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In vitro cytogenic studies in chronic myeloid leukemia (CML) Dubé, Ian David 1984

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IN VITRO CYTOGENETIC STUDIES IN CHRONIC MYELOID LEUKEMIA (CML) by IAN DAVID DUBE B.Sc, The University of B r i t i s h Columbia, 1977 M.Sc, The Univ e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Medical Genetics) We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF A p r i l © Ian David BRITISH COLUMBIA 1984 Dube, 1984 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 o f the r equ i r ements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Co lumb i a , 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 s tudy . 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 copy i ng o f 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 g r an t ed by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s unde r s tood t h a t copy ing 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 g a i n s h a l l no t be a l l owed w i t hou t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 1956 Main Mall Vancouve r , Canada V6T 1Y3 - i i -ABSTRACT In chronic myeloid leukemia (CML), neoplastic transformation occurs i n a si n g l e p l u r i p o t e n t hemopoietic stem c e l l . The d i f f e r e n t i a t e d progeny of t h i s clone predominate i n vivo so that i n most patients a l l the p r o l i f e r a t i n g bone marrow c e l l s bear the unique chromosomal marker of the transformed clone; the 1 Philadelphia chromosome (Ph ). This observation has r a i s e d questions about the fate of those p l u r i p o t e n t stem c e l l s that are not involved i n the leukemic clone. In t h i s work, the hypothesis that nonleukemic stem c e l l s e x i s t i n the hemopoietic system of most patients with CML but are suppressed from p r o l i f e r a -t i n g i n vivo, was tested. A method was developed that allowed from two to more than 50 metaphases to be obtained from most erythroid and granulopoietic colonies harvested i n d i v i d u a l l y . When t h i s technique was applied to myeloid colonies derived from progenitors i n marrow and blood of patients i n the chronic phase of CML, chromosomally normal, and presumably nonleukemic, hemo-p o i e t i c progenitors were detected i n a minority of p a t i e n t s . As a group, these were among those patients studied most recently a f t e r diagnosis and with lower than average white blood c e l l counts. I t i s possible that i n these cases, the leukemic clone had not expanded to such a degree so as to preclude the detection of r e s i d u a l normal progenitors based on the analysis of reasonable numbers of colonies. The f a c t that chromosomally normal progenitors were observed i n these patients at l e v e l s equivalent to that t y p i c a l of normal i n d i v i d u a l s when there was no evidence for the p r o l i f e r a t i o n of t h e i r progeny i n d i r e c t preparations, suggests that the p r o l i f e r a t i o n of nonleukemic progeni-t o r s was suppressed i n vivo. Hemopoietic progenitors from patients i n chronic phase were also cultured i n long-term marrow cultures f o r several weeks before they were removed and - i i i -assayed i n methylcellulose cultures. The long-term marrow cultures s e l e c t i v e l y maintained chromosomally normal hemopoietic progenitors i n most, but not a l l , p a t i e n t s . Using t h i s assay, i t was possible to demonstrate the existence of s i g n i f i c a n t numbers of chromosomally normal myeloid progenitors i n both newly diagnosed patients and i n patients treated for over one year. The f a c t that progenitors i n long-term cultures from a minority of patients remained 1 e x c l u s i v e l y Ph - p o s i t i v e suggests that heterogeneity e x i s t s among patients with respect to the a b i l i t y of the leukemic clone to dominate hemopoiesis _in vivo. A l t e r n a t i v e l y , some marrow aspirates may, i n f a c t , be devoid of normal progeni-t o r s . The extent to which the Ph^-positive clone dominates hemopoiesis i n the acute phase of CML was also studied. The r e s u l t s show persistence of the Ph"*-p o s i t i v e clone, even i n cases where newer sub-clones containing a d d i t i o n a l abnormalities were predominant j_n vivo. The maturational block that character-i z e s the acute phase of CML was also studied in. v i t r o . I t appeared that c e r t a i n mutations, sometimes v i s i b l e as gross chromosomal aberrations, were not compatible with normal myeloid d i f f e r e n t i a t i o n in v i t r o . F a i l u r e to detect chromosomally normal myeloid progenitors i n the acute phase of CML does not nece s s a r i l y imply that normal progenitors were absent since the chemotherapy used was designed to non-selectively k i l l both leukemic and nonleukemic c e l l s . The o v e r a l l r e s u l t s demonstrate the existence of chromosomally normal myeloid progenitors i n most patients i n the chronic phase of CML and further i l l u s t r a t e how dynamic i n t e r a c t i o n s between normal and leukemic progenitor populations may be studied _in. v i t r o . - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x i CHAPTER 1 CHROMOSOMES IN CANCER Introduction 1 The S i g n i f i c a n c e of Chromosomal Changes i n Human Cancer 20 P r a c t i c a l Applications of Cytogenetic Studies i n Cancer 25 Conclusion 27 CHRONIC MYELOID LEUKEMIA The Hemopoietic System 27 CML - A Stem C e l l Disorder 33 Cytogenetic Changes i n the Course of Ph - P o s i t i v e CML 40 Ph -Negative C e l l s i n Ph - P o s i t i v e CML 43 Present objective 45 References 47 CHAPTER 2 A METHOD FOR OBTAINING HIGH QUALITY CHROMOSOME PREPARATIONS FROM SINGLE HEMOPOIETIC COLONIES ON A ROUTINE BASIS Introduction 58 Materials and Methods 61 Results 66 Discussion 71 Summary 75 References 77 CHAPTER 3 CYTOGENETIC STUDIES OF EARLY MYELOID PROGENITOR COMPARTMENTS IN CHRONIC PHASE Ph -POSITIVE CML. METHYLCELLULOSE ASSAYS REVEAL THE PERSISTENCE OF Ph -NEGATIVE COMMITTED PROGENITORS THAT ARE SUPPRESSED FROM DIFFERENTIATING IN VIVO Introduction 80 Materials and Methods 85 Results 87 Discussion 92 Summary 99 References 101 - V -CHAPTER 4 CYTOGENETIC STUDIES OF EARLY MYELOID PROGENITOR COMPARTMENTS IN CHRONIC PHASE Ph -POSITIVE CML. LONG-TERM CULTURE REVEALS Ph -NEGATIVE COMMITTED PROGENITORS IN A MAJORITY OF PATIENTS Introduction 105 Materials and Methods 107 Results 111 Discussion 117 Summary 129 References 130 CHAPTER 5 CYTOGENETIC STUDIES OF EARLY MYELOID PROGENITOR COMPARTMENTS IN ACUTE PHASE Ph -POSITIVE CML. METHYLCELLULOSE ASSAYS REVEAL PERSISTENCE OF Ph -POSITIVE COMMITTED PROGENITORS Introduction 133 Materials and Methods 135 Results 137 Discussion 146 Summary 151 References 153 CHAPTER 6 SUMMARY AND CONCLUSION 155 APPENDIX IA Procedure f o r the Preparation of Hemopoietic Stem C e l l s f o r P l a t i n g from Fresh Marrow Aspirate 163 APPENDIX IB Procedure f o r the Preparation of Hemopoietic Stem C e l l s f o r P l a t i n g from Peripheral Blood 164 APPENDIX IIA Procedure f o r the Preparation of the Culture Medium f o r Hemopoietic Stem C e l l s 165 APPENDIX IIB Preparation of Methylcellulose 166 APPENDIX IIC Preparation of Standard PHA Conditioned Media 167 APPENDIX I I I Chromosome Banding Procedures 168 APPENDIX IV Procedures f o r Obtaining Bone-marrow Metaphases 171 APPENDIX V Procedure f o r Obtaining Metaphase Chromosomes from Peripheral Blood 173 APPENDIX VI Procedure f o r Obtaining Metaphase Chromosomes APPENDIX VII Procedure f o r D i f f e r e n t i a l Staining of Metaphase 174 APPENDIX VIII Procedures f o r I n i t i a t i o n , Maintenance and Analysis of Long-term .Marrow Cultures 176 - v i -APPENDIX IX APPENDIX X Cytogenetic Nomenclature Ode to the Colony Plucker - v i i -LIST OF TABLES Page TABLE I Percentage of c e l l s recovered when hemopoietic c e l l s grown i n standard methylcellulose cultures were har-vested f o r chromosomes using s l i d e s pretreated with p o l y l y s i n e . 64 TABLE II Results of karyotype analysis performed on metaphases obtained from si n g l e e r y t h r o i d colonies harvested from cultures containing colonies of both male and female o r i g i n . 67 TABLE III SCE frequency i n metaphases from e r y t h r o i d colonies harvested from 1- to 2-week-old methylcellulose cultures. Also shown for comparison are SCE frequencies i n epo-stimulated fresh marrow, PHA-stimulated peripheral blood, and skin f i b r o b l a s t s . Bloom syndrome values serve as a p o s i t i v e c o n t r o l . 72 TABLE IV Outcome of karyotype analysis performed on sing l e hemo-p o i e t i c colonies grown from: (a) a peripheral blood sample from a 40-year-old male with the c l i n i c a l diagnosis of polycythemia vera; (b) a perip h e r a l blood sample from a 47-year-old female with the c l i n i c a l diagnosis of chronic myelogenous leukemia. 73 TABLE V Results of cytogenetic studies of hemopoietic progenitors i n 7 patients with untreated, newly diagnosed Ph - p o s i t i v e CML i n whom no normal progenitors were found. 89 TABLE VI Results of cytogenetic studies i n 10 patients with treated, previously diagnosed, Ph - p o s i t i v e CML i n whom no normal progenitors were found. 90 TABLE VII Results of cytogenetic studies of hemopoietic progenitors i n 4 patients with Ph - p o s i t i v e CML i n whom normal progenitors were detected. 1 TABLE VIII Expected and observed Ph -negative BFU-E assuming persistence of normal progenitors at normal l e v e l s . 