e l l s using CAMAL-1 and i n d i r e c t immunoperoxidase. 1. Patient Group A l l patients attending the Hematology Department of Vancouver General Hospital (as i n - or out- patients) and who had BM aspirations 89 performed were included i n t h i s study. There were 701 patient samples examined during t h i s study. Thirty-seven percent of these samples were from 86 d i f f e r e n t ANLL patients while 63% were from any other i n d i v i d u a l s attending the Hematology Department of Vancouver General Ho s p i t a l . This l a t t e r group included patients with any other type of leukemia, hematologic malignancy or dyscrasia, and normals. This study reports the r e s u l t s from 34 of the 86 d i f f e r e n t ANLL patients examined during t h i s time. These 34 patients represent every ANLL patient f o r whom bone marrow samples were obtained both at diagnosis and post-chemotherapy (within 1.0 + 0.5 months). The remaining (n = 52) ANLL patients were e i t h e r i n i t i a l l y evaluated at l a t e r stages i n t h e i r disease (n = 35), had i n s u f f i c i e n t amounts of bone marrow aspirated to enable s l i d e preparation (n = 11) or were only seen at diagnosis (n = 6). Eighty percent of a l l ANLL patients that we examined i n i t i a l l y at diagnosis (n = 40) achieved complete remission (CR) during the study period. In the presently reported group of 34 patients, 92% achieved CR. Differences (80% versus 92%) were due to f a i l u r e of 5/6 remaining patients, examined i n i t i a l l y at diagnosis, to a t t a i n CR. No follow up samples were received from these patients. The median patient age was 48.4 years f o r the t o t a l ANLL group (n = 86) and 49.3 years f o r the group reported herein (n = 34). 2. Analysis Groups Slides made from patient samples were coded by successive numbering so that no patient information would be a v a i l a b l e to the p r i n c i p a l i n v e s t i g a t o r . The ANLL patients who had been examined both at diagnosis and post-chemotherapy were divided into 2 groups. 90 Groups 1 and 2 were compared by the product-limit method (Kaplan-Meier estimate) using log-rank s t a t i s t i c s to determine differences i n s u r v i v a l time p r i o r to relapse. 1. ANLL patients at i n i t i a l presentation whose CAMAL BM values decreased post-chemotherapy. 2. ANLL patients at i n i t i a l presentation whose CAMAL BM values increased or stayed the same post-chemotherapy. 3. S t a t i s t i c a l Analysis We have defined CAMAL BM values which had not changed s i g n i f i c a n t l y pre- and post-chemotherapy as being those which changed < 5.0%. This f i g u r e was chosen from r e s u l t s of an analysis 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 selected from a population of 700 s l i d e s so that the complete range of true proportions of recorded CAMAL BM values would be represented. In no instance did the observed CAMAL BM value of a given s l i d e d i f f e r by > 4.5%, hence the f i g u r e 5.0% was chosen to indica t e a s i g n i f i c a n t change. The absolute magnitude of the residu a l s (differences between the f i v e recordings f o r the same s l i d e and the mean of these recordings) never exceeded + 2.4%. No d i v i s i o n of the patient group was performed f o r analysis of the data by the Cox proportional hazards model which examined the following covariates as they r e l a t e d to disease-free s u r v i v a l time p r i o r to relapse: sex, age, i n i t i a l CAMAL BM value, change i n CAMAL BM value (from diagnosis to post-chemotherapy). 91 K. Myeloid Progenitor C e l l Assay 1. General Protocol A system f o r studying the growth of myeloid progenitors ( s p e c i f i c a l l y colony-forming u n i t s i n culture or CFU-c) was established i n Dr. J . Levy's laboratory a f t e r examining three 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 of Dr. C. Eaves (Terry Fox Laboratory, Vancouver, B.C.), Dr. I. Bernstein (Fred Hutchinson Cancer I n s t i t u t e , Seattle, Washington) and Dr. H. Messner (Ontario Cancer I n s t i t u t e , Toronto, Ontario). While each of these systems has i t s advantages and disadvantages, the system based on Dr. H. Messner's protocol was chosen f o r these studies f o r the following reasons: (1) the use of human plasma allows the culture system to be r e s t r i c t e d to human or even autologous materials (no FCS i s used); (2) human plasma increases the v i s c o s i t y of the system and makes i t much easier to accurately determine colony numbers than methylcellulose systems using FCS; (3) colonies are e a s i l y plucked from methylcellulose, when desired, compared to agar-bound colonies; (4) human plasma promotes the growth of well-defined megakaryocyte colonies i n v i t r o ; and (5) human plasma contains protein that adsorbs s p e c i f i c a l l y to CAMAL-1 i n a f f i n i t y chromatography, allowing study of the e f f e c t of i t s depletion from the system. The colony-plucking and immunoperoxidase s t a i n i n g of the CGL sample described i n Chapter IV was ca r r i e d out on colonies obtained from Dr. C. Eaves (Terry Fox Laboratory, Vancouver, B.C.). Dr. Eaves also k i n d l y provided her laboratory's expertise i n t h i s system over a 92 period of 6 months i n order f o r t h i s i n v e s t i g a t o r to learn d e t a i l s of the i n v i t r o colony assay. The basic components of the myeloid progenitor assay w i l l be outlined i n d e t a i l . In general, to prepare duplicate cultures, the following components were added to 15 ml s t e r i l e t e s t tubes: _5 1.4 ml 2% methylcellulose (containing 5 x 10 M 2-mercaptoethanol) 0.9 ml human plasma 0.3 ml conditioned medium 6 0.3 ml c e l l s (at approximately 1 - 2 x 10 cells/ml) i n Iscove's medium The mixture was gently vortexed and 1.0 ml was delivered into duplicate 35 mm t i s s u e culture dishes (Lux) with an 18 ga needle and 3 cc syringe. The duplicate cultures were placed inside a large (150 x 15 mm) p e t r i d i s h with a 35 mm dish containing 5 ml s t e r i l e dH^O f o r humidification. Cultures were incubated at 37°C, 100% humidity and 5% CO^ f o r 14 days at which time they were read with an inverted microscope. Colonies containing > 20 c e l l s were scored. On two occasions during these studies, counts were v e r i f i e d b l i n d l y by another researcher. In addition, counts were also done b l i n d l y at i n t e r v a l s to v e r i f y r e s u l t s by t h i s i n v e s t i g a t o r . Detailed descriptions of the components involved i n the myeloid progenitor c e l l assay follow. 2. Preparation of Conditioned Medium Conditioned medium was used i n the myeloid progenitor c e l l assay as a source of colony stimulating a c t i v i t y f o r growth of myeloid progenitors i n v i t r o . 93 a. Placental Conditioned Medium (PCM) One e n t i r e placenta was obtained during a scheduled Caesarian-section at St. Paul's H o s p i t a l , Vancouver, B.C. The placenta was immediately placed into s t e r i l e PBS and transported to the Un i v e r s i t y of B r i t i s h Columbia. The placenta was washed free of contaminating blood with s t e r i l e PBS, the t i s s u e was teased apart and forced through a s t e r i l e screen to obtain a s i n g l e c e l l suspension. Placental c e l l s were washed 2 times i n RPMI and resuspended i n RPMI (each 1.0 ml volume of packed c e l l s i n 20 mis) with 5% FCS. The c e l l s were incubated at 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 the supernatant was harvested. Three ml aliquots of pooled supernatant (placental conditioned medium or PCM) were stored at -70°C u n t i l used. b. Leucocyte Conditioned Medium (PHA-LCM) Heparinized peripheral blood from normal i n d i v i d u a l s was sedimented at 37°C f o r 40 - 50 min and the leucocyte-rich plasma was harvested. The leucocytes were then p e l l e t e d at 400 g f o r 10 min and resuspended i n Iscove's medium with 10% FCS and 1% 6 phytohemagglutinin (PHA) at a concentration of 1 x 10 c e l l s / m l . F i f t y ml volumes were incubated at 37°C and 5% C0 2 f o r 7 days a f t e r which the supernatants were harvested and stored i n aliquots at o -70 C u n t i l needed. To ensure potency of the PCM and PHA-LCM preparations, they were t i t r a t e d i n the myeloid progenitor c e l l assay using normal peripheral blood leucocytes from a volunteer whose c e l l s had previously been demonstrated to be capable of colony formation i n that assay. Both the PCM and PHA-LCM preparations were found to function optimally as 94 stimulators of myeloid colony formation when used at a concentration of 10%. A dose response curve f o r one batch of PHA-LCM i s shown i n Figure 2.2. 3. Preparation of 2% Stock Methylcellulose Twenty grams of methylcellulose (MC) powder was autoclaved i n a 2 l i t r e f l a s k . This was dissolved i n 500 ml s t e r i l e hot dH^O with constant s t i r r i n g . Another 500 ml of 2 X Iscove's medium was added a f t e r the s o l u t i o n had cooled to 20°C (approximately 3 hr) and t h i s s o l u t i o n was s t i r r e d overnight at 4°C. Aliquots (100 ml) of the 2% MC s o l u t i o n were prepared and stored at 20°C f o r 7 days when they were examined f o r contamination and discarded i f necessary. S t e r i l e o aliquots were stored at -20 C u n t i l needed. P r i o r to use (24 hr) -5 an a l i q u o t was thawed, 5 x 10 M 2-mercaptoethanol was added and mixed by vigorous shaking. The stock MC s o l u t i o n was used or the remaining discarded within 1 month of thawing. Before any batch of methylcellulose was used f o r experimentation, a t i t r a t i o n was performed using normal PBL to determine the optimal concentration of MC to be used i n the system. 4. Preparation of Human Plasma Samples of f a s t i n g human plasma prepared from heparinized peripheral blood samples ( c o l l e c t e d from normal volunteers) were tested f o r t h e i r growth promoting capacity i n the myeloid progenitor o c e l l assay. Appropriate samples were stored i n aliquots at -70 C u n t i l used. For standardization purposes required i n the experiments outined i n Chapter VI, plasma from a s i n g l e volunteer was obtained i n quantity (500-600 ml whole blood donation) on 3 separate occasions over 8 months. This plasma was always of high q u a l i t y with regard to 95 PERCENT CONDITIONED MEDIUM (PHA-LCM) F i g u r e 2 . 2 . Dose response curve 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 lood CFU-c . 96 growth promoting a b i l i t y i n the myeloid progenitor c e l l assay. Leucocytes from the same blood sample were always used to prepare PHA-LCM at the same time, to further standardize the system. P r i o r to use i n the assay, plasma samples were thawed, centrifuged to remove any f i b r i n c l o t s that 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 to reduce background p r e c i p i t a t e s . 5. Preparation of C e l l Samples Bone marrow or peripheral blood specimens were c o l l e c t e d i n preservative-free heparinized tubes. Mononuclear c e l l - e n r i c h e d preparations were obtained by layering samples over a Ficoll-Hypaque gradient as previously described. Leucocytes were washed twice i n Iscove's medium and resuspended at 1 x 10^ c e l l s / m l . Occasionally, c e l l s were plated at lower ( f o r CGL samples) or higher ( f o r ANLL samples) concentrations i n order to obtain appropriate numbers of colonies f o r assaying. To prepare non-adherent c e l l populations 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 populations by incubation (37 C, 5% CO^) overnight at 1 x 10 6 c e l l s / m l i n Iscove's medium with 5% FCS i n t i s s u e culture f l a s k s . 6. Plucking and Staining of Colonies On some occasions, colonies were plucked i n d i v i d u a l l y from the methylcellulose (while being examined through an inverted microscope) using a drawn-out c a p i l l a r y tube and a t i n y rubber bulb to aspirate c e l l s . These aspirated c e l l s were placed inside previously etched c i r c l e s on glass microscope s l i d e s and dispersed within the c i r c l e using a j e t of forced a i r from a pump. A f t e r a i r - d r y i n g , c e l l s were stained with a modified Papanicolaou technique or Wright's s t a i n and 97 examined by l i g h t microscopy. For the i n d i r e c t immunoperoxidase staining of the CGL colonies described i n Chapter VI, the technique used was the same as that already described herein except that i t was necessary to use an i n i t i a l immersion i n 10% H 0^ - methanol i n 2 2 order to adequately block endogenous peroxidase. 7. Preparation of Human Plasma f o r CAMAL Depletion Studies Normal human plasma was depleted of CAMAL by immunoadsorbence using a CAMAL-1 column. Between 10 and 20 ml of plasma was depleted i n t h i s manner at any given time. A 6.5 ml a f f i n i t y column was u t i l i z e d , plasma was passed over the column and c o l l e c t e d i n 1 ml f r a c t i o n s . Those cen t r a l f r a c t i o n s having equivalent O D23o readings were pooled and 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 before being stored i n aliquots at -70°C. A f t e r thoroughly washing the column with PBS u n t i l the unbound f i l t r a t e showed negative OD _ readings, the material adsorbed to the column 280 was eluted i n 1 ml f r a c t i o n s with 0.1 N HC1. Protein-containing f r a c t i o n s were pooled and stored. To prepare appropriate control plasma f o r these experiments, batches of plasma obtained from the same normal volunteer at the same time, were passed i n an i d e n t i c a l manner over a 6.5 ml BLV-1 (negative control MAb) a f f i n i t y column. Those cen t r a l f r a c t i o n s having OD readings equivalent to the plasma before column 280 passage were pooled, f i l t e r - s t e r i l i z e d and stored. 8. Preparation of Conditioned Medium f o r CAMAL Depletion Studies Conditioned medium (PCM and PHA-LCM) was depleted of CAMAL by passage over a CAMAL-1 immunoaffinity column i n the same manner as that j u s t described f o r plasma. Appropriate control conditioned 98 media was also prepared by passage over a BLV-1 immunoaffinity column. Conditioned medium so prepared was f i l t e r - s t e r i l i z e d and o stored i n aliquots at -70 C u n t i l used. 9. CAMAL Addition Studies Preparation of p u r i f i e d CAMAL or negative control proteins have been described previously. These proteins were maintained at pH 7.2 i n phosphate buffered s a l i n e at 4°C u n t i l u t i l i z e d . The solutions had been f i l t e r s t e r i l i z e d and Biorad p r o t e i n assays performed on an ali 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 studies, equivalent amounts ( 0 - 4 0 ug/ml) of CAMAL or negative control proteins were added to routine c e l l cultures 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 of p l a t i n g mixture were maintained at routine volume (equivalent to 1.0 ml/plate) by replacing portions of the c e l l s o l u t i o n (Iscove's medium) with PBS with or without CAMAL or negative c o n t r o l proteins. The preparations were plated and cultured i n a routine manner. Three separately prepared CAMAL pro t e i n samples were u t i l i z e d i n these studies. Each sample came from a CGL patient's c e l l s . Each sample was tested both on normal and myeloid leukemic c e l l s to ensure that non-specific t o x i c i t y of i n d i v i d u a l preparations was not occurring. Three separately prepared negative control protein samples were s i m i l a r l y u t i l i z e d . L. S t a t i s t i c a l Analyses Two-sided Student's t - t e s t analyses were performed to compare the data within Chapter I I I and within Chapter VI. Comparisons were made, between myeloid leukemics and normals (or other hematologic 99 malignancies), of the average percent CAMAL-1 p o s i t i v e c e l l s i n BM and PB. The r e s u l t s of CFU-c growth i n control and CAMAL depleted cultures were also compared i n myeloid leukemics and normals using the Students' two-sided t - t e s t . S t a t i s t i c s u t i l i z e d f o r the data presented i n Chapter V included the Cox s u r v i v a l analysis model i n the BMDP2L computer program to determine i f any of patient sex, age, CAMAL BM value at diagnosis or change i n CAMAL BM value post-chemotherapy were s i g n i f i c a n t f a c t ors i n the p r e d i c t i o n of which ANLL patients would have longer remission times. The Kaplan-Meier estimate with log rank s t a t i s t i c a l analysis (512) was used to compare s u r v i v a l time p r i o r to relapse f o r ANLL patients with (a) decreasing or (b) increasing/unchanging CAMAL BM values post-chemotherapy. 