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The analysis of human myelogenous leukemia cells in the fluorescence-activated cell sorter Malcolm, Andrew James 1983

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THE ANALYSIS OF HUMAN MYELOGENOUS LEUKEMIA CELLS IN THE FLUORESCENCE-ACTIVATED CELL SORTER By ANDREW JAMES MALCOLM B.Sc. (Hons.), Bishop's U n i v e r s i t y , 1975 M.Sc. (Immunology), U n i v e r s i t y of Manitoba, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT 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 req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1983 (S) Andrew James Malcolm, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT A c e l l surface p r o t e i n from human acute myelogenous leukemia (AML) c e l l s has been p u r i f i e d . (Al-Rammahy et a l . , Cancer Immunol. Immunother. 9:181, 1980; Malcolm et a l . , J . Immunol. 128:2599, 1982). This m a t e r i a l was used to immunize r a b b i t s . The r e s u l t i n g antiserum (anti-AML) showed myelogenous leukemia s p e c i f i c i t y i n that i t reacted w i t h myelogenous leukemia c e l l e x t r a c t s and d i d not react w i t h c e l l e x t r a c t s of normal i n d i v i d u a l s or p a t i e n t s w i t h non-myelogenous leukemia or other malignant d i s o r d e r s i n the enzyme-linked immunosorbent assay (ELISA). Bone marrow and p e r i p h e r a l blood leucocytes (PBL) from e i t h e r p a t i e n t s w i t h myelogenous leukemia, other d i s o r d e r s or normal i n d i v i d u a l s were analysed i n the f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t e r (FACS IV) a f t e r l a b e l l i n g w i t h anti-AML, normal r a b b i t serum (NRS), or antiserum r a i s e d to normal human membrane antigens. Of 40 c e l l samples from p a t i e n t s w i t h AML, 39 reacted s t r o n g l y w i t h the anti-AML. S i m i l a r l y , a l l of 15 specimens from p a t i e n t s w i t h chronic myelogenous leukemia (CML) reacted w i t h the anti-AML. When 42 bone marrow or PBL samples from p a t i e n t s w i t h a v a r i e t y of l y m p h o p r o l i f e r a t i v e d i s o r d e r s were examined, only 2 specimens reacted w i t h the antiserum, both from i n d i v i d u a l s w i t h diagnoses of acute lymphocytic leukemia (ALL). None of the 14 normal bone marrow or PBL i i i donor specimens t e s t e d reacted w i t h the anti-AML. I t was a l s o found that e s s e n t i a l l y a l l samples from p a t i e n t s i n c l i n i c a l remission from AML had high numbers of c e l l s r e a c t i v e w i t h the anti-AML. When c e l l s from such i n d i v i d u a l s were l a b e l l e d and sorted on the FACS IV, i t was found that the c e l l population f l u o r e s c i n g s t r o n g l y w i t h the anti-AML contained c e l l s of both myeloid and lymphoid o r i g i n . The AML antigen was used to produce AML s p e c i f i c monoclonal antibody. Spleens from AML-antigen immunized Balb/c mice were fused to NS-1 myeloma p a r e n t a l c e l l s and a myelogenous leukemia s p e c i f i c monoclonal antibody was s e l e c t e d from the h y b r i d c o l o n i e s produced. This monoclonal antibody (MAL-1) as w e l l as the r a b b i t anti-AML has been used to i d e n t i f y myelogenous leukemia p a t i e n t samples i n the FACS IV. In a d d i t i o n , t h i s monoclonal a l s o demonstrates p o s i t i v e fluorescence binding to HL-60 (a promyelocytic leukemia c e l l l i n e ) , w h i l e there i s no binding to lymphocytic leukemia c e l l l i n e s , CCRF-SB-ALL-B and CCRF-CEM-ALL-T. The MAL-1 monoclonal has been shown to be s p e c i f i c f o r myelogenous leukemia c e l l e x t r a c t s i n the ELISA and has been s u c c e s s f u l l y used as an immunoadsorbent f o r the i s o l a t i o n of the AML antigen from c e l l e x t r a c t s . No equivalent antigen was found when c e l l e x t r a c t s from normal c e l l s , lymphocytic leukemia c e l l s and lymphoma c e l l s were s i m i l a r l y absorbed. These f i n d i n g s i n d i c a t e that both the r a b b i t anti-AML serum and MAL-1 monoclonal show s p e c i f i c i t y f o r an antigen a s s o c i a t e d w i t h myelogenous leukemia c e l l s . ACKNOWLEDGEMENTS I would l i k e to thank my su p e r v i s o r , Dr. J u l i a G. Levy f o r her support and help w i t h t h i s p r o j e c t . I am als o indebted f o r the as s i s t a n c e of my co-workers, Dr. P a t r i c i a M. Logan and Mr. Robert C. Shipman. I am g r a t e f u l to Dr. J.W. Thomas and Dr. J . Denegri, Vancouver General H o s p i t a l , and Drs. C. and A. Eaves, Cancer C o n t r o l Agency of B r i t i s h Columbia, f o r t h e i r co-operation i n supplying p a t i e n t m a t e r i a l f o r analyses. I am als o indebted to Mr. Charles S y l v e s t e r and Mr. Dan Z e c c h i n i f o r t h e i r e x p e r t i s e and t h e i r time spent i n operating the FACS IV. I would a l s o l i k e to thank the B.C. Health Research Foundation and the N a t i o n a l Cancer I n s t i t u t e of Canada which support the present work i n Myelogenous Leukemia Research. V TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS i i i TABLE OF CONTENTS V LIST OF TABLES v i i i LIST OF FIGURES x CHAPTER I - I n t r o d u c t i o n 1 The Stem C e l l - Self-Renewal and D i f f e r e n t i a t i o n 1 C e l l Surface Antigens.on Myelogenous Leukemia 5 CHAPTER I I - A n a l y s i s of Human Myelogenous Leukemia C e l l s i n the F l u o r e s c e n c e - a c t i v a t e d C e l l S orter Using a Tumor-Specific Antiserum 8 I n t r o d u c t i o n 9 M a t e r i a l s and Methods .' 11 Prepa r a t i o n of Membrane E x t r a c t s 11 Immunoadsorbent 11 Polyacrylamide Gel E l e c t r o p h o r e s i s (PAGE) 12 E l u t i o n of Antigen 12 Antiserum 13 ELISA 13 Subjects 13 v i Page C e l l Line 14 Fluorescent Antibodies 14 C e l l s and C e l l L a b e l l i n g 15 FACS IV A n a l y s i s 16 FACS IV C e l l S o r t i n g Technique 17 Results 19 P r o p e r t i e s of the AML Antigen and Antiserum 19 R e a c t i v i t y of the Rabbit-anti-AML-Serum i n FACS IV Analyses 27 Dis c u s s i o n 59 CHAPTER I I I - Monoclonal Antibody (MAL-1) S p e c i f i c f o r Myelogenous Leukemia 65 I n t r o d u c t i o n 66 M a t e r i a l s and Methods 70 Immunization and Fusion Procedures 70 C e l l s and C e l l L a b e l l i n g 71 A f f i n i t y Chromatography 72 I s o l a t i o n and E l u t i o n of Antigen by A f f i n i t y Chromatography 72 Polyacrylamide Gel E l e c t r o p h o r e s i s (PAGE) 73 v i i R esults 74 R e a c t i v i t y of MAL-1 i n the ELISA 74 R e a c t i v i t y of MAL-1 i n FACS IV Analyses 74 Immunochemical P r o p e r t i e s of MAL-1 86 Disc u s s i o n 98 CHAPTER IV - A Comparison of the Binding A b i l i t y to Myelogenous Leukemia C e l l s of the Rabbit Anti-AML-Serum w i t h the Binding A b i l i t y of the MAL-1 Monoclonal 102 Summary Dis c u s s i o n 103 References 109 v i i i LIST OF TABLES Page 1. FACS analyses of bone marrow c e l l s from untreated p a t i e n t s w i t h acute myelogenous leukemia 28 2. FACS analyses of p e r i p h e r a l blood leucocyte c e l l s from untreated p a t i e n t s w i t h acute myelogenous leukemia 32 3. FACS analyses of bone marrow c e l l s from p a t i e n t s i n c l i n i c a l remission from acute myelogenous leukemia a f t e r chemotherapy 34 4. FACS analyses of p e r i p h e r a l blood leucocyte c e l l s from p a t i e n t s i n c l i n i c a l remission from acute myelogenous leukemia a f t e r chemotherapy 35 5. FACS analyses of bone marrow c e l l s from an acute myelomonocytic leukemia p a t i e n t w i t h a c t i v e disease, while i n r e m i s s i o n , and during r e l a p s e . 37 6. Microscopic a n a l y s i s of c e l l s from a remission p a t i e n t w i t h APML a f t e r l a b e l l i n g w i t h e i t h e r anti-normal human or anti-AML and a f t e r a n a l y s i s and s o r t i n g on the FACS 39 7. FACS analyses of c e l l s from relapsed p a t i e n t s w i t h acute myelogenous leukemia f o l l o w i n g chemotherapy 40 8. FACS analyses of bone marrow c e l l s from p a t i e n t s i n the chronic phase of chronic myelogenous leukemia 41 9. FACS analyses of p e r i p h e r a l blood leucocyte c e l l s from p a t i e n t s i n the chronic phase of chronic myelogenous leukemia 42 10. FACS analyses of bone marrow c e l l s from p a t i e n t s w i t h d i s o r d e r s other than AML 43 11. FACS analyses of p e r i p h e r a l blood leucocyte c e l l s from i n d i v i d u a l s w i t h leukemias of non-myelogenous o r i g i n 47 i x Page 12. FACS analyses of c e l l s from normal i n d i v i d u a l s 48 13. FACS analyses of lymphocyte-monocyte and granulocyte-enriched populations from a normal i n d i v i d u a l 49 14. FACS analyses of c e l l s from a p a t i e n t w i t h APML and her i d e n t i c a l twin 53 15. FACS analyses of e i t h e r bone marrow c e l l s or PBL from p a t i e n t s w i t h AML f o l l o w i n g bone marrow t r a n s p l a n t a t i o n 56 16. FACS analyses on c e l l s from three p a t i e n t s whose c e l l s y i e l d e d anomalous r e s u l t s w i t h regard to t h e i r diagnosis 58 17. FACS analyses of human c e l l l i n e s 77 18. FACS analyses of c e l l s from acute myelogenous leukemia p a t i e n t s and from a normal i n d i v i d u a l 79 19. FACS analyses of c e l l s from acute and chronic myelogenous leukemia p a t i e n t s 80 20. FACS analyses of c e l l s from a chronic myelogenous leukemia p a t i e n t 82 21. FACS analyses of non-AML i n d i v i d u a l s 83 22. FACS analyses of HL-60 c e l l s t e s t e d at d i f f e r e n t c e l l growth d e n s i t i e s 85 23. FACS analyses of the sandwich f l u o r e s c e n t technique on the HL-60 c e l l l i n e 89 24. Percent p o s i t i v e fluorescence of c e l l samples from p a t i e n t s w i t h myelogenous leukemia when analysed using e i t h e r the monoclonal MAL-1 or the conventional r a b b i t anti-AML serum 104 25. Percent p o s i t i v e fluorescence of c e l l samples from p a t i e n t s w i t h myelogenous leukemia which only bound the conventional r a b b i t anti-AML serum 106 X LIST OF FIGURES Page 1. Ontogeny of bone marrow derived c e l l lineages 3 2. A n a l y t i c a l non-reducing g e l p a t t e r n from membrane e x t r a c t s of normal human PBL and b l a s t c e l l s from an AML p a t i e n t 21 3. ELISA a n a l y s i s of anti-AML antiserum to band 3 23 4. Immunoprecipitation of *2!>I-labelled AML-1 w i t h r a b b i t antiserum 26 5. Fluorescence i n t e n s i t y p r o f i l e s of bone marrow c e l l s from two AML p a t i e n t s 31 6. Fluorescence i n t e n s i t y p r o f i l e s of bone marrow c e l l s from p a t i e n t s w i t h d i s o r d e r s other than AML 46 7. Fluorescence i n t e n s i t y p r o f i l e s of Ficoll-Hypaque-enriched populations of e i t h e r lymphocyte-monocyte c e l l s or granulocytes from a normal i n d i v i d u a l 51 8. Fluorescence i n t e n s i t y p r o f i l e s of p e r i p h e r a l blood leucocytes from a p a t i e n t w i t h APML and her i d e n t i c a l twin 55 9. ELISA r e s u l t s w i t h h y b r i d to myelogenous leukemia 76 10. Dot d i s p l a y s of the sandwich f l u o r e s c e n t technique on the HL-60 c e l l l i n e 88 11. A Sepharose-4B-MAL-l column used to i s o l a t e band 1 (AML-Ag) 92 12. ELISA r e s u l t s of an a f f i n i t y p u r i f i e d AML antigen 94 13. ELISA r e s u l t s of an a f f i n i t y p u r i f i e d CML antigen 96 1 CHAPTER I I n t r o d u c t i o n The Stem C e l l - Self-Renewal and D i f f e r e n t i a t i o n Haemopoietic stem c e l l s are defined as a small population of marrow c e l l s that are able to give r i s e to a l l other haemopoietic c e l l s ( p l u r i p o t e n t i a l c a p a c i t y ) as w e l l as being able to regenerate themselves ( s e l f - r e n e w a l c a p a c i t y ) . The prominent importance of the stem c e l l s i s e x e m p l i f i e d by t h e i r unique a b i l i t y to regenerate a f u n c t i o n a l haemopoietic system a f t e r d e p l e t i o n ( i . e . i r r a d i a t i o n ) . Loss of the s e l f - r e n e w a l c a p a c i t y seems to occur concomitantly w i t h the i r r e v e r s i b l e commitment of the stem c e l l to a more r e s t r i c t e d p o t e n t i a l f o r d i f f e r e n t i a t i o n . A proposed scheme of bone marrow-derived c e l l l ineages i s shown i n Figure 1. Since the development of a procedure to q u a n t i t a t e haemopoietic p l u r i p o t e n t i a l c e l l s w i t h the colony forming assay ( T i l l and McCulloch, 1961) many i n v e s t i g a t o r s have t r i e d to determine the f a c t o r s r e g u l a t i n g the d i f f e r e n t i a t i o n of these c e l l s (CFU-S). However, the r o l e of e x t e r n a l or i n t e r n a l r e g u l a t o r y f a c t o r s i n determining the d e c i s i o n to produce d i f f e r e n t i a t i o n progeny remains unknown. The p l u r i p o t e n t stem c e l l i s n a t u r a l l y of i n t e r e s t when studying disease s t a t e s such as myelogenous leukemia. Chronic myelogenous leukemia (CML) has been shown to be a c l o n a l l y - d e r i v e d disease; the leukemia c e l l s c h a r a c t e r i s t i c of CML are d e r i v e d from a s i n g l e stem c e l l i n the bone 2 Figure 1. Ontogeny of bone marrow der i v e d c e l l l i n e a g e s . Pre-G = g r a n u l o c y t i c progenitors CFU-C as defined by colony assays Pre-M = megakaryocyte progenitors CFU-M as defined by colony assays Pre-E = e r y t h r o i d progenitors p r i m i t i v e BFU-E, mature BFU-E and CFU-E as defi n e d by colony assays CFU-S = colony forming u n i t ( s ) i n the spleen D i f f e r e n t i a t i o n Process End C e l l s PRE-E r e d b l o o d c e l l s 4 marrow which has become n e o p l a s t i c (Barr and Fialkow, 1973; K o e f f l e r and Golde, 1981). CML i s c h a r a c t e r i z e d by the presence of a chromosomal abnormality c a l l e d the P h i l a d e l p h i a chromosome, Ph' (which normal a r i s e s , 90 per cent of cases, because of a t r a n s l o c a t i o n of chromosomal m a t e r i a l from the long arm of one of the 22d chromosomes to the long arm of one of the No. 9 chromosomes). The Ph' chromosome has been found i n n e u t r o p h i l , monocyte, e r y t h r o c y t e , p l a t e l e t and p o s s i b l y b a s o p h i l precursors ( K o e f f l e r and Golde, 1981). This a l t e r n a t i o n occurs i n haemopoietic stem c e l l s of p a t i e n t s w i t h CML. The c l o n a l nature of CML i s a l s o suggested by the enzyme stu d i e s w i t h glucose-6-phosphate dehydrogenase, which i s coded by the X chromosome (Fialkow et a l . , 1978). Women wi t h CML who are heterozygous f o r t h i s enzyme only have Type A (Gd A) or B (Gd B) enzyme i n granulocytes, monocytes, e o s i n o p h i l s , b a s o p h i l s , p l a t e l e t s and e r y t h r o c y t e s , suggesting a c l o n a l o r i g i n from a common p l u r i p o t e n t stem c e l l . Fialkow et a l . (1978, 1982) a l s o demonstrated that B lymphocytes and B-lymphoblastoid c e l l l i n e s e s t a b l i s h e d from a Gd A/Gd B CML p a t i e n t d i s p l a y e d only one type of G6PD enzyme, i n d i c a t i n g that some lymphoid c e l l s a r i s e from the leukemic clone. I t i s p o s t u l a t e d that leukemic stem c e l l s replace the normal stem c e l l s and u l t i m a t e l y give r i s e to leukemic blood c e l l s (Wiggans et a l . , 1978). Current t h e o r i e s of the o r i g i n s of human cancers are v a r i e d , but i t i s g e n e r a l l y thought that c l o n a l diseases, such as CML, may o r i g i n a t e from i r r a d i a t i o n or a r a r e oncogenic event, such as somatic mutation or v i r a l i n t e g r a t i o n next to a c e l l u l a r oncogene a c t i n g on a s i n g l e stem c e l l (Fialkow et a l . , 1973; Wiggans et a l . , 1978). With acute myelogenous 5 leukemia (AML) however, the p i c t u r e i s l e s s c l e a r . There are no c h a r a c t e r i s t i c chromosomal abnormalities unique to AML c e l l s . Although the c l o n a l nature of AML i s not c l e a r , Fialkow found a single-enzyme G6PD phenotype i n b l a s t c e l l s suggestive of a c l o n a l d i s o r d e r (Fialkow, 1982). C l e a r l y however, t h i s question remains unresolved. C e l l Surface Antigens on Myelogenous Leukemias Myelogenous leukemias are at present c l a s s i f i e d by h i s t o c h e m i c a l r e a c t i v i t y and the morphologic appearance of the malignant c e l l s . W i t h i n the l a s t decade, the i d e n t i f i c a t i o n , 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 c e l l surface antigens s p e c i f i c f o r human acute leukemia c e l l s have now been developed and are of p o t e n t i a l c l i n i c a l s i g n i f i c a n c e f o r both the diagnosis and treatment of these diseases. There i s considerable evidence i n the l