91 97 TABLE IX C l i n i c a l and hematological data f o r the nine cases included i n the long-term culture study. 1 TABLE X Ratios of cyto g e n e t i c a l l y normal:Ph - p o s i t i v e metaphases i n d i r e c t bone marrow aspirates and r a t i o s of c y t o g e n e t i c a l l y normal:Ph - p o s i t i v e colonies i n methylcellulose assays before and af t e r long-term culture of progenitors. 1 1 TABLE XI Number of Ph - p o s i t i v e and Ph -negative p r i m i t i v e BFU-E i n the adherent layer of long-term CML cultures. 108 113 116 - v i i i -Page TABLE XII C l i n i c a l and hematological data on the 6 patients studied i n the acute phase. 138 TABLE XIII Results of cytogenetic studies on hemopoietic progenitors i n 6 patients i n the acute phase. 141 1 TABLE XIV Percentage of c e l l s bearing the Ph -chromosome, with or without a d d i t i o n a l chromosomal abnormalities, among marrow metaphases and myeloid progenitors f o r the 6 patients studied i n the acute phase. 147 - i x -LIST OF FIGURES FIGURE 1 Schematic i l l u s t r a t i o n of non-random chromosome changes occurring i n P h i l a d e l p h i a - p o s i t i v e CML. A survey of 67 cases. Numbers i n parentheses i n lower part of diagram i n d i c a t e number of cases showing each karyotypic v a r i a n t . (From Reference 13.) Page FIGURE 2 Histograms showing the percentage d i s t r i b u t i o n of chromo-some aberrations i n v o l v i n g the d i f f e r e n t human chromosomes i n 86 cases of PV, 496 cases of AML, 30 cases of CLL, and 26 cases of EL. (From Reference 37.) 14 FIGURE 3 Outline of hemopoiesis as defined by colony assays of stem c e l l s and committed progenitors. 28 FIGURE 4 Schematic i l l u s t r a t i o n of the methodology used i n c u l t u r -i n g hemopoietic colonies from blood and marrow progenitors i n semi-solid media. 31 FIGURE 5 Schematic i l l u s t r a t i o n showing the involvement of the Phil a d e l p h i a chromosome i n the hemopoietic lineages. Numbers r e f e r to references. 37 FIGURE 6 A schematic i l l u s t r a t i o n demonstrating the r a t i o n a l e of using clonogenic assays f o r p r i m i t i v e precursor c e l l s . 60 FIGURE 7 A G-banded metaphase and karyotype obtained from a single e r y t h r o i d colony. 68 FIGURE 8 A d i f f e r e n t i a l l y stained metaphase from a s i n g l e eryth-r o i d colony. 69 FIGURE 9 A chromomycin A3-methyl green reverse banded metaphase from a granulocyte colony from a male with Ph -p o s i t i v e CML. 70 FIGURE 10a A graph showing the elevation i n the number of erythroid progenitors as a function of the white blood c e l l count. 94 FIGURE 10b A graph showing the elevation i n the number of granulo-cyte progenitors as a function of the white blood c e l l count. 95 FIGURE 11 Schematic i l l u s t r a t i o n of the methodological approach used i n Chapter Four. 110 FIGURE 12a Nucleated c e l l s per non-adherent layer as a function of the number of weeks i n long-term culture. Numbers r e f e r to patients. 118 FIGURE 12b Granulocyte progenitors per non-adherent layer as a function of the number of weeks i n long-term cu l t u r e . Numbers r e f e r to pat i e n t s . FIGURE 12c Granulocyte progenitors per adherent layer as a function of the number of weeks i n long-term cu l t u r e . Numbers r e f e r to patients. FIGURE 12d Mature e r y t h r o i d progenitors per non-adherent layer as a function of the number of weeks i n long-term c u l t u r e . Numbers re f e r to pat i e n t s . FIGURE 12e Mature e r y t h r o i d progenitors per adherent layer as a function of the number of weeks i n long-term cu l t u r e . Numbers r e f e r to pat i e n t s . FIGURE 12f P r i m i t i v e e r y t h r o i d progenitors per non-adherent layer as a function of the number of weeks i n long-term culture. Numbers r e f e r to pa t i e n t s . FIGURE 12g Primitive e r y t h r o i d progenitors per adherent layer as a function of the number of weeks i n long-term c u l t u r e . Numbers r e f e r to patients. FIGURE 12h Prim i t i v e e r y t h r o i d progenitors (giving r i s e to more than 8 cl u s t e r s ) per adherent layer as a function of the number of weeks i n long-term c u l t u r e . Numbers r e f e r to pa t i e n t s . - x i -ACKNOWLEDGEMENTS I wish to express my sincere appreciation : to my supervisor, D r . C J . Eaves (Department of Medical Genetics, the Uni v e r s i t y of B r i t i s h Columbia and the Terry Fox Laboratory, B.C. Cancer Research Centre) and to Dr. D.K. Kalousek (Departments of Medical Genetics and Pathology, The Un i v e r s i t y of B r i t i s h Columbia) f o r t h e i r enthusiastic support and c r i t i c a l guidance throughout my project; to Dr. A.C. Eaves (Director, Terry Fox Laboratory; Departments of Medicine and Pathology, The Univ e r s i t y of B r i t i s h Columbia) f o r h i s enthusiasm and help i n obtaining s u i t a b l e patient material; to members of my Advisory Committee - Dr. P. Baird (Head, Department of Medical Genetics), Dr. F. D i l l (Department of Medical Genetics) and Dr. T. G r i f f i t h s (Department of Botany); to Dr. Laure Coulombel f o r our provocative discussions and to the s t a f f and students of the B.C. Cancer Research Centre for providing an i n v i g o r a t i n g s c i e n t i f i c environment i n which to study; to Josephine Siu, Dr. C. Gupta, Dianne Reid, Ann-Marie McDougall, Marjorie Hutchison, Agnes Leung, Fred Bauder and Nancy Norris f o r expert t e c h n i c a l assistance; to Betty Andres f o r t i r e l e s s s e c r e t a r i a l assistance during the preparation of t h i s t h e s i s ; and f i n a l l y , to the National Cancer I n s t i t u t e of Canada f o r t h e i r f i n a n c i a l support. - x i i -Good Work; F i r s t , to provide necessary and u s e f u l goods and services. Second, to enable everyone of us to use and thereby perfect our g i f t s l i k e good stewards. Third, to do so i n service to, and i n cooperation with others, so as to l i b e r a t e ourselves. E.F. Schumacher, author of Small i s B e a u t i f u l 1 C H A P T E R O N E CHROMOSOMES IN CANCER INTRODUCTION Almost 70 years ago, Theodor Boveri's t r e a t i s e on the question of the o r i g i n of malignant tumors was published.^ Boveri understood that the chromosomes control the l i f e of the c e l l , i t s functions, and i t s a b i l i t y to div i d e . He r e a l i z e d that "hereditary u n i t s " are located i n the chromosomes and that an equilibrium among them i s a p r e r e q u i s i t e f o r the normal function of c e l l s and therefore of the en t i r e organism. He hypothesized that the c e l l of a malignant tumor has an abnormal chromosome c o n s t i t u t i o n and that any event leading to an abnormal chromosome c o n s t i t u t i o n w i l l r e s u l t i n a malignant 2 tumor. This hypothesis, put forward over 40 years before advances i n cytogenetic methodology would permit a s c i e n t i f i c study of the problem, has become a cornerstone i n the advancing f i e l d of cancer cytogenetics. Boveri's hypothesis provided the o r i g i n a l impetus f or the study of chromosomes i n cancer. To evaluate and t e s t Boveri's hypothesis, many inve s t i g a t o r s chose to work with human cancerous t i s s u e . However, i t was not u n t i l the mid 1950's that reproducible methods for obtaining well-spread metaphases from mammalian c e l l s were developed, thereby allowing e x i s t i n g controversies about the correct 3 number of chromosomes i n normal human c e l l s to be conclusively l a i d to r e s t . Cytogenetic data on human c e l l s before 1956 must therefore be in t e r p r e t e d with caution. The observations during the years 1956-1970, p r i o r to the development of banding techniques that permit accurate chromosome i d e n t i f i c a t i o n , i n d i c a t e d that the number of chromosomes and the chromosome pattern i n cancer c e l l s was 2 quite v a r i a b l e and thus the notion that chromosome changes were random, i r r e l e v a n t , and secondary phenomena seemed to be supported by the a v a i l a b l e data. There was, however, one notable exception. In 1960, Nowell and Hungerford reported the f i r s t consistent chromosome abnormality i n human 4 cancer. They observed an unusually small chromosome i n leukemic c e l l s from two patients with chronic myeloid leukemia (CML). This chromosome, which appeared to have l o s t about one-half of i t s long arm, i s now known as the 1 Philadelphia (Ph ) chromosome i n recognition of the c i t y where i t was f i r s t observed. Approximately 85% of patients with c l i n i c a l l y t y p i c a l CML were found to have the Ph^ chromosome i n t h e i r leukemic bone marrow c e l l s while remaining 5 6 k a r y o t y p i c a l l y normal i n a l l other c e l l types. ' In the course of the l a s t decade, widespread use of new and improved cytogenetic techniques has r e s u l t e d i n the accumulation of large amounts of data on the chromosomal c o n s t i t u t i o n of tumors. An overview of some s i g n i f i c a n t findings w i l l now be given. Marker Chromosomes and Karyotype Evolution as Features of Malignancy I t i s now apparent that many human tumors show deviations from the normal 7 d i p l o i d number of chromosomes. In general, two s t r i k i n g features of tumor c e l l s are the presence of marker chromosomes, and a tendency towards a s p e c i f i c pattern of karyotype evolution. Chromosomal markers are the r e s u l t of rearrangements that give r i s e to new chromosomes not found i n