100 CHAPTER III 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 of human leukemias using immunologic c r i t e r i a , p a r t i c u l a r l y s e r o l o g i c a l examination of c e l l surface antigenic markers, has gained widespread acceptance over the l a s t decade. C e l l surface marker phenotyping has i contributed s i g n i f i c a n t l y to our understanding of the nature of human acute leukemias, including the 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 hematologic malignancies and the r e l a t i o n s h i p of c e r t a i n c e l l surface phenotypes to c l i n i c a l prognosis. This subject has been investigated extensively and reviewed (469-472). Chapter I of t h i s thesis outlined antigens of 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 . This o u t l i n e emphasized the problems of antigenic heterogeneity i n ANLL which complicate diagnosis and po s s i b l y treatment of t h i s disease. Although there i s much evidence that leukemia-associated antigens, unique or not, e x i s t i n ANLL, there has been l i m i t e d major success at actual i s o l a t i o n and chara c t e r i z a t i o n of such antigens (452-464). We have previously reported on rabbit heteroantisera r a i s e d to p u r i f i e d CAMAL antigen derived from sonication of pooled peripheral blood leucocytes from ANLL patients with high leukemic c e l l counts (513,514). We have shown by ELISA, i n d i r e c t immunoflourescence i n the fluorescence-activated c e l l sorter and by immunoprecipitation 101 that t h i s anti-CAMAL serum reacts strongly with PBL and BM c e l l s from patients with ANLL (acute phase or remission) or CGL and not at detectable l e v e l s with c e l l s from normal i n d i v i d u a l s or most patients with lymphoproliferative disorders. More recently, we reported the development of a monoclonal antibody, CAMAL-1, derived from a fusion of NS-1 c e l l s and Balb/c splenocytes from mice immunized with the same p u r i f i e d antigen (515). While i n i t i a l r e s u l t s using FACS analysis were promising, i t soon became apparent that t h i s MAb did not react, i n many instances, with ANLL patients' c e l l s i n surface fluorescence assays. This MAb, however, reacted very strongly with sonicates of ANLL c e l l s i n the ELISA and was shown to be capable of p u r i f y i n g CAMAL from such crude sonicates using a f f i n i t y chromatography. Since we wished to use t h i s MAb to study the presence of CAMAL on leukemic c e l l s , we developed a simple and rapid method of doing t h i s using an i n d i r e c t immunoperoxidase procedure on s i n g l e c e l l s l i d e preparations. This chapter describes the cumulative r e s u l t s from more than three years of i n v e s t i g a t i o n using the immunoperoxidase s t a i n i n g procedure which was d e t a i l e d i n Chapter I I . We examined a l l patients [and normal bone marrow transplant (BMT) donors] who had peripheral blood and/or bone marrow aspirations performed through the Department of Hematology, Vancouver General Hospital as well as some normal PB from volunteers at the U n i v e r s i t y of B r i t i s h Columbia. For over two years, these studies have been performed b l i n d l y ; these r e s u l t s were comparable to those gathered before the b l i n d study outlined i n Chapter I I . The data presented here show c l e a r l y that the CAMAL-1 MAb reacts s p e c i f i c a l l y with PB or BM c e l l s from patients with 102 nonlymphoblastic leukemia and that most of these i n d i v i d u a l s have s i g n i f i c a n t l y greater numbers of CAMAL-positive c e l l s than do normals or patients with lymphoid malignancies. This s l i d e t e s t has also been shown to be of great s i g n i f i c a n c e f o r the study of other myeloid-associated antigens, using other MAb's, some of which ( l i k e CAMAL-1) performed poorly i n i n d i r e c t immunofluorescence c e l l surface antigen assays. RESULTS A. Comparative Immunoperoxidase R e a c t i v i t y Between Monoclonal and  Rabbit Antibodies Studies using the r a b b i t antibody with i n d i r e c t immunoperoxidase have shown that the l e v e l of detection of CAMAL-positive c e l l s i n samples from patients with ANLL i s comparable to r e s u l t s described previously by us using a s i m i l a r antiserum and FACS analysis (514). Another study (515) and subsequent investigations using CAMAL-1 i n FACS analysis indicated that t h i s MAb showed, i n general, lower (and often no) r e a c t i v i t y with ANLL or CGL c e l l s than did the r a b b i t antiserum. Results from the i n d i r e c t immunoperoxidase assay support these findings and i n d i c a t e that the differences i n r e a c t i v i t y r e s u l t from an often s t r i k i n g d i f f e r e n c e i n p r e f e r e n t i a l membrane (by the rabbit antibody) versus i n t r a c e l l u l a r (by CAMAL-1) antigen recognition. This point i s i l l u s t r a t e d i n Figure 3.1 a,b where BM c e l l s from the same ANLL patient were labeled with rab b i t antibody and CAMAL-1 res p e c t i v e l y . This general d i f f e r e n c e between the rabbit antibody and CAMAL-1 has been observed i n a large number of samples tested. These d i s t i n c t i o n s may be of importance i n d i s t i n g u i s h i n g 103 F i g u r e 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 monoclonal ant ibody on ANLL bone marrow c e l l s . (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 s t a i n i n g ; (b) CAMAL-1 MAb l a b e l e d , showing s t rong i n t e r n a l s t a i n i n g . a 105 those c e l l s that synthesize or i n t e r n a l i z e t h i s antigen from those that may only adsorb small amounts of soluble CAMAL on t h e i r plasma membrane. C e l l surface antigen modification may e f f e c t i v e l y mask the determinant uniquely recognized by the CAMAL-1 MAb. I t should be noted that the same a f f i n i t y - p u r i f i e d CAMAL preparation was used to produce both of these a n t i s e r a and that they competitively i n h i b i t each other i n the ELISA when tested against p u r i f i e d CAMAL (Dr. Robert Shipman, personal communication). A comparison of the actual percentage r e a c t i v i t i e s of myelogenous leukemia samples with both the rabbi t antibody and CAMAL-1 using immunoperoxidase i s shown i n Table IV. While r e a c t i v i t i e s with these samples are often higher with the rabbi t antibody, there are also a higher number of samples that show background a r t i f a c t u a l s t a i n i n g with the negative c o n t r o l r a b b i t serum than with the MAbs. The ease of standardization, v i r t u a l lack of a r t i f a c t u a l s t a i n i n g , and acceptance by other in v e s t i g a t o r s i n the f i e l d were the major factors involved i n choosing CAMAL-1 f o r the study presented here. B. CAMAL-1 Immunoperoxidase S l i d e Test The photomicrographs shown i n Figure 3.2 a-h i l l u s t r a t e many of the advantages of the i n d i r e c t immunoperoxidase s i n g l e c e l l s t a i n i n g technique (with monoclonal antibody l a b e l i n g ) , as well as a number of t y p i c a l s t a i n i n g patterns found i n various patient groups and normals. Figure 3.2 a,b i l l u s t r a t e a number of points important i n the t e s t . BM c e l l s from an ANLL patient at diagnosis are shown, counterstained with hematoxylin and Wright's r e s p e c t i v e l y . These photomicrographs show a d i r e c t comparison between r e s u l t s obtained with these two counterstains on the same labeled c e l l sample. Figure 106 Table IV. Comparison of myelogenous leukemia c e l l r e a c t i v i t y with anti-CAMAL and CAMAL-1 using immunoperoxidase Percentage r e a c t i v i t y  Diagnosis Sample CAMAL-1 Rabbit anti-CAMAL ANLL, i n i t i a l BM 31 49 presentation PB 2 12 ANLL, i n i t i a l BM 4 95 presentation ANLL remission (3 months p r i o r to relapse) PB 0 90 ANLL, relapse BM * 29 82 ANLL, relapse BM 13 21 CGL, chronic phase PB 23 43 CGL, chronic phase PB 19 45 * I l l u s t r a t e d i n Figure 3.1 a,b. Approximately 25,000 c e l l s were analyzed i n each case, with each antiserum. 107 Figure 3.2. Immunoperoxidase s i n g l e c e l l s l i d e t e s t with CAMAL-1. BM c e l l s from a newly diagnosed ANLL patient counterstained with (a) hematoxylin and (b) Wright's s t a i n s ; (c) same c e l l sample labeled with negative control MAb; (d) ANLL remission BM sample, counterstained with methyl green; (e) chronic phase CGL pe r i p h e r a l blood c e l l s ; (f) BM c e l l s from a patient with preleukemia; (g) normal BM; (h) normal peripheral blood c e l l s . 108 109 110 112 3.2 b indicates that, regardless of the q u a l i t y of the s l i d e preparation i t s e l f , p o s i t i v e c e l l s are exceptionally well delineated. The extreme v a r i a b i l i t y i n antigenic d i s t r i b u t i o n (cytoplasmic, perinuclear, nuclear, 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 the same c e l l sample labeled with the negative control monoclonal antibody. In a l l cases appropriate negative controls were performed. As i n Figure 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 e l l s ; consequently, further photomicrographs of such negative controls w i l l not be presented here. In Figure 3.2 d, a BM sample from an ANLL remission patient labeled with CAMAL-1 and counterstained with methyl green i s shown. A very s i g n i f i c a n t number of 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 remission patients, i n d i c a t i n g that we may be detecting a marker of an underlying abnormality which i s s t i l l occuring i n these pa t i e n t s , even though no morphologically i d e n t i f i a b l e malignant c e l l s are present. In Figure 3.2 e, a t y p i c a l s t a i n i n g pattern i s i l l u s t r a t e d when PBL from a CGL (chronic phase) patient were labeled with CAMAL-1. Immature and mature granulocytes comprised 95% of these CAMAL-1 re a c t i v e c e l l s , one of which demonstrates antigen-rich membrane blebbing. The a b i l i t y to d i r e c t l y determine the morphology of 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 of t h i s t e s t and has confirmed observations with fluorescence-activated c e l l s o r t i n g that c e l l s that are CAMAL-positive include many nonblast c e l l s (discussed i n Chapter IV). 113 Figure 3.2 f shows a si n g l e intensely p o s i t i v e mononuclear c e l l from the BM of a preleukemic patient labeled with CAMAL-1. Granular cytoplasmic s t a i n i n g , an intense perinuclear r i n g and prominent clumped intranuclear antigen d i s t r i b u t i o n are evident i n t h i s c e l l . Many samples, a f t e r extensive processing, s t a i n less r o u t i n e l y with any counterstain; however, the p o s i t i v e c e l l s are remarkable i n t h e i r s t a i n i n g pattern and stand out bo l d l y as seen i n t h i s photomicrograph. Approximately 75% of the 31 normal BM samples tested have revealed a very small number of CAMAL-1 p o s i t i v e c e l l s (average 0.6 % ) , one of which i s i l l u s t r a t e d i n Figure 3.2 g. This was the case whether Ficoll-Hypaque or buffy coat-separated samples were examined. Previous methods of c e l l analysis using the FACS did not detect t h i s very low number of CAMAL-expressing c e l l s i n normal BM samples. Figure 3.2 h i l l u s t r a t e s that normal PBL labeled with CAMAL-1 show no (or < 0.1%) p o s i t i v e l y - r e a c t i n g c e l l s . A large number of patients' c e l l s have now been examined i n t h i s way using CAMAL-1, and the r e s u l t s are summarized i n Tables V -VIII. These r e s u l t s demonstrate that s i g n i f i c a n t numbers of CAMAL-expressing c e l l s are present i n BM and PB of myeloid leukemia patients and i l l u s t r a t e a simple and rapid diagnostic s l i d e t e s t i n which the CAMAL-1 MAb may be employed to detect the presence of t h i s antigen. Table V shows a summary of r e s u l t s obtained when BM and PBL from patients with ANLL i n various stages of t h e i r disease were tested with CAMAL-1 i n the i n d i r e c t immunoperoxidase s l i d e t e s t . I t can be seen that a s i g n i f i c a n t number of CAMAL-1 p o s i t i v e c e l l s are present 114 Table V. Summary of immunoperoxidase st a i n i n g r e s u l t s of c e l l s from acute nonlymphoblastic leukemia patients labeled with CAMAL-1 Diagnosis Sample Number Percent CAMAL-positive c e l l s tested + SEM and range ANLL, at BM 75 14.3 + 2, .1 (0. 1 - 80.0) diagnosis PB 50 4.4 + 1. .8 (0 - 80.0) ANLL, post- BM 29 18.7 + 4, .9 (0 - 100.0) chemotherapy PB 24 12.1 + 4, ,7 (0 - 80.0) ANLL, remission BM 120 13.6 + 1, .7 (0. 1 - 100.0) PB 104 6.5 + 1, .3 (0 - 80.0) ANLL, relapse BM 39 14.4 + 2 .4 (0. 2 - 80.0) PB 31 8.6 + 3, .9 (0 - 100.0) 115 i n BM or PBL of most ANLL pat i e n t s , whether i n acute phase of t h e i r disease, post-chemotherapy or during remission. Furthermore, i t has been determined (Chapter V) that many patients showing low numbers of p o s i t i v e c e l l s at diagnosis subsequently display very high values post-chemotherapy and during remission. As mentioned previously, these r e s u l t s have been gathered over the past 3 1/2 years and therefore include s l i d e examinations performed during the b l i n d study described i n Chapter V as well as those previously 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 obtained before and a f t e r the b l i n d study indicated that r e s u l t s were, i n most cases, very s i m i l a r i n terms of average % p o s i t i v e c e l l s i n various patient groups. For example, average values f o r ANLL patient BM samples at diagnosis were found to be 13.6 before and 14.3% a f t e r the b l i n d study. Table VI summarizes r e s u l t s obtained when CGL patient BM and PB c e l l s were examined. Very high numbers of CAMAL-positive c e l l s are present i n CGL samples i n chronic or accelerated phases of the disease. We were interested i n determining i f CAMAL was s t i l l present i n or on c e l l s from CGL patients when they entered an acute b l a s t c r i s i s state and i f there might be a differ e n c e i n antigen expression depending on the nature of the b l a s t i c transformation (myeloid vs lymphoid). A l l patients i n myeloid b l a s t c r i s i s (CGL MBC) showed very s i g n i f i c a n t numbers of CAMAL-positive c e l l s i n t h e i r BM and PB; two patients i n lymphoid b l a s t c r i s i s (CGL LBC), however, had comparatively few p o s i t i v e PBL. BM and PB c e l l s from patients with myelodysplastic syndromes (preleukemia/MDS including r e f r a c t o r y anemia with excess b l a s t s i n 116 Table VI. Summary of immunoperoxidase s t a i n i n g r e s u l t s of c e l l s from patients, with chronic granulocytic leukemia Diagnosis Sample Number Percent CAMAL-positive c e l l s tested + SEM and range CGL, chronic BM 26 22.4 + 5.1 ( 0.8 - 100, .0) phase PB 40 23.0 + 4.2 ( 0 - 100, .0) CGL, accelerated BM 3 24.0 + 9.2 (12.0 - 42, .0) phase PB 4 5.5 + 2.9 ( 1.8 - 14, .0) CGL MBC * BM 13 23.8 + 6.7 ( 3.0 - 80. .0) PB 5 24.6 + 8.7 (10.0 - 50, .0) CGL LBC ** PB 2 0.3 + 0.2 ( 0.1 - 0. .5) * MBC = myeloid b l a s t c r i s i s ** LBC = lymphoid b l a s t c r i s i s 117 transformation or RAEBIT) were found to have high numbers of CAMAL-positive c e l l s , comparable to those found i n myeloid leukemics (Table VII). Since the CAMAL antigen i s common to a l l forms of myeloid leukemia and since these disorders often progress to that end, t h i s was not an unexpected f i n d i n g . Table VIII shows r e s u l t s obtained with ALL, CLL and lymphoma patients as well as normals. With the exception of c e r t a i n ALL remission patients, a l l of these samples showed very low average numbers of CAMAL-positive c e l l s i n t h e i r BM or PB and very narrow ranges of p o s i t i v i t y , i n d i c a t i n g a dramatic d i f f e r e n c e between these samples and the myeloid leukemics or myelodysplastics. These differences i n average numbers of CAMAL-positive c e l l s were found to be hi 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 , with a l l p values < 0.001. I t was i n t e r e s t i n g to note that the CAMAL-positive c e l l s found i n approximately h a l f of the newly diagnosed ALL pat i e n t s , while c o n s t i t u t i n g a very small f r a c t i o n of the t o t a l c e l l population when compared to myeloid leukemics, were nevertheless often present at l e v e l s s l i g h t l y higher than those found i n normal BM or PB samples. The s i g n i f i c a n c e of t h i s observation, i f any, remains unclear but may be r e l a t e d to some basic abnormality i n hemopoiesis. Some previous r e s u l t s from FACS analysis with r a b b i t anti-CAMAL serum showed an occasional ALL patient with high numbers of CAMAL-positive BM or PB c e l l s (514). Recently there has been much evidence published to support findings such as t h i s ; a number of investigators have found simultaneous expression of myeloid and lymphoid antigens on leukemic c e l l s (516-527). Lineage i n f i d e l i t y i n some leukemia patients' c e l l s apparently does e x i s t and t h i s discovery has helped 118 Table VII. Summary of immunoperoxidase s t a i n i n g r e s u l t s of c e l l s from patients with preleukemia/MDS including RAEBIT* Diagnosis Sample Number Percent CAMAL-positive c e l l s tested + SEM and range Preleukemia/ MDS RAEBIT BM PB BM PB 25 19 6 7 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) * MDS = myelodysplastic syndrome RAEBIT = r e f r a c t o r y anemia with excess b l a s t s i n transformation 119 Table VIII. Summary of immunoperoxidase s t a i n i n g r e s u l t s of c e l l s from patients with lymphoid malignancies or normals Diagnosis Sample Number Percent CAMAL-positive c e l l s tested + SEM and range ALL, at BM 41 1.2 + 0.3 (0 - 7.5) diagnosis PB 19 0.3 + 0.1 (0 - 2.0) ALL, remission BM 11 12.4 + 4.8 (0.1 - 50.0) PB 8 5.2 + 2.2 (0.4 - 18.0) CLL * BM 8 1.1 + 0.5 (0 - 3.8) PB 22 0.2 + 0.1 (0 - 1.5) Lymphoma BM 77 2.3 + 0.2 (0 - 9.4) PB 8 1.6 + 0.7 (0 - 5.5) Normal BM 31 0.6 + 0.1 (0 - 2.5) PB 38 <0.1 + <0.1 (0 - <0.1) * CLL = chronic lymphocytic leukemia 120 to widen our understanding of both leukemic c e l l o r i g i n s and hemopoiesis. Greaves et a l . suggested that such lineage promiscuity most l i k e l y represents a normally transient stage of gene expression i n m u l t i - or b i p o t e n t i a l precursors which has been "arrested" or frozen i n the acute leukemic state (528). The observation that approximately h a l f of the ALL remission BM and PB samples examined showed l e v e l s of CAMAL-positive c e l l s comparable to those found i n ANLL samples was not an expected r e s u l t . Nearly h a l f (5/11) of the ALL remission BM samples had > 8 % CAMAL-1 p o s i t i v e c e l l s (6/11 had < 4% p o s i t i v e ) . In comparison, none of 41 newly diagnosed ALL BM samples had > 8 % p o s i t i v e c e l l s ; i n f a c t only 2/41 had > 4 % p o s i t i v e . The peripheral blood revealed a s i m i l a r s i t u a t i o n . Half (4/8) of the ALL remission PB samples had > 5 % CAMAL-1 p o s i t i v e c e l l s (4/8 had < 0.6 % ) . In comparison, none of 19 newly diagnosed ALL PB samples had over 2 % p o s i t i v e c e l l s . The great majority of CAMAL-1 re a c t i v e c e l l s found i n the strongly p o s i t i v e ALL remission samples were of the myeloid lineage. Figure 3.3 i l l u s t r a t e s t h i s point i n an ALL remission BM sample that was 10.5 % p o s i t i v e . None of the ALL remission samples with high numbers of p o s i t i v e c e l l s were obtained from the ALL patients examined i n Table VIII at diagnosis, therefore i t i s not possible to determine i f any or a l l of these patients had high values at diagnosis as well as during remission. This, however, i s very u n l i k e l y considering the r e s u l t s from the newly diagnosed ALL group. Another explanation for these r e s u l t s i s more probable, based on the following data. In a study published i n 1985 (529), which demonstrated the presence of CAMAL-positive c e l l s p r i o r to relapse i n PB of acute leukemia 121 Figure 3.3. ALL remission bone marrow cel l s labeled by CAMAL-1 monoclonal antibody. Immature and mature myeloid cel l s are positively labeled. 122 patients who had undergone BMT, one of the patients who died i n relapse a f t e r transplantation (and who had 50% p o s i t i v e c e l l s present 3 months p r i o r ) was o r i g i n a l l y diagnosed as an ALL. S i m i l a r l y , we have found, i n two other ALL remission patients, that high numbers of CAMAL-positive c e l l s (25 and 30%) were present i n t h e i r BM one month p r i o r to relapse. I t i s p o s s i b l e , therefore, that i n some ALL patients an underlying p a t h o l o g i c a l mechanism i s occurring which may be signaled by the CAMAL marker and which may be r e l a t e d to imminent relapse. We know t h i s to be the case with some ANLL patients (Chapter V) and only further investigations with greater numbers of ALL patients can determine i f the same holds true f o r any patients i n t h i s 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 Test to the  Study of Other Myeloid C e l l Markers A group of four investigators i n Dr. J u l i a Levy's laboratory took part i n the Second International Workshop on Human Leukocyte D i f f e r e n t i a t i o n Antigens to determine the e f f i c a c y of applying the i n d i r e c t immunoperoxidase s l i d e t e s t described herein to the study of other antigens on malignant myeloid c e l l s using a panel of MAbs (476). This study showed that the s l i d e t e s t was extremely u s e f u l i n such investigations and could provide information not e a s i l y obtained using surface immunofluorescence assays, such as i n t r a c e l l u l a r antigenic d i s t r i b u t i o n and the morphology of antigen-expressing c e l l s . Comparisons of immunoperoxidase methods with FACS analysis have shown close c o r r e l a t i o n s f o r the detection of c e l l surface antigens (511,530). Of s p e c i a l i n t e r e s t , and r e l a t e d to t h i s t h e s i s , 123 was the discovery that 10/14 MAbs which ( l i k e CAMAL-1) showed no or minimal r e a c t i v i t y with ANLL c e l l l i n e s or patient samples using FACS c e l l surface antigenic a n a l y s i s , did s t a i n s i g n i f i c a n t numbers of c e l l s i n at le a s t some of these samples using i n d i r e c t immunoperoxidase. Figure 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 the a p p l i c a t i o n of t h i s t e s t to other myeloid-specific MAbs. I t can c l e a r l y be seen that the s l i d e t e s t allows e f f e c t i v e v i s u a l i z a t i o n of the antigens f o r which these MAbs are s p e c i f i c . Figure 3.4 c shows immunoperoxidase s t a i n i n g of HL60 c e l l s (a human promyelocytic c e l l l i n e ) with MAb 110, which was one of a group of MAbs that were not supposed to react strongly with ANLL c e l l l i n e s . We found 20.0% r e a c t i v i t y with HL60 and MAb 110; furthermore t h i s MAb showed intense p o s i t i v e s t a i n i n g with immunoperoxidase. Over the l a s t few years, there has been increased i n t e r e s t i n the use of t h i s technique f o r studying antigens on s i n g l e c e l l preparations. We believe that the technique o f f e r s s i g n i f i c a n t advantages over the routine surface immunofluorescence assays that have become so popular, and we have shown that i t can be very u s e f u l f o r the study of antigens using a v a r i e t y of MAbs which do not 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 ana l y s i s . 