i t e r a t u r e f o r the presence of tumor-associated antigens which may be common to myelogenous leukemia c e l l s . A number of procedures and animal species have been used to r a i s e xenoantisera to AML c e l l s , a l l of which have y i e l d e d a n t i s e r a that a f t e r appropriate absorptions, demonstrate v a r y i n g degrees of s p e c i f i c i t y f o r AML c e l l s (Baker et a l . , 1973, 1974, 1981; Durantez et a l . , 1976; Thranhardt et a l . , 1978, 1979). U n f o r t u n a t e l y , to render these a n t i s e r a s p e c i f i c , i t was necessary to ex h a u s t i v e l y absorb them with c e l l s of d i f f e r e n t c e l l u l a r l i n e a g e s , u s u a l l y r e s u l t i n g i n a g r e a t l y decreased r e a c t i v i t y to the myeloid leukemia c e l l s . In these studies s p e c i f i c i t y of the a n t i s e r a were a l l assessed by means of complement-mediated c y t o t o x i c i t y assays. More d e f i n i t i v e a n t i s e r a w i t h marked s p e c i f i c i t y f o r AML c e l l antigens have 6 been r a i s e d i n nonhuman primates (Mohanakumar et a l . , 1978, 1979). In these studies a l s o , c y t o t o x i c i t y assays were used to define the s p e c i f i c i t i e s of the t e s t a n t i s e r a , and i n d i c a t e d the presence of common AML-associated antigens. With the development of hybridoma derived monoclonal antibodies against c e l l surface antigens, i t seems the d i f f i c u l t i e s experienced w i t h h e t e r o a n t i s e r a may be overcome. Previous work i n t h i s l a b o r a t o r y reported on the production of an a n t i s e r a r a i s e d i n r a b b i t s to AML membrane e x t r a c t s which, a f t e r a bsorption, appeared to have s p e c i f i c i t y f o r AML c e l l e x t r a c t s as assessed by the enzyme-linked immunosorbent assay, ELISA (Al-Rammahy et a l . , 1980). We t e s t e d r a b b i t antiserum prepared against AML c e l l e x t r a c t s by use of a mixture of anti-normal human c e l l serum and AML KCL-extracted p r o t e i n (feedback procedure, Al-Rammahy and Levy, 1979) i n the ELISA against both the pooled normal antigen and the AML e x t r a c t . As the unabsorbed antiserum had a considerable amount of r e a c t i v i t y towards normal c e l l antigens, the antiserum was passed twice over an immunoadsorbent column to which normal c e l l antigens had been attached. Absorption removed e s s e n t i a l l y a l l the r e a c t i v i t y to normal c e l l antigens, while a considerable r e a c t i v i t y to the AML c e l l e x t r a c t was maintained. Indeed, a s e r i e s of t e s t s were run i n which t h i s absorbed antiserum was used i n the ELISA w i t h KCL e x t r a c t s of 16 AML and 12 normal c e l l e x t r a c t s . This antiserum reacted w i t h a l l 16 AML e x t r a c t s , while i t showed no r e a c t i v i t y w i t h e x t r a c t s of normal c e l l s . As i t showed no r e a c t i v i t y to e x t r a c t s of normal donors' c e l l s the p o s s i b i l i t y that t h i s r e a c t i v i t y was d i r e c t e d to HLA antigens i s exceedingly u n l i k e l y . 7 Furthermore, the antiserum showed no r e a c t i v i t y to PHA b l a s t c e l l e x t r a c t s , or to e x t r a c t s of ALL b l a s t c e l l s i n d i c a t i n g that i t was not rec o g n i z i n g an antigen common to b l a s t c e l l s . As the antiserum was not r e a c t i v e w i t h CLL c e l l s (known to be high i n Ia) the p o s s i b i l i t y that i t was r e c o g n i z i n g a l a - l i k e component was a l s o u n l i k e l y . The p o s s i b i l i t y that the antiserum was d e t e c t i n g a d i f f e r e n t i a t i o n antigen not found on lymphoid c e l l s was i n v e s t i g a t e d by t e s t i n g e x t r a c t s of bone marrow c e l l s , on the assumption that t h i s population would cont a i n b l a s t c e l l s of the myelocytic l i n e a g e . The antigen was not present i n detectable l e v e l s i n these populations e i t h e r , regardless of whether the marrow specimens came from apparently normal i n d i v i d u a l s or from those w i t h various malignant c o n d i t i o n s ; t h i s suggests that the r e a c t i v i t y was not d i r e c t e d toward d i f f e r e n t i a t i o n antigens found on normal b l a s t c e l l s of lymphocytic or myelogenous l i n e a g e (Al-Rammahy et a l . , 1980). This feedback procedure r e s u l t e d i n the production, i n r a b b i t s , of an antiserum that d i s p l a y e d s p e c i f i c r e a c t i v i t y to e x t r a c t s of AML p a t i e n t s * c e l l s . The s e n s i t i v i t y of the ELISA allows d e t e c t i o n of antigen i n nanogram q u a n t i t i e s ( K e l l y et^ a l . , 1979); the r e s u l t s obtained w i t h t h i s assay i n our l a b o r a t o r y suggest that there may be one unique (or more) but common leukemia-associated antigen on AML c e l l s . The r e s u l t s presented i n Chapters I I and I I I of t h i s t h e s i s , i n which t h i s antiserum was used to i s o l a t e an antigen f o r the production of a more s p e c i f i c r a b b i t anti-AML-serum and a mouse monoclonal antibody d i r e c t e d to AML, f u r t h e r support these f i n d i n g s . CHAPTER I I A n a l y s i s of Human Myelogenous Leukemia C e l l s i n Fluorescence-Activated C e l l S o r t e r Using a Tumour-Specific Antiserum 9 I n t r o d u c t i o n A considerable amount of work has been done on the c h a r a c t e r i z a t i o n of c e l l surface-markers which may c l a s s i f y leukemia c e l l s (Maheu et a l . , 1981; Foon et a l . , 1982). The m a j o r i t y of those described c o n s t i t u t e normal surface markers which may be expressed w i t h greater frequency or d e n s i t y on the surface of leukemia c e l l p opulations. A l t e r a t i o n s i n membrane carbohydrates, such as decreased complex g a n g l i o s i d e s (Hildebrand et a l . , 1972) have been reported w i t h leukemia c e l l s . Some leukemias d i s p l a y unique marker c h a r a c t e r i s t i c s ; f o r example, g l y c o l i p i d a s i a l a GMI i s found on c e l l s from p a t i e n t s w i t h acute lymphoblastic leukemia (ALL) but not on c e l l s from p a t i e n t s w i t h other forms of leukemia (Nakahara et a l . , 1980) and the i n t r a c e l l u l a r enzyme, t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e (TdT) i s detectable i n ALL but not AML c e l l s (Foon et a l . , 1982) . In s tudies on acute non-lymphocytic leukemias (myelogenous), there has been some evidence that antigens unique f o r the leukemia c e l l s may al s o be present. Baker and co-workers (1979) have demonstrated a leukemia a s s o c i a t e d antigen (LAA) on myelogenous leukemia c e l l s using a s p e c i f i c antiserum r a i s e d i n mice. They were able to detect imminent relapse of remission p a t i e n t s by demonstrating an increased number of s e r o p o s i t i v e c e l l s i n bone marrow a s p i r a t e s , detected by immunofluorescence. S i m i l a r l y , simian h e t e r o a n t i s e r a have been used to i d e n t i f y what appear to be LAA on myelogenous leukemia c e l l s (Mohanakumar et a l . , 1980). 10 As s t a t e d i n the previous chapter, our l a b o r a t o r y produced a r a b b i t antiserum w i t h apparently absolute s p e c i f i c i t y f o r membrane e x t r a c t s of human AML c e l l s and no r e a c t i v i t y f o r equivalent preparations of e i t h e r p e r i p h e r a l blood or bone marrow c e l l s from e i t h e r normal i n d i v i d u a l s or p a t i e n t s w i t h a v a r i e t y of non-myelogenous d i s o r d e r s . Monitoring f o r s p e c i f i c i t y of t h i s antiserum was c a r r i e d out using the ELISA. This chapter reports on the use of t h i s s p e c i f i c antiserum as a probe f o r the i s o l a t i o n of a t u m o r - s p e c i f i c component from polyacrylamide g e l s . This i s o l a t e d m a t e r i a l was used to r a i s e a second antiserum i n r a b b i t s which subsequently showed apparent s p e c i f i c i t y f o r AML c e l l e x t r a c t s i n the ELISA. Studies using a f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t e r (FACS IV) show that t h i s antiserum, when developed with f l u o r e s c e i n - l a b e l l e d goat a n t i - r a b b i t IgG, r e a c t s s t r o n g l y w i t h c e l l s from bone marrow a s p i r a t e s and p e r i p h e r a l blood leucocytes of AML p a t i e n t s and of chronic myelogenous leukemia (CML) p a t i e n t s . When c e l l s from p a t i e n t s w i t h a v a r i e t y of l y m p h o p r o l i f e r a t i v e d i s o r d e r s (ALL, CLL, lymphomas) were t e s t e d , there was no apparent r e a c t i v i t y w i t h the antiserum. S i m i l a r l y t h i s antiserum d i d not r e a c t w i t h c e l l s from normal i n d i v i d u a l s . The data reported show that t h i s antiserum i s s p e c i f i c f o r c e l l s of p a t i e n t s w i t h myelogenous leukemias. I t i s p o s s i b l e that the recognized antigen may c o n s t i t u t e a "malignancy marker" f o r a malignant clone i n p a t i e n t s w i t h myelogenous leukemias, since i t was found to be present, not only on b l a s t c e l l s but a l s o on other d i f f e r e n t i a t e d c e l l populations both from p a t i e n t s w i t h a c t i v e disease and i n remission. 11 M a t e r i a l s and Methods Prep a r a t i o n of Membrane E x t r a c t s AML or normal p e r i p h e r a l blood leucocyte (PBL) membranes were prepared by s o n i c a t i o n of c e l l s w i t h four 15 second bursts over i c e at a s e t t i n g of 60 on a B r o n w i l l B i o s o n i k Sonicator ( B r o n w i l l S c i e n t i f i c , Rochester, N.Y.). C e l l d ebris was removed by centr3,fugation of sonicates at 400 x g f o r 10 min. Membranes were p e l l e t e d from the supernatants by u l t r a c e n t r i f u g a t i o n at 110,000 x g f o r 90 min. Membrane p e l l e t s were resuspended i n minimal volumes of PBS (0.01 M pH 7.4) and sonicated again as described. Sonicates were d i l u t e d to 5.0 ml i n PBS and t o t a l p r o t e i n determined by the method of Bradford (1976). Immunoadsorbent Antiserum r a i s e d i n r a b b i t s to pooled normal PBL membrane e x t r a c t s from 15 i n d i v i d u a l s (Al-Rammahy et a l . , 1980) was coupled to Sepharose 4B (Pharmacia) w i t h cyanogen bromide (Avremeus and Ternynck, 1969; Cuatrecasas, 1970). P r i o r to use the anti-normal column was e q u i l i b r a t e d w i t h b o r a t e - s a l i n e , pH 8.5. Samples were l a y e r e d onto the column and c y c l e d over twice using borate s a l i n e . Both the AML and normal PBL• membrane e x t r a c t s were t r e a t e d i n t h i s way p r i o r to subsequent t e s t i n g . This treatment was found to remove approximately 90% of the p r o t e i n present i n the membrane e x t r a c t s . The f a l l through f r a c t i o n s were used f o r f u r t h e r s t u d i e s . 12 Polyacrylamide Gel E l e c t r o p h o r e s i s (PAGE) E l e c t r o p h o r e s i s was performed as p r e v i o u s l y described (Laemmli, 1970; Gold et a l . , 1976) except that no SDS or 2-mercaptoethanol was used. Samples of absorbed membrane e x t r a c t s were a p p l i e d to 7.5% polyacrylamide gels f o r both a n a l y t i c a l and prep a r a t i v e procedures, and electrophoresed at 50V f o r 5-6 h. Pr e p a r a t i v e gels were run using a one-tooth comb and loadi n g approximately 250 yg of p r o t e i n onto the g e l . Molecular weight determinations were c a r r i e d out by co-electrophoresing samples with low molecular weight p r o t e i n standards, no SDS (Pharmacia). Gels were s t a i n e d according to the method of Fairbanks et a l . (1971). For pr e p a r a t i v e g e l s , a s e c t i o n of the g e l was removed and s t a i n e d , the remainder of the g e l being stored at -70°C u n t i l i t was r e q u i r e d f o r s l i c i n g and e l u t i o n . E l u t i o n of Antigen Stained and unstained sections of prep a r a t i v e gels were l i n e d up and i l l u m i n a t e d from below on a l i g h t box. Bands were cut from regions of the unstained g e l corresponding to l o c a t i o n s shown on the sta i n e d s e c t i o n to contain detectable p r o t e i n . Gel s l i c e s were placed i n small tubes (Falcon 2003) and crushed with a spa t u l a . Two ml of PBS were added to each tube and e l u t i o n was e f f e c t e d by end-over-end mixing of the tubes i n a Labquake o ( L a b i n d u s t r i e s , Berkely, C a l i f . ) at 4 C f o r 48 h. El u t e d p r o t e i n was c o l l e c t e d by f i l t r a t i o n over vacuum and p r o t e i n determinations were c a r r i e d out on a l l f r a c t i o n s . Testing of e l u t e d m a t e r i a l s f o r a n t i g e n i c a c t i v i t y was c a r r i e d out using a r a b b i t antiserum p r e v i o u s l y shown to have s p e c i f i c i t y f o r AML c e l l e x t r a c t s (Al-Rammahy et a l . , 1980) i n the ELISA. 13 Antiserum F r a c t i o n s of e l u t e d p r o t e i n shown to have a n t i g e n i c a c t i v i t y i n the ELISA w i t h p r e v i o u s l y prepared AML-specific a n t i s e r a were used to immunize young adult female a l b i n o r a b b i t s . The e l u t e d m a t e r i a l was mixed 1:1 w i t h complete Freund's adjuvant to a t o t a l volume of 1.0 ml and i n j e c t e d i n t r a m u s c u l a r l y i n t o four d i s t a l s i t e s . Animals were given another immunization 4 weeks l a t e r and b l e d 7 days f o l l o w i n g the second immunization. We have e s t a b l i s h e d that the antigen used to r a i s e the anti-AML antiserum appears to be a homogeneous p r o t e i n which produces a s i n g l e spot on two-dimensional g e l e l e c t r o p h o r e s i s , has a p i of 7.1 - 7.2 and a molecular weight of 68,000 daltons (Shipman, Malcolm, and Levy, B r i t i s h J . Cancer, In p r e s s ) . Thus, the antiserum used here was prepared against a homogeneous pure p r o t e i n , according to the standard biochemical c r i t e r i a . ELISA A n t i s e r a were assayed by a standard ELISA procedure described p r e v i o u s l y i n d e t a i l ( K e l l y et a l . , 1979). Subjects A t o t a l of 40 p a t i e n t s w i t h acute myelogenous leukemia and 15 p a t i e n t s w i t h chronic myelogenous leukemia were st u d i e d . Of the AML p a t i e n t s 19 were t e s t e d when newly diagnosed, 16 p a t i e n t s were s t u d i e d when i n c l i n i c a l r e m i s s i o n , and 7 as relapsed AML p a t i e n t s . Two other AML p a t i e n t s were analysed post bone marrow t r a n s p l a n t a t i o n . When a t t a i n a b l e , 14 samples from the same AML p a t i e n t as newly diagnosed AML p a t i e n t , as a remission p a t i e n t , and/or as an AML i n relapse were t e s t e d . Forty-two p a t i e n t s w i t h leukemias of non-myelogenous o r i g i n or w i t h other malignant and non-malignant c o n d i t i o n s were used as c o n t r o l s . Fourteen l a b o r a t o r y personnel and bone marrow t r a n s p l a n t donors were al s o used as normal c o n t r o l s . C e l l L i n e The human promyelocytic c e l l l i n e HL-60 was obtained from Dr. R.C. G a l l o , N.C.I., Bethesda, Md. I t was maintained i n DME supplemented w i t h 10% f o e t a l c a l f serum i n a h u m i d i f i e d 37°C incubator i n 10% CO^. C e l l s were harvested when they reached 1 x 10 6 per ml, washed i n medium and l a b e l l e d f o r FACS IV a n a l y s i s as d e s c r i b e d above. Fluorescent Antibodies F l u o r e s c e i n - l a b e l l e d DEAE-purified goat a n t i - r a b b i t IgG was prepared according to a standard procedure (Wofsy et a l . , 1980). B r i e f l y , the goat antibody at 10 mg/ml was d i a l y z e d e x h a u s t i v e l y against 0.15 M NaCl, a f t e r which i t was d i a l y z e d f o r 4 h against 0.5 M bicarbonate-buffered s a l i n e , pH 8.5, and f i n a l l y against 0.05 M bicarbonate-buffered s a l i n e , pH 9.2 f o r 3 h, a l l at 4°C. The IgG was then d i a l y z e d f o r 24 h against 100 ug/ml of f l u o r e s c e i n i s o t h i o c y a n a t e (FITC, BBL) i n a 0.05 M bicarbonate-buffered s a l i n e at pH 9.2. The r e a c t i o n was stopped by d i a l y s i s against 0.02 M PBS, pH 7.0, at 4°C f o r 4 h. Unbound FITC was removed from FITC-Ig conjugates by passage of the m a t e r i a l over Sephadex G-25. A r a t i o of 15 fluorochrome to p r o t e i n (F:P) of 4 was determined by spectrophotometry a n a l y s i s (Wells et a l . , 1966). C e l l s and C e l l L a b e l l i n g Bone marrow a s p i r a t e s and p e r i p h e r a l blood samples were obtained from the D i v i s i o n of Haematology, Vancouver, General H o s p i t a l or the Cancer C o n t r o l Agency of B r i t i s h Columbia (Terry Fox Laboratory). Samples obtained were from p a t i e n t s whose diagnoses (AML, CML, or otherwise as r e f e r r e d to i