normal c e l l s . In some cases, the marker chromo-some can be i d e n t i f i e d i n terms of the chromosomes that gave r i s e to i t , but i n others t h i s i s not p o s s i b l e . While markers are very commonly present i n tumors with hypodiploid chromosome numbers, t h e i r frequency tends to be lower i n hyperdiploid and d i p l o i d tumors. For example, i n one study markers were present i n each of 11 hypodiploid ovarian carcinomas, and i n 18 out of 21 with 3 modal numbers of 46 or more. S i m i l a r l y , they were present i n each of 12 hypodiploid c e r v i c a l carcinomas but i n only eight of 18 tumors with modes of 46 g or more. In a more recent report, karyotypic analysis of 189 cases of c a r c i n -omas of the bladder revealed marker chromosomes i n 65 out of 85 preparations 9 that y i e l d e d analyzable metaphases. In general, using currently a v a i l a b l e techniques, marker chromosomes are found i n tumor c e l l s from about 50% of a l l c a n c e r s J " Their morphology and number d i f f e r from case to case and even among metaphases from the same tumor. Karyotype evolution, a stepwise rearrangement of the karyotype occurring i n an apparently ordered fashion, i s a second common feature of tumor karyotypes. Each i n d i v i d u a l tumor appears to vary with respect to the d i r e c t i o n and extent of any change i n karyotype. The o v e r a l l tendency, however, sometimes becomes clear when the average extent to which chromosomes of each type are under- or overrepresented i n a s e r i e s of tumors i s calculated. An analysis of the chromosomal data obtained i n a series of patients with 1 chronic myeloid leukemia (CML), who also expressed a s p e c i f i c marker, the Ph chromosome, suggested that the c l i n i c a l progression of CML i s usually accompanied by concomitant progression of the karyotype, and that the l a t t e r may r e f l e c t prognostic aspects of the disease. The Ph^-positive condition i n which the Ph^-chromosome i s the only karyotypic aberration, appears to be an 1 e a r l y manifestation of CML. C l i n i c a l progression of the chronic phase of Ph -p o s i t i v e CML i s accompanied by gradual progression of the cytogenetic p i c t u r e , 11 12 e s p e c i a l l y i n the more terminal b l a s t i c stage of the disease. ' Mitelman and co-workers summarized the sequential changes i n 67 cases of 13 1 CML studied with chromosome banding. They found that the o r i g i n a l Ph carrying c e l l l i n e underwent various a d d i t i o n a l chromosome changes, three of which were predominant: 4 (1) an a d d i t i o n a l Ph chromosome i n 18 cases (27%), (2) an a d d i t i o n a l chromosome #8 i n 7 cases (10%), and (3) s u b s t i t u t i o n of an isochromosome f o r the long arm of chromosome #17 i n 9 cases ( 13%) . One or more of these changes were found i n 59 of the 67 cases (88%) i n which secondary changes occurred. The findings are summarized i n Figure 1. Meningiomas provide a group of tumors i n which the phenomenon of karyo-t y p i c evolution has been r e l a t i v e l y well studied. In h i s summary of the cyto-genetic findings i n over 200 meningiomas, Sandberg suggested that the i n i t i a l step i s usually the los s or deletion of a chromosome #22, and the subsequent steps a f f e c t chromosomes #8 and #9 i n group C ( i . e . , #6 to #12) and #14 and #15 14 i n group D ( i . e . , #13 to #15) i n p a r t i c u l a r , and also #1. A possible explan-ation f o r the evolution of karyotypic changes i n meningioma could be the following: normal d i p l o i d stem l i n e ( a l l karyotypes normal)—* appearance of a new var i a n t stem l i n e with a growth advantage and showing monosomy f o r chromo-some #22 (mixture of normal karyotypes and karyotypes with monosomy 22 —> increase i n the r e l a t i v e proportion of c e l l s with monosomy 22) —* subsequent karyotypic deviations with accompanying increase i n growth advantage (mixture of monosomy 22 with c e l l s bearing a d d i t i o n a l chromosomal changes). A C l a s s i f i c a t i o n of Chromosomal Changes i n Cancer In surveying the chromosome aberrations reported i n human tumor c e l l s , one i s struck by the heterogeneity of the chromosomal changes that can be associated with malignancy. Every chromosome has been described as being involved i n one or another type of karyotypic anomaly (translocation, deletion, d u p l i c a t i o n , trisomy, monosomy, e t c . ) . The s t r u c t u r a l l y changed chromosomes, the markers, a l l have undergone one or more chromosome breaks i n t h e i r develop-5 Norma! 46,Ph 1 Further karyotype changes Major route (88%) Minor routes (1296) 2 Ph 1 de) *8 (7! i(17q)(9) 2Ph1+8.i(17q)(6) Figure 1. Schematic Illustration Ot Non-Random Chromosome Changes O c c u r r i n g In Phi ladelphia-Posi t ive CML. ASurvey Ot 67 Cases . Numbers In Parentheses In Lower Part Of Diagram Indicate Number Of Cases Showing Each Karyotypic Var iant . (From Reference 13.) 6 ment. The breakages required for the development of observed marker chromo-somes appear to be of two types: e i t h e r ( i ) a breakage occurs co n s i s t e n t l y i n one or two places i n the karyotype so that i n many cases the marker chromosomes found i n the same type of tumor i n d i f f e r e n t patients are a l i k e , or ( i i ) the breakpoints are highly v a r i a b l e , thereby g i v i n g r i s e to d i f f e r e n t marker chromosomes i n d i f f e r e n t primary tumors of a given type. Observations of t h i s kind l e d Levan and Mitelman to hypothesize that chromosome aberrations associated with malignancy could be of 2 d i s t i n c t types, "15 16 each with a d i f f e r e n t mode of o r i g i n . ' According to t h i s hypothesis, one kind of aberration, the primary or active type, was viewed as a d i r e c t r e s u l t of the i n t e r a c t i o n of the ultimate carcinogen with the genetic material of the target c e l l . Primary aberrations would therefore be expected to be r e s t r i c t e d 1 to s p e c i f i c chromosome regions, the Ph chromosome being a t y p i c a l example. * The other kind of aberration, the secondary or passive type, was viewed as a r i s i n g by a chance disturbance of mitosis. Only those secondary changes that happen to enhance the e f f e c t of the primary changes are thought to have s e l e c t i v e value and, thus, the a b i l i t y to accumulate i n the population. The passive changes would, therefore, be expected to also e x h i b i t d i s t i n c t non-random patterns that r e f l e c t the primary and subsequent changes. This framework f o r the c l a s s i f i c a t i o n of chromosomal changes i n cancer w i l l now be used i n a discussion of the chromosome changes seen i n human cancer. Examples of hematological malignancies w i l l be used wherever possi b l e , since they are more relevant to the o v e r a l l t h e s i s . (a) Primary or Active Chromosome Aberrations ( i ) Somatic C e l l Aberrations. Chronic myeloid leukemia (CML), Bur k i t t ' s Lymphoma, meningioma, and retinoblastoma, are diseases i n which s p e c i f i c chromosomal aberrations have been repeatedly described i n the same (-.* See notation on page 19. ) 7 type of malignant c e l l from d i f f e r e n t i n d i v i d u a l s . CML i s a neoplastic disease of the marrow i n which the major c l i n i c a l manifestations r e l a t e to an abnormal overproduction of granulocytes. (A more d e t a i l e d discussion of t h i s disease i s given below.) The majority (>85%) of CML patients have a hemato-1 " 5 6 1 p o i e t i c (blood forming) c e l l l i n e that expresses the Ph . ' The Ph chromo-some i s r e s t r i c t e d to the hematopoietic c e l l s since other body tissues from 1 17 18 Ph - p o s i t i v e CML patients appear to be chromosomally normal. ' In the e a r l y 1970's, banding techniques were introduced and immediately applied to the cytogenetic study of leukemic c e l l s . One of the f i r s t important 1 observations was the c h a r a c t e r i z a t i o n of the Ph chromosome as a deletion of 19 20 #22. ' Soon a f t e r , a more complete p i c t u r e emerged when Rowley reported that the Ph^ chromosome was the r e s u l t of a t r a n s l o c a t i o n rather than a 2 1 de l e t i o n . She proposed that the abnormal chromosome of CML was the r e s u l t of an apparently balanced r e c i p r o c a l t r a n s l o c a t i o n i n which material deleted from the end of the long arm of one #22 was t r a n s f e r r e d to the end of the long arm of one #9. In no other known neoplastic condition i s there a stronger case for the d i r e c t involvement of chromosomal change i n the development of the 1 malignant state. This i s based on the consistency with which the Ph i s found i n the involved c e l l s even at early stages of the disease, and the p e r s i s t e n t 1 presence of the Ph chromosome throughout the course of t h i s disease. I t should, however, be noted that although the Ph^ chromosome i s reputed to be almost diagnostic f or CML, i t i s observed, at a much lower incidence, i n other 22 r e l a t e d but d i s t i n c t c l i n i c a l e n t i t i e s i n c l u d i n g acute lymphocytic leukemia, 23 24 acute myeloblastic leukemia, polycythemia vera, and e s s e n t i a l thrombocy-25 t o s i s . The f a c t that i n these other diseases, the hematopoietic system i s also involved r a i s e s the p o s s i b i l i t y that the Ph^ chromosome i s not as s p e c i f i c for CML as i s conventionally held but may be