124 F i g u r e 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 ther 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 lood 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 that the CAMAL antigen i s present i n or on a s i g n i f i c a n t number of BM and PB c e l l s of patients with nonlymphoblastic leukemia compared to normals or most i n d i v i d u a l s with lymphoid malignancies. CAMAL appears to be a very unusual leukemia-associated antigen i n that i t has been found to be present i n c e l l s from patients with many v a r i e t i e s of my e l o p r o l i f e r a t i v e disorders (ANLL, CGL, preleukemia/myelodysplastic syndrome). In ANLL, CAMAL i s found i n patients' c e l l s i n a l l stages of the disease and i n a l l FAB subgroups. The discovery that CAMAL was present i n ANLL remission patients' c e l l s implied that we were not studying an antigen r e s t r i c t e d to the malignant b l a s t c e l l s themselves. We wished to determine the nature of the c e l l s expressing t h i s antigen i n various stages of leukemia i n order to help c l a r i f y our findings and further our understanding of t h i s antigen. Such in v e s t i g a t i o n s w i l l be described i n t h i s chapter. I I . RESULTS A. Morphology of CAMAL-positive C e l l s Two approaches were taken i n order to determine the morphology of CAMAL-positive c e l l s . The f i r s t involved c o l l e c t i n g c e l l s that had shown strong p o s i t i v e fluorescence when labeled with the rabbit anti-CAMAL serum and analyzed i n the fluorescence-activated c e l l 127 sorter. The d e t a i l s of t h i s procedure have been described i n Chapter I I . The second approach involved using the CAMAL-1 MAb i n i n d i r e c t immunoperoxidase studies; these procedures have also been described. 1. FACS Sorting Studies Three myeloid leukemic patient groups were examined i n t h i s manner with the rabbit anti-CAMAL serum (newly diagnosed ANLL, ANLL remission, and CGL). In each instance, two c e l l populations were c o l l e c t e d : that 25% of the t o t a l labeled c e l l population showing the (1) lowest and (2) highest r e l a t i v e fluorescence i n t e n s i t y with the anti-CAMAL serum. The remaining 50% of the sample population was not c o l l e c t e d . These c e l l populations were transferred to glass s l i d e s , stained with Wright's and d i f f e r e n t i a l c e l l counts performed to determine the morphology of the c e l l s . These d i f f e r e n t i a l counts were v e r i f i e d by Dr. Sheldon Naiman, a senior hematologist at the Vancouver General H o s p i t a l . A l l samples analyzed and sorted were prepared by Ficoll-Hypaque separation (and these are, therefore, enriched f o r mononuclear c e l l s ) with the exception of patient 203, Table X, where c e l l s were prepared using the plasmagel separation technique and therefore contained a l l leucocyte types. Table IX shows r e s u l t s from two newly diagnosed ANLL patients who 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 labeled with the anti-CAMAL serum. In both cases, the majority of the malignant c e l l s were present i n the highest f l u o r e s c i n g population although, as the FACS analysis shows, almost a l l c e l l s present i n the t o t a l population d i d l a b e l p o s i t i v e l y with the anti-CAMAL serum. Erythrocytes (rbc) and lymphocytes also labeled p o s i t i v e l y , and while most were detected i n the lowest f l u o r e s c i n g 128 Table IX. FACS so r t i n g r e s u l t s from two newly diagnosed ANLL patients* p e r i p h e r a l blood samples 1) FACS analysis of peripheral blood c e l l s Antiserum l a b e l Number of c e l l s f l u o r e s c i n g * Patient 101 Patient 102 anti-normal human 21,050 23,846 normal rabb i t serum 5,687 5,744 anti-CAMAL 20,154 21,874 2) D i f f e r e n t i a l c e l l counts f o r sorted (anti-CAMAL reactive) samples C e l l types Percent of each c e l l type present Patient 101 Patient 102 LOW HIGH LOW HIGH Bl a s t 23 73 6 14 Promyelocyte 0 54 Myelocyte 2 2 Metamyelocyte 1 6 S t a f f 1 3 Neutrophil occ. 1 9 Lymphocyte 62 22 49 8 Monocyte 3 5 2 4 rbc 12 occ. 38 * approximately 25,000 c e l l s were analyzed i n each case ** patient 102 had acute promyelocytic leukemia 129 population, i t was c l e a r that CAMAL was detected on t h e i r membranes as w e l l . Moreover, since these two c e l l types are smaller than any of those l i s t e d i n the myeloid s e r i e s , i t might be expected that the majority would f a l l i n the lower f l u o r e s c i n g population. The photomicrograph shown i n Figure 4.1 depicts the c e l l s present i n the highl y fluorescent population from patient 101 (Table IX), showing the predominance of b l a s t c e l l s . The s t r i k i n g number of lymphocytes present i n t h i s population v e r i f i e d the presence of CAMAL on the membrane of non-myeloid c e l l s . Three ANLL remission p a t i e n t s , whose c e l l s showed p o s i t i v e fluorescence with the anti-CAMAL serum were also analyzed and sorted i n t h i s manner. Table X shows these r e s u l t s . I t should be emphasized that ANLL remission c r i t e r i a defines that no b l a s t c e l l s are present i n the perip h e r a l blood and less than 5% b l a s t s (considered to be within normal range) are present i n the bone marrow of these patients. I t was of s p e c i a l i n t e r e s t , therefore, to determine the c e l l types found to express the CAMAL antigen i n these populations. Samples from patients 201 and 202,.Table X, were processed by Ficoll-Hypaque separation, providing a mononuclear c e l l - e n r i c h e d sample. In both of 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 consisted mainly of lymphocytes. Even i n the sample from patient 203, processed by plasmagel separation to obtain a t o t a l leucocyte population, a large percentage of lymphocytes were were present, even i n the high l y fluorescent c e l l population. Neutrophils comprised the majority of c e l l s , however, i n t h i s population i n d i c a t i n g that more CAMAL was present on the surface of these c e l l s than on lymphocytes. 130 F igure 4 . 1 . P e r i p h e r a l b lood c e l l s from an ANLL p a t i e n t at d i agnos i s sor ted on the b a s i s of s t rong r e a c t i v i t y w i t h r a b b i t anti-CAMAL serum. Approximately 3/4 of these c e l l s were b l a s t s a l though numerous c e l l s w i t h lymphoid morphology were p re sent . 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 (Table I X ) . 131 Table X. FACS so r t i n g r e s u l t s from three ANLL remission patients* peripheral blood samples 1) FACS analysis of peri p h e r a l blood c e l l s Antiserum l a b e l Number of c e l l s f l u o r e s c i n g * Patient 101 Patient 202 Patient 203 anti-normal human normal rabb i t serum anti-CAMAL 20,347 3,151 20,956 23,536 403 9,333 24,644 89 22,734 2) D i f f e r e n t i a l c e l l counts f o r sorted (anti-CAMAL reactive) samples C e l l types Percent of each c e l l type present Patient 201 Patient 202 Patient 203 LOW HIGH LOW HIGH LOW HIGH Myelocyte 2 Metamyelocyte 4 1 St a f f 3 5 Neutrophil 3 2 10 5 55 Eosinophil occ. 1 Lymphocyte 97 92 83 80 78 28 Monocyte 3 5 1 7 1 1 rbc 14 2 7 8 * approximately 25,000 c e l l s were analyzed i n each case 132 Two CGL patient samples were examined and r e s u l t s are shown i n Table XI. In both samples highly fluorescent populations were gre a t l y enriched f o r immature myeloid c e l l s , i n p a r t i c u l a r , metamyelocytes. Again, lymphocytes were present i n t h i s population i n d i c a t i n g that not only myeloid c e l l s had the CAMAL antigen on t h e i r surface. Figure 4.2 a,b 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 highly fluorescent population from patients 301 and 302. By c o l l e c t i n g two c e l l populations (lowest 25% and highest 25% fluorescing) f o r each patient i n Tables 4.1, 4.2 and 4.3, we hoped to determine i f there was a s p e c i f i c pattern of r e a c t i v i t y with the anti-CAMAL serum f o r any given c e l l type. In general t h i s did not seem to be the case. Two points, however, were c l e a r from the comparison. a. Lymphoid c e l l s were present i n both high and low f l u o r e s c i n g populations of the myeloid leukemia samples examined, whether they were from ANLL or CGL patients. This was most remarkable i n the ANLL remission patient peripheral blood samples, where they comprised most of the p o s i t i v e mononuclear c e l l population. b. The r e l a t i v e s i z e of the c e l l type, f o r example, small (erythrocyte and lymphocyte) or large (most of the myeloid ser i e s including neutrophil and promyelocyte) was probably the major determining f a c t o r f o r differences seen i n t h e i r numbers i n low and high populations. However, t h i s could not be the only f a c t o r i n a l l cases. For instance, patient 101, Table IX, had 23% b l a s t s present i n the low population, a l l of which would have been larger than the 22% lymphocytes present i n the high population. These lymphocytes 133 Table XI. FACS so r t i n g r e s u l t s f o r CGL patients' p e r i p h e r a l blood c e l l s 1) FACS analysis of peripheral blood c e l l s Antiserum l a b e l Number of c e l l s f l u o r e s c i n g * Patient 301 Patient 302 anti-normal human 19,330 16,025 normal r a b b i t serum 698 1,095 anti-CAMAL 11,164 12,206 2) D i f f e r e n t i a l c e l l counts of sorted (anti-CAMAL reactive) samples C e l l types Percent of each c e l l type present Patient 301 Patient 302 LOW HIGH LOW HIGH Bl a s t 1 1 1 Promyelocyte 5 14 1 10 Myelocyte 24 15 35 17 Metamyelocyte 36 42 22 39 Staf f 18 7 10 Neutrophil 2 5 7 Eosinophil 1 Basophil 1 Lymphocyte 32 9 7 8 Monocyte 3 Normoblast 2 1 Late normoblast 21 2 * approximately 25,000 c e l l s were analyzed i n each case ** I l l u s t r a t e d i n Figure 4.2a (patient 301) and b (patient 302) 134 F i g u r e 4 . 2 . P e r i p h e r a l b lood c e l l s from two CGL p a t i e n t s s o r t e d on the ba s i s of s t rong 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 Table XI ) were immature myelo id c e l l s . 135 b 136 must have had a greater concentration of membrane bound CAMAL than did the b l a s t s i n the low population. 2. Immunoperoxidase Studies a. Lack of 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 that many c e l l s besides b l a s t s contain the CAMAL marker; t h i s was most obvious i n the case of ANLL remission samples. Even i n newly diagnosed ANLL patients, no c o r r e l a t i o n seemed to e x i s t between the number of b l a s t s and the number of CAMAL-1 p o s i t i v e c e l l s (CAMAL BM value). Indeed, a number of ANLL patients at i n i t i a l presentation showed very low CAMAL BM values (Chapter V) even though there were many malignant c e l l s present. Table XII shows t h i s lack of c o r r e l a t i o n between the number of b l a s t c e l l s present i n ANLL pat i e n t s ' BM and t h e i r CAMAL BM value using immunoperoxidase. For example, while patient sample 1302 showed very s i m i l a r % b l a s t s and % < CAMAL-positive c e l l s (9 and 7%), sample 1150 (0 and 35%) and sample 1089 (59 and 1.3%) showed that, i n general, no such c o r r e l a t i o n existed and CAMAL'BM values might be much higher or much lower than % N marrow b l a s t s . Even i n the same patient, no consistent p o s i t i v e or negative c o r r e l a t i o n was found to e x i s t (see patients a, b, c and d, Table XII) using the immunoperoxidase assay. b. Lack of C o r r e l a t i o n Between Regenerative or A p l a s t i c BM and  CAMAL BM Value I t has been speculated that CAMAL expression i n c e l l s may simply be a marker of regenerating BM, p a r t i c u l a r l y because of the remission patient data. This has c l e a r l y been shown not to be the case; Tables XIII and XIV summarize CAMAL BM r e s u l t s from 26 regenerating and 15 Table XII. Lack of c o r r e l a t i o n between the number of b l a s t c e l l s present i n bone marrow and the CAMAL BM value Patient Code % BM b l a s t s CAMAL BM value number (% p o s i t i v e c e l l s ) 1302 9 7.0 1311 32 5.5 1021 23 10.0 1125 occ. 75.0 1089a 59 1.3 1557a 18 2.0 1577 20 100.0 1060 1 8.6 1524 36 3.0 1053b 53 6.9 1425b 15 31.0 1031c 2 5.4 1150c 0 35.0 1194c 13 20.0 1015 1 62.5 1372d 55 0.8 1406d 1 20.0 a ) b ) r e f e r to samples examined f o r the same 4 patients (a,b,c,d) at c ) d i f f e r e n t times 138 non-regenerating BM samples. For these Tables, only samples that were found to be i n act i v e states of regeneration (Table XIII) and t o t a l l y non-regenerative or a p l a s t i c states (Table XIV) were included. The expression of CAMAL by c e l l s , therefore, appeared to 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 or negative, with regeneration or lack thereof. c. The Morphology of CAMAL-positive C e l l s by Immunoperoxidase As we (510) and others (530) have reported, the immunoperoxidase method of detecting c e l l u l a r antigens allows straightforward microscopic v i s u a l i z a t i o n of antigen-positive c e l l s and, i n addition, provides information regarding the d i s t r i b u t i o n of antigen on or within i n d i v i d u a l c e l l s . The morphology of ra b b i t anti-CAMAL-positive c e l l s has been discussed using the FACS method of c e l l c o l l e c t i o n . Immunoperoxidase l a b e l i n g with 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 s t a i n p o s i t i v e l y when c e l l s are labeled with CAMAL-1 MAb. As mentioned previously, t h i s may be of some importance i n i d e n t i f y i n g c e l l s that produce CAMAL i n comparison to c e l l s that may adsorb (possibly v i a s p e c i f i c receptors) CAMAL on t h e i r c e l l surface. The majority of CAMAL-positive c e l l s (by immunoperoxidase labeling) i n ANLL patients BM at diagnosis or relapse include malignant b l a s t c e l l s and very immature myeloid c e l l s , as already i l l u s t r a t e d i n Chapter I I I (Figure 3.2 a,b). This i s not at a l l s u r p r i s i n g since, i n many instances, these c e l l s are present at extremely high r e l a t i v e proportions. In ANLL remission patient BM, CAMAL-expressing c e l l s include myeloid c e l l s at 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 of maturation 139 Table XIII. CAMAL BM values i n regenerating bone marrows Patient Status CAMAL BM value (% p o s i t i v e c e l l s ) 501 ANLL remission with good 3.6 regeneration 502 ANLL remission, regenerating 16.5 bone marrow 503 ANLL remission, t r i l i n e a r 13.7 regeneration occurring 504 ANLL remission with active 2.5 granulocytic regeneration 505 Lymphoma post-BMT with excellent 1.5 t r i l i n e a r regeneration Summary: For 26 patients with regenerating BM (23 ANLL, 1 ALL, 1 lymphoma and 1 post-BMT lymphoma) the mean CAMAL BM value was 11.4%. 140 Table XIV. CAMAL BM values i n non-regenerating bone marrows Patient Status CAMAL BM value (% p o s i t i v e c e l l s ) 601 ANLL; hypoce l l u l a r with absence of myeloid and erythroid precursors 35.0 602 ANLL post-chemotherapy; a p l a s i a 0.1 603 ANLL post-chemotherapy; a p l a s i a 35.5 604 Biphenotypic (ALL and ANLL) leukemia post-chemotherapy; a p l a s i a 6.0 605 ANLL post-chemotherapy; a p l a s i a 0.2 Summary: For 15 patients (12 ANLL, 1 biphenotypic [ALL/ANLL], 1 ALL and 1 MDS) with non-regenerative bone marrows, the mean CAMAL BM value was 12.6%. 141 of maturation, from b l a s t s to end stage polymorphonuclear leucocytes. P o s i t i v e c e l l s from the granulocyte (Figure 4.3 a,b), monocyte, megakaryocyte (Figure 4.3 c) and erythroid lineages have a l l been i d e n t i f i e d i n ANLL remission patient BM. In general, fewer c e l l s i n the peripheral blood of remission patients s t a i n p o s i t i v e l y . These include granulocytes and monocytes p r i m a r i l y , but i n a number of cases have included a very high number of lymphocytes (Figure 4.4). The pos s i b l e s i g n i f i c a n c e of t h i s w i l l be discussed. Observed differences i n CAMAL d i s t r i b u t i o n within c e l l s have been profound. As photomicrographs i n Chapter I I I , Figure 3.2, and i n Figure 4.3 i l l u s t r a t e , most of t h i s p rotein i s located 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 of cytoplasmic s t a i n i n g patterns from d i f f u s e to strongly granular. Intense perinuclear s t a i n i n g of myeloid c e l l s has been observed with great frequency (Figures 3.1 b, 3.2 a,b,d and 4.3 a,b). The s i g n i f i c a n c e , i f any, of apparent antigen shedding has not been determined (Figures 3.2 e, 4.3 a). I t has been commonly observed i n chronic stage CGL peri p h e r a l blood preparations. Indeed, a very frequent pattern of CGL PB l a b e l i n g i s i l l u s t r a t e d i n Figure 4.5 where a he a v i l y granular s t a i n i n g of PB leucocytes i s seen. In many cases t h i s granular l a b e l has been i d e n t i f i e d outside, as well as within, these c e l l s . CGL (BM or PB) samples r o u t i n e l y contain greater percentages of CAMAL-positive c e l l s than any other myeloid leukemia group examined. CGL c e l l s at v i r t u a l l y a l l stages of myeloid maturation l a b e l p o s i t i v e l y with CAMAL-1. This general pattern of r e a c t i v i t y with a l l stages of c e l l s within the myeloid lineage holds true i n BM samples from 142 F i g u r e 4 . 3 . Morphology of CAMAL-1 p o s i t i v e mye lo id 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 . (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 granulocytes 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 b lood p o s i t i v e l y l a b e l e d by CAMAL-1. 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 of CGL p e r i p h e r a l b lood c e l l s by CAMAL-1. Th 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 c e l l s . 