n the Tables) were e s t a b l i s h e d e i t h e r p r i o r to or f o l l o w i n g fluorescence t e s t i n g . The c l i n i c a l diagnoses were made by haematologists a t , or a f f i l i a t e d w i t h , the Vancouver General H o s p i t a l . The t e s t s used included: morphological examination of p e r i p h e r a l blood and bone marrow smears, biochemical t e s t s on bone marrow (Sudan black, PAS, combined ester a s e s , and a c i d phosphatase t e s t s when necessary), colony growth s t u d i e s , and chromosomal analyses ( i n cases of CML). Lymphocyte surface Ig and r o s e t t e t e s t s were performed on samples from p a t i e n t s w i t h lymphoid leukemias. Buffy coats were c o l l e c t e d from bone marrow samples by sedimentation at 1 g, and from the h e p a r i n i z e d p e r i p h e r a l blood samples using Plasmagel ( L a b o r a t o i r e Roger B e l l o n , N e u i l l y , France) or by the Ficoll-Hypaque technique (Boyum, 1976; E n g l i s h and Anderson, 1974). In the case of p e r i p h e r a l blood samples, s i m i l a r r e s u l t s were obtained t o those of Boyum (1976) and E n g l i s h and Anderson (1974) regarding the r e l a t i v e p u r i t i e s of mononuclear c e l l - e n r i c h e d (967o) and granulocyte-enriched (98%) c e l l suspensions recovered r e s p e c t i v e l y . The c e l l s were washed i n PBS c o n t a i n i n g 5% FCS and the c e l l p e l l e t was 16 suspended i n 0.14M ammonium c h l o r i d e and 0.017 M t r i s b u f f e r at pH 7.2 to l y s e contaminating e r y t h r o c y t e s , f o l l o w e d by a subsequent wash. Then 6 10 c e l l s were incubated f o r 1.5 h on i c e i n 0.2 ml of antiserum (anti-normal human, anti-AML or normal r a b b i t serum (NRS)) d i l u t e d to 1/10 i n PBS. The normal r a b b i t serum (NRS) used i n these studies was taken from unimmunized r a b b i t s which were used subsequently f o r immunization with the AML antigen. Consequently, they served as an appropriate negative c o n t r o l i n these experiments. A l l a n t i s e r a used were c e n t r i f u g e d b r i e f l y before use at 20,000 x G to e l i m i n a t e aggregates. C e l l s were washed three times i n PBS and 0.2 ml of f l u o r e s c e i n a t e d goat a n t i - r a b b i t IgG at 1/20 was added to each c e l l p e l l e t . C e l l s were incubated another 1.5 h at 0°C, washed once i n PBS, and then c e n t r i f u g e d through 100% FCS. They were f i n a l l y suspended i n 1.0 ml PBS and 5% FCS f o r FACS IV a n a l y s i s . FACS IV A n a l y s i s Twenty-five thousand c e l l s from each samples were analysed on a Becton-Dickinson FACS IV using the 488-nm wavelength of the Spectra Physics Model 164-05 Argon l a s e r at a power s e t t i n g of 400 mW. The standard f i l t e r f o r FITC a n a l y s i s was used (520-long pass f i l t e r ) . The FACS IV was standardized by using g l u t a r a l d e h y d e - f i x e d chicken red blood c e l l s (Herzenberg et a l . , 1976) and f l u o r e s c e n t monodispersed carboxymethylated microspheres (d = 1.75 m ± 0.02 SD; cat. no. 9847, Polysciences Inc., Warrington, PA, 18976). The r e s u l t s reported here include a l l those samples which were analysed and which gave acceptable 17 r e s u l t s i n the FACS IV w i t h the p o s i t i v e (anti-normal human) and negative (NRS) c o n t r o l s . O c c a s i o n a l l y , i t was impossible to o b t a i n samples or book FACS time u n t i l the specimens were 48 hours o l d . Sometimes these samples y i e l d e d e i t h e r extremely high backgrounds w i t h NRS or d i d not react s t r o n g l y w i t h the p o s i t i v e c o n t r o l antiserum. Thus, i t was impossible i n these circumstances to evaluate the r e a c t i v i t y of c e l l s w i t h the anti-AML, and these r e s u l t s were discounted. A c e r t a i n degree of f l u c t u a t i o n i n negative t e s t samples was observed when the c e l l s f l u o r e s c i n g were compared to the NRS c o n t r o l . The anti-AML f r e q u e n t l y reacted at a lower l e v e l w i t h such c e l l s than d i d the NRS. I t was a s c e r t a i n e d that v a r i a t i o n i n the range of ± 5% i n comparison to NRS r e s u l t s was w i t h i n the margins of e r r o r f o r these s t u d i e s . FACS IV C e l l S o r t i n g Technique An APML (promyelocytic) remission sample that had been analysed as s i g n i f i c a n t l y p o s i t i v e w i t h anti-AML, was sorted using the FACS IV. Two so r t windows were determined, one contained the 25% of the sample showing no or minimal r e l a t i v e fluorescence and one contained the 25% of the sample showing the highest r e l a t i v e f luorescence. The two c e l l populations were c o l l e c t e d i n separate tubes c o n t a i n i n g 1 ml PBS/FCS. The c e l l s between these two windows, representing the remaining 50% of the sample c e l l p o pulation were discarded i n t o the r e s e r v o i r f l a s k . The head d r i v e frequency was set at 36 KHz, and 2,000 v o l t s were a p p l i e d 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 18 run at 5 d r o p l e t s per d e f l e c t i o n pulse w i t h the abort on; the d r o p l e t 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. The c e l l s were then washed once i n PBS to remove p r o t e i n i n the FCS and the sample loaded on a c y t o s p i n (John's S c i e n t i f i c ) at 800 rpm f o r 8 minutes to o b t a i n s l i d e p r e parations. The s l i d e s were s t a i n e d r o u t i n e l y w i t h Wright's s t a i n and examined by l i g h t microscopy. 19 Results P r o p e r t i e s of the AML Antigen and Antiserum Membrane e x t r a c t s of PBL from p a t i e n t s i n AML b l a s t c e l l c r i s i s and from pooled normal PBL were adsorbed twice on an immunoadsorbent co n t a i n i n g r a b b i t a n t i b o d i e s to normal PBL membrane components. The adsorbed m a t e r i a l s ( i . e . m a t e r i a l s not removed from the preparations by the adsorbent) were run on an a n a l y t i c nonreducing g e l and were s t a i n e d . Representative r e s u l t s are shown i n Figure 2. The AML m a t e r i a l contained four d i s t i n c t bands that d i d not appear to be present i n the equivalent normal p r e p a r a t i o n . The c o n s t i t u e n t s were i s o l a t e d by e l u t i o n of each band from p r e p a r a t i v e g e l s . The f o u r preparations were t e s t e d i n the ELISA f o r r e a c t i v i t y w i t h the AML-specific r a b b i t antiserum described p r e v i o u s l y (Chapter I ) . Band 1, 2 and 3 showed r e a c t i v i t y i n the ELISA, band 3 being the most pronounced (data shown i n R. Shipman's M.Sc. Thesis, 1982). The m a t e r i a l i n band 3 was used to r a i s e an antiserum i n r a b b i t s . The r e s u l t i n g a n t i s e r a , when t e s t e d i n the ELISA w i t h the absorbed AML e x t r a c t and the e q u i v a l e n t normal PBL e x t r a c t , showed marked s p e c i f i c i t y f o r the AML p r e p a r a t i o n and v i r t u a l l y no r e a c t i v i t y w i t h the normal PBL m a t e r i a l (Figure 3). To determine whether the three bands represented m a t e r i a l s w i t h s i m i l a r a n t i g e n i c p r o p e r t i e s , the antiserum was t e s t e d i n the ELISA w i t h bands 1 and 2. The r e s u l t s showed that e s s e n t i a l l y t o t a l c r o s s - r e a c t i v i t y e x i s t e d between the i n d i v i d u a l bands (R. Shipman, M.Sc. T h e s i s , 1982). Because these components were i s o l a t e d from nonreducing 20 Figure 2. A n a l y t i c a l non-reducing g e l pa t t e r n from membrane e x t r a c t s of normal human PBL and b l a s t c e l l s from an AML p a t i e n t . Lane a: Molecular weight standards. Lane b: Normal human PBL membrane e x t r a c t . Lane c: AML b l a s t c e l l membrane e x t r a c t . Band 1 = 62,000 daltons M.W.; Band 2 = 89,000 daltons M.W.; Band 3 = 140,000 daltons M.W. Both c e l l membrane preparations had been absorbed twice on an immunoadsorbent column c o n t a i n i n g anti-normal human antibody, p r i o r to PAGE. Gel s t a i n e d i n Coomassie Blue according to Fairbanks et a l . (1971). 67,000 • 43,000 • 30,000 • 20,100 • 22 Figure 3. ELISA a n a l y s i s of anti-AML antiserum to band 3. Antigen c o n c e n t r a t i o n was at 300 ng/ml and readings were made 45 min a f t e r s u b s t r a t e was added to the p l a t e s . • r e a c t i o n of the antiserum w i t h the AML e x t r a c t ; 0 0, r e a c t i o n of the antiserum w i t h noraml PBL e x t r a c t . 24 g e l s , i t was p o s s i b l e that a l l the bands contained the same antigen. This was subsequently e s t a b l i s h e d by demonstrating that absorbed e x t r a c t s of c e l l s from a v a r i e t y of AML p a t i e n t s , when run on reducing PAGE, a l l demonstrated the presence of band 1 (the unique component with the lowest molecular weight) but d i d not show the presence of e i t h e r band 2 or 3 (Shipman, Malcolm and Levy, B r i t i s h J . Cancer, In p r e s s ) . S e r o l o g i c a l l y , t h i s observation was s u b s t a n t i a t e d when we observed that a n t i s e r a r a i s e d i n r a b b i t s to bands 1 and 2 each demonstrated complete cross r e a c t i v i t y i n the ELISA w i t h each other as w e l l as band 3 (R. Shipman, M.Sc. Thesis, 1982). S i m i l a r l y , complete i d e n t i t y between the three a n t i s e r a was observed when they were compared f o r t h e i r a b i l i t y to react w i t h markers on the surface of c e l l s from AML p a t i e n t s as demonstrated by FACS IV a n a l y s i s . Because band 1 (AML-1) antigen appeared to be the simplest common antigen, a n t i s e r a was r a i s e d to i t . This antiserum was used i n the f o l l o w i n g study of marker d e t e c t i o n on the surface of AML and CML p a t i e n t s ' c e l l s by FACS IV a n a l y s i s . The a b i l i t y of anti-AML-1 to p r e c i p i t a t e AML-1 antigen was t e s t e d by itumunoprecipitation (Kurth et a l . , 1979). Iodinated AML-1 was p r e c i p i t a t e d with r a b b i t a n t i AML-1 and p r o t e i n A and run on a polyacrylamide g e l (Laemmli et a l . , 1970; Gold et a l . , 1976). The r e s u l t s (Figure 4) show th a t the antiserum p r e c i p i t a t e s predominantly AML-1 m a t e r i a l and i s a c t i v e up to a d i l u t i o n of 1:8000. 25 Figure 4. Immunoprecipitation of I - l a b e l l e d AML-1 w i t h r a b b i t antiserum. Lane 1 - normal r a b b i t serum + 50,000 cpm of antigen 2 - 1:2 d i l u t i o n of anti-AML-1 + 50,000 cpm of antigen 3 - 1 : 8 d i l u t i o n of anti-AML-1 + 50,000 cpm of antigen 4 - 1:32 d i l u t i o n of anti-AML-1 + 50,000 cpm of antigen 5 - 1:128 d i l u t i o n of anti-AML-1 + 50,000 cpm of antigen 6 - 1:512 d i l u t i o n of anti-AML-1 + 50,000 cpm of antigen 7 - 1:2048 d i l u t i o n of anti-AML-1 + 50,000 cpm of antigen 8 - 1:8192 d i l u t i o n of anti-AML-1 + 50,000 cpm of antigen 9 - AML-1 alone, 50,000 cpm. 10 - AML-1 alone, 25,000 cpm. 26 27 R e a c t i v i t y of the Rabbit-anti-AML-Serum i n FACS IV Analyses. To t e s t the s p e c i f i c i t y of t h i s antiserum on c l i n i c a l m a t e r i a l , a s e r i e s of experiments were c a r r i e d out using bone marrow a s p i r a t e s from p a t i e n t s w i t h a v a r i e t y of d i s o r d e r s . The data shown i n Table 1 are the r e s u l t s of FACS IV a n a l y s i s of bone marrow samples from p a t i e n t s shown to have AML. The r e s u l t s of r e p r e s e n t a t i v e i n d i v i d u a l t e s t s as w e l l as the averaged r e s u l t s of the t o t a l number of t e s t s run are shown. Fluorescence p r o f i l e s of these c e l l s from two r e p r e s e n t a t i v e p a t i e n t s are shown i n Figure 5, and the numbers corresponding to those p r o f i l e s are shown i n Table 1. These r e s u l t s were obtained by determining the number of c e l l s f l u o r e s c i n g beyond the poi n t at which the p r o f i l e of c e l l s c o n s t i t u t i n g the negative c o n t r o l (NRS) i n t e r s e c t e d the p r o f i l e of c e l l s t r e a t e d w i t h the p o s i t i v e c o n t r o l serum. I t can be seen that the number of c e l l s f l u o r e s c i n g w i t h the t e s t (anti-AML) antiserum was f r e q u e n t l y almost as high as the number f l u o r e s c i n g w i t h the p o s i t i v e c o n t r o l (anti-normal) antiserum, and i s always s i g n i f i c a n t l y higher than the negative c o n t r o l (NRS). The r e s u l t s of PBL c e l l s from a number of p a t i e n t s presenting w i t h AML i s shown i n Table 2. I t can be seen that i n a l l cases, a high percentage of the c e l l s f l u o r e s c e d p o s i t i v e l y w i t h the anti-AML-serum. The r e s u l t s obtained w i t h the bone marrow a s p i r a t e s and PBL were e s s e n t i a l l y the same (Tables 1 and 2). I t i s i n t e r e s t i n g to note that the number of c e l l s f l u o r e s c i n g w i t h anti-AML showed no c o r r e l a t i o n w i t h the number of b l a s t c e l l s found i n e i t h e r bone marrow or PBL but i s u s u a l l y c o n s i s t e n t l y higher. Furthermore, r e l a t i v e b l a s t c e l l numbers d i d not c o r r e l a t e to r e l a t i v e f l u o r e s c e n c e , sample 5 of Table 1 w i t h an 8% b l a s t Table 1. FACS analyses of bone marrow c e l l s from untreated patients with acute myelogenous leukemia Diagnosis * blasts Number of c e l l s fluorescing with various treatments* X positive Anti-normal human NRS Anti-AML c e l l s * * 1. AML-M2**** A3 23,920 l a 1,553 22.299 1 6 92.7 2. AML-M2 13 18,008 513 12,183 66.7 3. AML-M2 15 24,446 623 10,366 40.9 A. AML-M2 13 23,276 4,116 15,083 57.2 5. AML-M2 8 15,096 983 15,457 >100 6. AML-M2 74 17,266 2,395 16,191 92.9 7. AML-M2 76 20,635 4,725 12,762 50.5 8. AKL-M2 11 19,670 2 a 2,332 21,628 2 b >100 9. APML-M3 8 16,789 3,444 15,096 87.3 10. APML-M3 6 23,395 1,275 7,577 28.5 11. AMML-M4 49 22,435 2,338 14,609 61.1 12. AMM1-M4 10 23,413 2,492 19,672 91.7 * A t o t a l of 25,000 c e l l s were analysed in each case. ** Percent p o s i t i v e = number of c e l l s fluorescing with anti-AML - number fluorescing with NRS JQQ number of c e l l s fluorescing with anti-normal - number fluorescing with NRS *** Averaged results + standard deviation of 12 bone marrow samples. **** FAB c l a s s i f i c a t i o n (see over). la,lb,2a,2b: See fluorescence i n t e n s i t y p r o f i l e s in F i g . 5. FAB C l a s s i f i c a t i o n The FAB (Franco-American B r i t i s h Co-operative Group) c l a s s i f i c a t i o n i s shown on a l l the t a b l e s . C l a s s i f i c a t i o n C e l l type 1. AML-M1 my e l o b l a s t i c without maturation 2. AML-M2 my e l o b l a s t i c w i t h maturation 3. APML-M3 hypergranular p r o m y e l o c y t e 4. AMML-M4 myelomonocytic 5. AML-M5 6. AML-M6 monocytic erythroleukemia 7. AML i n the cases when no FAB c l a s s i f i c a t i o n could be given by the p a t h o l o g i s t 30 Figure 5. Fluorescence i n t e n s i t y p r o f i l e s of bone marrow c e l l s from two AML p a t i e n t s . V e r t i c a l a x i s , r e l a t i v e number of c e l l s i n l o g s c a l e . H o r i z o n t a l a x i s , r e l a t i v e fluorescence i n t e n s i t y . N o n s p e c i f i c fluorescence i s demonstrated by the c e l l s t r e a t e d w i t h NRS and these p r o f i l e s are shown i n a and b i n a l l cases. Superimposed upon the NRS c o n t r o l s are the fluorescence i n t e n s i t y p r o f i l e s of c e l l s t r e a t e d w i t h anti-normal human antiserum ( p o s i t i v e c o n t r o l ) shown i n a and the fluorescence i n t e n s i t y p r o f i l e s of the same c e l l s t r e a t e d w i t h anti-AML antiserum, shown i n b. 32 Table 2. FACS analyses of peripheral blood leucocyte c e l l s from untreated patients with acute myelogenous leukemia Diagnosis % blasts Number of c e l l s fluorescing with various treatments* Anti-normal human NRS Anti-AML % po s i t i v e c e l l s 1. AML-M2 55 23,368 1,641 22,275 91.9 2. AML-M2 40 24,895 38 22,685 91.1 3. AML-M2 26 21,052 5,687 20,154 94.2 4. AML-M2 77 20,900 4,840 8,252 21.2 5. AML-M2 45 21,283 1,213 17,680 82.0 6. APML-M3 93 23,120 280 21,021 90.8 7. AMML-M4 60 20,705 2,790 20,480 98.7 8. AMML-M4 23 18,796 4,306 10,476 42.6 9. AMML-M4 50 24,541 937 23,803 96.8 10. AML-M5 60 18,806 914 22,608 >100 AML - - -80.93+ 26.8** 11. HL-60*** APML-M3 c e l l l i n e 21,304 1,764 17,844 82.3 * A t o t a l of 25,000 c e l l s were analysed in each ci ase. ** Averaged res u l t s + standard deviation of 10 PBL samples. *** Acute promyelocytic leukemia c e l l l i n e . 