more generally i n d i c a t i v e of 8 transformation i n a plu r i p o t e n t hematopoietic stem c e l l . In B u r k i t t ' s lymphoma, B-lymphocytes occur i n s o l i d tumors that a r i s e predominantly i n the f a c i a l (African type) or abdominal (non-African type) regions. In t h i s malignancy, there also i s evidence for a highly s p e c i f i c chromosome defect i n the malignant c e l l s . Two c h a r a c t e r i s t i c marker chromo-somes seen i n B u r k i t t ' s lymphoma are: (1) chromosome #8 deleted f o r a s p e c i f i c part of the long arm, and (2) chromosome #14 with a d d i t i o n a l material added to i t s long arm. In one report of 32 cases studied by chromosome banding, 23 out of 24 A f r i c a n B u r k i t t ' s lymphoma biopsies and c e l l l i n e s had one or both of the 2 6 c h a r a c t e r i s t i c markers. Twenty-one of the cases were due to a s p e c i f i c t r a n s l o c a t i o n i n v o l v i n g chromosomes #8 and #14. Four out of 9 non-African type Bu r k i t t ' s lymphomas were also p o s i t i v e for the same t r a n s l o c a t i o n . Cases of Bu r k i t t ' s lymphoma have been described i n which the t r a n s l o c a t i o n partner of 22 chromosome #8 i s not #14 and i t appears that the important change i s the dele t i o n of a s p e c i f i c region i n chromosome #8. (See below f o r a more complete discussion of the s i g n i f i c a n c e of t h i s translocation.) Meningiomas are tumors composed of s p e c i a l i z e d arachnoidal l i n i n g c e l l s . Chromosomal preparations of d i r e c t or cultured tumor material from over 200 meningiomas in d i c a t e that these tumors have a remarkable tendency to develop hypodiploid stem l i n e s . The i n i t i a l step i s usually a loss or deletion of 14 chromosome #22. Retinoblastoma i s a malignant eye tumor of c h i l d r e n which i s i n h e r i t e d i n some cases. One cytogenetic study of 5 patients with nonhereditary r e t i n o -blastoma showed normal karyotypes i n mitoses from PHA stimulated per i p h e r a l blood, but revealed a s p e c i f i c chromosomal abnormality i n c e l l s derived from the tumor. Hashem and K h a l i f a reported that i n a l l 5 cases, abnormal chromo-somes were discovered i n some of the mitoses prepared d i r e c t l y from the tumor 9 27 c e l l s . In 4 of the 5 patients, a consistent abnormality was the deletion of the long arm of one of the D group chromosomes ( i . e . , #13, #14 or #15). Recently, a more d e t a i l e d cytogenetic study of tumor t i s s u e i n patients with retinoblastoma and chromosomally normal lymphocytes was performed. In 5 out of 6 cases, Balaban and co-workers found a d e l e t i o n or rearrangement i n v o l v i n g the same s p e c i f i c chromosomal band i n the long arm of chromosome 28 #13. A s i m i l a r f i n d i n g was reported i n 2 out of 10 tumors studied by Gardner 29 and co-workers. These four examples (Ph 1 - p o s i t i v e CML, Burkitt's lymphoma, meningioma, and nonhereditary retinoblastoma) are good examples of human neoplastic conditions i n which the malignant c e l l s have c h a r a c t e r i s t i c chromosomal markers. The cytogenetic findings i n each case suggest that s p e c i f i c chromosomal regions play a r o l e i n the development of s p e c i f i c tumor types. Such s p e c i f i c i t y has also been demonstrated i n murine thymomas. Chan and colleagues used chromosome banding techniques to study the karyotypes of tumor c e l l s from thymic lymphomas induced by three d i f f e r e n t carcinogens: X-i r r a d i a t i o n , p o l y c y c l i c aromatic hydrocarbons and endogenous leukemogenic 30 v i r u s . Newborn mice of two d i f f e r e n t s t r a i n s were used. Of a t o t a l of 89 tumors studied, 76 (85%) were characterized by trisomy for chromosome #15. (Thymus, spleen and bone marrow c e l l s were k a r y o t y p i c a l l y normal i n these and/or control animals). The remaining 13 tumors were k a r y o t y p i c a l l y normal. The r e s u l t s obtained were independent of the carcinogenic agents and the s t r a i n of mouse used. In a more recent report, cytogenetic studies on T c e l l lympho-mas induced i n mice car r y i n g t r a n s l o c a t i o n chromosomes i n v o l v i n g centromeric fusion of chromosome #15 with chromosome #1, #5 or #6, also suggested that d u p l i c a t i o n of the gene(s) located on chromosome #15 i s of c r i t i c a l 31 importance. Thus, i n these Robertsonian translocations, even large chromo-10 somes such as #1 were forced i n t o a state of trisomy along with chromosomes #15 i n a s i g n i f i c a n t number of cases. These f i v e examples of conditions i n which unique chromosomal defects appear to be associated with p a r t i c u l a r malignancies i n otherwise chromosomally normal i n d i v i d u a l s have been invoked as support for the hypothesis that a s p e c i f i c a l t e r a t i o n i n the genome of a somatic c e l l , observable as a chromo-somal change, may be a primary event i n transformation. ( i i ) Germ C e l l Aberrations. Hereditary renal c e l l carcinoma, hereditary retinoblastoma, and Wilms tumor associated with a n i r i d i a are examples of diseases i n which i n d i v i d u a l s with a s p e c i f i c germ c e l l mutation, v i s i b l e at the chromosomal l e v e l i n a l l c e l l s , are predisposed to a s p e c i f i c type of malignancy. Hereditary renal c e l l carcinoma i s a malignancy of the kidney. In a unique report, Cohen and h i s associates described a family i n which members with an i n h e r i t e d chromosomal t r a i t were predisposed to renal 3 2 cancer. An apparently balanced t r a n s l o c a t i o n between chromosomes #3 and #8 was found i n per i p h e r a l leukocytes i n a l l eight of the patients with renal cancer who were karyotyped. No family member with a normal karyotype had renal cancer. The t r a n s l o c a t i o n was transmitted to approximately one-half of the l i v i n g o f f s p r i n g through three generations. The development of renal cancer i n such heterozygous persons with the t r a n s l o c a t i o n followed an autosomal dominant pattern of inheritance. The f i n d i n g of a del e t i o n of the long arm of chromosome #13 i n tumor c e l l s from chromosomally normal i n d i v i d u a l s with retinoblastoma has been noted above. I n t e r e s t i n g l y , i n a minority of retinoblastoma patients, cytogenetic analysis of p e r i p h e r a l leukocytes and f i b r o b l a s t s has revealed i n these c e l l s a l so the del e t i o n of a small but s p e c i f i c segment of the long arm of chromosome 33 # 1 3 . This f i n d i n g suggests that a p a r t i c u l a r region on chromosome #13 may 11 carry the dominant mutation i n some instances of f a m i l i a l retinoblastoma, and that the genes involved at the somatic l e v e l are the same as those involved at the germ c e l l l e v e l . (See below f o r a more in-depth discussion of t h i s concept). Wilms tumor i s a kidney cancer that occurs i n c h i l d r e n . I t i s sometimes associated with a n i r i d i a (lack of i r i s e s ) and i n these cases a consistent chromosomal abnormality has been found i n chromosome #11. There are now a s i g n i f i c a n t number of cases (>18) i n the l i t e r a t u r e i n which a del e t i o n of a s p e c i f i c band i n chromosome #11 has been reported to e x i s t i n nontumor tiss u e 34 35 36 (lymphocytes and/or f i b r o b l a s t s ) from these p a t i e n t s . ' ' These examples of hereditary retinoblastoma, hereditary renal c e l l carcinoma, and Wilms tumor, are good examples of instances i n which a s p e c i f i c primary chromosomal event occurring p r e z y g o t i c a l l y appears to predispose the i n d i v i d u a l to develop a s p e c i f i c tumor. (b) Secondary or Passive Chromosome Aberrations Polycythemia vera (PV), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL) are t y p i c a l and r e l a t i v e l y well studied examples of neoplasms where cytogenetic studies have revealed evidence of secondary or passive chromosome aberrations. The hetero-geneous cytogenetic changes seen i n these diseases are i n s t r i k i n g contrast to the s p e c i f i c i t y of the primary or active type of aberrations. These are summarized i n a survey of previously published and unpublished cases where cytogenetic findings f o r 86 PVs, 496 AMLs, and 30 CLLs i n which karyotypes were 37 studied with banding were reported. Polycythemia vera (PV) i s a chronic myeloproliferative disease i n which a single hyperplastic clone with a p r o l i f e r a t i v e advantage takes over the bone marrow. The disorder i s characterized c l i n i c a l l y p r i m a r i l y by an increased 12 red c e l l mass, which i s frequently accompanied by increased numbers of c i r c u l -a t i n g p l a t e l e t s and/or granulocytes. In 10-15% of PV patients, the clone undergoes fur t h e r changes r e s u l t i n g i n a c l i n i c a l p i c t u r e s i m i l a r to that seen 38 i n acute leukemia (see below). The incidence of c l o n a l chromosome abnormalities i n bone marrow c e l l s of patients with PV v a r i e s with the stage of the disease. Fewer than 15% of patients have abnormalities p r i o r to therapy with cytotoxic agents. A higher incidence of 35-40% has been reported for early samples from treated 39 p a t i e n t s . The chromosomes most commonly involved i n aberrations are numbers 1,8,9 and 20. The most d i s t i n c t i v e feature of t h i s disease i s a de l e t i o n of a part of the long arm of chromosome #20 which has been i d e n t i f i e d i n one quarter 40 of the cases with aberrations. The percentage d i s t r i b u t i o n of chromosome aberrations i n v o l v i n g the d i f f e r e n t human chromosomes i n 86 cases of treated and untreated PV studies with chromosome banding are shown i n Figure 2A. The acute leukemias are progressive, malignant diseases of the hematopoietic t i s s u e which, i f untreated, terminate f a t a l l y i n about 6 months. 