146 preleukemi.es/ myelodysplastics, some ALL remission patients (Figure 3.3) and normals, even though r e l a t i v e percentages of CAMAL-l-reactive c e l l s may often be very small i n comparison with myeloid leukemics. B. ANLL Remission Pathology In order to investigate the p o s s i b i l i t y that s i g n i f i c a n t l y elevated numbers of CAMAL-1 re a c t i v e c e l l s may represent an underlying pathology present i n apparently normal remission BM or PBL, we chose to study an ANLL remission patient whose malignant c e l l s possessed a c h a r a c t e r i s t i c karyotype (47,XY,+6). At i n i t i a l c l i n i c a l diagnosis (ANLL FAB M5), cytogenetic evaluation of a d i r e c t bone marrow preparation with G-banding performed by Dr. Dagmar Kalousek (Terry Fox Laboratory, Vancouver, B.C.) revealed the presence of two c e l l l i n e s (46,XY/47,XY,+6). Four months l a t e r morphological evaluation of t h i s patient's BM was normal, but cytogenetic analysis revealed one abnormal metaphase (48,XY,+6,+8) i n the 23 examined. At the same time, immunoperoxidase l a b e l i n g of BM showed 10% r e a c t i v i t y with CAMAL-1. Three weeks following these te s t s 74% b l a s t s were present i n the BM, 24/25 metaphases showed hyperdiploidy (48,XY,+6,+8), and a c l i n i c a l diagnosis of ANLL i n relapse was made. Another ANLL (FAB M4) patient, whose leukemia may have been secondary to previous chemotherapy f o r ovarian carcinoma, was investigated c y t o g e n e t i c a l l y at presentation f o r ANLL. G-banding chromosome analysis revealed a unique hypodiploid karyotype i n a l l metaphases studied [44,XX,-4,-17,-21,+MAR,7q~,t(5q;llp;13q), 147 t(9q;19q)]. An i d e n t i c a l twin BM transplant was performed i n acute phase. The patient's cytogenetic analyses 2 and 4 weeks post-transplant were normal (46,XX) i n d i c a t i n g that her malignant c e l l s had apparently been s u c c e s s f u l l y eradicated. However, at 4 weeks post-transplant we found that 100% of her PBL reacted with the rab b i t anti-CAMAL serum i n FACS analysis. When these labeled c e l l s were sorted to obtain that 25% of the population showing the highest r e l a t i v e fluorescence, we discovered that these c e l l s were normal by routine morphological c r i t e r i a . Figure 4.6 a,b shows that t y p i c a l c e l l s sorted out i n t h i s manner included both mature neutrophils and lymphocytes. There were no malignant c e l l s detectable i n e i t h e r her blood or BM at t h i s time. Four months l a t e r , t h i s p a tient suffered c l i n i c a l ANLL relapse with the emergence once again of her o r i g i n a l hypodiploid c e l l clone, which had undergone minor evolution. The observation that her normal c e l l s were p o s i t i v e f o r membrane CAMAL post-transplant could imply adsorption of soluble CAMAL by transplanted c e l l s and/or information exchange r e s u l t i n g i n CAMAL expression by the i d e n t i c a l twin donor c e l l s . The r e s u l t s from the two ANLL patients studied using immunoperoxidase and CAMAL-1 or FACS analysis and rabbi t anti-CAMAL, when correlated with morphological and cytogenetic analyses, revealed two important r e l a t e d f i n d i n g s : 1) morphological evaluation of remission may be incapable of accurately gauging remission state i n a l l cases, and 2) underlying seminal changes i n ANLL may involve (or be i d e n t i f i e d by) clone(s) of c e l l s that are normal both morphologically and cyt o g e n e t i c a l l y and that include terminally d i f f e r e n t i a t i n g l i n e s . 148 Figure 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 p a t i e n t ' s p e r i p h e r a l blood c e l l s . C e l l s l a b e l i n g s t r o n g l y w i t h r a b b i t anti-CAMAL serum 4 months p r i o r to leukemic relapse included 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) higher m a g n i f i c a t i o n of same. a 150 C. The Presence of CAMAL i n Plasma I t was poss i b l e that the presence of CAMAL-positive c e l l s i n ANLL remission patients, p a r t i c u l a r l y lymphoid c e l l s , resulted from adsorption of soluble CAMAL which had been produced and secreted by other c e l l s . Were t h i s to be the case, i t would be reasonable to assume that soluble CAMAL might be present i n the plasma of these i n d i v i d u a l s . 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 of a number of myeloid leukemia patients as well as normals using a f f i n i t y chromatography as described i n Chapter I I . A f f i n i t y chromatography extraction of CAMAL from plasma was performed a f t e r numerous unsuccessful attempts at quantitating serum or plasma CAMAL le v e l s using ELISA and radio-immunoassay. Table XV shows roughly estimated plasma l e v e l s of CAMAL i n myeloid leukemics and normals. CAMAL protein concentrations were estimated by Biorad protein assay of eluted material bound to a CAMAL-1 immunoadsorbent column a f t e r plasma had been passed over the column. There are obvious problems with t h i s method of estimating plasma CAMAL concentrations. These include the immunological c r o s s - r e a c t i v i t y of CAMAL-1 with human serum albumin (Shipman and Shellard, personel communication) and the f a c t that t h i s method would, at best, only estimate p r o t e i n concentration. With t h i s i n mind, the following estimates are reported, but i t i s conceded that these estimates may be q u a n t i t a t i v e l y quite inaccurate. Normal (n = 6) plasma l e v e l s were 21 ug/ml on average; l e v e l s i n myeloid or preleukemics (n = 4) averaged 52 ug/ml. I t i s not poss i b l e 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 differences between plasma CAMAL l e v e l s i n normals and myeloid 151 Table XV. Detection of CAMAL i n plasma by immunoaffinity chromatography Diagnosis Estimated Plasma CAMAL Levels (yg/ml) CGL 50.0 CGL 66.0 Preleukemia 47.0 ANLL remission 44.7 Normal 19.0 Normal 25.9 Normal 15.0 Normal 13.5 Normal 26.7 Normal 26.5 152 leukemics although t h i s may be the case. The eluted CAMAL (Figure 4.7) reacted to the same extent i n ELISA with CAMAL-1 MAb, whether p u r i f i e d from normals or myeloid leukemics (Figure 4.8a) and had the same m o b i l i t y i n PAGE, running as expected at 68 KD (Figure 4.8b). D. CAMAL Adsorption Studies I t has been demonstrated that p e r i p h e r a l blood can, on occasion, contain s i g n i f i c a n t l y high numbers of CAMAL-positive c e l l s even during c l i n i c a l remission i n ANLL. Moreover, i n a b l i n d study of PBL from acute leukemia patients who had undergone BMT (529), those ANLL patients who relapsed showed increased numbers of CAMAL-1 p o s i t i v e c e l l s (using immunoperoxidase) up to 3 months p r i o r to relapse (Figure 4.9). We wished to determine i f normal PBL would become CAMAL-1 re a c t i v e i f incubated i n the presence of excess CAMAL, p a r t i c u l a r l y since there was the p o s s i b i l i t y that myeloid leukemic plasma might contain higher l e v e l s of CAMAL than normals. Whole heparinized peripheral blood samples from two normal i n d i v i d u a l s were incubated overnight with increasing concentrations of soluble CAMAL derived from myeloid leukemia c e l l extracts (or equivalent amounts of i r r e l e v a n t p r o t e i n or PBS) as outlined i n Chapter I I . Following t h i s incubation, PBL were i s o l a t e d from these samples by Ficoll-Hypaque separation and s l i d e preparations 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 using CAMAL-1 or an i r r e l e v a n t MAb. In a l l of three normal samples tested i n t h i s manner, CAMAL-1 rea 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 of c e l l s which had been previously incubated with > 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. • • CAMAL-1 column eluate O O negative control MAb column eluate - L O G 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 O normal plasma 9 0 ANLL remission plasma 154b 1 2 3 4 5 6 7 8 Figure 4.8b. Polyacrylamide gel electrophoretic p r o f i l e of 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 molecular weight standards Lanes 2,7: 62/66 KD doublet of CAMAL, p u r i f i e d by a f f i n i t y chromatography, from myeloid leukemia c e l l lysates (62/66 KD doublet was consistently 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 myeloid leukemia plasma samples. 155 Figure 4.9. Immunoperoxidase staining of an ANLL post-bone marrow transplant patient's peripheral blood ce l l s , labeled with CAMAL-1, one month prior to relapse. 156 p o s i t i v e l y labeled c e l l s were seen i n s l i d e s labeled with the i r r e l e v a n t MAb or i n s l i d e s containing c e l l s pre-incubated with < 5 yg/ml CAMAL. Figure 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, incubated with increasing amounts of CAMAL and labeled with CAMAL-1 or the negative control MAb i n the immunoperoxidase assay, showing obvious l a b e l i n g of c e l l s ( p r imarily lymphoid and erythroid membranes) with 12.5 and 50.0 yg/ml excess CAMAL. Figure 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 , with rbc, lymphocytes, monocytes and granulocytes l a b e l i n g with CAMAL-1. These r e s u l t s i n d i c a t e that, at a c e r t a i n concentration of excess CAMAL (> 12.5 ug/ml), normal PBL can become CAMAL-positive. I t i s possibl e , therefore, that the increased number of CAMAL-positive PBL ( e s p e c i a l l y lymphoid c e l l s ) detected i n ANLL remission (Chapter V) or BMT patients p r i o r to relapse may be due to a s i m i l a r adsorption, r e s u l t i n g from increased amounts of soluble CAMAL i n the plasma of these i n d i v i d u a l s at such times. 157 F i g u r e 4 .10 . Normal p e r i p h e r a l b lood c e l l s , incubated w i t h i n c r e a s i n g 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. 158 b. 5 yg/ml CAMAL, labeled with CAMAL-1. c. 12.5 yg/ml CAMAL, labeled with CAMAL-1. i • % • + d. 12.5 yg/ml CAMAL, labeled with negative control MAb. 1 _ « • e. 50 yg/ml CAMAL, labeled with negative control MAb. 160 f. 50 ug/ml CAMAL, l a b e l e d with CAMAL-1. 161 F igure 4 . 1 1 . 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 lood sample a f t e r i n c u b a t i o n w i t h or wi thout CAMAL. (a) C e l l s incubated 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 mye lo id ( 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 incubated 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 ^  t • b 163 CHAPTER V SIGNIFICANCE OF CAMAL AS A PROGNOSTIC MARKER FOR REMISSION IN ACUTE NONLYMPHOBLASTIC LEUKEMIA I. INTRODUCTION Studies of myeloid lineage-associated antigens have contributed s i g n i f i c a n t l y to our present understanding of the complexity of c e l l types involved i n acute nonlymphoblastic leukemia. As mentioned previously, the attempt to c o r r e l a t e French-American-British (FAB) group c l a s s i f i c a t i o n s of ANLL with s p e c i f i c c e l l surface phenotypes (472-476) has met with l i m i t e d success, with the exception of c e r t a i n defined associations (M4/M5 with CD14 antigen, f o r example). Marked antigenic heterogeneity complicates diagnosis and treatment of ANLL, p a r t i c u l a r l y i n areas currently of widespread i n t e r e s t , namely diagnosis of r e s i d u a l disease and immuno-purging of bone marrow f o r autologous bone marrow transplantation purposes. ANLL without bone marrow transplantation (BMT) generally has a poor long term prognosis. Leukemic relapse continues to be a serious problem i n treated ANLL and at present there i s l i m i t e d u s e f u l information a v a i l a b l e concerning prognostic in d i c a t o r s i n ANLL. With regard to chronic granulocytic leukemia (CGL), studies have indicated that i t might be pos s i b l e to i d e n t i f y c e r t a i n p o t e n t i a l candidates f o r successful BMT i n the younger CGL patient group by examining a number of c l i n i c a l parameters including serum l a c t i c dehydrogenase (LDH) a c t i v i t y , sex, spleen s i z e , hematocrit, p l a t e l e t 164 count, c i r c u l a t i n g nucleated rbcs, as well as % marrow b l a s t s , basophils plus eosinophils (531). Longer remissions i n ANLL have been shown to be associated with such f a c t o r s (many of which are in 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 LDH or fibrinogen l e v e l s and rapid development of complete remission status (532). In v i t r o assays f o r hemopoietic progenitor c e l l s or t h e i r associated regulatory f actors have been examined f o r t h e i r prognostic implications i n ANLL (533-537). Unfortunately, very l i t t l e information has been well characterized concerning factors ( c e l l markers, serum proteins, etc.) that might serve as common prognostic i n d i c a t o r s i n ANLL during remission. Over a decade ago, i t was observed that the nature of myeloid colony-forming c e l l growth i n v i t r o could a s s i s t i n the monitoring of i n d i v i d u a l ANLL patients i n terms of p r e d i c t i n g the onset of remission or relapse (538,539). Baker et a l . were able to p r e d i c t relapse i n 21/26 ANLL remission patients' BM using murine heteroantiserum s p e c i f i c f o r a myeloblast antigen (461). In a co l l a b o r a t i v e b l i n d study using an i n d i r e c t immunoperoxidase s l i d e t e s t and the CAMAL-1 MAb, we were able to detect increased amounts of CAMAL i n peripheral blood leucocytes of BMT patients up to 3 months or more p r i o r to relapse (529). Although present i n ANLL b l a s t c e l l s , CAMAL i s not s t r i c t l y a b l a s t c e l l antigen and has been demonstrated i n or on many c e l l types within the myeloid lineage, as described i n Chapter IV. On occasion we have found detectable l e v e l s of CAMAL on the surface of lymphoid c e l l s i n ANLL remission patients as well (514, Ch.IV). While CAMAL 165 appears to be expressed at much higher l e v e l s i n PBL or BM c e l l s of myelogenous leukemia patients, i t has been established (Chapter III) that CAMAL i s also present i n normal BM c e l l s , a l b e i t at s i g n i f i c a n t l y lower l e v e l s . As described, normal BM contains approximately 1% CAMAL-1 re a c t i v e c e l l s by immunoperoxidase assay (510). The remarkable f i n d i n g that CAMAL was present, often at very high l e v e l s , i n BM c e l l s from ANLL remission patients, led us to question the possible prognostic s i g n i f i c a n c e of i t s presence. This chapter describes the r e s u l t s obtained from a b l i n d study c a r r i e d out with ANLL patients attending the Department of Hematology, Vancouver General H o s p i t a l . We wished to determine whether or not the number of CAMAL-1 re a c t i v e BM c e l l s (CAMAL BM value) r e f l e c t e d c l i n i c a l prognosis i n ANLL patients over the course of t h e i r disease. 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 with the CAMAL-1 MAb i n an attempt to determine any c o r r e l a t i o n between length of c l i n i c a l remission and changes i n patients' CAMAL BM values following chemotherapy. Results presented here ind i c a t e that t h i s change appears to be a u s e f u l prognostic i n d i c a t o r f o r remission i n ANLL. RESULTS De t a i l s of the b l i n d study protocol have been given i n Chapter I I ; some points 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 data from ANLL patients are presented here, although the b l i n d study included examination of over 700 samples from a l l patients (and BMT donors) attending the Department of Hematology, Vancouver General Hospital. A l l s l i d e s were prepared and coded numerically so that no patient 166 information was a v a i l a b l e to the s l i d e reader. ANLL patients (n = 34) whose BM had been s e r i a l l y examined at the appropriate times were divided into two groups f o r analysis by the product-limit method (Kaplan-Meier estimate): 1. ANLL patients at diagnosis whose CAMAL BM values decreased post-chemotherapy (within 1 + 0 . 5 months), and 2. ANLL patients at diagnosis whose CAMAL BM values increased or remained the same post-chemotherapy, A. Relationship Between Sur v i v a l Time P r i o r to Relapse and Change  i n CAMAL BM Values The e n t i r e group (n = 34) of ANLL patients was examined, using the Cox s u r v i v a l analysis model i n the BMDP2L biomedical computer program, i n order to determine i f there was any s i g n i f i c a n t v a r i a b l e ( s ) that could be us e f u l to pr e d i c t which patients would have longer remission times. Covariates analysed were patient age, sex, CAMAL BM value at diagnosis and change i n CAMAL BM value post-chemotherapy. The only s i g n i f i c a n t (p = 0.05) v a r i a b l e i n pr e d i c t i n g patients with longer remission times was the change i n the CAMAL BM value post-chemotherapy. B. C o r r e l a t i o n Between Decreasing CAMAL BM Values Post-chemotherapy  and Longer Remission Times Since the change i n CAMAL BM value (pre- to post-chemotherapy) had been shown to be s i g n i f i c a n t i n terms of prognosis, the t o t a l ANLL group was divided into two subgroups based on t h i s parameter. Group 1 (n = 10) consisted of ANLL patients whose CAMAL BM values 16 7 decreased s i g n i f i c a n t l y post-chemotherapy (Table XVI). The average remission length f o r t h i s group was 19.2 months, with 50% of these patients s t i l l i n f i r s t remission. This was compared to the remaining ANLL patients (Group 2) whose CAMAL BM values e i t h e r increased or remained unchanged post-chemotherapy (Table XVII). The average remission length f o r Group 2 was 6.8 months, with only 25% s t i l l i n f i r s t remission. A comparison of the s u r v i v a l time p r i o r to relapse was made between these two groups using the Kaplan-Meier estimate with log-rank s t a t i s t i c a l a n a l y s is. Figure 5.1 i l l u s t r a t e s the s u r v i v a l curves 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 very l i t t l e d i f f e r e n c e between remission lengths f o r patients whose values increased (x = 6.1 months) and those whose values remained unchanged (x = 7.4 months) post-chemotherapy. The improved prognosis was correlated only with a decreasing CAMAL BM value. Figure 5.2 i l l u s t r a t e s CAMAL BM values pre- and post-chemotherapy f o r two ANLL patients whose values dropped s i g n i f i c a n t l y following treatment. These patterns may be compared with those from three ANLL patients whose CAMAL BM values increased or were unchanged following chemotherapy (Figure 5.3). I t i s apparent from these examples that the absolute percentage increase (or decrease) does not seem to define p r e c i s e l y how long any i n d i v i d u a l remission w i l l be. However, the r e l a t i v e change (decrease or increase, +/-) post-chemotherapy i s d i r e c t l y correlated with average remission length. Table XVI. ANLL patients whose CAMAL BM values decreased post-chemotherapy (Group 1) Patient CAMAL BM value (percent p o s i t i v e c e l l s ) Remission At diagnosis Post-chemotherapy length (months) 101 21.0 0.7 0 102 18.9 7.5 6.5 103 80.0 0.1 10 104 31.0 13.0 14 105 31.0 0.1 17 106 50.0 11.0 20 108 21.0 2.5 32 107 12.3 6.3 24.5 109 14.2 0.5 32 110 21.0 0.1 36 To t a l number of patients = 10 Average remission