33 c e l l population had greater fluorescence i n t e n s i t y w i t h the anti-AML-serum (>100) than the anti-normal human serum, however sample 7, w i t h a b l a s t c e l l population of 75% showed only 507* r e l a t i v e fluorescence. These r e s u l t s i n d i c a t e that the antigen i s not only on b l a s t c e l l s but i s a l s o expressed on other c e l l p opulations. I t i s a l s o i n t e r e s t i n g to note that the human c e l l l i n e HL-60, described as promyelocytic leukemia l i n e , a l s o f l u o r e s c e s w i t h the anti-AML-serum (Table 2, sample 11). A s i m i l a r s e r i e s of analyses were c a r r i e d out on p a t i e n t s who were i n c l i n i c a l remission from acute myelogenous leukemia a f t e r chemotherapy. The r e s u l t s are shown i n Tables 3 and 4. I t can be seen that i n both bone marrow and PBL, there are s t i l l a s i g n i f i c a n t number of c e l l s showing p o s i t i v e fluorescence w i t h the anti-AML-serum, even though the c l i n i c a l s t a t e i n each case i s c l e a r l y one of remission. The v a r i a t i o n between cases i s somewhat greater than that seen i n c l i n i c a l disease. Whether or not t h i s i s r e l a t e d to prognosis i s u n c e r t a i n at t h i s time. This data i n d i c a t e s t h a t , i n remission p a t i e n t s , normally d i f f e r e n t i a t i n g c e l l s are expressing t h i s c e l l surface antigen (malignancy marker). One long-term remission p a t i e n t , #4 i n Table 3 wi t h a remission p e r i o d of 3-1/2 years at the time of t e s t i n g , does show a decrease from the average % p o s i t i v e anti-AML c e l l s (38.4% as opposed to 73.6% average). However, very few long-term remission p a t i e n t s have been a v a i l a b l e f o r t e s t i n g to date. I t i s c l e a r however, that the AML p a t i e n t 3k Table 3. FACS analyses of bone marrow c e l l s from patients in c l i n i c a l remission from acute myelogenous leukemia after chemotherapy Diagnosis % blasts Number of c e l l s with various fluorescing treatments* % p o s i t i v e Anti-normal human NRS Anti-AML c e l l s 1. AML-M1 3 16,176 1,573 7,325 39.4 2. AML-M2 2 17,852 480 13,362 72.2 3. AML-H2 1 24,106 1.197 6,067 21.3 4. AML-M2 0 17,940 1,338 7,716 38.4 5. AML-M2 1 14,405 935 15,254 >100 6. AML-M2 occasional 21,001 4,923 20,491 96.8 7. AML-M2 1 22,400 1,775 19,548 86.2 8. APML-M3 1 12,546 1,970 15,308 >100 9. APML-M3 3 11,845 2,160 12,141 >100 10. AHHL-M4 1 16,934 2,087 10,426 56.0 11. AMML-M4 0 18,426 5,541 15,308 75.8 12. AMML-M4 3 23,832 1,547 13,335 49.5 13. AML-M5 4 24,469 2,041 23,401 95.2 14. AML-M5 2 14,268 290 17,975 >100 AML - - - 73.6+ 27.7** * A t o t a l of 25,000 c e l l s were analysed in each case. ** Averaged results + standard deviation of 14 bone marrow samples from remission patients. 35 Table 4. FACS analyses of peripheral blood leucocyte c e l l s from patients in c l i n i c a l remission from acute myelogenous leukemia after chemotherapy. Diagnosis % blasts Number of c e l l s fluorescing with various treatments* Anti-normal human NRS Anti-AML % po s i t i v e c e l l s 1. AML-M2 0 19,980 2,437 12,060 54.8 2. AML-M2 occasional 21,283 1,213 17,680 82.0 3. AML-M2 0 14,967 1,080 5,192 29.6 4. APML-M3 0 20,347 3,151 20,956 >100 5. AMML-M4 occasional 23,827 2,277 11,775 44.1 6. AML-not FAB c l a s s i f i e d 0 22,028 1,783 11,364 47.3 AML - - - 59.63+ 26.2** * A t o t a l of 25,000 c e l l s were analysed in each case. Averaged results + standard deviation of 6 PBL samples from remission patients. 36 presents the "malignant" antigen both during the acute phase and remission p e r i o d . I have had the opportunity to t e s t one AML p a t i e n t over an extended p e r i o d of time, r e s u l t s shown i n Table 5. During the acute phase (10% b l a s t s ) he had 45.5% p o s i t i v e anti-AML c e l l s , 4 months l a t e r now being i n remission ( o c c a s i o n a l b l a s t s i n B.M.) he had 15.2% p o s i t i v e c e l l s , 2 months f o l l o w i n g t h i s , w h i l e s t i l l i n remission (no b l a s t s i n B.M.) he presented 23.8% p o s i t i v e c e l l s and 6 months l a t e r when i n relapse (with 9% b l a s t s i n B.M.) a 66.27o p o s i t i v e c e l l p o pulation was noted. I t i s c l e a r that many more p a t i e n t s (as they become a v a i l a b l e ) should be t e s t e d through the phases of the disease; however t h i s r e s u l t seems promising i n terms of i t s p o t e n t i a l usefulness i n p r e d i c t i n g imminent r e l a p s e . The observation that some PBL samples which are enriched f o r mononuclear c e l l s , a l s o showed a high number of f l u o r e s c i n g c e l l s suggested the p o s s i b i l i t y that lymphoid, as w e l l as myeloid c e l l s , might be expressing t h i s antigen. Since the involvement of lymphoid c e l l s has never been i m p l i c a t e d i n human acute myelogenous leukemia, Dr. P a t r i c i a Logan and myself wished to e s t a b l i s h the gross morphology of those c e l l s showing the highest r e l a t i v e p o s i t i v e fluorescence w i t h the anti-AML, and to determine whether t h i s p opulation d i d , i n f a c t , i n c l u d e lymphocytes. The FACS IV was used to c o l l e c t and s o r t PBL from a remission p a t i e n t whose c e l l s had been analysed as s i g n i f i c a n t l y p o s i t i v e , w i t h the anti-AML-serum. Cytology was performed on that 25% of the c e l l sample showing the lowest p o s i t i v e r e l a t i v e fluorescence and on that 25% of the same sample showing the highest p o s i t i v e r e l a t i v e f luorescence. 37 Table 5. FACS analyses of bone marrow c e l l s from an acute myelomonocytic leukemia patient with active disease, while in remission, and during relapse. Diagnosis % blasts Number of c e l l s fluorescing with various treatments*  Anti-normal NRS Anti-AML % po s i t i v e human c e l l s 1. AMML-M4 10 24,862 3,112 13,011 45.5 (active disease) 2. AMML-M4 occasional 19,055 1,371 4,056 15.2 (in remission) 3. AMML-M4 0 22,734 3,255 7,884 23.8 (in remission) 4. AMML-M4 9 19,099 2,880 13,622 66.2 (in relapse) * A t o t a l of 25,000 c e l l s were analysed in each case. 38 S i m i l a r s o r t s were c a r r i e d out with the p o s i t i v e c o n t r o l antiserum (anti-normal-human). The r e s u l t s shown i n Table 6, were obtained using PBL from the remission p a t i e n t , which had been subjected to a one-step Ficoll-Hypaque separation (mononuclear enriched). As can be seen, a m a j o r i t y of the c e l l s showing the highest l e v e l of fluorescence were lymphoid. These r e s u l t s i n d i c a t e that i n remission p a t i e n t s , c e l l s of both lymphoid and myeloid l i n e a g e are expressing t h i s antigen. When bone marrow a s p i r a t e s or PBL of p a t i e n t s i n relapse w i t h acute myelogenous leukemia were examined with the anti-AML-serum, i t was again seen that a m a j o r i t y of the c e l l s showed p o s i t i v e f l u o r e s c e n c e , and again, the % p o s i t i v e c e l l s d i d not c o r r e l a t e w i t h the number of b l a s t s present, i n d i c a t i n g that t h i s a n t i g e n i c marker i s present on c e l l s other than b l a s t s . The r e s u l t s are shown i n Table 7. A number of bone marrow and p e r i p h e r a l blood samples from p a t i e n t s w i t h CML were a l s o analysed. The r e s u l t s are shown i n Tables 8 and 9. I t can be seen that i n t h i s c o n d i t i o n a l s o , a high percentage of the c e l l s analysed show p o s i t i v e fluorescence. These r e s u l t s were somewhat s u r p r i s i n g because a l l these p a t i e n t s were being s u c c e s s f u l l y t r e a t e d at the time the bone marrow a s p i r a t e s were taken. The p o s s i b l e i m p l i c a t i o n s of these data and the observation that the numbers of b l a s t c e l l s i n the marrows of AML p a t i e n t s d i d not c o r r e l a t e w i t h the numbers of c e l l s f l u o r e s c i n g i s included i n the d i s c u s s i o n . Studies were c a r r i e d out on bone marrow c e l l s taken from p a t i e n t s w i t h d i s o r d e r s other than AML (shown i n Table 10). Representative fluorescence p r o f i l e s of c e l l s from p a t i e n t s w i t h acute lymphocytic 39 Table 6. Microscopic analysis of c e l l s from a remission patient with APML a f t e r l a b e l l i n g with e i t h e r anti-normal human or anti-AML and a f t e r analysis and sor t i n g on the FACS IV Test antiserum used Test performed Anti-normal human Anti-AML FACS IV analyses (number of c e l l s f l uorescing) 20, 347 20,956 ( 100%) FACS IV sort % c e l l s i n high r e l a t i v e * fluorescence population % c e l l s i n low r e l a t i v e * * fluorescence population % c e l l s i n high r e l a t i v e * fluorescence population % c e l l s i n low r e l a t i v e * * fluorescence population neutrophils 2 occ*** 3 -lymphocytes 95 98 92 97 monocytes 3 2 5 3 red blood c e l l s - occ. - -eosinophils - - occ. -* Those c e l l s sorted as the 25% of the population having the highest r e l a t i v e fluorescence. ** Those c e l l s sorted as the 25% of the population having the lowest r e l a t i v e fluorescence. *** Occasional. Table 7. FACS analyses of c e l l s from relapsed patients with acute myelogenous leukemia following chemotherapy. Diagnosis C e l l % blasts Number of c e l l s fluorescing population with various treatments* % pos i t i v e Anti-normal NRS Anti-AML c e l l AML-M2 bone marrow 13 19,167 5,903 15,753 74. 3 PBL 13 21,726 6,353 21,461 98. 2 AHL-H2 bone marrow 67 18,576 5,008 12,992 58. 8 PBL 34 23,276 775 17,218 73. ,0 AML-M2 bone marrow 40 24,826 2,834 22,298 88. .5 AML-M2 bone marrow 91 24,469 2,041 23,401 95 .2 AMML-M4 PBL 60 13,604 4,481 14,278 MOO AMML-M4 PBL 49 22,435 2,338 14,609 61 .0 AMML-M4 PBL 9 18,988 1,753 20,710 >100 * A t o t a l of 25,000 c e l l s were analysed in each case. Table 8. FACS analyses of bone marrow c e l l s from patients in the chronic phase of chronic myelogenous leukemia. Diagnosis Number of c e l l s fluorescing with various treatments* Anti-normal human NRS Anti-AML X p o s i t i v e c e l l s 1. CML 24,856 75 21,716 87.3 2. CML 23,703 341 15,607 65.2 3. CML 22,810 801 23,576 >100 4. CML 22,128 358 17,861 79.6 5. CML 23,411 900 24,601 >100 6. CML 18,189 911 12,583 67.6 7. CML 22,986 1,699 15,826 66.4 8. CML 24,583 1,375 21,103 85.0 9. CML 24,055 351 7,598 30.6 CML - - - 75.7+ 21.6** * A t o t a l of 25,000 c e l l s were analysed in each case. ** Averaged results + standard deviation of 9 bone marrow samples. Table 9. FACS analyses of peripheral blood leucocyte c e l l s from patients in the chronic phase of chronic myelogenous leukemia. Diagnosis Number of c e l l s fluorescing with various treatments*  Anti-normal NRS Anti-AML * positive human c e l l s 1. CML 22,418 2, ,005 13, ,607 57. ,0 2. CML 22,986 1, ,699 15, ,826 66. .3 3. CML 14,421 196 7,156 41. .9 4. CML 22,586 1 ,375 21, ,103 85. .0 5. CML 20,610 575 3, ,693 17. .9 6. CML 21,020 1 ,480 17, ,361 81 .3 7. CML 24,493 330 16, ,851 70 .1 8. CML 22,904 923 10 ,432 43 .3 9. CML 21,797 671 6 ,743 28 .7 CML - - - 54.6+ 23.3** * A t o t a l of 25,000 c e l l s were analysed in each case. ** Averaged results + standard deviation of 9 peripheral blood leucocyte samples. Table 10. FACS analyses of bone marrow c e l l s from patients with disorders other than AML Diagnosis Condition of Bone Marrow Number of c e l l s fluorescing with various antisera  Anti-normal human NRS Anti-AML % pos i t i v e c e l l s 1. ALL 2. ALL 3. ALL 4. ALL 5. ALL 6. CLL 7. CLL 8. CLL remission patient -normal marrow acute disease -82% blast c e l l s occasional blast c e l l s acute disease -92% blast c e l l s congenital -8% blast c e l l s lymphocytosis lymphocytosis, pleomorphic population consistent with CLL lymphocytosis, . abnormal c e l l s 23,657 l a 348 9. Lymphoma 6% blasts 10. lymphoma 11. Lymphoma 2% blast c e l l s , no evidence of major i n f i l t r a t i o n no i n f i l t r a t i o n , normal marrow 12. Hodgkins no i n f i l t r a t i o n , normal Lymphoma marrow 13. Hodgkins no i n f i l t r a t i o n , normal Lymphoma marrow 14. Myeloma no i n f i l t r a t i o n , normal marrow 15. Idiopathic thrombocytopenic purpura no blast 21,575 24,179 24,231 24,882 23,516 24,165 23,472 23,519 23,911 2 a 21,088 21,897 24,572 3 a 20,071 24,093 236 728 745 347 1,453 456 243 430 427 lb 2,118 3,535 687 753 457 3,242 3,112 5,168 4,070 199 309 2b 773 2,431 2,377 3,463 4,257 2,331 2,173 1,818 2,395 3b 0.3 0.03 <0 <0 6.2 0.32 0.46 <0 <0 0.5 <0 <0 3.8 *0 2.6 * A t o t a l of 25,000 c e l l s were analysed in each te s t . l a , l b , 2a, 2b, 3a, 3b: See fluorescence intensity p r o f i l e s in Fig . 6. 44 leukemia (ALL), lymphoma, and Hodgkins disease are shown i n Figure 6. The r e p r e s e n t a t i v e samples shown i n t h i s f i g u r e are designated i n Table 10. I t i s c l e a r from these experiments that the anti-AML d i d not bind s i g n i f i c a n t l y to the marrow c e l l s of any of these p a t i e n t s at measurable l e v e l s above background obtained w i t h NRS. I t appears that the anti-AML i s r e c o g n i z i n g a c e l l surface antigen common to AML and CML p a t i e n t s ' c e l l s , which i s not present i n detectable amounts on bone marrow c e l l s of p a t i e n t s w i t h other d i s o r d e r s . Further studies w i t h PBL from p a t i e n t s w i t h leukemias of non-myelogenous o r i g i n are shown i n Table 11. As seen w i t h the bone marrow samples, the anti-AML-serum d i d not bind to these c e l l s . To determine whether the antiserum was d e t e c t i n g markers on mature c e l l s , a s e r i e s of normal i n d i v i d u a l s * PBL were analysed on the FACS IV w i t h anti-AML-serum. None have shown any r e a c t i v i t y above background (Table 12). A number of normal bone marrow samples have a l s o been t e s t e d . These were obtained from p o t e n t i a l bone marrow donors or from p a t i e n t s w i t h suspected P. vera who were subsequently shown to have normal marrows. These r e s u l t s are a l s o presented i n Table 12, i n which i t can be seen that these m a t e r i a l s a l s o showed no s i g n i f i c a n t r e a c t i v i t y w i t h the anti-AML. A l s o , Ficoll-Hypaque-enriched populations of e i t h e r lymphocyte-monocyte c e l l s or granulocytes from normal i n d i v i d u a l s were examined. A r e p r e s e n t a t i v e r e s u l t i s shown i n Table 13 and confirms that the anti-AML-serum has no s i g n i f i c a n t r e a c t i v i t y w i t h these c e l l p o p u lations. The fluorescence i n t e n s i t y p r o f i l e s of these samples are shown i n Figure 7. 45 Figure 6. Fluorescence i n t e n s i t y p r o f i l e s of bone marrow c e l l s from p a t i e n t s w i t h d i s o r d e r s other than AML. Conditions are i d e n t i c a l to those described f o r Figure 5. a. P r o f i l e s of c e l l s t r e a t e d w i t h e i t h e r NRS or anti-normal human serum; b. p r o f i l e s of c e l l s t r e a t e d w i t h e i t h e r NRS or anti-AML serum. Sample 1 represents c e l l s taken from a p a t i e n t w i t h ALL, Sample 2, c e l l s from a lymphoma p a t i e n t and Sample 3 from a p a t i e n t w i t h Hodgkins. 46 3a 3b k7 Table 11. FACS analyses of peripheral blood leucocyte c e l l s from individuals with leukemias of non-myelogenous o r i g i n . Diagnosis Number of c e l l s fluorescing with various treatments*  Anti-normal human NRS Anti-AML % p o s i t i v e c e l l s 1. ALL-remission 23,733 1,413 1,538 2. ALL-remission 21,197 958 1,227 3. ALL-remission 24,416 327 593 4. ALL 27% blasts 15,627 1,108 2,178 5. ALL-remission (same patient when in remission) 22,310 1,157 1,140 6. Idiopathic thrombocytopenic purpura 19,788 597 370 7. CLL 18,608 1,421 983 0.5 1.3 0.1 7.4 <0 <0 <0 A t o t a l of 25,000 c e l l s were analysed in each case. Table 12. FACS analyses of c e l l s from normal individuals C e l l population Number of c e l l s fluorescing with various treatments* Anti-normal NRS Anti-AML % pos i t i v e human c e l l s 1. bone marrow 24,650 277 415 0. 5 2. bone marrow 16,105 91 101 0. 06 3. bone marrow 22,898 143 420 1. 2 4. bone marrow 14,082 2,463 2,023 <0 5. PBL** 24,604 263 712 1. 8 6. PBL 24,783 519 272 <0 7. PBL 24,827 83 282 0. 8 8. PBL 22,147 866 776 <0 9. PBL 19,870 354 196 <0 10. PBL 23,191 2,076 1,151 <o 11. PBL 22,541 1,768 1,675 <0 12. PBL 24,170 1,820 1,463 <Q 13. PBL 24,397 2,930 1,153 < o * A t o t a l of 25,000 c e l l s were analysed in each case. ** PBL = peripheral blood leucocyte c e l l s . 49 Table 13. FACS analyses of lymphocyte-monocyte and granulocyte-enriched population from a normal i n d i v i d u a l . C e l l population Number of c e l l s fluorescing with various treatments* Anti-normal NRS Anti-AML % pos i t i v e human c e l l s 1. Lymphocyte-monocyte 24,455 enriched 2. Granulocyte enriched 20,748 852 1,191 1 D 1.4 798 l , 5 5 3 2 b 3.8 * A t o t a l of 25,000 c e l l s were analysed in each case. l a , l b , 2a, 2b: See fluorescence i n t e n s i t y p r o f i l e s in F i g . 7. 