41 These diseases are also c l o n a l and are characterized c l i n i c a l l y by the presence of large numbers of immature and abnormal blood c e l l s i n the c i r c u l a t -i n g blood and/or bone marrow. As the diseases progress, production of normal blood c e l l s i s commonly decreased. Abnormal blood c e l l s i n f i l t r a t e and replace or damage to v a r i a b l e degrees the bone marrow, the lymph nodes, spleen and other organs i n c l u d i n g the c e n t r a l nervous system. The acute leukemias can be further subdivided on the basis of morphological and biochemical properties of the malignant c e l l s . In most patients with acute myeloblastic leukemia (AML), some morphologic evidence of maturation toward one or more of the myeloid c e l l l i n e s can be detected. In most patients with acute lymphoblastic leukemia (ALL), the malignancy r e s u l t s from the abnormal growth of lymphocytes of the B-13 c e l l or T - c e l l lineage. Chromosomal abnormalities i n bone marrow c e l l s from untreated patients with AML have t y p i c a l l y been found i n approximately 50% of cases, although one study reported an incidence of 100% when a more i n depth cytogenetic analysis 42 technique was used. The chromosomes most commonly involved are #5, #7, #8, 43 #17 and #21; a f f e c t e d i n 17%, 22%, 37%, 20% and 23% of cases, r e s p e c t i v e l y . (See Figure 2B) A more in-depth analysis of the data reveals a s t r i k i n g tendency for the same type of aberration to be associated with a given chromo-some. For example, when chromosome #5 i s involved, i t i s deleted i n the long arm 47% of the time and monosomic 31% of the time. Likewise, when chromosome #7 i s involved, i t i s deleted i n the long arm 22% of the time and i s monosomic 65% of the time. In contrast, loss of genetic material from chromosome #8 i s rare and amounts f o r only about 8% of the instances i n which i t i s involved while trisomy and translocations i n v o l v i n g t h i s chromosome occur i n 57% and 35% 44 r e s p e c t i v e l y , of the cases i n which i t i s involved. Chronic lymphocytic leukemia (CLL) i s characterized by an excessive number of small lymphocytes i n the blood and bone marrow. In most of these patients, excessive number of lymphocytes are also found i n lymph nodes, spleen, l i v e r and other organs. The underlying feature of CLL i s the abnormal growth of a clone of lymphocytes expressing B-type or, le s s often, T-type surface markers. Many of the major complications r e f l e c t immunologic aberrations and i n f e c t i o n 38 i s the major cause of death. Most cytogenetic studies of patients with CLL have been performed on phytohemagglutinin (PHA)-stimulated peripheral lymphocytes. Since approximately 90% of the CLL cases are of B - c e l l type, the study of PHA-stimulated lymphocytes usually reveals a normal karyotype since most d i v i d i n g c e l l s represent non-neoplastic T - c e l l s . Chromosome aberrations have, however, 14 100 100 1 5 10 15 20 xy 1 5 10 15 20 xy 100 100 1 10 15 20 xy 1 10 15 20 xy F i g u r e 2. H i s t o g r a m s S h o w i n g t h e P e r c e n t a g e D i s t r i b u t i o n o f C h r o m o s o m e A b e r r a t i o n s I n v o l v i n g t h e D i f f e r e n t H u m a n C h r o m o s o m e s i n 8 6 C a s e s o f P V , 4 9 6 C a s e s o f A M L , 3 0 C a s e s o f C L L a n d 2 6 C a s e s of B L ( f r o m r e f e r e n c e 3 7 ) . 15 been ascertained i n 30 cases, 23%, 37% and 27% of which involved chromosomes 44 #12, #14 and #17 r e s p e c t i v e l y . (See Figure 2C) The cytogenetic changes seen i n PV, AML, ALL and CLL are good candidates f o r the secondary or passive type of chromosomal aberration seen i n cancer c e l l s . Although there i s not enough data presently a v a i l a b l e to conclude that these aberrations ocur by chance disturbances of mitosis, as suggested by Levan and Mitelman, ' the heterogeneity of the types of aberrations seen i n any one disorder, as i l l u s t r a t e d i n Figures 2A,B,C are compatible with such a mechanism. The f a c t that c e r t a i n s p e c i f i c changes are seen i n a minority of 45 cases i n each disease (e.g., a deleted #20 i n PV, s p e c i f i c translocations 46 47 i n v o l v i n g chromosomes #8 and #21 and #15 and #17 i n AML ) support the hypothesis that these s p e c i f i c changes provide s e l e c t i v e advantages to the malignant clone and that the high p r o l i f e r a t i v e capacity of the transformed c e l l enhances the l i k e l i h o o d that c e r t a i n chromosomal aberrations w i l l dominate, depending on the c e l l type involved and i t s immediate environment. Oncogenes Chromosomal aberrations are ultimately the r e s u l t of disruptions i n the DNA molecule that makes up each chromosome. Therefore, a discussion of the chromosomal changes seen i n human cancer i s not complete without mention of some of the recent relevant findings i n molecular genetics. I t i s generally thought that some, i f not a l l , cancers are brought about as a r e s u l t of some s t r u c t u r a l a l t e r a t i o n to the DNA although the exact nature of the damage remains unknown. Most recent e f f o r t s aimed at deducing how DNA changes might r e s u l t i n cancerous growth have invoked the existence of "oncogenes". These are thought to be components of the normal c e l l u l a r genome or c l o s e l y r e l a t e d elements, whose a c t i v i t y can be unleashed or augmented by carcinogens of various kinds i n c l u d i n g v i r u s e s . Continued expression of these genes i n t h i s fashion 16 i s thought to be a possible f a c t o r responsible f o r sustaining the p r o l i f e r a t i v e behaviour of cancer c e l l s . The observations that l e d up to the "oncogene" hypothesis of carcinogenesis begun i n 1910 when Rous showed that a c e l l - f r e e f i l t r a t e from a type of chicken s o l i d tumor (sarcoma) could induce new sarcomas i n other 48 chickens. I t i s now well established that a tumor v i r u s was responsible for Rous' observation. We now know that the Rous sarcoma v i r u s (RSV) belongs to a family of viruses that have an RNA genome. During i n f e c t i o n , the v i r a l RNA i s reverse transcribed i n t o DNA that i s then integrated into the c e l l ' s genome. When t h i s occurs, the v i r a l genes are r e p l i c a t e d along with c e l l u l a r genes and 49 are expressed by the machinery of the c e l l . Using a temperature s e n s i t i v e mutant of the RSV, Martin showed that the transformed phenotype of c e l l s i n culture could only be observed at the 50 permissive temperature. This suggested that a s p e c i f i c gene, now c a l l e d src, was the putative cancer gene, or oncogene. This has been mapped to a segment of RNA near the 3' end of the v i r a l genome using deletion mutants of RSV.^ I t i s now evident that the src gene codes f o r a p r o t e i n kinase that 52 s p e c i f i c a l l y phosphorylates tryosine residues. (Phosphorylation of proteins i s thought to play a r o l e i n the regulation of c e l l p r o l i f e r a t i o n . ) Rohrschneider has shown that the src p r o t e i n i s concentrated on regions of the c e l l membrane of i n f e c t e d c e l l s that are necessary for adhesion to s o l i d 53 surfaces. This f i n d i n g suggests that the p r o t e i n kinase coded for by src may a f f e c t c e l l u l a r adhesion by phosphorylating one or more of the proteins involved i n t h i s process and thereby give r i s e to at l e a s t one of the i n v i t r o c h a r a c t e r i s t i c s of tumor c e l l s ; l o s s of contact i n h i b i t i o n . In an attempt to explain the induction of cancer by many d i f f e r e n t agents, Huebner and Todaro suggested that oncogenes may be a part of the genetic 17 baggage of a l l c e l l s and that such genes remain innocuous as long as they are 54 quiescent. However, when stimulated i n t o a c t i v i t y by a carcinogen they convert t h e i r host c e l l s into a pattern of cancerous growth. Recent support f o r t h i s theory has come from the demonstration of sequences of DNA c l o s e l y r e l a t e d to src (and other subsequently described r e t r o v i r a l transformation sequences) i n the genomic DNA of many vertebrates. Using a DNA probe synthesized from the RSV src gene, Stehelin and co-workers demonstrated 55 s p e c i f i c h y b r i d i z a t i o n to sequences i n the DNA of uninfected animals. More compelling evidence that the DNA sequence found i n uninfected animals was i n f a c t a c e l l u l a r gene came from the subsequent f i n d i n g that the protein-encoding information of the c e l l u l a r DNA i s divided i n t o several separate exons; a configuration not seen i n the v i r a l g e n o m e . I n addition, Wang and co-workers have shown that RSV RNA, deleted f o r as much as 75% of the src gene when 57 58 i n j e c t e d i n t o chickens r e s u l t e d i n the recovery of reconstituted src DNA. ' Apparently, genetic