length = 19.2 months Present status of a l l patients i n Group 1 n Percent 1st remission 5 50 2nd remission 1 10 Relapse 1 10 Dead 3 30 169 Table XVII. ANLL patients whose CAMAL BM values increased or remained the same post-chemotherapy (Group 2) Patient CAMAL BM value (percent p o s i t i v e c e l l s ) Remission At diagnosis Post-chemotherapy length (months) 201 0.5 7.0 0 202 80.0 100.0 0 203 0.1 25.0 1 204 5.0 20.0 1.0 205 2.5 4.0 2.5 206 0.5 2.9 3 207 4.5 12.3 3.5 208 2.5 45.5 3.5 209 11.0 6.5 3.5 210 1.5 62.5 5 211 5.5 4.9 5.5 212 3.0 6.3 6 213 5.5 1.5 7 214 4.2 6.0 7.0 215 8.0 6.5 8 216 1.4 0.5 8 217 1.8 2.5 8 218 2.2 0.7 8 219 0.1 21.0 10.0 220 0.8 20.0 13 221 0.1 2.2 14 222 2.2 16.5 14 223 0.7 13.7 16 224 1.3 3.5 16 Tota l number of patients = 24 Average remission length = 6.8 months Present status of a l l patients i n Group 2 n Percent 1st remission 6 25 2nd remission 1 4 Relapse 2 8 Dead 11 46 BMT 4 17 170 LU (A < _ l Ul < <2 > g 5 fc UJ o CC Q. 1.0 0.9 0.8 0.7-I 0.6 0.5 0.4-0.3-0.2 0.1 0 GROUP 2 _ l GROUP 1 "T r ~ i r 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 5 10 15 20 25 30 35 MONTHS POST CHEMOTHERAPY Figure 5.1. Kaplan-Meier s u r v i v a l curve showing s u r v i v a l time p r i o r to relaps e f o r ANLL p a t i e n t s i n b l i n d study. Group 1, ANLL p a t i e n t s (n = 10) whose CAMAL BM values decreased 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 =24) whose CAMAL BM values increased or remained unchanged f o l l o w i n g chemotherapy. MONTHS POST-CHEMOTHERAPY Figure 5.2. CAMAL BM values over time f o r two ANLL patients with s i g n i f i c a n t l y decreased values post-chemotherapy. Survival times p r i o r to relapse were (a) 10 months and (b) s t i l l i n f i r s t remission. L, acute leukemia; R, remission; (R), relapse; D, dead 172 Figure 5.3. CAMAL BM values over time f o r three ANLL patients whose values increased or were unchanged following treatment. Survival times p r i o r to relapse were (a) 1.3 months, (b) 10 months and (c) 8 months. L, acute leukemia; R, remission; (R), relapse; D, dead 173 C. CAMAL BM Values and Simultaneous BM Morphology Morphological examination of CAMAL-1 p o s i t i v e c e l l s at diagnosis revealed that, i n a l l ANLL patients examined, the 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 other very immature myeloid c e l l s as expected. However, when post-chemotherapy s l i d e s were examined, there were differences among the ANLL patients with respect to the morphology of CAMAL-1 p o s i t i v e c e l l s . A l l ANLL patients whose CAMAL BM values increased post-chemotherapy showed s i m i l a r kinds of p o s i t i v e c e l l s to those seen at diagnosis (primarily immature myeloid c e l l s , including b l a s t s ) . In general, t h i s was the case as well f o r patients whose CAMAL BM values did not change post-chemotherapy, although a few patients showed p o s i t i v e c e l l s at a l l stages of myeloid maturation. However, i n those patients whose CAMAL BM values 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 included myeloid c e l l s at a l l l e v e l s of maturation, some with a preponderance of mature polymorphonuclear leucocytes. D. Supportive Data from Other ANLL Patients The c o r r e l a t i o n between c l i n i c a l prognosis and r e l a t i v e CAMAL BM value pre- and post-chemotherapy was supported by r e s u l t s from ANLL relapse and BMT patients examined. 5/6 ANLL relapse patients whose CAMAL BM values increased s i g n i f i c a n t l y a f t e r treatment f a i l e d to a t t a i n 2nd remission. This was compared with 4/4 ANLL relapse patients with s i g n i f i c a n t l y decreased values post-chemotherapy who did achieve 2nd remissions. F i n a l l y there were 7 ANLL patients who underwent BMT and f o r whom CAMAL BM values had been recorded pre- and post-BMT. 5/7 of 174 these patients had decreased CAMAL BM values and a l l are s t i l l i n remission (at 6.5 - 34 months). 2/7 showed increased CAMAL BM values post-BMT; one of these patients relapsed at 3 months, the other suffered f o c a l uterine leukemic relapse at 8 months. E. Relationship Between F i r s t Remission Length and CAMAL BM Value  at Diagnosis I t was i n t e r e s t i n g to note that a l l patients i n Group 1 had elevated (well above normal) CAMAL BM values pre-treatment (n = 10, x = 30). These were the patients who fared better i n terms of s i g n i f i c a n t l y longer f i r s t remissions. Patients i n Group 2 who had, on average, s i g n i f i c a n t l y shorter remissions had much lower pre-treatment values (n = 24, x = 6). This led to the speculation that c e l l s i n t h i s state of antigen expression may be more responsive to successful treatment. However, when a Cox s u r v i v a l analysis was performd on a l l ANLL patients (n = 40) whose BM was examined at diagnosis, none of the following covariates were found to be s i g n i f i c a n t with regard to p r e d i c t i n g remission length: age, sex, or CAMAL BM value at diagnosis. A decrease i n the CAMAL BM value post-chemotherapy appeared to be the only 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. Increasing CAMAL BM Values and Relapse In a previous i n v e s t i g a t i o n (529) we showed that CAMAL PBL values increased p r i o r to relapse i n a BMT group studied. The present study also indicated that t h i s was the case when CAMAL BM values were monitored during remission. There were 9 patients whose 175 sequential CAMAL BM data showed an increase occurring during remission. For 4 of these pa t i e n t s , we had obtained samples within the few months immediately p r i o r to relapse. Table XVIII shows these 4 pa t i e n t s ' recorded CAMAL BM values over the course of t h e i r remission. In each case, these values were observed to increase 1.5 to 3 months p r i o r to relapse. We found that 78% (7/9) of patients who had increasing remission values suffered relapse while only 33% (3/9) of patients with decreasing remission values did. Since no samples were examined f o r these l a t t e r 3 patients f o r 5 - 8 months p r i o r to relapse, i t cannot be determined i f t h e i r CAMAL BM values had increased during that period. We were able to obtain very regular samples from one ANLL patient over the 19 month course of h i s disease. Figure 5.4 i l l u s t r a t e s t h i s patient's recorded CAMAL BM values over time. P r i o r to each of h i s 3 relapses, i t was observed that h i s CAMAL BM values increased s i g n i f i c a n t l y (5.0 - 20.0%, 0.8 - 12.0% and 33.5 - 75.0%). I t may be that remission PB samples w i l l prove to be as us e f u l as BM i n routine monitoring procedure using immunoperoxidase. At present, lack of reasonably sequential PB samples i n key patients has prevented us from making more d e f i n i t i v e statements on PB. Cer t a i n l y , the same trend as that seen with BM samples has been indicated thus f a r . The following ser i e s of photomicrographs c l e a r l y i l l u s t r a t e s these points. Figure 5.5 a, b, c shows immunoperoxidase s l i d e t e s t r e s u l t s of PB samples from an ANLL patient with increasing values (a) post-chemotherapy (0.2% p o s i t i v e ) , (b) 2 weeks l a t e r , i n remission (now 8.6% p o s i t i v e ) , and (c) another 2 weeks l a t e r , also during remission (32.0% p o s i t i v e ) . Within 2-1/2 months of the l a s t Table X V I I I . 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 va lues d u r i n g r e m i s s i o n P a t i e n t CAMAL BM value (percent 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 d i agnos i s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 301 8.5 20 - 30 - (R) 302 3.5 - - - - - 2.2 - - 1.0 - 20 - - (R) 303 8.6 32 - 5.5 (80) 304 - 5.4 - - - 35 27.5 (20) (R) or ( ) = re lapse 17 L R(R) R (R) T (R)D MONTHS Figure 5.4. CAMAL BM values f o r one ANLL p a t i e n t over the course of h i s disease. L, acute leukemia; R, rem 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 Figure 5.5. CAMAL-1 immunoperoxidase s l i d e t e s t s r e s u l t s of p e r i p h e r a l blood c e l l s from an ANLL remission 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 . (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 emission; (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 remission but 2-1/2 months p r i o r to re l a p s e . a 179 180 t e s t , t h i s patient was i n relapse. This may be compared to another ANLL patient whose PB values decreased during remission. Figure 5.6 a, b, c i l l u s t r a t e s immunoperoxidase PB values (a) 1 month post-chemotherapy, i n remission (12.0% p o s i t i v e ) , (b) 2 months l a t e r (1.3% p o s i t i v e ) , and (c) 1-1/2 months l a t e r , s t i l l during remission (0.5% p o s i t i v e ) . This patient has remained i n f i r s t remission f o r over 24 months. 181 Figure 5.6. CAMAL-1 immunoperoxidase s l i d e t e s t r e s u l t s of p e r i p h e r a l blood c e l l s from an ANLL rem i s s i o n p a t i e n t showing decreasing r e a c t i v i t y . (a) 12.0% p o s i t i v e 4 weeks post-chemotherapy, during c l i n i c a l remission; (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 during remission 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 to be a s i g n i f i c a n t marker both i n the p r e d i c t i o n of relapse i n ANLL BMT patients (529) and i n the prognostic assessment of remission i n ANLL, as shown i n the previous chapter. The observation that CAMAL BM (or PBL) values often increased p r i o r to relapse i n ANLL patients, led to the speculation that CAMAL might be involved i n some way i n the onset of abnormal myelopoiesis which must occur i n ANLL patients at some point before the c l i n i c a l diagnosis of relapse can be made. There i s evidence to ind i c a t e that during remission i n ANLL, abnormalities i n c e l l u l a r i n t e r a c t i o n s occur which may be r e l a t e d to the eventual re-emergence of the leukemic clone (536-538). This chapter outlines evidence to support the possible regulatory r o l e of CAMAL i n myelopoiesis including i t s i n h i b i t i o n of normal myelopoiesis. Such i n h i b i t i o n could contribute to the growth ( p r o l i f e r a t i v e ) advantage that leukemic c e l l s possess over normal c e l l s . I I . RESULTS A. CAMAL-1 P o s i t i v e Colonies i n CGL In order to determine whether or not myeloid progenitors (and the c e l l s i n the colonies that they formed) contained the CAMAL antigen, i n d i v i d u a l colonies were manually plucked out of methylcellulose cultures, spread 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 subjected to a modified i n d i r e c t immunoperoxidase assay using CAMAL-1, as outlined i n Chapter I I . Some d i f f i c u l t y was encountered with t h i s procedure but c l e a r r e s u l t s were obtained i n the s t a i n i n g of s l i d e s from a patient with chronic granulocytic leukemia. Seventy-five percent (18/24) of colonies plucked from methylcellulose showed p o s i t i v e s t a i n i n g with the CAMAL-1 MAb. Twenty-five percent (6/24) of colonies were p o s i t i v e with the rabbit anti-CAMAL serum. Furthermore, a l l c e l l s i n each of these colonies stained p o s i t i v e l y , i n d i c a t i n g that the antigen appeared to be present i n a l l c e l l s derived from a common progenitor. P o s i t i v e colony types included granulocyte, macrophage and erythroid bursts. No p o s i t i v e colonies were found i n 2 normals tested. Figure 6.1 a, 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 negative c e l l s stained with the immunoperoxidase procedure on colonies from the CGL p a t i e n t . This preliminary r e s u l t , and the previously described f i n d i n g that excess CAMAL could adsorb to normal PBL (Chapter IV), prompted the experimental procedures that w i l l now be described. B. Presence of CAMAL i n Conditioned Medium Conditioned medium f o r i n v i t r o myeloid progenitor studies contains hemopoietic growth factors required by these progenitors f o r t h e i r 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 . E f f e c t i v e conditioned media f o r human c e l l s may be prepared i n a number of ways including (1) placenta-conditioned medium (PCM) (199), peripheral blood leucocyte conditioned medium of which phytohemagglutinin-stimulated lymphocyte conditioned medium i s most common (PHA-LCM) (197), (3) other human organ conditioned medium (198,203) and (4) various human tumor c e l l 184 F igure 6 . 1 . C e l l s from CGL p e r i p h e r a l b lood 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 granulocyte colony 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 co lony 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 l i n e s (200-202). D e t a i l s of the preparation of PCM and PHA-LCM used i n the following studies have been given i n Chapter I I . In order to determine i f CAMAL was present i n conditioned medium (a s i t u a t i o n that might be expected i f , indeed, CAMAL played some r o l e i n myelopoiesis) conditioned media (CM) prepared by three d i f f e r e n t methods were passed over a CAMAL-1 immunoadsorbent column. Results showed that a l l 3 types of conditioned media (PCM, PHA-LCM and p e r i p h e r a l blood c e l l CM prepared by Dr. N. Denegri's laboratory) contained material that bound s p e c i f i c a l l y to the CAMAL-1 column. Figure 6.2 i l l u s t r a t e s that when PCM was passed over a CAMAL-1 column, the eluate contained detectable amounts of CAMAL but when the same amount of RPMI/5% FCS was passed over the same column, no detectable material had adsorbed. The only d i f f e r e n c e between these two preparations was the conditioning of the former by pl a c e n t a l c e l l s . To v e r i f y these f i n d i n g s , another preparation of PCM (PCM-2) was passed over a CAMAL-1 immunoadsorbent column with s i m i l a r r e s u l t s . At the same time an equal amount of PCM-2 was passed over an i r r e l e v a n t (negative control) MAb column; Figure 6.3 a shows that material i n the PCM-2 bound s p e c i f i c a l l y to the CAMAL-1 and not to the negative control column. Another source of CM, PHA-LCM, was prepared and subjected to immunoadsorbence with the CAMAL-1 and negative control MAb columns. Figure 6.3 b i l l u s t r a t e s that material present i n t h i s source of CM also bound s p e c i f i c a l l y to the CAMAL-1, and not the negative c o n t r o l , column. Protein assays were performed on the pooled, 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 the CAMAL-1 column using the Biorad p r o t e i n 187 0.10-o •+ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 S 6 7 8 9 10 11 12 13 14 COLUMN FRACTION Figure 6.2. E l u t i o n p r o f i l e of ma t e r i a l bound to a CAMAL-1 immunoadsorbent column from p l a c e n t a l conditioned medium. • — • PCM run over CAMAL-1 column • • same volume of RPMI/5% FCS run over 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 with BSA as the standard. Results from these assays determined that the following estimated amounts of CAMAL were adsorbed from the various sources of CM. Type of CM CAMAL concentration 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 C. CAMAL Depletion Studies Since i t had been demonstrated that both the human plasma (Chapter IV) and the conditioned medium ( j u s t described) used i n the i n v i t r o myeloid clonogenic progenitor assay appeared to contain CAMAL, experiments were performed to determine the e f f e c t , i f any, of depletion of CAMAL from these two sources on the growth of myeloid progenitors. CAMAL was depleted from both plasma and CM by immunoadsorbence using a CAMAL-1 column. The e f f e c t of t h i s depletion was assayed on normal BM and PB, ALL BM, CGL PB and ANLL BM and PB. Appropriate controls f o r these experiments included (1) the use of plasma and CM that had not been treated i n any way, and (2) the use of plasma and CM that had been subjected to immunoadsorbence with an i r r e l e v a n t MAb (BLV-1) column. In addition to these i n t e r n a l controls, a number of "system" controls were performed to ensure that the e f f e c t s observed were not due to tec h n i c a l problems with t h i s system. These system controls included the following: 190 1. the use of two separately prepared CAMAL-1 columns f o r depletion which ensured that the e f f e c t s seen were not due to a problem with a s i n g l e column 2. the use of two d i f f e r e n t types (PCM and PHA-LCM) and two (PCM) or three (PHA-LCM) d i f f e r e n t batches of conditioned medium to ensure that the e f f e c t s seen were of a general nature and not due to conditions p a r t i c u l a r to any given CM source or preparation 3. the use of plasma from two d i f f e r e n t normal volunteers and samples of plasma c o l l e c t e d at four d i f f e r e n t times from the same normal volunteer to again ensure that the e f f e c t s seen were of a general nature. Normal PB was used i n the myeloid progenitor assay to examine the p o s s i b i l i t y of a r t i f a c t s using the controls j u s t described. Once i t had been established that the system d i d not have inherent a r t i f a c t u a l problems, the experiments that w i l l now be described were performed. For a l l of these experiments, plasma from the same normal i n d i v i d u a l (tested f o r i t s high q u a l i t y i n the assay) and PHA-LCM prepared from PHA-stimulated lymphocytes from the same i n d i v i d u a l were u t i l i z e d i n order to further standardize the system. 1. E f f e c t of CAMAL Depletion on Normal Bone Marrow Bone marrow samples from 10 normal BMT donors were used i n these experiments. A complete l i s t of the r e s u l t s from these experiments are presented i n the Appendix (Table XIX-A) following Chapter VII. For purposes of c l a r i t y three examples of these r e s u l t s and a summary are included here (Table XIX). Table XIX. E f f e c t of CAMAL depletion on normal bone marrow Patient Code Number of CFU-c % % I n h i b i t i o n per 10-> c e l l s 1 0 268 + 3 100 0 B 255.5 + 4.5 95.3 4.7 C 210 + 4 78.4 21.6 2 0 74 + 1 100 0 B 72 + 0 97.3 2.7 C 56 + 2 75.7 24.3 3* 0 127.5 + 12 100 0 B 131 + 8 102.4 -2.4 C 95 + 7.5 74.3 25.7 Average of r e s u l t s from 10 samples : Mean % B*s: 96.8% Mean % C s : 76.4% Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column * Nonadherent c e l l s were plated 192 A l l of 10 normal BM samples tested showed a decrease i n the number of CFU-c when CAMAL was depleted from the plasma and CM. I n h i b i t i o n of these normals ranged from 15.7 to 32.2% (average 23.6%) of the untreated controls, or 14.0 to 27.6% (average 20.4%) of the negative MAb column-treated c o n t r o l s . When a two-sided Student's t - t e s t analysis was performed to compare the r e s u l t s of the negative MAb column-treated controls and the CAMAL-1 column-treated (CAMAL-depleted) CFU-c, the di f f e r e n c e between these r e s u l t s was found to be highly s i g n i f i c a n t with p < 0.001. The r e s u l t s shown f o r patient 3, Table XIX, using non-adherent c e l l s indicated that the i n h i b i t o r y e f f e c t seen i n normal BM was not due to an i n d i r e c t action mediated through adherent c e l l s (known to be responsible f o r production of various regulatory 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 of a population of normal myeloid progenitors by CAMAL depletion indicated that t h i s p r o t e i n may be involved i n some regulatory manner i n normal myelopoiesis. The presence of CAMAL i n t h i s system appeared to be a requirement f o r the growth of at le a s t some normal myeloid clonogenic progenitors. Whether t h i s i n h i b i t i o n observed with CAMAL depletion was due to i n h i b i t i o n of a p a r t i c u l a r subpopulation of CFU-c or due to a generalized i n h i b i t i o n has not been determined as yet. Both the number and average s i z e of marrow CFU-c grown under CAMAL-depleted conditions were often reduced i n comparison to control cultures. 