50 Figure 7. Fluorescence i n t e n s i t y p r o f i l e s of Ficoll-Hypaque-enriched populations of e i t h e r lymphocyte-monocyte c e l l s or granulocytes from a normal i n d i v i d u a l . Conditions are i d e n t i c a l to those described f o r Figure 5. a. P r o f i l e s of c e l l s t r e a t e d w i t h e i t h e r NRS or anti-normal human serum; b, p r o f i l e s of c e l l s t r e a t e d w i t h e i t h e r NRS or anti-AML-serum. Sample 1 represents the lymphocyte-monocyte enriched p o p u l a t i o n , Sample 2 represents the granulocyte enriched p o p u l a t i o n . lb 2b 52 Table 14 shows the r e s u l t s of analyses of a bone marrow a s p i r a t e and p e r i p h e r a l blood leucocytes from an APML (acute promyelocytic leukemia) p a t i e n t and the p e r i p h e r a l blood leucocytes from her normal i d e n t i c a l twin. The APML bone marrow a s p i r a t e , p e r i p h e r a l blood leuocyte sample, and the Ficoll-Hypaque-enriched populations of e i t h e r mononuclear of g r a n u l o c y t i c c e l l s (samples 1,2,3 and 4) a l l showed a high percentage of c e l l s r e a c t i n g p o s i t i v e l y w i t h the anti-AML-serum. Hence, both bone marrow and the p e r i p h e r a l blood c e l l s from t h i s APML p a t i e n t have the AML malignancy marker. I t i s c l e a r , however, that the anti-AML d i d not bind to the p e r i p h e r a l blood l e u c o c y t e s , nor to the enriched mononuclear or granulocyte populations (samples 5, 6 and 7 r e s p e c t i v e l y ) of the normal i d e n t i c a l twin. Representative fluorescence p r o f i l e s of p e r i p h e r a l blood leucocytes from the APML p a t i e n t (sample #2) and from the normal i d e n t i c a l twin (sample #5) are shown i n Figure 8. This t e s t demonstrates that the normal i d e n t i c a l twin s i s t e r d i d not have the AML antigen and hence would have been an i d e a l bone marrow donor f o r the APML s i b l i n g (58 y r , ^  ), age being the l i m i t i n g f a c t o r . This normal i n d i v i d u a l a l s o represented an i d e a l c o n t r o l . The r e s u l t s of f l u o r e s c e n t t e s t i n g of c e l l s from two p a t i e n t s who re c e i v e d a l l o g e n e i c bone marrow t r a n s p l a n t s from t i s s u e matched s i b l i n g donors as a treatment f o r AML are shown i n Table 15. Donor c e l l s had not been t r e a t e d i n any way to reduce the mature T c e l l p o p u l a t i o n . C e l l s from p a t i e n t 1 were taken 20 months a f t e r t r a n s p l a n t a t i o n and the p a t i e n t was doing w e l l w i t h no disease recurrence. The c e l l s from t h i s p a t i e n t d i d not show s i g n i f i c a n t fluorescence w i t h the t e s t anti-AML-serum. The 53 Table IA. FACS analyses of c e l l s from a patient with APML and her i d e n t i c a l twin Diagnosis Number of c e l l s fluorescing with various treatments*  * Anti-normal NRC Anti-AML % pos i t i v e blasts human c e l l s APML-M3 1 bone marrow 7.0 2 peripheral blood 7.0 3 peripheral blood (mononuclear enriched-96% pure) N.D. 4 peripheral blood (granulocyte enriched-98% pure) N.D. 23,610 3,136 72,052 92.3 21,807 l a 2,842 14,796 l b 63.0 23,578 5,260 11,415 23,385 1,030 16,573 33.6 69.5 Normal (Identical twin) 5 peripheral blood 6 peripheral b l l o d (mononuclear enriched-96% pure) 7 peripheral blood (granulocyte-enriched-98% pure) 20,774 2 a 667 524 2 b 24,455 20,748 852 1,191 798 1,553 <0 1.4 3.6 * A t o t a l of 25,000 c e l l s were analysed in each case. l a , l b , 2a, 2b: See fluorescence intensity p r o f i l e s in F i g . 8. 54 Figure 8. Fluorescence i n t e n s i t y p r o f i l e s of p e r i p h e r a l blood leucocytes from a p a t i e n t w i t h APML (1) and her i d e n t i c a l twin (2). V e r t i c a l a x i s , r e l a t i v e number of c e l l s i n l o g s c a l e . H o r i z o n t a l a x i s , r e l a t i v e fluorescence i n t e n s i t y . N o n s p e c i f i c fluorescence i s demonstrated by the c e l l s t r e a t e d w i t h NRS and these p r o f i l e s are shown i n a and b i n a l l cases. Superimposed upon the NRS c o n t r o l s are the fluorescence i n t e n s i t y p r o f i l e s of c e l l s t r e a t e d w i t h anti-normal human serum ( p o s i t i v e c o n t r o l ) shown i n a and the fluorescence i n t e n s i t y p r o f i l e s of the c e l l s t r e a t e d w i t h anti-AML-serum, shown i n b.. 55 56 Table IS. FACS analyses of either bone marrow c e l l s or PBL from patients with AML following bone marrow transplantation. Diagnosis Time after Condition Number of c e l l s fluorescing % po s i -transplant with various treatments* t i v e c e l l s Anti-normal NRS Anti-AML 1. AML bone marrow 20 months remission 13,177 208 634 3.2 2. AML-M5 PBL 2 weeks remission (no blast c e l l s ) 23,761 2,128 23,759 99.9 PBL 4 months relapse 23% blasts 18,828 4,695 11,496 48.1 bone marrow 4 months relapse 23% blasts 20,736 2,439 14,618 66.5 3 Normal donor s i b l i n g of - normal 14,082 2,463 2,023 <0 patient #2 (bone marrow) * A t o t a l of 25,000 c e l l s were analysed in each case. 57 c e l l s from p a t i e n t 2 were fo l l o w e d from s h o r t l y a f t e r t r a n s p l a n t a t i o n . At no time ( e i t h e r 2 weeks a f t e r t r a n s p l a n t a t i o n , when the p a t i e n t was i n c l i n i c a l remission or 4 months l a t e r , when the p a t i e n t was i n relapse) d i d c e l l s show negative r e a c t i v i t y w i t h the anti-AML-serum. The donor (sample 3 ) , on the other hand, had no measurable r e a c t i v e c e l l s i n h i s bone marrow. By and l a r g e , the anti-AML-serum under t e s t here has shown absolute s p e c i f i c i t y w i t h regard to r e a c t i v i t y w i t h c e l l s of p a t i e n t s w i t h myelogenous leukemia. In a l l specimens t e s t e d , only 3 cases have not shown the expected r e a c t i o n s . These are shown i n Table 16. P a t i e n t 1 was diagnosed as having ALL. At the time the bone marrow a s p i r a t e was t e s t e d , the p a t i e n t was i n c l i n i c a l remission. However, a high percentage of the p a t i e n t s ' c e l l s f l u o r e s c e d s t r o n g l y w i t h the anti-AML-serum. We have no explanation f o r t h i s observation. P a t i e n t 2 was diagnosed as ALL. However, the c l i n i c a l r e p o r ts show that while bone marrow colony growth suggested ALL, p e r i p h e r a l blood growth was suggestive of some type of g r a n u l o c y t i c disease. I t would appear that t h i s p a t i e n t ' s diagnosis may be somewhat questionable. P a t i e n t 3 i s a j u v e n i l e (10 year old) who had been diagnosed as having AML (see d i s c u s s i o n ) . 58 Table 16. FACS analyses or c e l l s from three patients whose c e l l s yielded anomalous results with regard to t h e i r diagnosis. Number of c e l l s fluorescing with Diagnosis % blasts C e l l population various treatments*  Anti-normal NRC Anti-AML % pos i t i v e human c e l l s 1. ALL** remission (no blasts) bone marrow 23,207 1,992 13,777 55.6 2. ALL*** 92 bone marrow 20,505 1,683 17,142 82.2 41 PBL 24,375 301 18,606 76.3 3. AML-M2**** 42 bone marrow 18,244 4,495 4,718 1.6 42 PBL 19,628 6,911 6,311 <0 * A t o t a l of 25,000 c e l l s were analysed in each case. ** This patient has been diagnosed as a t y p i c a l ALL in remission. *** This patient record shows that although bone marrow growth suggests ALL, peripheral blood c e l l colony growth better f i t s some type of granulocytic disease. **** This patient i s a 10 year old juvenile AML case. 59 D i s c u s s i o n There are a number of questions which are r a i s e d by the r e s u l t s reported here. I t has been shown that an antiserum r a i s e d i n r a b b i t s to an antigen p u r i f i e d on p r e p a r a t i v e polyacrylamide gels from membrane e x t r a c t s from c e l l s of AML p a t i e n t s reacts s t r o n g l y i n FACS IV a n a l y s i s w i t h a c e l l surface antigen found on 39 of 40 samples taken from p a t i e n t s diagnosed as having acute myelogenous leukemias. C e l l s taken from i n d i v i d u a l s w i t h no known d i s o r d e r s , d i d not react w i t h t h i s antiserum. I t would thus appear that the i s o l a t e d antigen represents what could be termed a tumor a s s o c i a t e d antigen (TAA) or malignancy marker i n t h a t i t does not appear to be present, at detectable l e v e l s , on normal c e l l s . This i s i n agreement with previous f i n d i n g s i n t h i s l a b o r a t o r y , and as reported i n Chapter I , that the antigen could not be detected i n e x t r a c t s from normal c e l l membranes when t e s t e d w i t h the antiserum i n the ELISA (Al-Rammahy §_t a l . , 1980). Whether or not the antigen i s present on a small population of normal bone marrow c e l l s i s the subject of ongoing st u d i e s i n the l a b o r a t o r y . I t was a l s o reported here that t h i s AML-associated antigen i s a l s o detectable on the c e l l s of p a t i e n t s w i t h CML. Of 15 samples taken from p a t i e n t s w i t h CML, a l l samples 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 (at l e a s t 20% p o s i t i v e ) i n both bone marrow and PBL m a t e r i a l s . These r e s u l t s i n d i c t e that a common TAA i s present on the c e l l s of p a t i e n t s diagnosed as having e i t h e r AML or CML. These r e s u l t s 60 may imply a common event i n the onset of both of these c o n d i t i o n s . This has not been suggested i n previous s t u d i e s , even though both c o n d i t i o n s i n v o l v e p r o l i f e r a t i v e d i s o r d e r s of n y e l o i d c e l l s . I t has been a c o n s i s t e n t observation i n these studies that the numbers of c e l l s r e a c t i n g w i t h the anti-AML-serum i n e i t h e r bone marrow a s p i r a t e s or PBL i n AML p a t i e n t s do not c o r r e l a t e w i t h the number of b l a s t c e l l s present i n the sample. This would imply that the antigen i s present on c e l l s other than b l a s t s . This i s e x p l i c i t l y demonstrated by the observation that p a t i e n t s i n c l i n i c a l remission from AML (Tables 3 and 4) showed a high percentage 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 . These f i n d i n g s are i n agreement with those of Metzgar and Mohanakumar (1978) who found that simian anti-AML a n t i s e r a reacted p o s i t i v e l y w i t h 40% of c e l l s from a p a t i e n t i n remission from AMML. In p a t i e n t s w i t h a c t i v e disease, these f i n d i n g s i n d i c a t e that t h i s marker (antigen) may be present on a p l u r i p o t e n t stem c e l l . This f i n d i n g a l s o supports the p o s s i b i l i t y that there may be c l o n a l dominance of a p o t e n t i a l l y malignant stem c e l l , probably p l u r i p o t e n t i a l , i n the bone marrow and p e r i p h e r a l blood of AML p a t i e n t s (Wiggans e t a l . , 1978; Fialkow et a l . , 1979). The obse r v a t i o n , i n remission p a t i e n t s , that lymphoid as w e l l as myeloid c e l l s may be expressing t h i s antigen, suggests t h a t : a p l u r i p o t e n t stem c e l l i s expressing the antigen, and c e l l s expressing t h i s antigen can d i f f e r e n t i a t e , a l b e i t t r a n s i e n t l y , i n normal f a s h i o n . Indeed, Fialkow (1982), i n h i s isoenzyme s t u d i e s w i t h glucose-6-phosphate dehydrogenase, demonstrated a single-enzyme G6PD phenotype i n leukemic b l a s t c e l l s , suggestive of a c l o n a l d i s o r d e r . When studying remission 61 p a t i e n t s , however, there was a r e t u r n of a normal double-enzyme phenotype i n g r a n u l o c y t i c c e l l s . This observation suggests that normal stem c e l l s were present, but not expressed during the acute phase of the leukemia. At f i r s t , one might conclude that Fialkow's f i n d i n g s c o n t r a d i c t the AML remission date reported here. However, i t may be that the malignancy marker i n remission p a t i e n t s e x i s t s on p o t e n t i a l l y malignant and normal c e l l s . At t h i s time, however, i t would be premature t o conclude c l o n a l dominance i n AML. I t i s e s s e n t i a l , i n i t i a l l y , t hat a l a r g e number of remission p a t i e n t s be s t u d i e d , to determine i f predominance of t h i s marker on normally d i f f e r e n t i a t i n g c e l l s i s an i n d i c a t o r of the length of remission. At t h i s stage, long term remission p a t i e n t s would be of great i n t e r e s t , but such i n d i v i d u a l s are r a r e , and i t has been d i f f i c u l t to accumulate enough data to evaluate the prognostic value of t h i s marker. The question could be r a i s e d as to whether the antigen, detected on lymphocytes of remission p a t i e n t s , i s a c t u a l l y synthesized by these c e l l s or whether i t i s adsorbed from other sources. At t h i s time, there i s no d e f i n i t e proof as to the o r i g i n of the antigen on lymphocytes. Because the p a t i e n t s are i n c l i n i c a l remission i t i s u n l i k e l y t h a t the amounts of antigen observed i n these c e l l s are derived from a few o c c u l t malignant c e l l s . However, the p o s s i b i l i t y that the antigen i s d e r i v e d from a normally d i f f e r e n t i a t i n g myelocytic population cannot be r u l e d out. The r e s u l t s of extensive FACS IV s o r t i n g studies on p e r i p h e r a l blood and bone marrow c e l l s from p a t i e n t s w i t h m y e l o p r o l i f e r a t i v e d i s o r d e r s i s one of the subjects of Dr. Logan's d o c t o r a l programme. 62 In 39 of 40 diagnosed cases of AML, the anti-AML used here detected antigen on the surface of a s i g n i f i c a n t number of e i t h e r bone marrow c e l l s or PBL of these p a t i e n t s . The one case of diagnosed AML whose c e l l s d i d not react w i t h the antiserum, was a 10 year o l d j u v e n i l e p a t i e n t . There i s no explanation f o r t h i s , however, i t may be that a small number of AML p a t i e n t s do not express antigen on t h e i r bone marrow c e l l s , or i t may be tha t AML i n c h i l d r e n i n v o l v e s a s e r i e s of c e l l u l a r changes d i s t i n c t from the adult disease. I t i s hoped that subsequent s t u d i e s , as p a t i e n t samples become a v a i l a b l e , w i l l c l a r i f y these p o s s i b i l i t i e s . Fialkow (1979, 1982) has shown d i f f e r e n c e s i n e l d e r l y and j u v e n i l e AML p a t i e n t s . Single-enzyme phenotypes were found i n b l a s t c e l l s , e r y t h r o c y t e s and p l a t e l e t s of e l d e r l y p a t i e n t s , which i s suggestive of c l o n a l dominance. With the j u v e n i l e p a t i e n t s , the single-enzyme was found only i n c e l l s w i t h r e s t r i c t e d d i f f e r e n t i a t i v e a b i l i t y . These f i n d i n g s may r e f l e c t age-related v a r i a t i o n s i n c l i n i c a l f e a t u r e s , or that d i f f e r e n t oncogenic events may be in v o l v e d . I t was al s o found that c e l l s from two p a t i e n t s diagnosed as ALL had c e l l s ( e i t h e r bone marrow or PBL) which reacted s t r o n g l y with the antiserum. One p a t i e n t , who had a s i g n i f i c a n t number of b l a s t c e l l s i n both bone marrow and PBL had a disease p i c t u r e which was somewhat ambiguous i n that bone marrow c u l t u r e s i n d i c a t e d ALL, whereas PBL c u l t u r e s i n d i c a t e d a m y e l o p r o l i f e r a t i v e d i s o r d e r . The second p a t i e n t was diagnosed as ALL but was i n c l i n i c a l remission at the time that c e l l s were t e s t e d . At t h i s time, i t i s only p o s s i b l e to s t a t e that the antigen i s present on c e l l s from a high percentage of p a t i e n t s diagnosed as AML (or other 63 m y e l o p r o l i f e r a t i v e leukemias) and may be expressed on c e l l s of a small number of p a t i e n t s w i t h ALL. I t has not been observed on c e l l s of p a t i e n t s w i t h other lymphoid d i s o r d e r s . To date, c e l l s from two p a t i e n t s who have re c e i v e d bone marrow t r a n s p l a n t s as treatment f o r AML have been examined. One p a t i e n t who was doing w e l l and was s t i l l i n c l i n i c a l remission a f t e r 20 months had e s s e n t i a l l y no c e l l s i n a bone marrow a s p i r a t e which reacted w i t h the anti-AML-serum. The second p a t i e n t , who was followed from 2 weeks a f t e r t r a n s p l a n t a t i o n , at a l l times showed a s i g n i f i c a n t number of c e l l s r e a c t i v e w i t h the antiserum. This p a t i e n t relapsed at 4 months p o s t - t r a n s p l a n t . I t i s p o s s i b l e that such monitoring may be of prognostic value i n t h i s form of treatment. I t has been shown that the antiserum described h e r e i n i s d e t e c t i n g an antigen which i s present on the surface of a high percentage of both bone marrow c e l l s and PBL of most p a t i e n