material from the c e l l u l a r gene can recombine with the deleted v i r a l genome to give r i s e to a f u l l y f u n c t i o n a l src oncogene. In the l a s t 3-4 years, at l e a s t 17 oncogenes have been i d e n f i f i e d from studies of d i f f e r e n t RNA tumor viruses and 16 are known to have c l o s e l y r e l a t e d sequences i n the normal genomes of many vertebrate species. The c e l l u l a r src gene i s t r a n s c r i b e d and t r a n s l a t e d to give a protein which, l i k e i t s v i r a l counterpart, phosphorylates tyrosine residues. In v i r a l l y transformed c e l l s , the l e v e l of the src protein i s greatly elevated. This observation has l e d to the "dosage hypothesis" of oncogenesis by s r c . According to t h i s hypothesis, the r e t r o v i r a l oncogenes act by overburdening c e l l s with normal proteins. To t e s t t h i s hypothesis, v i r a l promotors were attached to presumptive c e l l u l a r oncogenes. In each case, the v i r a l promoter was a DNA-encoded s i g n a l thought to help i n regulation of the expression of a 18 nearby gene. In accordance with the dosage hypothesis, when the c e l l u l a r - s r c -promoter complex was introduced i n t o c e l l s , some of the c e l l s were transformed as i f they had received a v i r a l oncogene and large quantities of the proteins 58 encoded by the genes were obtained. S i m i l a r findings have been made for other oncogenes. For example, the human c e l l u l a r myc oncogene (c-myc) i s the homologue of the transforming gene of the avian myelocytomatosis v i r u s (MC 29). In most B - c e l l lymphomas induced i n chickens by the avian leukosis v i r u s , oncogenesis i s associated with the occasional chance i n t e g r a t i o n of an avian leukemia provirus near to the c-myc gene.~^'^ As a r e s u l t of t h i s event, a v i r a l promoter apparently activates the c e l l u l a r oncogenes and neoplasia r e s u l t s from an increased c o n s t i t u t i v e expression of the c-myc gene. That t h i s gene may be involved i n human cancer comes from the recent f i n d i n g that the human c-myc homologue was amplified i n 61 62 c e l l s derived from a patient with promyelocytic leukemia. ' In malignant 63 c e l l s from t h i s p a t i e n t , increased l e v e l s of myc messenger RNA were cor-r e l a t e d i n the presence of 20 to 30 copies of the myc gene. (See below for a discussion of the possible r o l e of c-myc i n human B - c e l l lymphomas.) Recently, gene transf e r experiments have demonstrated the existence of oncogenes that are not r e t r o v i r u s associated but are c e l l associated. For example, the DNA of a human bladder carcinoma c e l l l i n e was found to induce f o c i on mouse f i b r o b l a s t monolayers. C e l l s of the f o c i grew out into 64 fibrosarcomas when inoculated i n t o young mice. Several other active tumor oncogenes have been i d e n t i f i e d t h i s way and i s o l a t e d by molecular cloning. These include the H-ras oncogene of the T24/EJ human bladder carcinoma c e l l 6 5™6 7 l i n e , the B-lym oncogenes of a chicken lymphoma and a Burkitt's 68 69 lymphoma, and one from a human neuroblastoma that has been also found i n leukemias and s a r c o m a s . ^ I n each case, the oncogene i s r e l a t e d to a 19 counterpart DNA sequence i n the normal c e l l u l a r genome. I t i s f a s t becoming evident that more than one oncogene may be involved i n the development of a tumorigenic phenotype. For example, i t seems that both ( 7 2 ) c-myc and B-lym are ac t i v a t e d i n chicken lymphomas and B u r k i t t ' s lymphomas 73 and that ras could induce tumorgenicity when aided by a v i r a l oncogene. These studies support the idea that carcinogenesis i s a process i n v o l v i n g multiple, independent steps and that events leading to the a c t i v a t i o n of oncogenes r e l a t e d to r e t r o v i r a l and/or c e l l u l a r DNA are good candidates f o r 74 stages m the multistep process. * Notation i n reference to the use of the word "secondary". The word "secondary" i s used i n t h i s t h e s i s to characterize abnormalities that occur a f t e r the i n i t i a l step i n the pathogenesis of the disease and may or may not be important steps i n oncogenesis. 20 THE SIGNIFICANCE OF CHROMOSOMAL CHANGES IN HUMAN CANCER The enthusiasm with which inv e s t i g a t o r s have attempted to elucidate the ro l e of chromosomal changes i n human cancer has waxed and waned over the past decades. The excitement a r i s i n g i n the ea r l y '60s from the discovery that CML i s strongly associated with the Ph^ chromosome diminished when i t became cl e a r that few other cancers e x h i b i t such s p e c i f i c chromosomal aberrations. Recent improvements i n chromosome banding and the a p p l i c a t i o n of molecular genetic techniques have, however, r e s u l t e d i n the re-emergence of the chromosome at the center of cancer biology. The recent r e s u l t s and how they may help towards a better understanding of the chromosomal changes seen i n human cancer w i l l now be discussed. (a) Chromosomal Deletions S p e c i f i c chromosomal deletions have been found i n tumor c e l l s from 2 7 ™ 2 9 34™3675 76 patients with retinoblastoma, Wilm's tumor, ' neuroblastoma, and 77 small c e l l carcinoma of the lung. In some patients with retinoblastoma and Wilm's tumor, the i d e n t i c a l chromosomal dele t i o n seen i n tumor c e l l s has been i d e n t i f i e d i n a l l c e l l s analyzed from unaffected t i s s u e s and must, therefore, be of germ c e l l o r i g i n . These observations l e d Knudson to propose a two-hit • 78 79 model of carcinogenesis. ' According to t h i s model, the " f i r s t h i t " can be a r e s u l t of the i n h e r i t e d chromosomal deletion while the "second h i t " involves the mutation (deletion or otherwise) of the one remaining f u n c t i o n a l a l l e l e at the locus on the non-deleted chromosome i n a somatic c e l l at the eventual tumor s i t e . Accordingly, i n the case of a retinoblastoma i n an i n d i v i d u a l with a c o n s t i t u t i o n a l d e l e t i o n i n one chromosome #13, a r e t i n a l c e l l would become cancerous when the remaining copy of the pertinent gene(s) i s also damaged and can no longer compensate f o r the defect i n the other chromosome #13. Recently, Sparkes et a l have provided evidence supporting Knudson's 21 8 0 hypothesis. Evaluation of three f a m i l i e s with hereditary retinoblastoma demonstrated close linkage of the gene for t h i s tumor with the genetic locus 8 1 f o r esterase D. Using a biochemical assay f o r l e v e l s of t h i s ubiquitous enzyme the authors screened red blood c e l l s from new cases of retinoblastoma on the premise that decreased l e v e l s of the enzyme would be i n d i c a t i v e of a germ c e l l mutation i n the immediate region of the locus responsible f o r the pr e d i s p o s i t i o n to retinoblastoma seen i n the hereditary form. Only a f t e r the biochemical assay provided evidence for a genetic mutation were cytogenetic studies undertaken i n order to determine i f a chromosomal deletion i n v o l v i n g the c r i t i c a l region seen i n retinoblastoma was responsible f o r the low l e v e l of esterase D. Using t h i s system, a patient was discovered with retinoblastoma who exhibited only 50% of normal esterase D a c t i v i t y i n somatic c e l l s . High r e s o l u t i o n chromosome banding techniques f a i l e d to reveal a deletion i n chromosome #13 i n the region of the esterase D and retinoblastoma l o c i . The authors concluded that t h i s was a case i n which a submicroscopic d e l e t i o n had occured p r e z y g o t i c a l l y . When cytogenetic studies were performed on tumor c e l l s , a common feature of a l l metaphases studied was monosomy f o r chromosome #13. When esterase D l e v e l s were assayed, tumor c e l l s showed no a c t i v i t y . This f i n d i n g suggested that the chromosome #13 l o s t from the tumor c e l l s was the one without the submicroscopic d e l e t i o n . This has been recently confirmed using independent, non-linked, r e s t r i c t i o n length fragment polymorphisms 82 s p e c i f i c f o r chromosome #13. That the normal chromosome #13 was l o s t from the tumor c e l l s i s of considerable t h e o r e t i c a l importance because i t implies that at the tumor l e v e l , there was a t o t a l l o s s of genetic information within the chromosomal region containing the locus f o r gene(s) responsible f o r the i n h e r i t e d p r e d i s p o s i t i o n 22 to develop retinoblastoma. These findings are compatible with the hypothesis that a gene(s) involved i n the normal development of the r e t i n a i s located i n a s p e c i f i c region of chromosome #13. When one copy of t h i s gene(s) i s mutated, normal r e t i n a l development may s t i l l proceed. However, when both copies of the gene(s) are non-functional i n a r e t i n a l c e l l , then factors responsible f o r normal development cease to function and a retinoblastoma develops. Applying Knudson's hypothesis to t h i s case of retinoblastoma, the " f i r s t h i t " must have occurred i n one of the parent's germ c e l l s (since both parents were unaffected and esterase D l e v e l s were normal) and resu l t e d i n a submicroscopic c o n s t i t u t i o n a l deletion i n the af f e c t e d c h i l d . The "second h i t " occurred i n a r e t i n a l c e l l when the one remaining