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 at l e a s t three weeks i n v i t r o . Figure 6.4 i l l u s t r a t e s t y p i c a l CFU-c from (a) control and (b) CAMAL-depleted cultures of the same normal BM sample. 193 Figure 6.4. Normal marrow CFU-c from (a) c o n t r o l and (b) CAMAL depleted c u l t u r e s , showing a decrease i n s i z e and number of CFU-c. 194 2. E f f e c t of CAMAL Depletion on Normal Peripheral Blood Table XX shows r e s u l t s from two of the four experiments performed on normal PB samples. As was the case with the normal BM, CAMAL depletion caused a s i g n i f i c a n t (p < 0.005) decrease i n the number of CFU-c when compared to controls. A complete data f i l e i s presented i n the Appendix (Table XX-A) for^review i f desired. The i n h i b i t i o n observed ranged from 26.3 to 45.1% of con t r o l values. While t h i s may appear to be of greater magnitude than that observed f o r normal BM, the increased i n h i b i t i o n was only seen f o r two normal PB samples from the same i n d i v i d u a l (routine and non-adherent c e l l s were tested). I t was i n t e r e s t i n g , and may eventually prove to be s i g n i f i c a n t , that these two samples represented a completely autologous s i t u a t i o n ; the c e l l s , plasma and conditioned medium were a l l from the same i n d i v i d u a l i n t h i s case. 3. E f f e c t of CAMAL Depletion on ALL Bone Marrow Two bone marrow samples from ALL patients were tested to determine the e f f e c t of CAMAL depletion on t h e i r myeloid colony formation. The r e s u l t s from these experiments are shown i n Table XXI. These r e s u l t s show very s i m i l a r l e v e l s of i n h i b i t i o n with CAMAL depletion to those observed i n normals (14.9 and 22.2 % i n h i b i t i o n compared to negative column-treated control values), implying that s i m i l a r CAMAL regulatory controls may be i n e f f e c t f o r both normals and at l e a s t some ALL patients. This i s a reasonable assumption because, u n l i k e the s i t u a t i o n i n ANLL, normal myeloid progenitors are not suppressed i n ALL and t h e i r colony numbers i n v i t r o r e f l e c t the l e v e l of malignant c e l l i n f i l t r a t i o n i t s e l f (391). Table XX. E f f e c t of CAMAL depletion on normal peripheral blood Patient Code Number of CFU-c % % I n h i b i t i o n per 10-* c e l l s 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 of 4 normal peri p h e r a l blood samples : Mean % B*s: 88.6 % Mean % C*s: 53.1 % Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column 196 Table XXI. E f f e c t of CAMAL depletion on acute lymphoblastic leukemia (ALL) bone marrow Patient Code Number of CFU-c per 10^ c e l l s % I n h i b i t i o n (remission) 2 (not i n remission) 0 B C 0 B C 168 153 128 ± 9 ± 4 + 5 121.5 + 3.5 111.5 + 1.5 84.5 + 4.5 100 91.1 76.2 100 91.8 69.6 0 8.9 23.8 0 8.2 30.4 Mean % B's: Mean % C*s: 91.5 % 72.9 % Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column 197 4. E f f e c t of CAMAL Depletion on Chronic Granulocytic Leukemia  Peripheral Blood Having established that 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 depletion of CAMAL from the myeloid progenitor assay i n both normals and ALL patients, a se r i e s of s i m i l a r experiments were performed using CGL PBL to determine i f t h i s e f f e c t held true f o r myeloid leukemics as well. Data from 2 of 7 such experiments are i l l u s t r a t e d i n Table XXII; a complete l i s t of a l l experimental r e s u l t s are given i n the Appendix (Table XXII-A). In no case was any e f f e c t observed by CAMAL depletion i n these CGL samples, whether routine or non-adherent c e l l s were plated. Both CFU-c numbers and s i z e were in d i s t i n g u i s h a b l e i n CAMAL depleted cultures from those i n control cultures. S t a s t i s t i c a l analysis by Student's two-sided t - t e s t v e r i f i e d that 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 these cultures. This lack of e f f e c t i n CAMAL depleted cultures was not due to the use of a d i f f e r e n t batch of prepared plasma or CM; t h i s p o s s i b i l i t y was tested a number of times (using the same plasma and CM preparations shown to 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 normals). Indeed, i t appeared that myeloid clonogenic progenitors were not influenced i n any way by CAMAL depletion. I t has been demonstrated that CGL PB c e l l s contain s i g n i f i c a n t l y increased amounts of CAMAL compared to normals (Chapter I I I ) , and yet i t appeared that CAMAL depletion had no e f f e c t on these c e l l s i n terms of t h e i r myeloid progenitor 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 . 5. E f f e c t of CAMAL Depletion on ANLL Four ANLL samples were examined to determine i f t h e i r myeloid progenitors were unresponsive to CAMAL depletion as i n the case of 198 Table XXII. E f f e c t of CAMAL depletion on chronic granulocytic leukemia per i p h e r a l blood Patient Code Number of CFU-c per 5 x 10^ c e l l s % % I n h i b i t i o n 0 B C 91 + 1 84 + 1 87 + 2.5 100 92.6 96 0 7.4 4 0 B C 193 194 204 + 7 ± 11 + 14 100 100.5 105.7 0 -0.5 -5.7 Summary of 7 CGL PB samples tested Mean % B's: Mean % C*s: 102.2 107.7 Code: 0 B C = no treatment of plasma or CM = plasma and CM passed over negative (BLV-1) MAb column = plasma and CM passed over CAMAL-1 column 199 the CGL samples tested. Table XXIII shows r e s u l t s from two such experiments on recently diagnosed ANLL patients. Again no e f f e c t was observed f o r any ANLL cultures depleted of CAMAL, both i n the two ANLL patients shown i n Table XXIII and i n an ANLL remission patient tested (Table XXIII-A, Appendix), whether routine or non-adherent c e l l s were plated. 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 depleted and cont r o l cultures when analyzed by the 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 patients shown to have high r e l a t i v e CAMAL BM and PB values compared to normals were unaffected by the i n h i b i t o r y e f f e c t that had been observed f o r CAMAL i n normals. Table XXIV and Figure 6.5 summarize the e f f e c t of CAMAL depletion on CFU-c i n the sample groups described. 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 negative column-treated control ("B") cultures i n myeloid leukemics versus normals/ALL; 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 depleted ("C") cultures i n myeloid leukemics versus 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 very d i f f e r e n t CAMAL-mediated regulatory controls are i n operation i n myelopoiesis i n these two groups or that the c e l l s responsive to t h i s regulation are not present i n the myeloid leukemics tested. D. CAMAL Addition Studies Once i t had been established that CAMAL depletion i n the myeloid progenitor assay had no e f f e c t on myeloid leukemics tested but caused s i g n i f i c a n t i n h i b i t i o n of normals, experiments were designed to determine the e f f e c t , i f any, of addition of excess CAMAL to these cultures. Preliminary experiments which showed that CAMAL was 200 Table XXIII. E f f e c t of CAMAL depletion on ANLL Patient Code Number of CFU-c per 10-> c e l l s % % I n h i b i t i o n 1* (PB) 0 B C 63 + 2 70 + 1 69 + 9 100 111 109.5 0 -11 - 9.5 2 (BM) 0 B C 35 + 2 30 + 0.5 38.5 + 1.5 100 85. 110 0 14.3 -10 Average of 4 ANLL samples tested Mean % B's: 99.2 % Mean % C s : 108.2 % Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column *The colony numbers shown f o r patient 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 colony numbers were low) at 10^ c e l l s plated. 201 Table XXIV. Summary of CAMAL depletion on myeloid colony growth Sample Number Mean % CFU-c i n B* Mean % CFU-c number i n C* % Difference (B - C) Normal BM Normal PB ALL CGL ANLL 10 4 2 7 4 96.8 88.6 91.5 102.2 99.2 76.4 53.1 72.9 107.7 108.2 20.4 35.5 18.6 -5.5 -9.0 * = compared to untreated control cultures B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column Student's t - t e s t r e s u l t s : a) B cultures: no s i g n i f i c a n t d i f f e r e n c e between myeloid leukemics vs normal/ALL b) C cultures: s i g n i f i c a n t d i f f e r e n c e (p < 0.025) 202 in K UJ ~ CO o S O 2 60-40-u. O o ce o 20-m <# z o CO u z < U i S NORMALS 5 = 13 MYELOID LEUKEMICS x = 10 Figure 6 . 5 . Summary of the e f f e c t of CAMAL depletion on CFU-c from normals and myeloid leukemics. 0 = no treatment of plasma or conditioned medium (CM) B = plasma and CM passed over negative co n t r o l MAb column C = plasma and CM passed over CAMAL-1 column 203 present i n the plasma of both normals and myeloid leukemics (Chapter IV) indicated that there may be a differe n c e i n the amount of t h i s p r o t e i n i n the plasma of these two groups, with greater amounts of CAMAL being present i n the myeloid leukemic plasma. I t was speculated that t h i s might be of some b i o l o g i c a l s i g n i f i c a n c e with respect to leukemogenesis, p a r t i c u l a r l y since addition of excess (between 5.0 and 12.5 yg/ml) CAMAL to normal peri p h e r a l blood had been shown to r e s u l t i n adsorption of the pro t e i n to normal leucocytes (Chapter IV). D e t a i l s of the tec h n i c a l procedures f o r these experiments are given i n Chapter I I . B r i e f l y , excess (leukemia-derived) p u r i f i e d CAMAL (or PBS or i r r e l e v a n t protein) was added to routine cultures of normal or myeloid leukemic c e l l s and CFU-c counts were performed. 1. E f f e c t of CAMAL Addition on Normal Myeloid Colony Growth Three normal samples were tested (two BM and one PB sample) i n these experiments. Table XXV shows the r e s u l t s of addition of excess CAMAL to these samples. In a l l cases shown here, excess CAMAL (> 10 yg/ml) caused massive i n h i b i t i o n of normal myeloid colonies with an average i n h i b i t i o n of 74.8 + 13.9 (SEM) % at l e v e l s of 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 to 40 yg/ml ad 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 i n one normal BM and one normal PB sample tested. I n h i b i t i o n appeared to occur r a p i d l y once l e v e l s of approximately 15 yg/ml excess CAMAL were added. Of in t e r e s t i s the previous demonstration of CAMAL adsorption by normal leucocytes at l e v e l s of 12.5 yg/ml excess CAMAL addition. These and previous r e s u l t s indicated that, while a c e r t a i n amount of CAMAL appeared to be required f o r normal myelopoiesis to occur, an 204 Table XXV. E f f e c t of CAMAL addition on normal myeloid colony growth Sample yg/ml excess p r o t e i n Number of CFU-c per 10 5 c e l l s (BM), per 2 x 10 5 c e l l s (PB) % % i n h i b i t i o n 1 0 18.5 + 1.5 100 0 (BM) 10 25 + 2 135.1 -35.1 CAMAL 20 0.5 + 0.5 2.7 97.3 40 0.5 + 0.5 2.7 97.3 10 27 + 1 145.9 -45.9 NEG PROT 20 19 + 1 102.7 - 2.7 40 22 + 3 118.9 -18.9 2* 0 148 + 0 100 0 (BM) 1 148.5 + 13.5 100.3 - 0.3 5 154.5 + 9.5 104.4 - 4.4 CAMAL 10 137 + 5 92.6 7.4 15 98.5 + 0.5 66.6 33.4 20 75 + 2 50.7 49.3 3 0 22.5 + 0.5 100 0 (PB) 1 22 + 1 97.8 2.2 CAMAL 10 24.5 + 5 108.9 - 8.9 15 7.5 1.5 33.3 66.7 20 5 + 0 22.2 77.8 Nonadherent c e l l s were plated 205 excessive amount of t h i s p r o t e i n (or possibly, an alte r e d form of CAMAL) was capable of causing s i g n i f i c a n t shut-down of normal myelopoiesis. Figure 6.6 i l l u s t r a t e s the r e s u l t s obtained from one normal BM (patient 2, Table XXV) examined i n t h i s manner; t h i s 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% of control values at 20 yg/ml excess CAMAL. This, i n f a c t , was the lowest l e v e l of i n h i b i t i o n observed i n the normals at t h i s CAMAL concentration. One 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 (97.3%) at t h i s concentration. 2. E f f e c t of CAMAL Addition on Myeloid Leukemia Patients' Colony  Growth The same experimental protocol was performed on three myeloid leukemia patients. Two of these patients were i n act i v e stages of t h e i r disease (one chronic phase CGL, one recently diagnosed ANLL) and one was an ANLL BMT patient with normal BM who had been i n remission post-BMT f o r 17 months and had a normal CAMAL BM value (by i n d i r e c t immunoperoxidase). Table XXVI shows the r e s u l t s of these experiments. The ANLL BMT patient showed s i g n i f i c a n t i n h i b i t i o n of myelopoiesis at l e v e l s of 10 and 15 yg/ml excess CAMAL (patient 1, Table XXVI and Figure 6.7 a), s i m i l a r to that observed with the normals. No i n h i b i t o r y e f f e c t whatsoever on myelopoiesis by addition of up to 20 yg/ml excess CAMAL, was seen i n the remaining ANLL (Figure 6.7b, patient 3, Table XXVI) and the CGL patient (Figure 6.8, patient 2, Table XXVI). Since the same CAMAL preparation was used i n both the experiments on normals and on the myeloid leukemics, there was no p o s s i b i l i t y that the preparation i t s e l f was of a nonspecific t o x i c nature. Furthermore, the negative control p r o t e i n 206 Ul U in o O (J CC Ul CD s 3 2 160 • 1 4 0 1 2 0 100 8 0 6 0 4 0 2 0 0 0 1 5 10 15 2 0 /ug/ml C A M A L ADDED Figure 6 .6 . I n h i b i t i o n of normal marrow CFU-c by addition of p u r i f i e d leukemia-derived CAMAL at l e v e l s of 15 - 20 ug/ml. 207 Table XXVI. E f f e c t of CAMAL addition on myeloid leukemic colony growth Patient vg/ml excess Number of CFU-c* % % i n h i b i t i o n p r o t e i n 1 0 35.5 + 0.5 100 0 (BM) 10 19.5 + 1.5 54.9 45.1 CAMAL 15 5.5 + 0.5 15.5 84.5 10 31 + 4 87.3 12.7 NEG PROT 15 33 + 1 93 7 2** 0 195 + 5 100 0 (PB) 1 192 + 13 98.5 1.5 5 198 + 11 101.5 - 1.5 CAMAL 10 201 + 6 103.5 - 3.5 15 211.5 + 1.5 108.5 - 8.5 20 196 + 8.5 100.5 - 0.5 3 0 15 + 2 100 0 (PB) 5 20.5 + 2.5 136.7 -36.7 CAMAL 10 20 + 8 133.3 -33.3 20 20 + 1 133.3 -33.3 5 14.5 + 0.5 96.7 3.3 NEG PROT 10 14.5 + 0.5 96.7 3.3 20 16.5 + 1.5 110 -10 * = patient 1 c e l l s plated at 10 3 c e l l s per dish patient 2 c e l l s plated at 7.5 x 10^ c e l l s per dish patient 3 c e l l s plated at 1.5 x 10-> c e l l s per dish ** = nonadherent c e l l s were plated 208 25-20--h rh ill rh /ug/ml /ug/ml NEG PROTEIN C A M A L A D D E D A D D E D /ug/ml N E G P R O T E I N A D D E D fig/ml C A M A L A D D E D Figure 6.7. E f f e c t of addi t i o n of p u r i f i e d leukemia-derived CAMAL on CFU-c from ANLL pat i e n t s . (a) ANLL bone marrow transplant (17 months post-BMT) patient's marrow CFU-c, showing i n h i b i t i o n at le v e l s of 10 and 15 yg/ml excess CAMAL; (b) newly diagnosed ANLL patient's PB CFU-c, showing no i n h i b i t i o n at l e v e l s up to 20 yg/ml excess CAMAL. 209 2 O o u o CC UJ CO S 3 2 I jug/ml NEG PROTEIN ADDED /ug/i ml CAMAL ADDED Figure 6.8. Lack of i n h i b i t i o n of CGL perip h e r a l blood CFU-c by addition of p u r i f i e d leukemia-derived CAMAL. No i n h i b i t i o n was observed at lev e l s up to 20 ug/ml excess CAMAL. 210 added was prepared i n an i d e n t i c a l manner to the CAMAL protein , using immunoadsorption, and no i n h i b i t o r y e f f e c t was observed f o r those con t r o l cultures. The r e s u l t s from the myeloid leukemia patients c l e a r l y indicated that patients i n whom act i v e disease, and therefore abnormal myelopoiesis, were occurring were t o t a l l y unaffected i n terms of t h e i r CFU-c growth i n the presence of l e v e l s of leukemia-derived CAMAL that were i n h i b i t o r y to normal myelopoiesis. The implications of these r e s u l t s are obvious with regard to a poss i b l e mechanism by which myeloid leukemic c e l l s can cause the shut-down of normal myeloid progenitors and thus a f f o r d themselves a s i g n i f i c a n t growth advantage over normal c e l l s . This w i l l be discussed fur t h e r i n the following Chapter. E. Evidence that Normal and Leukemia-derived CAMAL May Not be the Same Soluble CAMAL, p u r i f i e d from leukemic c e l l s , was added to normal PB CAMAL-depleted c e l l cultures i n order to determine i f such treatment would "reverse" the apparent i n h i b i t o r y e f f e c t of CAMAL depletion on normal CFU-c growth. CAMAL, at 10 yg/ml, was added to both CAMAL depleted and negative MAb column-treated controls (Table XXVII); t h i s amount was calculated to be approximately equal to the amount of CAMAL depleted from the plasma and conditioned medium u t i l i z e d i n t h i s assay. CAMAL depletion 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 to control cultures, s i m i l a r to that previously reported. As Table XXVII demonstrates, addition of 10 ug/ml leukemia-derived CAMAL resul t e d i n approximately 36% i n h i b i t i o n of 211 Table XXVII. E f f e c t of CAMAL addition on normal peripheral blood CAMAL depleted cultures Code Average number % % I n h i b i t i o n of CFU-c B cultures 54.5 + 3.5 100 0 C cultures (CAMAL depleted) 46.5+3.5 85.3 14.7 Addition of leukemia- (B 34.5+4.0 63.3 36.7 derived CAMAL (10 ug/ml) (C 29.5+0.5 63.4 36.6 Code: B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column 212 CFU-c i n both "B" (control) and "C" (CAMAL depleted) cultures. These data imply the following: 1. the same colonies that are s e n s i t i v e to CAMAL (normal) -depletion are probably also CAMAL (leukemic) -a d d i t i o n s e n s i t i v e , since there was no dif f e r e n c e between the % i n h i b i t i o n of CFU-c i n B versus C cultures. 2. there are 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) -depletion s e n s i t i v e CFU-c, since greater % i n h i b i t i o n was observed i n the cultures to which leukemia-derived CAMAL was added. An alternate and, i n the author's opinion, more probable, explanation i s that CAMAL (normal) was not t o t a l l y depleted from plasma and CM. 3. Leukemia-derived CAMAL may not be i d e n t i c a l to normal CAMAL, since i t was not poss i b l e to reconstitute CAMAL (normal) depletion by CAMAL (leukemic) addition. 213 CHAPTER VII DISCUSSION AND SUMMARY I. DISCUSSION A. Evaluation and Diagnostic Implications of a Rapid S l i d e Test f o r CAMAL The development of an i n d i r e c t immunoperoxidase s l i d e t e s t , using the CAMAL-1 MAb to demonstrate the presence of the CAMAL marker i n or on i n d i v i d u a l blood or bone marrow c e l l s , has resu l t e d i n a rapid, simple and inexpensive assay f o r the detection of CAMAL. Such an assay could e a s i l y be transferred to a routine c l i n i c a l t e s t i n g laboratory since, i n i t s present form, i t requires no s p e c i a l i z e d s k i l l s to perform beyond those already i n use 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 at d i f f e r e n t times and i n four d i f f e r e n t laboratories i n the procedure outlined herein. Advantages of t h i s type of assay f o r the detection of antigenic markers include, i n addition to those mentioned above: 1. 