t s w i t h AML (FAB Ml to M5) or CML, whether they are presenting w i t h the disease, i n remission or i n r e l a p s e . I t i s not present, at dete c t a b l e l e v e l s , on normal bone marrow c e l l s or normal PBL, nor i s i t present, at detectable l e v e l s , on the c e l l s of most p a t i e n t s w i t h l y m p h o p r o l i t e r a t i v e d i s o r d e r s . I t i s p o s s i b l e that t h i s antigen may be expressed on a small population of normal c e l l s of bone marrow o r i g i n . Because i n FACS IV a n a l y s i s , we observe v a r i a t i o n of backgrounds ± 5% against NRS c o n t r o l s , we w i l l not be able to determine i f a small number of normal c e l l s do have the antigen on t h e i r surfaces. Microscopic examination of i n d i v i d u a l c e l l populations using e i t h e r f l u o r e s c e n t l a b e l l i n g or immunoperoxidase s t a i n i n g are the subject of 64 o n g o i n g s t u d i e s i n t h e l a b o r a t o r y . N o n e t h e l e s s , i t may be c o n c l u d e d t h a t t h i s marker ( a n t i g e n ) may be o f c l i n i c a l s i g n i f i c a n c e i n b o t h p r o g n o s i s and d i a g n o s i s o f mye logenous l e u k e m i a . CHAPTER I I I Monoclonal Antibody (MAL-1) S p e c i f i c f o r Myelogenous Leukemia 66 I n t r o d u c t i o n U n t i l r e c e n t l y , there have been r e l a t i v e l y few reports on monoclonal antibodies r e a c t i v e w i t h membrane determinants of myeloid or myelogenous leukemia c e l l s . In c o n t r a s t , there are a l a r g e number of p u b l i c a t i o n s on monoclonal antibodies w i t h r e a c t i v i t y f o r lymphocyte and lymphocytic leukemia surface antigens; i . e . common acute lymphocytic leukemia antigen (CALLA) (Greaves et a l . , 1980, 1981a, 1981b; L i s z k a et a l . , 1981; Navarrete et a l . , 1981; Newman et a l . , 1981; Mulder et a l . , 1981; Lebacq et a l . , 1982; Boucheix et a l . , 1982). Katz et a l . (1981) have s t u d i e d the chromosomal c o n t r o l of s e v e r a l monoclonal antibody-defined ALL c e l l s urface antigens. Procedures have been developed f o r the sub-typing of ALL w i t h monoclonals ( n u l l ALL, common ALL-cALL, pre-B-ALL, B-ALL and T-ALL) (Greaves et a l . , 1981; P o u l i k et a l . , 1981; Foon et a l . , 1982). Non-lymphoid c e l l s can a l s o be c h a r a c t e r i z e d w i t h monoclonal antibodies d i r e c t e d against c e l l surface d i f f e r e n t i a t i o n antigens. Knapp et a l . (1981) have a number of monoclonal antibodies d i r e c t e d against myeloid d i f f e r e t i a t i o n antigens which have v a r i e d s p e c i f i c i t i e s and may prove of use i n the c h a r a c t e r i z a t i o n and s u b d i v i d i n g of myelocytic leukemias. Some i n v e s t i g a t o r s have used HL-60 c e l l s as immunogens to produce myelocyte-specif i c monoclonals. One group (Peng §_t a l . , 1982) has produced a monoclonal which recognizes a d i f f e r e n t i a t i o n antigen on e a r l y haemotopoietic c e l l s of the myeloid and e r y t h r o i d lineages (monoclonals Pro-Im 1, Pro Im 2). Another monoclonal (HL-C5) shows r e a c t i v i t y 67 r e s t r i c t e d to the myeloid l i n e a g e ( G i r a r d e t et a l . , 1982). This monoclonal may be of p a r t i c u l a r use f o r the diagnosis of AML i n that i t reac t s s t r o n g l y w i t h M3 (hypergranular promyelocytic AML-FAB c l a s s i f i c a t i o n ) l e s s s t r o n g l y w i t h M2 (me y l o b l a s t i c w i t h maturation AML) and not at a l l w i t h Ml (m y e l o b l a s t i c without maturation AML). Als o a monoclonal which was produced by immunizing with AMML (M4) c e l l s and demonstrates AMML s p e c i f i c i t y has been i s o l a t e d by Uchanska-Ziegler et a l . (1982). Reading et a l . (1983) have produced human monoclonal antibodies r e a c t i v e w i t h human AML c e l l s , but not with remission c e l l s . They used an Epstein-Barr V i r u s (EBV) transformed B c e l l p o pulation from an AML p a t i e n t i n complete remission. This p a r t i c u l a r remission AML p a t i e n t was pr e v i o u s l y found to have serum antibodies which were r e a c t i v e w i t h h i s own AML c e l l s which had been stored at p r e s e n t a t i o n , but unreactive w i t h h i s own bone marrow c e l l s s t ored a f t e r he reached remission. Seven c e l l l i n e s were e s t a b l i s h e d from the EBV transformed c u l t u r e s , and a l l continued to produce the an t i b o d i e s . Only one of these antibodies reacted w i t h the p a t i e n t ' s remission bone marrow c e l l s i n the ELISA. Studies l o o k i n g at the s e l e c t i v e l o s s or diminished expression of antigens recognized by monoclonals on the surface of AML c e l l s may als o be of some d i a g n o s t i c value (Navarrete et a l . , 1982; Rumpold et a l . , 1982). I t i s c l e a r that monoclonals may have s i g n i f i c a n t a p p l i c a t i o n s i n the diagnosis of AML. The d i f f i c u l t i e s i n d i s t i n g u i s h i n g the u n d i f f e r e n t i a t e d forms of AML from ALL may be e l i m i n a t e d . As has been s t a t e d e a r l i e r , w i t h AML, the dominant c e l l type may r e f l e c t a l e v e l of matu r a t i o n a l a r r e s t , however t h i s a r r e s t i s not n e c e s s a r i l y at the l e v e l at which the malignant 68 transformation has occurred. Monoclonal Ab might c l a r i f y the scheme of normal myeloid and malignant d i f f e r e n t i a t i o n . Already some i n t e r e s t i n g work has been done on the staging of myeloid d i f f e r e n t i a t i o n using monoclonal i d e n t i f i c a t i o n markers (Andrews §_t a l . , 1982; B a l l and Fanger, 1983). I t may a l s o be p o s s i b l e to increase the accuracy of measuring haematologic remission w i t h monoclonals to s p e c i f i c markers. Indeed, the i d e n t i f i c a t i o n of leukemia c e l l types may lea d to a new immunotherapy regime, i n which the o b j e c t i v e would be to encourage maturation of leukemia c e l l s as has been done with the HL-60 c e l l l i n e w i t h DMSO and phorbol e s t e r s ( C o l l i n s et a l . , 1978). Myelogenous leukemia s p e c i f i c monoclonals may i n the f u t u r e be used to t r e a t remission bone marrow c e l l s and hence have an even greater t h e r a p e u t i c value i n autologous t r a n s p l a n t s ( F i t c h e n et a l . , 1981). Indeed, J5 monoclonal Ab (anti-CALLA) has been used to s u c c e s s f u l l y t r e a t the bone marrow of ALL p a t i e n t s w i t h no histocompatible donor. In one study, 7 of 13 p a t i e n t s t r e a t e d t h i s way have remained i n a second remission f o r over 2 years (reported by Dr. J . R i t z , Sidney Farber Cancer I n s t i t u t e , at the UCLA Symposium, Recent Advances i n Bone Marrow T r a n s p l a n t a t i o n , Feb., 1983). This method of purging the bone marrow has some very p o s i t i v e advantages over bone marrow t r a n s p l a n t a t i o n i n that p a t i e n t s without a histocompatible donor can be t r e a t e d ; i t avoids c l i n i c a l g r a f t vs. host disease, and i t allows f o r the end of chemotherapy, t h e r e f o r e e l i m i n a t i n g the immunosuppression which occurs i n post bone marrow t r a n s p l a n t a t i o n . 69 This chapter reports on the production and c h a r a c t e r i z a t i o n of a monoclonal antibody r a i s e d to band 1 (AML-Ag). Data i s presented which shows the s p e c i f i c i t y of t h i s monoclonal i n an immunofluorescence t e s t i n the FACS IV, the ELISA and by polyacrylamide g e l e l e c t r o p h o r e s i s of the a f f i n i t y p u r i f i e d AML antigen. 70 M a t e r i a l s and Methods Immunization and Fusion Procedures Balb/c mice were given an i n t r a p e r i t o n e a l ( i . p . ) primary immunization of 20 yg of PAGE p u r i f i e d (described i n Chapter I I ) myelogenous leukemia s p e c i f i c Ag (AML-Ag, band 1) i n 50% complete Freund's adjuvant (CFA) and then re-immunized i n one month by three consecutive d a i l y intravenous ( i . v . ) i n j e c t i o n s of the AML-Ag alone i n PBS. The NS-1 myeloma c e l l l i n e was c u l t u r e d i n DME medium supplemented with 0.163 M sodium bicarbonate, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 u n i t s / m l p e n i c i l l i n , 50 yg/ml streptomycin, 5 mM hepes, and 20 % FCS i n a h u m i d i f i e d 10% CO^ incubator at 37°C. To ensure that the NS-1 c e l l s were i n log-phase growth at the time of the f u s i o n , they were used at a d e n s i t y of about 10 5 c e l l s / m l . The spleens were removed from the primed mice the day f o l l o w i n g the l a s t i . v . i n j e c t i o n and fused to NS-I myeloma p a r e n t a l c e l l s w i t h a 50% polyethylene g l y c o l s o l u t i o n (Serva Feinbiochemica, Heidelberg). The c o n d i t i o n s f o r f u s i o n were the same as those described by Oi and 8 Herzenberg (1971). A c e l l r a t i o of approximately 10 immune spleen c e l l s to 2 x 10 7 NS-I c e l l s were used f o r the f u s i o n . A 0.1 ml volume of the f u s i o n products (2 x 10 5 t o t a l c e l l s per w e l l ) , plus newborn outbred mouse thymocytes (2 x 10"* t o t a l c e l l s per w e l l ) to act as a feeder l a y e r , were p l a t e d i n 96 - w e l l t i s s u e c u l t u r e p l a t e s (Costar #3596). The s e l e c t i o n procedure, to y i e l d s t a b l e monoclonal antibody-producing h y b r i d c e l l l i n e s was based on the w e l l known 71 L i t t l e f i e l d * s (1964) hypoxanthine-aminopterin-thymidine (HAT) s e l e c t i v e medium. The day a f t e r the f u s i o n 0.1 ml of 2X HAT s e l e c t i v e medium was added to each w e l l . Supernatants from these primary t i s s u e c u l t u r e s of fused spleen w i t h myeloma c e l l s were screened f o r antibody production using the ELISA ( K e l l y et a l . , 1979). C e l l s from w e l l s i n which supernatant m a t e r i a l reacted p o s i t i v e l y w i t h the AML-Ag (band 1) on the ELISA p l a t e were cloned. Again outbred mouse thymocytes were used as a feeder l a y e r . These cloned c e l l s were again t e s t e d a f t e r 7 days and p o s i t i v e w e l l s , when confirmed to be only one clone by microscopic examination, were grown up i n c u l t u r e i n DME + 20% FCS. An a l i q u o t 6 (10 c e l l s ) of the antibody-producing c u l t u r e c e l l s were i n j e c t e d i n t o p r i s t a n e t r e a t e d Balb/c mice. These mice produced a s c i t e s u s u a l l y w i t h i n 2 weeks which was subsequently c o l l e c t e d from the p e r i t o n e a l c a v i t y of these mice. The a s c i t e s c e l l s were e i t h e r i n j e c t e d i n t o p r i s t a n e t r e a t e d s u b l e t h a l l y i r r a d i a t e d (600 rads) outbred mice (these mice r o u t i n e l y produced more a s c i t e s than Balb/c mice) or prepared f o r f r e e z i n g at -70°C i n DME c o n t a i n i n g 20% FCS and 10% DMSO. The a s c i t e s f l u i d c o l l e c t e d from these mice was again screened f o r s p e c i f i c i t y w i t h the ELISA and a l s o s e l e c t e d f o r s p e c i f i c i t y i n the FACS IV (methodology described i n next s e c t i o n ) . C e l l s and C e l l L a b e l l i n g The HL-60 c e l l l i n e was c u l t u r e d as described i n Chapter I I . The acute lymphocytic leukemia c e l l l i n e , CCRF-SB-ALL-B was maintained i n DME plus 20% FCS, w h i l e the CCRF-SB-ALL-T c e l l l i n e was grown i n MEM plus 20% 72 FCS i n a h u m i d i f i e d 37 C incubator i n 10% C0 2. Bone marrow and p e r i p h e r a l blood samples from p a t i e n t s w i t h leukemia and other malignant diseases were p u r i f i e d as described i n Chapter I I . 6 For l a b e l l i n g , c e l l p e l l e t s (10 c e l l s ) from e i t h e r bone marrow, p e r i p h e r a l blood, or c e l l l i n e samples were incubated f o r 1-1/2 h on i c e w i t h 0.2 ml of a s c i t e s monoclonal antibody d i l u t e d to 1/100 i n PBS. A monoclonal d i r e c t e d to an u n r e l a t e d antigen ( f e r r e d o x i n ) used at the same conc e n t r a t i o n as the AML-monoclonal was employed as a negative c o n t r o l . The c e l l s were subsequently washed three times i n PBS and 0.2 ml of f l u o r e s c e i n a t e d goat-anti-mouse-IgG (Cappel) at a d i l u t i o n of 1/20 w i t h PBS (+ 27o FCS) was added to each c e l l p e l l e t . C e l l s were incubated f o r o another 1.5 h at 0 C, washed once i n PBS, and then c e n t r i f u g e d through 100% FCS. They were f i n a l l y suspended i n 1.0 ml PBS and 5% FCS f o r FACS IV a n a l y s i s (FACS IV A n a l y s i s was c a r r i e d out as described i n Chapter I I ) . A f f i n i t y Chromatography The anti-AML monoclonal was f i r s t p u r i f i e d over DEAE-Sephacel and then coupled to Sepharose-4B using the methods described by Cuatrecasas (1970). I s o l a t i o n and E l u t i o n of Antigen by A f f i n i t y Chromatography This Sepharose-4B-anti-AML monoclonal Ab column was used to i s o l a t e band 1 (AML-Ag) from high speed supernatants of AML membrane sonicate p r e p a r a t i o n s . The column was f i r s t e q u i l i b r a t e d w i t h s e v e r a l washes of 73 b o r a t e - s a l i n e (pH 8.5). The sample (at 4 mg/ml) was loaded onto the column (7 ml column volume) and r e - c y c l e d over t h i s column f o r 30 min. Borate s a l i n e was washed e x h a u s t i v e l y over the column to remove unbound m a t e r i a l and the bound m a t e r i a l was then e l u t e d w i t h 0.1 N HC1. E l u t e d m a t e r i a l s were immediately n e u t r a l i z e d and each f r a c t i o n was monitored f o r p r o t e i n content by the Lowry technique (Lowry et a l . , 1951) or by measuring the absorbance at 280 nm of the 1.0 ml f r a c t i o n s c o l l e c t e d . Equivalent preparations of membrane e x t r a c t s from normal pooled PBL were run over the immunoadsorbent i n the same manner. I n d i v i d u a l samples from e l u t e d f r a c t i o n s were t e s t e d at various concentrations i n the ELISA f o r the d e t e c t i o n of m a t e r i a l r e a c t i v e w i t h the monoclonal antibody. Polyacrylamide Gel E l e c t r o p h o r e s i s (PAGE) E l e c t r o p h o r e s i s was performed as described i n Chapter I I except that SDS and 2-mercaptoethanol were used. The g e l was s i l v e r s t a i n e d according to the method of Wray et a l . (1981). 74 Results R e a c t i v i t y of MAL-1 i n the ELISA Eight s i n g l e - c e l l d e r i v e d monoclonal Ab-producing clones which had r e a c t i v i t y w i t h band 1 i n the ELISA were i s o l a t e d . Only one of these however, reacted c o n s i s t e n t l y s t r o n g l y and q u i c k l y ( w i t h i n 20 minutes) on ELISA p l a t e s coated w i t h band 1 or on p l a t e s coated w i t h whole membrane e x t r a c t s of AML b l a s t s . The r e a c t i v i t y of t h i s monoclonal, on an AML e x t r a c t and w i t h e x t r a c t s of the CCRF-SB-ALL-B or the CCRF-SB-ALL-T c e l l l i n e s (the preparation procedures as described i n Chapter I I ) i s shown i n Figure 9. This monoclonal ( h e r e a f t e r c a l l e d MAL-1) showed binding to band 1 Ag and to an AML c e l l e x t r a c t w i t h readable t i t r e s (absorption at -6 405 nm) to a 10 d i l u t i o n and i t showed no r e a c t i v i t y to ALL-B or ALL-T Ag e x t r a c t s even at high concentrations. I t a l s o showed no r e a c t i v i t y w i t h ELISA p l a t e s coated w i t h normal human PBL e x t r a c t s and demonstrated r e a c t i v i t y to a HL-60 (promyelocytic) c e l l l i n e e x t r a c t . R e a c t i v i t y of MAL-1 i n FACS IV Analyses To t e s t f u r t h e r the s p e c i f i c i t y of MAL-1 on these c e l l l i n e s , FACS IV a n a l y s i s experiments were c a r r i e d out. The data i n Table 17 shows these r e s u l t s , the number of c e l l s f l u o r e s c i n g w i t h the monoclonal on a HL-60 c e l l sample i s ten f o l d greater than the negative c o n t r o l (the f e r r e d o x i n monoclonal, c o n t r o l MoAb). The heterologous r a b b i t a n t i s e r a were run as c o n t r o l s f o r the experiment. The anti-AML-serum bound 43.3% of the HL-60 75 Figure 9. ELISA r e s u l t s w i t h h y b r i d to myelogenous leukemia (MAL-1). T i t r a t i o n curves of MAL-1 monoclonal Ab to myelogenous leukemia i n the ELISA. Antigen con c e n t r a t i o n was at 300 ng/ml and readings were made 45 minutes a f t e r substrate was added to the p l a t e s . A A r e a c t i o n of MAL-1 monoclonal w i t h an AML e x t r a c t , 0- 0 r e a c t i o n of MAL-1 to an ALL-T c e l l l i n e e x t r a c t , It A r e a c t i o n of MAL-1 to an ALL-B c e l l l i n e e x t r a c t . The MAL-1 monoclonal a l s o demonstrated r e a c t i v i t y to a HL-60 (promyelocytic) c e l l l i n e e x t r a c t , and no r e a c t i v i t y to normal human PBL e x t r a c t s ( r e s u l t s not shown). 