normal chromosome #13 was l o s t . Extrapolating to other cases of retinoblastoma, these findings r a i s e the I p o s s i b i l i t y that i n the i n h e r i t e d form, i t i s simply the p r e d i s p o s i t i o n to tumor development that i s transmitted i n a dominant manner and that a second event must occur i n the appropriate c e l l before the phenotype (tumor growth) i s expressed. In the sporadic, non-hereditary form, i t i s possible that both events occur i n the same r e t i n a l c e l l and occasionally t h i s can be v i s u a l i z e d as a chromosomal dele t i o n i n tumor c e l l s only. The findings described above further our understanding of the role of chromosomal changes i n human cancer. As further i n v e s t i g a t o r s report t h e i r f i n d i n g s , i t may turn out that the deletion seen i n retinoblastoma i s a prototype of a clas s of human cancer genes characterized by a loss of genetic information at the c o n s t i t u t i o n a l or tumor l e v e l and that i n some cases (probably the minority), t h i s l o s s can be v i s u a l i z e d as a microscopic chromosomal change. (b) Chromosomal Translocations With the widespread use of chromosome banding techniques i n the l a s t 83 decade, several consistent and apparently r e c i p r o c a l translocations have been discovered that are r e l a t i v e l y s p e c i f i c a l l y associated with p a r t i c u l a r types of human leukemia and lymphoma. These include the translocations 2 1 i n v o l v i n g chromosomes #9 and #22 i n CML, #8 and #21 i n acute myeloblastic 84 85 leukemia, #15 and #17 i n acute promyelocytic leukemia, and the long arm of • 86 #11 i n acute monocytic leukemia. In addition, translocations i n v o l v i n g chromosome #8 and e i t h e r #14, #2 or #22 have been observed i n B-lymphoid 87-90 malignancies. The recent a p p l i c a t i o n of recombinant DNA technology to human gene mapping has l e d to the e l u c i d a t i o n of the actual sequences of DNA involved i n some tra n s l o c a t i o n s . In the case of the B - c e l l malignancies, t h i s information has turned out to be of considerable t h e o r e t i c a l i n t e r e s t and may have wider ranging i m p l i c a t i o n s . This enthusiasm stems from the l o c a l i z a t i o n of the three immunoglobulin l o c i , f o r the heavy chains, the kappa l i g h t chain and the lambda l i g h t chain on the three chromosomes involved with #8 i n the translocations that characterize the B - c e l l malignancies, i . e . , #14, #2 and #22 9 1-93 r e s p e c t i v e l y . Further, using i n s i t u h y b r i d i z a t i o n , the kappa l i g h t chain genes were shown to reside i n the subchromosomal region involved i n one of the 92 t r a n s l o c a t i o n s . Recently, Lenoir et a l have shown that not only are the general chromosomal regions containing the immunoglobulin genes involved i n the t r a n s l o c a t i o n s but that the expression of these genes themselves are 94 involved. They reported on the complete concordance of karyotype with the immunoglobulin secreted by the malignant c e l l s , i . e . , tumors with a 2;8 t r a n s l o c a t i o n secreted kappa l i g h t chains while those with an 8;22 t r a n s l o c a t i o n secreted lambda l i g h t chains. The involved sequences on chromosome #8 have also been elucidated. D a l l a -Favera e_t a l have found that the human c e l l u l a r oncogene (c-myc) homologous to 24 the transforming gene of the avian mylocytomatosis v i r u s (MC 29) i s located i n -95 the region of chromosome #8 that i s translocated i n B u r k i t t ' s lymphoma c e l l s . The same group have further shown that the c-myc locus i s frequently rearranged i n c e l l s from u n d i f f e r e n t i a t e d B - c e l l lymphomas and that i n some patients, the • 96 rearranged region contains the j o i n i n g segment between chromsome #8 and #14. I t appears that as a r e s u l t of the t r a n s l o c a t i o n , the c-myc gene i s brought i n t o juxtaposition with the heavy chain immunoglobulin gene locus. Although t h i s was not the case i n a l l tumors examined and although the mere bringing together of a c e l l u l a r oncogene with a sequence of DNA that i s p o t e n t i a l l y transcribable i s not s u f f i c i e n t evidence for a p o s i t i o n e f f e c t (indeed, the l o c i appear to be l i n k e d i n a head-to-head f a s i o n leading to opposite 96 d i r e c t i o n s of t r a n s c r i p t i o n and the chromosome #14 involved appears to be the 97 one a l l e l i c a l l y excluded i n the development of an immunocompetent B - c e l l ), these preliminary observations do provide an i n s i g h t i n t o the type of events that may underlie the s p e c i f i c chromosomal translocations seen i n human cancers. The frequent involvement of immunoglobulin genes i n chromosomal transloc a t i o n s c h a r a c t e r i s t i c of B-lymphocyte tumors r a i s e s the p o s s i b i l i t y that some chromosomal abnormalities may be recombinational mistakes occurring i n genomic regions characterized by high l e v e l s of DNA recombination. These events may be r e l a t i v e l y frequent but only detectable when the genetic rearrangement confers a s e l e c t i v e growth advantage to the c e l l i n which i t occurs. Such a growth advantage may a r i s e by the a c t i v a t i o n or increased expression of genes involved i n the regulation of c e l l p r o l i f e r a t i o n such as oncogenes. 25 PRACTICAL APPLICATIONS OF CYTOGENETIC STUDIES IN CANCER P r a c t i c a l a p p l i c a t i o n s of cytogenetic studies i n cancer have not been f u l l y explored or r e a l i z e d . This i s i n large part due to the f a c t that i t has been only r e l a t i v e l y recently that advances i n methodology, i n c l u d i n g techniques f o r the a c q u i s i t i o n of p r o l i f e r a t i n g c e l l s from s o l i d tumors, have permitted an in-depth study of chromosomes i n cancer. There has, however, already been some progress i n the a p p l i c a t i o n of cytogenetic studies to the diagnosis and therapy of cancer. This w i l l now be b r i e f l y discussed as well as future possible a p p l i c a t i o n s of cytogenetic studies to cancer. U t i l i z a t i o n of karyotypic studies i n cancer may become an important t o o l 1 i n the diagnosis of neoplasia. Reference has already been made to the Ph chromosome as being a s p e c i f i c aberration found i n bone marrow of patients with CML. Similar consistent cytogenetic abnormalities are being found i n other human cancers (e.g., t r a n s l o c a t i o n (t)(8;21) i n acute myeloblastic leukemia, t(15;17) i n acute promyelocytic leukemia, t(8;14) i n B - c e l l malignancies, d e l t i o n i n chromosome #3 i n s m a l l - c e l l lung cancer, etc.) as a r e s u l t of attempts to c o r r e l a t e some markers with c e r t a i n tumors. Perhaps newly a v a i l -able methodologies f or the banding of human chromosomes and d i f f e r e n t approaches i n the c l a s s i f i c a t i o n of cancer sub-types w i l l reveal several cyto-genetic features of c e r t a i n cancers that may be of considerable help i n t h e i r diagnosis. The a p p l i c a t i o n of cytogenetic findings to the therapeutic approaches to cancer needs to be further evaluated. In the case of CML, i t has been shown that those cases characterized by the PtJ chromosome i n t h e i r bone marrow c e l l s 98 have a more favourable prognosis than those that do not. I t i s apparent that the cytogenetic findings i n t h i s instance can be included i n the approach to therapy. In acute lymphocytic leukemia, a recent study has shown that the 26 chromosome number of the leukemic clone was the strongest single p r e d i c t o r of outcome and was the only v a r i a b l e that added s i g n i f i c a n t prognostic information 99 to leukocyte count. I t seems reasonable that as more c o r r e l a t i o n s are made between c l i n i c a l course, response to therapy, and cytogenetic findings i n various human cancers, the r o l e of cytogenetic studies i n the therapy of cancer w i l l become more s i g n i f i c a n t . The a p p l i c a t i o n of cytogenetic studies to cultures of tumor c e l l s provides a p a r t i c u l a r l y u s e f u l approach to the i d e n t i f i c a t i o n of c e l l s belonging to the transformed clone. For example, some progress i n the c u l t u r i n g of c e l l s from human tumor t i s s u e has been recently reported. The use of chromosomal analysis has been p a r t i c u l a r l y important i n assessing the nature of the c e l l s p r o l i f e r a -t i n g i n v i t r o . "* ^  Nevertheless, i t must be remembered that although karyotypic change may accompany or even correspond to the primary event i n some cases, i n others (probably the majority), the i n i t i a l mutation may be so small that i t i s not v i s i b l e by conventional cytogenetic techniques. In such cases, changes v i s i b l e at the chromosomal l e v e l would represent l a t e r events. In t h i s s i t u a -t i o n , apparently chromosomally normal and abnormal members of the sane malig-nant population might coexist, and the u t i l i t y of karyotype analysis to d i s t i n g u i s h normal and malignant c e l l s would be correspondingly l i m i t e d . I t should a l s o be noted that even i n the case of CML some recent observa-tions have l e d to the suggestion that the a c q u i s i t i o n of the Ph^ chromosome may be an event secondary to transformation. Using a biochemical marker f o r the neoplastic clone, the existence of c e l l s belonging to the neoplastic clone that di d not bear the chromosomal marker was demonstrated. (This i s discussed i n more d e t a i l i n Chapter III.) In t h i s regard, the use of cytogenetic markers for the transformed clone i n CML may also be l i m i t e d . 