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 (cytoplasmic, perinuclear, or nuclear) of the antigen within c e l l s as well as on the membrane 2. a permanent record i n the form of stained, mounted s l i d e s , very u s e f u l i n retrospective analyses and f o r teaching purposes 3. the a b i l i t y to store or s t o c k p i l e s l i d e s i n an unlabeled state f o r up to at l e a s t 3 months ( f o r CAMAL) p r i o r to performing the 214 assay, allowing more f l e x i b i l i t y with regard to a v a i l a b i l i t y of time and personnel 4. the a b i l i t y to detect 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 of the morphology of antigen-positive c e l l s . The complexity of antigenic heterogeneity i n ANLL has been discussed (Chapter I ) . L i t e r a t u r e review has indicated that several putative leukemia-associated antigens (LAA) e x i s t on myeloid leukemia c e l l s . Studies u t i l i z i n g a panel of absorbed heteroantisera defining myeloid LAA revealed that some LAA were common to both ANLL and CGL c e l l s , some were present on only c e r t a i n i n d i v i d u a l ' s c e l l s , while some malignant b l a s t s were unreactive with any of the sera examined (464). More recent studies have indicated that no i n d i v i d u a l myeloid-specific (or associated) MAbs or cytochemical markers recognized the malignant b l a s t c e l l s of every ANLL patient (466-472). Even within i n d i v i d u a l patients, there was a considerable degree of heterogeneity with respect to c e l l u l a r expression of the markers examined. D e f i n i t i v e evidence i s lacking to implicate e i t h e r true genetic heterogeneity or the existence of asynchronous l i m i t e d d i f f e r e n t i a t i o n as the explanation f o r t h i s i n a l l cases of ANLL, as mentioned previously. A great deal of supportive evidence has indicated that leukemic c e l l phenotypes are l i k e l y to be the r e s u l t of a combination of the expression of normal genes (not n e c e s s a r i l y expressed i n a normal qu a n t i t a t i v e manner) and abnormal, asynchronous d i f f e r e n t i a t i o n . The r e s u l t s of the CAMAL-1 i n d i r e c t immunoperoxidase t e s t have established that CAMAL i s present i n a s i g n i f i c a n t l y increased number of BM or peripheral blood c e l l s from i n d i v i d u a l s with myelogenous 215 leukemia (ANLL, acute phase or remission and CGL) when compared to very low numbers of c e l l s expressing CAMAL i n normal i n d i v i d u a l s or most patients with lymphoid malignancies. From a diagnostic standpoint, CAMAL appears to be a u s e f u l marker i n BM f o r the dis c r i m i n a t i o n of ANLL from ALL at diagnosis, although l i k e a l l other myeloid LAAs, t h i s i s not the case f o r every ANLL patient. As Table V showed, not a l l ANLL patients expressed s i g n i f i c a n t l y increased numbers of CAMAL-positive c e l l at diagnosis. The f a c t that increased numbers of CAMAL-positive c e l l s were present i n BM and perip h e r a l blood of preleukemic, myelodysplastic and chronic granulocytic leukemia patients implied a common hemopoietic abnormality (possibly r e l a t e d to the l i k e l i h o o d of development of acute leukemia) e x i s t i n g i n these conditions. This was also indicated by the "common" nature of CAMAL expression i n a l l FAB subgroups of ANLL. I t further implied that increased CAMAL expression was an early event i n the onset of acute leukemia. Were t h i s to be the case, ANLL patients at diagnosis with low numbers of CAMAL-positive c e l l s may be i n d i v i d u a l s with more advanced disease, having passed beyond the stage of expression of t h i s marker. This implication w i l l be discussed i n more d e t a i l i n a l a t e r section. There i s no cl e a r explanation yet f o r the observation that CAMAL BM or PB values i n some patients with lymphoid malignancies (ALL at diagnosis or lymphoma), while s i g n i f i c a n t l y lower than most myeloid leukemics, were nonetheless increased somewhat above normal BM or 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 the observation that h a l f of the ALL remission BM and PB samples examined demonstrated numbers of CAMAL-expressing myeloid 216 c e l l s comparable to those found i n many ANLL patients. As discussed previously, two ALL remission patients examined during the b l i n d study showed high numbers (25 and 30%) of CAMAL-positive BM c e l l s one month p r i o r to relapse and an ALL BMT patient demonstrated a CAMAL PB value of 50% three months p r i o r to relapse (529). A larger group of ALL remission patients needs to be s e r i a l l y examined before d e f i n i t i v e remarks can be made concerning the p o s s i b i l i t y that increased CAMAL expression may s i g n a l the onset of relapse 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 that the CAMAL-expressing remission c e l l s i n ALL BM are d e f i n i t e l y of myeloid morphology. I t may be important at t h i s point to remember that stimulated lymphoid c e l l s or c e l l l i n e s are a major source of myelopoietic colony-stimulating factors (GM-CSF, G-CSF, -» EPA) as well as other important regulators of granulopoiesis (interferon-gamma, t r a n s f e r r i n ) and that there i s an enormously complex i n t e r p l a y between these regulators, the c e l l s that produce them and t h e i r target c e l l s . In l i g h t of t h i s , i t i s conceivable that during the onset of abnormal lymphopoiesis occurring p r i o r to relapse i n ALL, abnormal stimulation of and in t e r a c t i o n s between myeloid regulatory factors could occur and be "marked" by increased CAMAL expression by myeloid c e l l s . This speculation i s offered only as food f o r thought; s p e c i f i c research could be performed to t e s t t h i s p o s s i b i l i t y . Alternate explanations, such as lineage i n f i d e l i t y previously discussed (Chapter III) could be invoked f o r a l i m i t e d number of ALL patients at diagnosis expressing both CAMAL and (primarily) lymphoid antigens, but t h i s could c e r t a i n l y not explain the s i t u a t i o n i n ALL remission patients. 217 In conclusion, i t appears that CAMAL i s a us e f u l adjunctive diagnostic marker at diagnosis f o r ANLL. The main value of determining the number of CAMAL-positive BM c e l l s at diagnosis, however, appears to be i n i t s changing expression post-chemotherapy and during remission, as w i l l be discussed i n a l a t e r section. B. CAMAL Expression i n Leukemia C e l l l a b e l i n g studies with r a b b i t anti-CAMAL serum ( i n the fluorescence activated c e l l sorter) and CAMAL-1 MAb ( i n immunoperoxidase) allowed i d e n t i f i c a t i o n of the morphology of CAMAL-positive c e l l s i n myeloid leukemic patients. CAMAL-1 p o s i t i v e c e l l s included myeloid c e l l s at a l l l e v e l s of maturation, from b l a s t s (predominant at diagnosis i n ANLL) to end stage granulocytes and monocytes. In ANLL remission peripheral blood samples, CAMAL-1 p o s i t i v e c e l l s included mature myeloid c e l l s and, i n a number of samples with high numbers of p o s i t i v e c e l l s (up to 80%), lymphocytes were strongly labeled as we l l . In general, r a b b i t anti-CAMAL serum (using i n d i r e c t immunofluorescence) labeled more numerous but s i m i l a r c e l l types i n myeloid leukemia patients. Many more c e l l s with lymphoid morphology were labeled by the heteroantiserum. This probably r e l a t e s to the p r e f e r e n t i a l recognition of membrane bound CAMAL by the rabbi t anti-CAMAL serum, which presumably recognizes many more antigenic determinants displayed by c e l l surface CAMAL than does the CAMAL-1 MAb. The question remains as to whether or not the morphologically mature CAMAL-expressing c e l l s are a c t u a l l y derived from the o r i g i n a l malignant clone. G-6-PD and chromosomal analyses of ANLL have 218 indicated that i t i s a disease 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 involvement has not been established c a t e g o r i c a l l y and i s complicated by apparent heterogeneity i n t h i s regard. While i t i s generally believed that the GM precursor i s most commonly involved i n the stem o r i g i n of ANLL, some investigators have c l e a r l y implicated much more p r i m i t i v e c e l l s (with erythroid and B lymphoid p o t e n t i a l i n addition to GM) or occasionally, monocyte-restricted precursors (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 malignant blood c e l l s and number of CAMAL-positive c e l l s at diagnosis. Moreover the lack of c o r r e l a t i o n of CAMAL expression with e i t h e r a c t i v e l y regenerating or a p l a s t i c BM argues against a c e l l c y cle dependent expression of t h i s marker, which might have explained why only some of the malignant c e l l s were CAMAL-positive. The demonstration of a small proportion of normal CAMAL-positive BM c e l l implied that not a l l c e l l s , even i n myeloid leukemics, that express CAMAL were n e c e s s a r i l y derived from the malignant clone. The ANLL remission pathology studies outlined i n Chapter IV, where i n one case i t was determined that 100% of peripheral blood c e l l s with normal morphology and karyotype labeled strongly CAMAL p o s i t i v e , implied that these CAMAL-expressing c e l l s were not derived from the o r i g i n a l hypodiploid leukemic clone. Since relapse followed within four months (with re-emergence of the o r i g i n a l clone), i t i s obvious that, i n f a c t , underlying pathology was indeed s t i l l present nonetheless. The r e s u l t s of the CAMAL adsorption studies, wherein normal PBL became CAMAL-positive a f t e r incubation with excess CAMAL, suggested that i t was p o s s i b l e f o r normal PBL, including lymphocytes, 219 to bind excess amounts of soluble CAMAL on t h e i r membrane. I labeled-CAMAL membrane binding studies indicated non-saturable l a b e l i n g of normal and CGL c e l l s by p u r i f i e d CAMAL, of s i m i l a r magnitude to the 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 to i s o l a t e s p e c i f i c CAMAL receptors, i f they e x i s t , from c e l l membrane extracts by a f f i n i t y chromatography using a CAMAL column. I t i s conceivable, and consistent with these r e s u l t s , that increased amounts of CAMAL i n the plasma of myeloid leukemics p r i o r to relapse (when increased numbers of CAMAL-expressing c e l l s have been demonstrated) can account f o r the increased appearance of p o s i t i v e c e l l s at these times. Furthermore, the ALL remission samples showing the presence of p o s i t i v e myeloid c e l l s also indicated that c e l l s other than those involved i n the o r i g i n a l malignant clone may express the CAMAL marker. Evidence has been presented i n t h i s t h e s i s that points to a r e l a t i o n s h i p between the increased expression of CAMAL and leukemic pathology. C. S i g n i f i c a n c e of CAMAL as a Prognostic Marker f o r Remission i n ANLL The marked antigenic heterogeneity, v a r i a b l e chromosomal abnormalities and uncertainty concerning the l e v e l of stem c e l l involvement i n ANLL are r e f l e c t e d i n the lack of us e f u l common prognostic i n d i c a t o r s or markers f o r t h i s disease, e s p e c i a l l y during remission. I t i s c l e a r that routine monitoring protocols (consisting p r i m a r i l y of morphological and cytochemical evaluation of BM c e l l s ) f a l l f a r short of 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 to adequately detect 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. Usually by 220 the time that i t becomes obvious that r e s i d u a l leukemia i s present, relapse i s well established and with i t , an extremely poor long term prognosis. The i n a b i l i t y to as c e r t a i n the presence of underlying pathology complicates the establishment of r a t i o n a l therapeutic regimens as well as the evaluation of t h e i r e f f i c a c y a f t e r i n i t i a l c o n solidation chemotherapy. The i n a b i l i t y to p r e d i c t which patients w i l l have long or short remissions further increases the d i f f i c u l t y of patient management decisions regarding the im p l i c a t i o n of novel forms of treatment i n selected patients. The preliminary r e s u l t s presented from the 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 regard. The r e l a t i v e CAMAL BM value, before and a f t e r consolidation chemotherapy, does appear to be of prognostic s i g n i f i c a n c e . In t h i s study, a c l e a r l y better (p < 0.025) prognostic trend was indicated i n the ANLL group whose CAMAL BM values dropped s i g n i f i c a n t l y post-chemotherapy. For patients i n t h i s group, the average remission length was 2.8 times longer than those ANLL patients whose values increased or remained the same post-chemotherapy. I t i s v a l i d to argue that patients with low (< 5%) i n i t i a l values could not show a s i g n i f i c a n t decrease post-treatment. Nonetheless, i t i s equally v a l i d to say that patients with i n i t i a l values of < 100% ( r e a l l y , < 95%) could show e i t h e r an increase or a decrease. The data has indicated that t h i s was not the trend seen. I t has been c l e a r l y demonstrated that the only 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 increased s u r v i v a l time p r i o r to relapse was the change (decrease) i n the CAMAL BM value pre- to post-chemotherapy. A much greater proportion of CAMAL-1 p o s i t i v e mature myeloid c e l l s were 221 observed post-chemotherapy i n those patients whose values decreased, perhaps i n d i c a t i n g that the CAMAL-1 p o s i t i v e c e l l population at diagnosis had undergone a greater 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 tient group. Increasing 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 could, therefore, account f o r decreased numbers of c e l l s expressing CAMAL post-chemotherapy i n t h i s p a tient group. Now that three-quarters of adult ANLL patients achieve complete remission following intensive chemotherapy, the problem of maintaining these patients i n remission state assumes much greater importance than ever before. Procedures such as maintenance chemotherapy during remission to prevent relapse have been demonstrated to be i n e f f e c t i v e i n t h i s regard, p o s s i b l y due to our i n a b i l i t y to determine the times at which such treatment might be c r i t i c a l on an i n d i v i d u a l patient basis. Very recently, the polymerase chain reaction (PCR) technique has been u t i l i z e d i n the detection of minimal r e s i d u a l disease i n human 5 f o l l i c u l a r lymphoma (540). As l i t t l e as 1 i n 10 c e l l s 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 lymphoma cases was detectable using t h i s technique, exceeding the detection l i m i t s of other reported methods (flow cytometry or Southern b l o t analysis f o r c l o n a l immunoglobulin gene rearrangements). Synthetic oligonucleotides flanking the crossover s i t e s of the t(14;18) t r a n s l o c a t i o n acted as primers f o r a PCR, allowing DNA sequences flanking the crossover s i t e s to be amplied exponentially, and thus to be made e a s i l y detectable. This e x c i t i n g new technique would not, however, be p r a c t i c a l i n diseases such as ANLL where even though c e r t a i n nonrandom chromosomal abnormalities occur, they are multiple 222 and would require the preparation of d i f f e r e n t primers f o r each i n d i v i d u a l genetic change on the basis of DNA sequencing. Moreover, many ANLL patients demonstrate no chromosomal abnormalities using conventional techniques. The PCR technique would be reasonable f o r the monitoring of minimal residue disease i n Ph' chromosome p o s i t i v e ,CGL patients r e c e i v i n g allogeneic bone marrow transplants. In ANLL, e a r l i e r i n vestigations by Baker and colleagues using anti-myeloblast heteroantiserum i n immunofluorescence experiments, demonstrated that r e a c t i v e c e l l s were present i n the BM of many ANLL remission patients several months p r i o r to relapse (461,462). Neither the nature, of the r e a c t i v e c e l l s nor the antigen(s) detected were described and i t i s p o s s i b l e that these in 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, or some r e l a t e d antigen. We have previously demonstrated the presence of CAMAL-positive PB c e l l s i n 6/6 ANLL BMT patients up to several months p r i o r to relapse (529). In contrast, 14/14 acute leukemia BMT patients dying of causes unrelated to relapse, and 13/15 patients who remained i n remission f o r the 12 month study period, showed no s i g n i f i c a n t numbers of CAMAL-1 re a c t i v e c e l l s (by immunoperoxidase). In the 3 year b l i n d study reported herein (Chapter V), a more complex s i t u a t i o n existed than that of the acute leukemia BMT patients, since no 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 of age (obviously a much wider range was examined) or d i f f e r e n t treatment protocols on CAMAL expression. While the r e s u l t s presented are l i m i t e d and preliminary, there are c l e a r i n d i c a t i o n s that increasing numbers of CAMAL-positive c e l l during remission may s i g n a l the onset of relapse within the following few months. Indeed, when 223 we obtained s u f f i c i e n t l y sequential samples from an i n d i v i d u a l patient, i t was cl e a r that t h i s pattern held true (Figure 5.4 and Table XVIII). Recent data has also indicated that over 83% of ANLL patients given chemotherapy when t h e i r CAMAL BM values were high (> 10%) responded more s u c c e s s f u l l y to chemotherapy (had longer remission times) than those patients treated when t h e i r values were < 10%. The r e s u l t s from t h i s b l i n d study may prove to have implications i n the following s i t u a t i o n s : 1. determination of "true" remission status i n ANLL (the maintenance of low or decreasing CAMAL BM values) and, r e l a t e d to t h i s , 2. determination of optimal times f o r performing BMT on ANLL remission patients (while i n "true" remission state) 3. development and evaluation of novel therapeutic protocols including: a. e a r l y a d d i t i o n a l treatments f o r ANLL patients with predictably shorter remission lengths, i n p a r t i c u l a r those demonstrating a large increase i n CAMAL BM value post-chemotherapy b. administration of remission chemotherapy to i n d i v i d u a l ANLL patients based on a s i g n i f i c a n t increase i n CAMAL BM value c. administration of 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 increase i n CAMAL BM value. These applications are only suggested here, based on preliminary fi n d i n g s . We are planning a na t i o n a l 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) values and c l i n i c a l prognosis i n a large group of c l o s e l y monitored ANLL patients at three leukemia centers. This t r i a l should e s t a b l i s h whether or not these preliminary findings are v a l i d . D. The Possible Role of CAMAL i n Myelopoiesis I t has now been established that CAMAL can act as a s i g n i f i c a n t marker i n the p r e d i c t i