1 2 3 4 5 6 7 8 9 - L O G OF DILUTION 77 Table 17. FACS analyses of human c e l l l i n e s . Number of c e l l s fluorescing with various treatments*  C e l l Anti-normal NRS Anti-AML Control MoAb** MAL-1 Population human 1. HL-60 24,680 190 16,248 2,555 21,680 (promyelocyte) 2. ALL-B 24,233 561 524 3,797 3,381 3. ALL-T 24,757 237 235 2,185 1,822 * A t o t a l of 25,000 c e l l s were analysed in each case. ** Control irrelevant monoclonal antibody. 78 c e l l s above background fluorescence. The monoclonal however, d i d not bind the ALL-B or ALL-T c e l l l i n e . S i m i l a r l y , the r a b b i t anti-AML-serum showed negative fluorescence w i t h these two ALL c e l l l i n e s . These FACS a n a l y s i s are i n agreement with the r e s u l t s found i n the ELISA using MAL-1. To t e s t the s p e c i f i c i t y of MAL-1 on c l i n i c a l m a t e r i a l a s e r i e s of experiments were c a r r i e d out using bone marrow and p e r i p h e r a l blood samples. Table 18 shows the r e s u l t s of a n a l y s i s of an AML ( i n a c t i v e disease w i t h 50% b l a s t s ) p e r i p h e r a l blood sample. With MAL-1 a considerable amount of bi n d i n g above background was observed, approximately a 6 f o l d increase i n r e l a t i v e f luorescence. As expected the anti-AML-serum bound these AML c e l l s (87.4%). The bone marrow sample from t h i s p a t i e n t a l s o e x h i b i t e d high MAL-1 b i n d i n g . A good negative c o n t r o l f o r the s p e c i f i c i t y of MAL-1 i s the normal bone marrow from a bone marrow t r a n s p l a n t donor (sample 3 i n Table 18). A one year post-AML 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 sample (#4) a l s o shows negative b i n d i n g . In a l l cases the monoclonal showed a s i m i l a r r e a c t i v i t y as the r a b b i t anti-AML-serum. Table 19 shows the r e s u l t s obtained w i t h MAL-1 on f u r t h e r t e s t i n g of AML and some CML samples. Again, as w i t h the r a b b i t anti-AML-serum, MAL-1 reacted s u b s t a n t i a l l y above background with these p e r i p h e r a l blood and bone marrow c e l l s . I t should be noted that MAL-1 reacted w i t h both AML c e l l s from p a t i e n t s i n the a c t i v e stage of the disease ( p a t i e n t samples 1 and 2) as w e l l as w i t h c e l l s from AML p a t i e n t s i n remission (samples 3 and 4). I t a l s o reacted w i t h CML c e l l s from chronic phase p a t i e n t s . The f a c t that MAL-1 reacted to remission c e l l s shows that i t s a c t i v i t y does not 79 Table 18. FACS analyses of c e l l s from acute myelogenous leukemia patients and from a normal i n d i v i d u a l . Number of c e l l s fluorescing with various treatments*  Diagnosis % blasts Anti-normal NRS Anti-AML Control MAL-1 human MoAb 1. AML-M2 50 21,183 2,122 18,791 2,684 16,860 (PBL sample) 2. AML-M2 50 17,096 2,538 17,617 2,486 13,294 (BM sample) 3. Normal BM 23,559 1,629 1,859 1,575 1,540 (a BMT** donor) 4. AML - 1 year post-BMT re c i p i e n t 0 24,903 105 2,585 300 329 (PBL sample) * A t o t a l of 25,000 c e l l s were analysed in each case. ** Bone marrow transplant. 80 Table 19. FACS analyses of c e l l s from acute and chronic myelogenous leukemia patients. Number of c e l l s fluorescing with various treatments* Diagnosis % blasts Anti-normal NRS Anti-AML Control MAL-1 human MoAb 1. APML-M3-active disease (BM sample) 15 21,987 4,266 22,500 4,103 17,602 2. APML-M3-active disease (PBL sample) 3 - - - 627 6,104 3. AML-M2-in remission (BM sample) 0 - - - 722 5,938 4. AML-M2- in remission (PBL sample) 0 20,124 1,174 10,567 2,006 3,611 5. CML (BM sample) chronic phase - - - 1,788 5,875 6. CML (PBL sample)-both granulocyte & lymphocyte populations *, 22,146 287 18,044 6,857 20,544 7. CML (BM sample) II 23,003 281 22,041 4,530 21,722 8. CML (PBL sample) II 24,703 48 18,765 534 6,934 * A t o t a l of 25,000 c e l l s were analysed in each case. 81 c o r r e l a t e w i t h the number of b l a s t c e l l s . Thus, MAL-1 shows s i m i l a r b i n d i n g p r o p e r t i e s to those e x h i b i t e d by the heterologous anti-AML r a b b i t antiserum. In Table 20 the mononuclear and granulocyte separated f r a c t i o n s of a CML p a t i e n t (method as described i n Chapter I I ) were l a b e l l e d . MAL-1 bound both the granulocyte f r a c t i o n and the mononuclear f r a c t i o n . This r e s u l t i s a l s o s i m i l a r to that found with the r a b b i t anti-AML-serum. Furthermore, as p r e v i o u s l y demonstrated w i t h the r a b b i t anti-AML-serum, MAL-1 a l s o bound the granulocyte f r a c t i o n s more than i t d i d the mononuclear sample. I t i s c l e a r that MAL-1 r e a c t s w i t h other c e l l types ( i . e . granulocytes, lymphocytes) and not only w i t h the leukemic b l a s t c e l l p o p u l a t i o n . This phenomenon was a l s o demonstrated w i t h the r a b b i t anti-AML-serum, p a r t i c u l a r l y w i t h the FACS s o r t i n g study. In Table 21, t y p i c a l normal c e l l sample r e s u l t s are shown. MAL-1 e x h i b i t e d no or very l i t t l e b inding to any of these samples. The low amount of fluorescence e x h i b i t e d by some of these c e l l s above background ( i . e . sample 3) i s probably due to n o n - s p e c i f i c s t i c k i n g to the monocyte c e l l p o p u l a t i o n . A l s o , as seen w i t h the ALL c e l l l i n e s , a bone marrow and a PBL sample from an ALL p a t i e n t showed no fluorescence. The p o s s i b i l i t y s t i l l e x i s t s , however, that MAL-1 may recognize a d i f f e r e n t i a t i o n antigen that may be expressed on only a small number of normal bone marrow c e l l s , which the FACS IV a n a l y s i s assay i s not s e n s i t i v e enough to recognize (see D i s c u s s i o n ) . 82 Table 20. FACS analyses of c e l l s from a chronic myelogenous leukemia patient. Number of c e l l s fluorescing with various treatments*  C e l l Anti-normal NRS Anti-AML Control MoAb MAL-1 Population human 1. Mononuclear 24,703 48 18,765 6,985 15,151 enriched -96% pure 2. Granulocyte 24,726 37 21,434 5,713 21,251 enriched -98% pure A t o t a l of 25,000 c e l l s were analysed in each case. 83 Table 21. FACS analyses from non-AML in d i v i d u a l s . Number of c e l l s fluorescing with various treatments* Diagnosis Anti-normal NRC Anti-AML Control MoAb MAL-1 human  1. ALL-BM** sample 18,775 3,017 2,210 1,670 1,543 99% blasts 2. ALL-PBL sample 18,460 2,898 2,610 3,480 3,642 90% blasts 3. Normal-PBL 22,121 629 302 2,776 993 sample 4. Normal-PBL 23,820 414 235 5,833 6,225 sample 5. Normal-PBL 23,442 1,615 1,166 909 1,334 sample 6. Normal BM 18,847 1,303 1,487 1,260 1,232 (for BMT) 7. Normal BM 23,840 276 330 586 427 (for BMT) 8. Hodgkin's N.D. N.D. N.D. 1,478 6,227 disease-BM sample * A t o t a l of 25,000 c e l l s were analysed in each case. ** BM = bone marrow c e l l s . 84 The monoclonal a l s o showed binding to bone marrow c e l l s of two Hodgkin's disease p a t i e n t s . In both cases the bone marrow reports were negative f o r Hodgkin's disease. The reports showed however, a marked h y p e r c e l l u l a r marrow w i t h an increased myeloid to e r y t h r o i d r a t i o , o c c a s i o n a l promyeloblasts and b l a s t c e l l s . The f a c t that there were promyeloblasts and b l a s t s present i n these two c e l l populations may be of some s i g n i f i c a n c e . I t i s of course premature t o speculate about t h i s p o s i t i v e MAL-1 b i n d i n g , which may indeed be a t o t a l l y n o n s p e c i f i c phenomenon. I t w i l l be i n t e r e s t i n g to have follow-up bone marrow samples from these p a t i e n t s . MAL-1 has shown l e s s consistency i n binding than the r a b b i t antiserum. MAL-1 showed e r r a t i c and on occasion no binding to AML c e l l s which bound the r a b b i t anti-AML-serum (data not shown). The reason f o r t h i s i s unclear. I t may be, however, that the epitope that MAL-1 i s d i r e c t e d t o , i s i n t e r n a l i z e d during a c e r t a i n phase of the c e l l c y c l e , or i t may be masked by other c e l l surface p r o t e i n s or g l y c o p r o t e i n s . To i n v e s t i g a t e the p o s s i b i l i t y that c e l l d e n s i t y or growth might have some e f f e c t on the b i n d i n g a b i l i t y of MAL-1, HL-60 c e l l s were l a b e l l e d from c e l l c u l t u r e s at d i f f e r e n t c e l l d e n s i t i e s ( i . e . a c t i v e l y d i v i d i n g , log-phase growth, and at maximum d e n s i t y ) . Table 22 shows the r e s u l t s of these FACS IV a n a l y s i s . I t i s c l e a r from these r e s u l t s that the HL-60 c e l l s expressed the epitope at a l l stages of c e l l growth. A sandwich antibody was added to the i n d i r e c t antibody technique to t r y to i n t e n s i f y and b e t t e r c l a r i f y the binding of MAL-1. As a t e s t f o r t h i s technique, MAL-1 was added to HL-60 c e l l s , then the c e l l s were 85 Table 22. FACS analyses den s i t i e s . of HL-60 c e l l s tested at d i f f e r e n t c e l l growth Number of c e l l s fluorescing with various treatments* C e l l population Anti-HLA** (tve control) Control MoAb MAL-1 1. HL-60 5 X 1QA c e l l s / m l 24,332 736 15,608 2. HL-60 5 X 10 s c e l l s / m l 22,414 1,423 14,932 3. HL-60 1.5 X 10 6 c e l l s / m l 23,009 2,001 16,101 * A t o t a l of 25,000 c e l l s were analysed in each case. ** Anti-HLA = monoclonal antibody W6/32, against the 43,000 dalton molecular weight chains of HLA-A, B and C antigens. (Barstable, C.J., Bodmer, W.F., Brown, G., Galfre, G., M i l s t e i n , C., Williams, A.F. and Z i e f l e r , A. (1978). Production of monoclonal antibodies to group A erythrocytes, HLA and other human c e l l surface antigens - New tools for genetic analysis. C e l l 14:9.) 86 sandwiched w i t h r a b b i t anti-mouse Ig (RaHIg) and f i n a l l y w i t h g o a t - a n t i - r a b b i t - F I T C . This d i d s u c c e s s f u l l y i n t e n s i f y MAL-1 bindi n g as demonstrated i n Figure 10 and Table 23. Although t h i s p a r t i c u l a r s e r i e s of experiments i n d i c a t e d that the c e l l l i n e HL-60 expressed the antigen i n a form recognized i n c e l l suspension by MAL-1 at a l l stages of i t s growth; and that surface fluorescence could be i n t e n s i f i e d using a double sandwich, these r e s u l t s were not 100% r e p r o d u c i b l e . On occasion, MAL-1 would show no bi n d i n g to HL-60 w i t h or without the double sandwich technique. This i n c o n s i s t e n c y i n c e l l surface b i n d i n g by MAL-1 was al s o observed w i t h some of the p a t i e n t m a t e r i a l w i t h which i t was t e s t e d . At t h i s time, we have no d e f i n i t i v e understanding of these observations but various p o s s i b i l i t i e s w i l l be discussed (see D i s c u s s i o n ) . I t was f e l t t h at s u f f i c i e n t evidence was a v a i l a b l e to i n d i c a t e that the epitope recognized by MAL-1 i s e x h i b i t e d on the surface of at l e a s t some myelogenous leukemia c e l l s , and does not appear to be detectable on the surface of c e l l s from normal i n d i v i d u a l s or p a t i e n t s w i t h l y m p h o p r o l i f e r a t i v e diseases. Immunochemical P r o p e r t i e s of MAL-1 MAL-1 was i d e n t i f i e d by immunodiffusion and a P r o t e i n A column as an Ig G l . In order to i n v e s t i g a t e the nature of the bindi n g p r o p e r t i e s of the monoclonal MAL-1, and the p o s s i b i l i t y that i t might bind m a t e r i a l s found i n normal c e l l s , an a f f i n i t y column was prepared i n which DEAE p u r i f i e d MAL-1 was conjugated to Sepharose-4B v i a cyanogen bromide. This immunoadsorbent column was used to absorb antigen from a membrane e x t r a c t 87 Figure 10 Dot d i s p l a y s of the sandwich f l u o r e s c e n t technique on the HL-60 c e l l l i n e . # 1 (negative c o n t r o l ) = HL-60 c e l l s + c o n t r o l MoAb + r a b b i t anti-mouse + goat a n t i - r a b b i t - FITC. # 2 ( p o s i t i v e c o n t r o l ) = HL-60 c e l l s + anti-HLA + r a b b i t anti-mouse + goat a n t i - r a b b i t - FITC. 9 3 (MAL-1 i n d i r e c t fluorescence) = HL-60 c e l l s + MAL-1 + r a b b i t anti-mouse - FITC. # 4 (MAL-1 sandwich fluorescence assay) = HL-60 c e l l s + MAL-1 + r a b b i t anti-mouse + goat a n t i - r a b b i t - FITC. 88 89 Table 23. FACS analyses of the sandwich fluorescent technique on the HL-60 c e l l l i n e . Number of c e l l s fluorei scinR with various treatments* C e l l population Ant i-HLA (tve control) Control MoAb MAL-1 1. HL-60 c e l l s i n d i r e c t assay 24,720 733 17,028 2. HL-60 c e l l s sandwich assay 23,078 2,188 24,893 A t o t a l of 25,000 c e l l s were analysed in each case. 90 prepared from b l a s t c e l l s of an AML p a t i e n t . Absorbed m a t e r i a l was e l u t e d w i t h 0.1 N HC1 i n 1.0 ml f r a c t i o n s . Each f r a c t i o n was n e u t r a l i z e d and an a l i q u o t run on reducing PAGE. The r e s u l t s , shown i n Figure 11, demonstrate that the major component e l u t i n g from the MAL-1 column present i n the tubes i s i n d i s t i n g u i s h a b l e from the band 1 AML antigen to which the hybridoma was r a i s e d . No m a t e r i a l was e l u t e d or observed on the g e l when an e q u i v a l e n t p r e p a r a t i o n of normal human PBL sonicate was run over the column. In order to f u r t h e r determine whether the MAL-1 immunoadsorbent column would react s p e c i f i c a l l y w i t h m a t e r i a l prepared from normal c e l l membranes, another experiment was run. In t h i s case, an immunoadsorbent column, c o n t a i n i n g 7.0 ml of MAL-l-Sepharose was used to absorb 2.0 ml of AML-cell membrane e x t r a c t at 7.5 mg per ml. Absorbed m a t e r i a l was e l u t e d i n 0.1 N HC1 i n 1.0 ml a l i q u o t s and n e u t r a l i z e d immediately. Over the same column, an equivalent preparation of normal c e l l membranes was absorbed. E l u t e d f r a c t i o n s were analysed f o r p r o t e i n content and each f r a c t i o n t e s t e d i n the ELISA at 500 ng per ml w i t h a standard d i l u t i o n of 1:5000 of p u r i f i e d MAL-1. The r e s u l t s (Figure 12) show that the antigen w i t h which MAL-1 r e a c t s , while being present i n the AML e x t r a c t s i s not d e t e c t a b l e by t h i s procedure i n normal c e l l e x t r a c t s . Further absorptions were c a r r i e d out using membrane e x t r a c t s from c e l l s of p a t i e n t s w i t h CML, ALL or lymphnode c e l l s from a p a t i e n t w i t h a lymphoma. Only m a t e r i a l e l u t i n g from absorptions of CML membrane e x t r a c t s gave p o s i t i v e r e a c t i v e m a t e r i a l e i t h e r i n the ELISA or on PAGE (Figure 13). 91 Figure 11. A sepharose-4B-MAL-l column used to i s o l a t e band 1 (AML-Ag). High speed c e n t r i f u g e d supernatants of AML and normal human PBL membrane sonicate preparations were run over the column. The bound m a t e r i a l was e l u t e d w i t h 0.1 N HC1. E l e c t r o p h o r e s i s was performed according to Laemmli et a l . , 1970. Lane A : low molecular weight standards. Lane B : p u r i f i e d AML antigen, band 1. Lane C : f r a c t i o n s 2-5 of AML membrane e x t r a c t s e l u t e d from the MAL-1 column Lane D : equivalent f r a c t i o n s e l u t e d from the MAL-1 column a f t e r normal PBL e x t r a c t had been passed over i t . 93 Figure 12. ELISA r e s u l t s of an a f f i n i t y p u r i f i e d AML antigen. • • r e a c t i o n of MAL-1. monoclonal with antigen e l u t e d from the Sepharose-4B column when an AML membrane sonicate was run over the column. X K r e a c t i o n of MAL-1 monoclonal w i t h m a t e r i a l e l u t e d from the Sepharose-4B column when a normal human e x t r a c t was run over i t . 95 Figure 13. ELISA r e s u l t s of an a f f i n i t y p u r i f i e d CML antigen. A A r e a c t i o n of MAL-1 monoclonal w i t h antigen e l u t e d from the Sepharose-4B column when a CML e x t r a c t was run over the column. A A r e a c t i o n of MAL-1 monoclonal with m a t e r i a l e l u t e d from the Sepharose-4B column when an ALL e x t r a c t was run over i t . Negative ELISA r e s u l t s were al s o obtained when an e x t r a c t of lymph node c e l l s of a lymphoma p a t i e n t was run over the column (not shown). 96 97 These r e s u l t s show c l e a r l y that MAL-1 recognizes and binds w e l l to the major antigen detected by the heterologous r a b b i t antiserum described p r e v i o u s l y . The immunoadsorbent studies imply that t h i s antigen i s present at r e l a t i v e l y high l e v e l s on and/or i n c e l l membranes of p a t i e n t s w i t h myelogenous leukemia but that i t i s not present at detectable l e v e l s i n e q u ivalent normal, ALL or lymphoma c e l l p r e p arations. I t should be emphasized that the t e s t s reported here do not preclude the p o s s i b i l i t y t h a t a small population of normal c e l l s produces t h i s antigen but that our methods may not be s u f f i c i e n t l y s e n s i t i v e to detect i t s presence i n normal pop u l a t i o n s . 