o n of remission length and i n the onset of relapse i n ANLL patients. The question now surfaces as to whether CAMAL i s simply a convenient antigenic marker, associated i n d i r e c t l y with myeloid leukemogenesis, or whether CAMAL may be involved i n a more d i r e c t b i o l o g i c a l manner. Due to the complexity of in t e r a c t i o n s between well and poorly characterized p o s i t i v e and negative myelopoietic regulators, t h e i r target c e l l s or molecules, and the c e l l s producing them, pre c i s e answers to t h i s question become very d i f f i c u l t to approach experimentally, l e t alone resolve with c e r t a i n t y . Some headway has now been made towards t h i s end and future experimental approaches have become bett e r defined. D e f i n i t i v e answers concerning the poss i b l e r o l e of CAMAL i n myelopoiesis await molecular analysis of t h i s p r o t e i n i n both normals and myeloid leukemics. For the 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 of important factors need to be considered i n experimental approaches aimed at demonstrating e i t h e r stimulation or i n h i b i t i o n of hemopoietic colony formation by any b i o l o g i c a l l y -derived substance. F i r s t of a l l , there i s always a problem with i n t e r p r e t a t i o n of the detection and ch a r a c t e r i z a t i o n of putative 225 regulatory factors using an i n v i t r o t i s s u e culture system. Such systems are inherently a r t i f i c i a l i n terms of i n vivo c e l l concentrations and other natural microenvironmental influences. However, the s i m p l i c i t y and capacity to standardize such systems can o f f e r advantages impossible to duplicate otherwise. Furthermore the i n v i t r o myeloid progenitor c e l l assay has been demonstrated to be a us e f u l and relevant system f o r such i n v e s t i g a t i o n s . Other important considerations include the presence of a putative regulatory f a c t o r i n vivo at l e v e l s s i m i l a r to those required to demonstrate e f f e c t s i n v i t r o , demonstration of p h y s i o l o g i c a l l y relevant i n vivo f l u c t u a t i o n s of the fa c t o r , and the a b i l i t y to e f f e c t i v e l y p u r i f y the f a c t o r from relevant sources. CAMAL has been demonstrated to be present i n vivo, both i n human plasma and i n BM and PB c e l l s . Given that methods used to quantify CAMAL plasma l e v e l s are not conclusive, i t i s more d i f f i c u l t to j u s t i f y any statements concerning relevant i n vivo l e v e l s . However, CAMAL adsorption studies indicated that approximately 12 ug/ml (or greater) a f f i n i t y - p u r i f i e d CAMAL bound to normal PB leucocytes, and t h i s l e v e l i s remarkably s i m i l a r to those demonstrated to cause i n h i b i t i o n of normal myeloid colony growth. With regard 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 vivo, i t has been shown that numbers of CAMAL-positive c e l l s f l u c t u a t e i n vivo, and that 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 of c l i n i c a l prognosis. F i n a l l y , CAMAL can be p u r i f i e d e f f e c t i v e l y by immunoadsorption. Soluble extracts of leukemic c e l l s provided s u f f i c i e n t q u a n t i t i e s f o r i n v i t r o assays. S p e c i f i c antibodies coupled to a f f i n i t y columns have been u t i l i z e d by others to s u c c e s s f u l l y p u r i f y both M-CSF and IL-2 (128,175). 226 As discussed i n Chapter I, serum (or plasma) contains numerous components with known growth promoting a c t i v i t i e s f o r cultured hemopoietic c e l l s . The s e l e c t i v e , r e s t r i c t i v e (or absent) growth and p r o l i f e r a t i o n of some or a l l hemopoietic c e l l s i n v i t r o , depending on the batch of f e t a l c a l f serum or plasma u t i l i z e d , are testimony for the major regulatory r o l e that some of these components can play. I t has been well established that the only consistent method f o r demonstration of any p o s s i b l e e f f e c t ( s ) of a serum or plasma component i s the use of s e l e c t i v e depletion (234). When CAMAL was 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 and conditioned medium used i n the myeloid progenitor c e l l assay, a decrease i n the number (and often size) of normal CFU-c was observed. Reconstitution attempts using CAMAL derived from leukemic c e l l s were unsuccessful, as were s i m i l a r attempts using human serum albumin (up to 40 yg/ml), a growth-promoting serum component with s i m i l a r molecular weight and some immunological c r o s s - r e a c t i v i t y with CAMAL. Depletion of CAMAL from the same normal human plasma and conditioned medium f a i l e d to i n h i b i t CFU-c development i n myeloid leukemics. These r e s u l t s implied that, i n normal myelopoiesis, CAMAL may be involved i n some p o s i t i v e regulatory manner, but that i n myeloid leukemia, such regulation (or the c e l l s responsive to i t ) do not e x i s t . Future studies aimed at 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 include comparative morphological examination of colonies from depleted and c o n t r o l cultures i n order to determine i f a s p e c i f i c subpopulation(s) of CFU-c was affected. The decrease i n s i z e of many colonies from depleted marrow cultures could r e l a t e to 227 more rapid d i f f e r e n t i a t i o n (and therefore, decreased p r o l i f e r a t i v e capacity) within these colonies. I t i s poss i b l e that, i n normal myelopoiesis, CAMAL may be involved i n the t r a n s i t i o n between these two processes. One very important aspect concerning the ch a r a c t e r i z a t i o n of CAMAL which has not been c l e a r l y established, r e l a t e s to the c e l l ( s ) responsible f o r producing i t . As these studies are part of other i n v e s t i g a t o r s ' research objectives, experiments into the nature of c e l l s producing CAMAL have not been attempted here. The presence of CAMAL i n human serum CAMAL and plasma implied that CAMAL i s released from i t s c e l l ( s ) of o r i g i n . The presence of CAMAL i n human placental and leucocyte conditioned media implied that these c e l l s e i t h e r a c t i v e l y produce and secrete CAMAL or release CAMAL from i n t r a c e l l u l a r stores. Recently i t has been demonstrated that the human promyelocytic c e l l l i n e HL-60 produces CAMAL-1 re a c t i v e material when grown i n serum-free medium (Shellard, personal communication), i n d i c a t i n g that myeloid c e l l s are the probable source of CAMAL. Whether myeloid c e l l s of a l l l e v e l s of maturation (known to contain CAMAL) a c t u a l l y continue to produce i t , or merely store and/or secrete i t , has not been established, nor do we know i f the same c e l l type(s) are responsible f o r CAMAL production i n normals and leukemics. When the e f f e c t of addition of excess CAMAL to cultures was investigated, leukemic (CGL) c e l l s were used as a convenient source f o r obtaining s u f f i c i e n t amounts of p u r i f i e d CAMAL f o r t e s t i n g . I t was discovered that addition of > 10 - 15 v&/ml excess leukemia-derived CAMAL resu 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 of normal CFU-c but the same preparation had no e f f e c t on colony growth i n 228 myeloid leukemia patients with a c t i v e disease. The i n h i b i t o r y e f f e c t d i d not appear to be i n d i r e c t l y mediated through adherent accessory c e l l s , a s i t u a t i o n s i m i l a r to that i n the previous CAMAL depletion studies. While these r e s u l t s at f i r s t seemed discrepant with the previous studies, the observation that leukemia-derived CAMAL could not re c o n s t i t u t e CAMAL depleted 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 present i n normals and that i n myeloid leukemics. The existence of a hemopoietic f a c t o r (multi-CSF) derived from malignant c e l l s (WEHI-3B) that i s d i f f e r e n t from i t s normal counterpart has been documented previously (114,115). A si n g l e amino acid d i f f e r e n c e between these two molecules apparently accounts f o r decreased fu n c t i o n a l a c t i v i t y of the WEHI-3B-derived multi-CSF i n the stimulation of hemopoietic c e l l p r o l i f e r a t i o n (182). Future studies using CAMAL derived from normal sources are indicated, as i s determination of the primary amino acid sequence of CAMAL from normal and leukemic sources. A number of a d d i t i o n a l questions 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 the target c e l l ( s ) or molecule(s) f o r CAMAL? 2. Is CAMAL from myeloid leukemia patients "non-functional" by i t s e l f but capable of blocking receptors f o r other required factors (factors which normal c e l l s require but myeloid leukemics do not)? 3. Does leukemic CAMAL bind some important regulatory molecule i n conditioned medium or plasma, and can i t s e f f e c t be overcome by addition of increased CSF? 229 4. Does addition of CAMAL to PHA-stimulated leucocyte cultures cause any change i n production of CSF? 5. Does leukemia-derived CAMAL need to be present throughout the culture period i n order to exert i t s e f f e c t , or would preincubation or delayed addition also be e f f e c t i v e ? 6. Is a s i m i l a r molecule present i n mice or i s human CAMAL fun c t i o n a l i n the murine system, allowing i n vivo testing? 7. What i s the molecular nature of the mechanism by which CAMAL exerts i t s i n h i b i t o r y e f f e c t on normal hemopoietic c e l l s ? 8. Does CAMAL cause an increase or decrease i n the production and/or release of well characterized negative regulators (LF, AIF, IFN, TF, PGE)? The r e l a t i o n s h i p , i f any, of leukemic CAMAL to defined negative regulators would be important to e s t a b l i s h . The most prominant s i m i l a r i t y appears to be the di f f e r e n c e i n responsiveness to negative re g u l a t i o n by leukemic c e l l s compared to t h e i r normal counterparts. Myeloid leukemia patients' c e l l s have been shown to have decreased responsiveness to the suppressive or i n h i b i t o r y e f f e c t s of LF, AIF, PGE and CAMAL. In the case of AIF and PGE, t h i s i s re l a t e d to t h e i r Class II antigen target c e l l s p e c i f i c i t y . Since there i s a def i c i e n c y of Class II antigens on myeloid progenitors of leukemia pa t i e n t s , these regulators have decreased e f f e c t s on c e l l s from these patients. Indeed, AIF i s present i n elevated amounts i n leukemic c e l l s (261,268), a somewhat s i m i l a r s i t u a t i o n to that of CAMAL. The p o s s i b i l i t y of Class II (HLA-DR) antigen-positive target c e l l s p e c i f i c i t y of CAMAL could be examined experimentally. 230 The v i r t u a l l y t o t a l suppression of normal hemopoietic progenitor c e l l s i n the face of the emerging malignant clone i n ANLL i s a l l the more remarkable i n the absence of conclusive explanations f o r i t s occurrence. Normal hemopoietic c e l l s have profound regenerative ca p a c i t i e s i n s i t u a t i o n s of consumption or l o s s . Moreover, the c e l l c y cle time of normal hemopoietic c e l l s i s shorter than that of leukemic c e l l s . And yet the s t r i k i n g puzzle remains concerning the a b i l i t y of the malignant clone to gain a foothold i n terms of increased s e l f generation over normal c e l l s . Long-term marrow cultures of both CGL and newly diagnosed ANLL patients* c e l l s have demonstrated that normal hemopoietic progenitor c e l l s are s t i l l present, even though suppressed, i n vivo (381). I t has been established that myeloid leukemia c e l l s (ANLL and CGL) remain completely dependent on CSF f o r growth i n v i t r o . Large di f f e r e n c e s i n responsiveness to CSF by myeloid leukemia c e l l s compared to normals have not been indicated. Possible mechanisms f o r c l o n a l dominance by myeloid leukemic c e l l s include f a i l u r e to i n t e r a c t appropriately with stromal c e l l s , stromal c e l l dysfunction and/or abnormal CSF production i n the immediate v i c i n i t y of the leukemic clone. The most convincing explanations f o r the emergence and dominance of the leukemic clone involve the profound decrease i n responsiveness to negative regulation that these c e l l s e x h i b i t . This has been discussed previously i n d e t a i l . Such a mechanism provides an i d e a l method f o r gaining a p r o l i f e r a t i v e advantage over normal c e l l s . Experimental r e s u l t s have indicated that t h i s mechanism may be the manner i n which CAMAL plays an operational r o l e i n abnormal myelopoiesis. I t i s apparent that r e s i d u a l leukemic c e l l s may e x i s t 231 at very low or undetectable l e v e l s i n ANLL during remission. I t i s apparent that CAMAL-expressing c e l l s can also e x i s t at low l e v e l s i n the same s i t u a t i o n . The observation that increasing numbers of c e l l s (whether part of the malignant clone or not) expressing CAMAL appear to herald the onset of relapse may r e l a t e to the suppressive e f f e c t of increasing amounts of leukemic CAMAL on normal hemopoietic c e l l s . This event would, by i t s nature, be required to take place p r i o r to the c l i n i c a l re-emergence of the malignant clone, and t h i s does indeed coincide with increasing CAMAL expression. The observation that increasing CAMAL BM values post-chemotherapy correspond to shorter remission lengths than do decreasing values, i s also i n accordance with t h i s i n t e r p r e t a t i o n . Many questions and speculations concerning the nature of the CAMAL marker remain to be examined. In addition to those already 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 myelopoiesis, which we have ascribed as being mediated by the p u r i f i e d 68 KD protein , may be the r e s u l t of some other molecule, c l o s e l y associated and c o - p u r i f i e d with CAMAL. U n t i l the gene encoding CAMAL i s i s o l a t e d and subsequent recombinant p r o t e i n i s produced, t h i s p o s s i b i l i t y may be very d i f f i c u l t to examine i n d e t a i l . The p o s s i b i l i t y that normal and leukemia-derived CAMAL may not be i d e n t i c a l must also 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 modifications of the protein may occur i n leukemic c e l l s that a l t e r CAMAL*s normal expression as well as i t s function. Such p o s s i b i l i t i e s are now being addressed by other i n v e s t i g a t o r s . 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 cell 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 cell 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 first 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 utilizing 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 resulted i n s i g n i f i c a n t i n h i b i t i o n of normal CFU-c growth but had no e f f e c t on myeloid leukemic colony growth. Addition of > 10 ug/ml leukemia-derived excess CAMAL caused profound i n h i b i t i o n of normal CFU-c but had no e f f e c t on CFU-c growth from myeloid leukemia patients i n act i v e disease states. These r e s u l t s suggested differences i n hemopoietic regulation by normal and leukemic c e l l s that have not been previously examined. A mechanism whereby myeloid leukemic c e l l s can cause the i n h i b i t i o n of 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 differences i n CAMAL expression by leukemic c e l l s compared to normals have been established which may r e l a t e to leukemogenesis and increase our understanding of the pathology underlying t h i s complex hemopoietic malignancy. 234 APPENDIX A complete l i s t of data pertaining to Chapter VI (c, d, and e) i s presented here. The following Tables i l l u s t r a t e a l l data accumulated from which selected examples were chosen i n Chapter VI. Table XIX-A: E f f e c t of CAMAL depletion on normal bone marrow Patient Code Number of CFU-c % % I n h i b i t i o n per 10 5 c e l l s 1 0 268 + 3 100 0 B 255.5 + 4.5 95.3 4.7 C 210 + 4 78.4 21.6 2 0 74 + 1 100 0 B 72 + 0 97.3 2.7 C 56 + 2 75.7 24.3 3* 0 128 + 12 100 0 B 131 + 8 102.4 -2.4 C 95 + 7.5 74.3 25.7 4* 0 170 + 2 100 0 B 163 + 16 95.6 4.4 C 121 + 17 71.2 28.8 5 0 123 + 3 100 0 B 123.5 + 0.5 100.4 -0.4 C 95.5 + 2.5 77.6 22.4 6 0 186.5 + 3.5 100 0 B 178 + 11 95.4 4.6 C 126.5 + 9.5 67.8 32.2 7 0 70 + 7 100 0 B 69.5 + 7.5 99.3 0.7 C 59 + 5 84.3 15.7 8 0 132.5 + 2.5 100 0 B 132 + 1 99.1 0.9 C 112 + 6 81 19.0 235 Patient Code Number of CFU-c % % I n h i b i t i o n per IO-* c e l l s 9 0 135 + 10.1 100 0 B 121.4 + 9.5 89.9 10.1 C 102.4 + 7.8 75.9 24.1 10 0 131.5 + 9.5 100 0 B 122 + 5.5 92.8 7.2 C 102 + 1.5 77.9 22.1 Average r e s u l t s from above experiments : Mean % B's: 96.8% Mean % C s : 76.4% Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) column C = plasma and CM passed over CAMAL-1 column *Nonadherent c e l l s were plated i n these samples Table XX-A. E f f e c t of CAMAL depletion on normal peripheral blood Patient Code Number of CFU-c % % I n h i b i t i o n per 10-> c e l l s 1 0 34 + 0 100 0 B 25 + 4 87 .7 12.3 C 17.5 + 2.5 61 .4 38.6 2* 0 22.5 + 1.5 100 0 B 21 + 0 93 .3 6.7 C 11.5 + 2.5 51 .1 48.9 3* 0 15.5 + 1.5 100 0 B 14 + 2 90 .3 9.7 C 7 + 0 45 .2 54.8 4 0 26.5 + 3.5 100 0 B 22 + 0 83 17 C 14.5 + 1.5 54 .7 45.3 Average of 4 normal peripheral blood samples : Mean % B's: 88.6 % Mean % C s : 53.1 % Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column *Nonadherent c e l l s were plated. Table XXII-A. E f f e c t of CAMAL depletion on chronic granulocytic leukemia peripheral blood Patient Code Number of CFU-c % % I n h i b i t i o n per 5 x 10^ c e l l s 1 0 91 + 1 100 0 B 84 + 1 92.6 7.4 C 87 + 2.5 96 4 2 0 193 + 7 100 0 B 194 + 11 100.5 - 0.5 C 204 + 14 105.7 - 5.7 2* 0 210 + 2 100 0 B 206 + 1 98.1 1.9 C 205 + 5 97.6 2.4 3 0 24.5 + 1.5 100 0 B ND C 31 + 3 126 -26 3* 0 22 + 2 100 0 B 29.5 + 2.5 134.1 -34.1 C 28.5 + 2.5 129.5 -29.5 4 0 180 + 4 100 0 B 169 + 9.5 93.9 6.1 C 191 + 1.5 106.1 - 6.1 4* 0 193 + 6.5 100 0 B 180 + 3 93.8 6.2 C 178 + 7 92.7 7.3 Summary of 7 samples tested : Mean % B's: 102.2 % Mean % C's: 107.7 % Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column * = nonadherent c e l l s were plated i n these experiments ND = not determined Table XXIII-A. E f f e c t of CAMAL depletion on ANLL Patient Code Number of CFU-c % % I n h i b i t i o n per 1()5 c e l l s 1 0 63 ± 2 100 0 (PB) B 70 + 1 111 -11 C 69 ± 9 109.5 - 9.5 2 0 35 ± 2 100 0 (BM) B 30 + 0.5 85.7 14.3 C 38.5 + 1.5 110 -10 3 0 ND (PB) B 7.5 + 1.3 100 0 C 8.5 + 3.3 113.3 -13.3 3* 0 ND B 6.8 + 2.5 100 0 C 6.8 + 3.2 100 0 Summary of samples tested : Mean % B's: 99.2 % Mean % C s : 108.2 % Code: 0 = no treatment of plasma or CM B = plasma and CM passed over negative (BLV-1) MAb column C = plasma and CM passed over CAMAL-1 column * = nonadherent c e l l s were plated ND = not determined Patients 1 and 2 were recently diagnosed ANLLs; patient 3 was an ANLL remission patient. Patient 3 experiments were performed i n quadruplicate. 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 leukocytes i n health and disease. Semin Hematol 4:359, 1967. 2. Cartwright GE, Athens JW, Wintrobe MM: The k i n e t i c s of granulopoiesis i n normal man. 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