98 D i s c u s s i o n I t has been shown that MAL-1, r a i s e d to an antigen p u r i f i e d on p r e p a r a t i v e gels from membrane e x t r a c t s of AML p a t i e n t s ' c e l l s , r eacts i n the FACS IV with a c e l l surface antigen found on c e l l s of p a t i e n t s diagnosed as having AML. This AML-associated antigen was a l s o detected by MAL-1 on the c e l l s of p a t i e n t s w i t h CML. I t d i d not appreciably react i n the FACS IV with c e l l s from normal i n d i v i d u a l s . This f i n d i n g i s i n agreement with that reported i n Chapter I I with the anti-AML-serum i n which t h i s antiserum reacted with most p a t i e n t c e l l s w i t h AML or CML and not w i t h c e l l s from normal i n d i v i d u a l s or other p a t i e n t s . In a d d i t i o n , as seen with the r a b b i t anti-AML-serum, t h i s monoclonal demonstrates p o s i t i v e fluorescence binding to the HL-60 c e l l l i n e and no binding to the ALL-B and ALL-T c e l l l i n e s . The MAL-1 monoclonal has a l s o been shown to be s p e c i f i c i n the ELISA and to be able to i s o l a t e the band 1 Ag when used on an immunoadsorbent column. The f a c t t h a t MAL-1 maintains i t s band 1 bi n d i n g c a p a c i t y on an immunoadsorbent column i s e s p e c i a l l y u s e f u l f o r i s o l a t i n g l a r g e amounts of band 1 Ag. Under equivalent c o n d i t i o n s , no such m a t e r i a l was demonstrated when e x t r a c t s from normal c e l l s were absorbed on the MAL-1 column. An ongoing study i n t h i s l a b o r a t o r y i s to c a r r y out sequencing s t u d i e s on t h i s antigen and to clone the p u t a t i v e malignancy marker gene. 99 The f a c t that MAL-1 bound to some Hodgkin's disease c e l l s i n the FACS IV study (Table 21, sample 7) i s somewhat confusing. This was not seen w i t h the r a b b i t anti-AML-serum. The fluorescence observed may be due to n o n - s p e c i f i c binding ( i . e . s t i c k y c e l l s , Fc r e c e p t o r s ) . In the absence of more extensive data on e i t h e r these p a r t i c u l a r p a t i e n t s or a l a r g e r number of Hodgkin's disease p a t i e n t s , i t i s impossible at t h i s stage to i n t e r p r e t these r e s u l t s . As pointed out i n the r e s u l t s , these p a t i e n t s had promyeloblasts present i n the bone marrow a s p i r a t e s , t h i s may have some e f f e c t on the MAL-1 binding p r o p e r t i e s . As monoclonal technology i s a r e l a t i v e l y new science, there are many things that are not as yet understood about t h e i r r e a c t i v i t y . Many monoclonals which are commercially a v a i l a b l e bind a multitude of other c e l l s and c e l l t i s s u e s as w e l l as those against which they were i n i t i a l l y s t i m u l a t e d . One problem which e x i s t s with MAL-1 i s that i t does not show c o n s i s t e n t binding p r o p e r t i e s i n terms of i t s a b i l i t y to bind to c e l l s i n suspension as determined by FACS IV analyses. The f i n d i n g that MAL-1 acts as a s u i t a b l e antibody f o r immunoadsorbent p u r i f i c a t i o n of the antigen i n d i c a t e s that MAL-1 i t s e l f has a r e l a t i v e l y strong a f f i n i t y f o r the antigen; t h e r e f o r e , i t i s u n l i k e l y that poor binding to c e l l surfaces i s a consequence of low a f f i n i t y . The determinants recognized by the r a b b i t and monoclonal reagents are probably not carbohydrate since treatment of the AML antigen w i t h mixed g l y c o s i d a s e s , or w i t h p e r i o d i c a c i d , or neuraminadase, or when reduced and a l k y l a t e d , does not a f f e c t the b i n d i n g a b i l i t y of a MAL-1 column to the AML antigen (R. Shipman, personal communication). 100 The p o s s i b i l i t y that the nature of the AML antigen i t s e l f may c o n s t i t u t e the problem must be considered. The AML antigen i s a r e l a t i v e l y h y d r o p h i l i c molecule with a molecular weight of 68,000 daltons and a p l of 7.1 - 7.2 (Shipman et a l . , B r i t i s h J . Cancer, In p r e s s ) . This p r o t e i n does not possess the expected c h a r a c t e r i s t i c s of a membrane antigen i n that i t i s r e a d i l y s o l u b l e i n aqueous s o l u t i o n s , contains a high percentage of a l i p h a t i c amino a c i d s , and has been shown to be h e a v i l y g l y c o s y l a t e d . I t i s p o s s i b l e , t h e r e f o r e , that the antigen i t s e l f , w h i l e being membrane a s s o c i a t e d , i s not deeply embedded i n the membrane. On t h i s b a s i s , i t i s p o s s i b l e to suggest that a heterologous antiserum d i r e c t e d to i t ( i . e . , the r a b b i t antiserum), r e c o g n i z i n g two or more epitopes, could e f f e c t i v e l y " f r e e z e " or f i x t h i s antigen to the c e l l s u r f a c e , thus making i t e a s i l y d e t e c t a b l e . A l t e r n a t e l y , a monoclonal antibody, such as MAL-1, while r e c o g n i z i n g the same antigen, can only bind a s i n g l e epitope, and t h i s i n i t s e l f could serve to d e - s t a b i l i z e and r e l e a s e the antigen from the membrane. Indeed, Baker et a l . (1982) have shown that AML c e l l s shed compounds i n v i t r o which can be p r e c i p i t a t e d w i t h t h e i r a n t i - m y e l o b l a s t i c serum. Further s t u d i e s , i n v o l v i n g combinations of various monoclonal antibodies d i r e c t e d to the AML antigen, w i l l address the p o s s i b i l i t y that t h i s may be o c c u r r i n g under some circumstances. A l t e r n a t e l y , i t i s p o s s i b l e that the epitope recognized by MAL-1 i s not r e a d i l y a c c e s s i b l e on the c e l l membrane, i n that i t may be masked by other membrane components. At t h i s time, i t i s impossible to define more p r e c i s e l y the problems with o b t a i n i n g reproducible membrane s t a i n i n g w i t h 101 MAL-1. Studies i n progress i n t h i s l a b o r a t o r y are to look at the d i s t r i b u t i o n of t h i s epitope by the immuno-gold s t a i n i n g method (DeWaele et a l . , 1981) and peroxidase d i r e c t s l i d e s t a i n i n g (McMillan et a l . , 1981). I t i s hoped that monoclonal an t i b o d i e s such as MAL-1 can be used as "magic b u l l e t s " f o r cancer therapy. One monoclonal, anti-M-1 which i s d i r e c t e d to a tumor s p e c i f i c antigen on the DBA/2J rhabdomyosarcoma Ml, has been coupled with the photochemical haematoporphyrin and has been shown to e x h i b i t s i g n i f i c a n t in. v i v o anti-tumor a c t i v i t y without any v i s i b l e side e f f e c t s (Mew et a l . , 1983). MAL-1 when coupled w i t h haematoporphyrin may show a s i m i l a r s p e c i f i c i t y w i t h myelogenous leukemia c e l l s , or a l t e r n a t e l y such an assay may a l l o w f o r a b e t t e r understanding of the r e a c t i v i t y p r o p e r t i e s of MAL-1. U n t i l the d i f f e r e n t i a t i o n process i s more p r e c i s e l y d e f i n e d , i t i s very d i f f i c u l t to speculate on the nature, whether i t be embryonic or d i f f e r e n t i a t i o n or malignant, of the antigen which MAL-1 recognizes. Indeed, monoclonals such as MAL-1 may e l u c i d a t e the d i f f e r e n t i a t i o n process i n haemopoietic c e l l s . MAL-1 may be employed to detect a relapse i n an AML remission p a t i e n t or relapse i n bone marrow t r a n s p l a n t p a t i e n t s . I t i s c l e a r that MAL-1 shows s p e c i f i c r e a c t i v i t y w i t h myelogenous leukemia c e l l s . The s p e c i f i c i t y of MAL-1 on ELISA p l a t e s and i t s a b i l i t y to i s o l a t e band 1 from AML b l a s t c e l l and CML c e l l e x t r a c t s and not from normal, ALL or lymphoma c e l l preparations on an immunoadsorbent column has been unequivocally shown. CHAPTER IV A Comparison of the Binding Leukemia C e l l s of the Rabbit Binding A b i l i t y of the A b i l i t y to Myelogenous Anti-AML-Serum with the MAL-1 Monoclonal 103 Summary Dis c u s s i o n In t h i s s e c t i o n , the binding a b i l i t y of MAL-1 w i l l be compared to the r e a c t i v i t y found with the r a b b i t anti-AML-serum. This comparison w i l l ,be based on the percent p o s i t i v e c e l l s r e a c t i n g w i t h MAL-1 or w i t h the r a b b i t anti-AML-serum. For the purpose of t h i s d i s c u s s i o n , i n the absence of a " p o s i t i v e c o n t r o l " f o r the monoclonal s e r i e s ( i . e . , a monoclonal which would react w i t h e s s e n t i a l l y a l l c e l l s being tested) the percent of c e l l s r e a c t i n g w i t h MAL-1 was estimated on the number of c e l l s r e a c t i n g w i t h the r a b b i t anti-normal-serum. From Table 24 i t can be seen that i n most cases, both MAL-1 and the r a b b i t anti-AML-serum reacted b e t t e r w i t h bone marrow c e l l s than they d i d w i t h PBL. Both MAL-1 and the r a b b i t anti-AML d i d r e a c t w i t h e i t h e r AML or CML p a t i e n t s ' c e l l s regardless of status (acute, chronic or r e m i s s i o n ) . The degree of r e a c t i v i t y d i d not c o r r e l a t e w i t h the number of b l a s t c e l l s present; these antibodies are c e r t a i n l y not only r e a c t i n g w i t h the b l a s t c e l l p opulations. In the FACS s o r t study, the f a c t that both a mononuclear enriched c e l l population as w e l l as a granulocyte enriched population react e q u a l l y w e l l w i t h these a n t i b o d i e s f u r t h e r support the theory of an " u b i q u i t o u s " nature f o r t h i s malignancy marker. Furthermore, the f i n d i n g s of an ongoing FACS s o r t i n g study and immunoperoxidase c e l l s t a i n i n g study demonstrate the marker on v i r t u a l l y a l l c e l l types (Dr. P a t r i c i a Logan, personal communication). These st u d i e s may indeed give a b e t t e r understanding as to the c l o n a l nature of AML. 10k Table 24. Percent p o s i t i v e fluorescence of c e l l samples from patients with myelogenous leukemia when analysed using either the monoclonal MAL-1 or the conventional rabbit anti-AML serum. Sample Diagnosis % blasts % pos i t i v e X positive with with MAL-1* Rb-anti-AML 1. bone marrow AML-M2 50 73. 4 ^100 2. PBL AML-M2 50 76. 6 98. 5 3. bone marrow APML-M3 15 75. 5 >100 4. PBL AML-M2 remission 0 9. .5 49. 5 5. bone marrow CML chronic phase 87. .6 95. .8 6. PBL CML chronic phase 26 .4 75. .9 7. PBL CML chronic phase 89 .5 81 .2 8. PBL-mono-nuclear enriched CML chronic phase 76 .0 75 .9 9. PBL-granulocyte CML chronic phase 81 .2 86 .7 enriched * Calculated upon the 100X l e v e l being the anti-normal human value (minus non-specific background). 105 With most c e l l samples the r a b b i t anti-AML reacted w i t h a higher number of c e l l s i n the population than d i d MAL-1 (Table 24). This f i n d i n g i s not s u r p r i s i n g , MAL-1 i s d i r e c t e d to only one epitope, whereas the heterologous r a b b i t antiserum would recognize many epitopes. As discussed i n Chapter I I I , MAL-1 sometimes f a i l e d to react above background l e v e l s w i t h c e l l populations of CML or AML p a t i e n t s w i t h which the r a b b i t anti-AML reacted s t r o n g l y (see Table 25). These f i n d i n g s are a l s o not r e a l l y s u r p r i s i n g i n that monoclonal antibodies are e x q u i s i t e l y s p e c i f i c regarding the epitope w i t h which they r e a c t , and i f that epitope i s masked or u n a v a i l a b l e on the c e l l s u r face, the monoclonal w i l l f a i l to r e a c t . The r a b b i t anti-AML however, would cont a i n a n t i b o d i e s of v a r i e d a v i d i t y and s p e c i f i c i t y , which may have a greater chance of binding to an antigen which may be o r i e n t e d somewhat d i f f e r e n t l y i n some c e l l s i n comparison to others. Another p o s s i b l e explanation f o r the f a i l u r e of MAL-1 to bind to c e l l s w i t h which the r a b b i t anti-AML bound may be r e l a t e d to the s t a b i l i t y of the antigen-antibody complex. Because the monoclonal can bind only one epitope per molecule, t h i s k i n d of one:one r e a c t i o n may enhance sloughing o f f of antigens whereas m u l t i p l e binding and p o s s i b l y c r o s s - l i n k i n g by conventional antiserum may s t a b i l i z e the complexes on c e l l membranes. These p o s s i b i l i t i e s are c u r r e n t l y being i n v e s t i g a t e d by examining the a b i l i t y of MAL-1 to bind c e l l u l a r antigens i n and/or on f i x e d c e l l s , using the immunoperoxidase procedure. Of course, an i d e a l s i t u a t i o n would be to have a " c o c k t a i l " of monoclonal a n t i b o d i e s , that i s a mixture of monoclonals each d i r e c t e d to d i f f e r e n t epitopes of the AML malignancy marker. This would d e f i n i t e l y y i e l d more repr o d u c i b l e r e s u l t s 106 Table 25. Percent p o s i t i v e fluorescence of c e l l samples from patients with myelogenous leukemia which only bound the conventional rabbit anti-AML serum. Sample Diagnosis % blasts % p o s i t i v e % positive with with MAL-1* Rb-anti-AML 1. bone marrow AMML-M4 85 1.0 86.7 2. PBL AMML-M4 76.3 3. PBL AML-M2 35 0.06 67.8 4. PBL CML chronic phase 65.2 * Calculated upon the 100% l e v e l being the anti-normal human value (minus non-specific background). 107 and more information as to the nature and o r i e n t a t i o n of t h i s antigen. At t h i s time, the i n a b i l i t y to demonstrate the presence of t h i s antigen on and/or i n c e l l s other than those of p a t i e n t s w i t h myelogenous leukemia does not e s t a b l i s h the e x c l u s i v e s p e c i f i c i t y of t h i s antigen. I t i s indeed h i g h l y p o s s i b l e that when s i n g l e c e l l populations are examined e x t e n s i v e l y , using more s e n s i t i v e techniques than FACS a n a l y s i s such as immunoperoxidase s t a i n i n g , we w i l l be able to determine whether a small population of c e l l s i n normal i n d i v i d u a l s or p a t i e n t s w i t h other c o n d i t i o n s r e a c t w i t h MAL-1 or the r a b b i t anti-AML. I t i s c l e a r that both the r a b b i t anti-AML and MAL-1 show s p e c i f i c r e a c t i v i t y w i t h myelogenous leukemia c e l l s . In p a r t i c u l a r , the usefulness of a monoclonal such as MAL-1 i s indeed p o t e n t i a l l y great, both as a d i a g n o s t i c and as a p o s s i b l e t h e r a p e u t i c t o o l . We are now studying the f e a s i b i l i t y of using MAL-1 to purge bone marrow of AML candidates f o r t r a n s p l a n t a t i o n w i t h no histocompatible donors. In summary, the data reported h e r e i n r a i s e i n t e r e s t i n g questions regarding the events i n v o l v e d i n leukemogenesis, and i n d i c a t e some d i r e c t i o n s i n which f u t u r e research should be c a r r i e d out. Our f i n d i n g that c e l l s of p a t i e n t s i n remission from AML react (sometimes s t r o n g l y ) w i t h both the r a b b i t antiserum and MAL-1, show that the "malignancy marker" continues to be expressed on normally d i f f e r e n t i a t i n g c e l l s . P r e l i m i n a r y experiments w i t h c e l l s o r t i n g i n d i c a t e that both lymphocytes and granulocytes are expressing the antigen. These observations at l e a s t suggest the p o s s i b i l i t y that the i n i t i a l oncogenic event g i v i n g r i s e to 108 both AML and CML occurs i n a p l u r i p o t e n t stem c e l l . The f i n d i n g that the antigen continues to be expressed on normally d i f f e r e n t i a t i n g remission c e l l s has a number of i m p l i c a t i o n s . Are remission c e l l s t r u l y "normal" or do they c o n s t i t u t e , i n some p a t i e n t s , a dominant clone of malignant c e l l s , which have been induced by chemotherapy to d i f f e r e n t i a t e normally, at l e a s t f o r a time? Is the antigen expressed on the clonogenic c e l l i n the leukemia p a t i e n t , and i f so, w i l l i t be p o s s i b l e to e l i m i n a t e the cancer c e l l s from bone marrow c e l l s of p a t i e n t s who are subjects f o r autologous marrow t r a n s p l a n t a t i o n ? Do long term remission p a t i e n t s have a d i f f e r e n t d i s t r i b u t i o n of the marker than do the ones stud i e d so f a r (we have not had access to such p a t i e n t m a t e r i a l at t h i s time)? 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( S u b m i t t e d f o r p u b l i c a t i o n i n B l o o d ) . 

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