27 CONCLUSION Advances i n cytogenetics have been and continue to be at the mercy of methodology. Nevertheless, abnormal chromosome consti t u t i o n s i n tumor t i s s u e are now commonly observed. These findings support Boveri's hypothesis that the c e l l of a malignant tumor has an abnormal chromosome c o n s t i t u t i o n and that events leading to abnormal chromosome con s t i t u t i o n s can r e s u l t i n malignant transformation. Molecular genetic approaches to the problem w i l l undoubtedly help to elucidate the s i g n i f i c a n c e of chromosome changes i n cancers. CHRONIC MYELOID LEUKEMIA THE HEMOPOIETIC SYSTEM The majority of c e l l s that c i r c u l a t e i n the blood are highly d i f f e r e n t i a t e d , incapable of p r o l i f e r a t i o n , and r e l a t i v e l y s h o r t - l i v e d . Current concepts of hemopoiesis favour the existence of a hierarchy of committed progenitor c e l l types f o r each pathway with progressively decreasing capacity f o r p r o l i f e r a t i o n and l i m i t e d , i f any, self-renewal capacity. Erythroid, megakaryocytic, and granulopoietic progenitors are thought to have, i n turn, a common o r i g i n from a p l u r i p o t e n t stem c e l l that i s capable of s e l f -renewal but maybe r e s t r i c t e d to myelopoiesis. There i s also now some evidence f o r the persistence throughout adult l i f e of a more " p r i m i t i v e " stem c e l l that gives r i s e to lymphoid as well as myeloid progeny (see Figure 3). The experi-mental data supporting these concepts are discussed below. (a) Colony Assays f o r P r i m i t i v e C e l l Types. The f a c t that p r i m i t i v e hemopoietic c e l l s are rare and not morphologically d i s t i n c t has forced i n v e s t i g a t o r s to seek a l t e r n a t i v e methods to i d e n t i f y them. In 1961, T i l l and McCulloch showed that mouse marrow c e l l s , when i n j e c t e d into heavily i r r a d i a t e d syngenic mice, were capable of forming d i s c r e t e nodules of hematopoietic c e l l s ULTIMATE S T E M CELL • Lymphopoiesis ERYTHROID PROGENITORS Primitive BFUE large erythroid colony or burst Mature BFUE C F U E small erythroid colony or burst erythroid cluster RED CELLS self-renewal MYELOID STEM CELL (CFU-S, CFU-GEMS) mixed colony M E G A K A R Y O C Y T E PROGENITORS i CFU-M large megakaryocyte colony small megakaryocytic colony PLATELETS GRANULOCYTE PROGENITORS C F U C arge granulocytic colony small granulocytic colony GRANULOCYTES & MACROPHAGES Figure 3 . Outline Of Hemopoiesis AsDefined By Colony Assays Of Stem Cells And Committed Progenitors . (Courtesy of Dr. A.C. Eaves) 29 101 i n the spleens of these animals 8 t o 10 days a f t e r i n j e c t i o n . These nodules were found to contain erythroid, granulocytic, megakaryocytic, and undifferen-102 t i a t e d c e l l s , e i t h e r as pure populations or i n varying mixtures. D e f i n i t i v e proof of the c l o n a l nature of these nodules was provided by studies i n which marrow c e l l s containing s p e c i f i c stable chromosomal markers were i n j e c t e d i n t o i r r a d i a t e d , but otherwise chromosomally normal r e c i p i e n t s . Some colonies 103 showed uniquely abnormal karyotypes i n 95 to 99% of the metaphases. Evidence f o r the self-renewal p o t e n t i a l of the c e l l s that give r i s e to such colonies was demonstrated by i n j e c t i n g a suspension of c e l l s derived from a spleen colony i n t o another l e t h a l l y i r r a d i a t e d mouse and observing new spleen colonies of 104 undiminished s i z e 10 to 14 days l a t e r . Using t h i s method, c e l l s i n apparent-l y pure spleen colonies ( i . e . , only granulocytic or e r y t h r o i d c e l l s seen) were shown to be capable of g i v i n g r i s e to colonies of a l l c e l l types a f t e r retrans-p l a n t a t i o n . » 1 0 6 These studies showed that one marrow c e l l could form a colony containing a l l three myeloid c e l l types, and that t h i s colony forming c e l l could p r o l i f e r a t e and d i f f e r e n t i a t e extensively and undergo self-renewal. Studies by Wu and associates provided the f i r s t evidence f o r the existence of a p l u r i p o t e n t stem c e l l capable of g i v i n g r i s e to both myeloid and lymphoid 107 c e l l s . These i n v e s t i g a t o r s showed that when i r r a d i a t e d mice were transplanted with marrow containing unique radiation-induced chromosomal markers, the marker chromosomes were subsequently found i n the lymphoid as well as myeloid t i s s u e s . More recently, Prchal and h i s colleagues reported a case of s i d e r o b l a s t i c anemia i n a human female heterozygous f o r G6PD A and B a l l e l e s who showed the same, sing l e isoenzyme i n erythrocytes, granulocytes, and macrophages, as well as B and T lymphocytes, suggesting an o r i g i n of both 1 0 8 myeloid and lymphoid c e l l s from a single c e l l . G6PD heterozygosity was shown by the presence of both isoenzymes i n her f i b r o b l a s t s and c e l l u l a r s a l i v a r y 30 lysates. (b) In V i t r o Assays f o r Hemopoietic Progenitors. In v i t r o colony assays have been developed that permit the growth and maturation of c e l l s that give r i s e to a l l types of mature myeloid and lymphoid c e l l types. The f a c t that these p r i m i t i v e precursors are present at low frequency and do not possess d i s t i n c t morphological features precludes t h e i r d i r e c t recognition. The development of colonies containing c e l l s of a p a r t i c u l a r lineage (e.g., e r y t h r o i d c e l l s ) i s dependent on the addition to the culture medium of the corresponding s p e c i f i c stimulatory factors (e.g., e r y t h r o p o i e t i n ) . "Pure" colonies are thought to represnt the c l o n a l descendants of "committed" progenitors since they can be found i n cultures i n which more than one colony type i s obtained. "Mixed" colonies contain c e l l s of two or more d i s t i n c t types (e.g., granulocytes and macrophages) and generally contain more c e l l s than pure colonies. They are thought to represent the c l o n a l descendants of a more pr i m i t i v e c e l l i n which a s u f f i c i e n t number of c e l l d i v i s i o n s had not occurred i n vivo (before the i n i t i a t i o n of i n v i t r o growth) f or f i n a l determination (granulocyte vs. macrophage) but had occurred f o r e a r l i e r determination events (myeloid vs. lymphoid). In v i t r o colony assays f o r hemopoietic progenitors involve suspending marrow or blood c e l l s i n one of various types of semisolid media containing the appropriate n u t r i e n t s , serum and stimulatory factors (see Figure 4 and Chapter I I ) . A f t e r 1 to 3 weeks of incubation, e r y t h r o i d colonies can be i d e n t i f i e d by t h e i r red colour, i n d i c a t i v e of ongoing hemoglobin synthesis i n the e r y t h r o i d progeny that have reached t h e i r f i n a l stage of d i f f e r e n t i a t i o n . E r y t h r o i d colonies composed of 16 or more c l u s t e r s of c e l l s , each c l u s t e r containing about 50 c e l l s , are thought to represent the descendants of the most p r i m i t i v e 109 c e l l s which are committed to the e r y t h r o i d lineage. There i s a d i r e c t r e l a -31 V M A R R O W Unit Gravity S e d i m e n t e d Buf ly C o a t C e l l s OR F ico l l Hypaque S e d i m e n t e d N u c l e a t e d Ce l l s W a s h e d and Diluted S E M I - S O L I D M E T H Y L C E L L U L O S E MEDIUM B L O O D Incubate 9 - 1 2 D a y s I fe ftb 5^ 1 E r y t h r o i d , G r a n u l o c y t e , and M i x e d E r y t h r o i d / G r a n u l o c y t e C o l o n i e s F igure 4. S c h e m a t i c Illustration of the Methodo logy u s e d in Culturing Hemopoiet i c C o l o n i e s from B lood and Marrow Progen i tors in S e m i - S o l i d M e d i a . 32 tionship between the p r o l i f e r a t i v e capacity of the o r i g i n a l progenitor c e l l and the time taken to generate hemoglobin producing erythroblast progeny; the l a r g e r the colony, the longer before terminally d i f f e r e n t i a t e d c e l l s f i r s t appear. Evidence has been obtained to i n d i c a t e that the s i z e of colony formed i n v i t r o i s determined by the state of d i f f e r e n t i a t i o n of the colony forming 110 c e l l i i i vivo. Under i d e a l culture conditions, granulopoietic colonies can also be f a i r l y r e l i a b l y i d e n t i f i e d without f i x a t i o n and s t a i n i n g by v i r t u e of t h e i r c h a r a c t e r i s t i c morphology and lack of red c o l o r . Several l i n e s of evidence i n d i c a t e that the majority of colonies obtained under routine assay conditions are from single colony forming c e l l s . These include the micromanipulation of single colony forming c e l l s and the subsequent 111 observation of colonies; time-lapse photography of the developing 112 colonies, p h y s i c a l i s o l a t i o n of the c e l l s i n the culture dish by p l a s t i c 113 rings, and the c h a r a c t e r i z a t i o n of i n d i v i d u a l colonies i n cultures i n i t i a t e d 114 with c e l l s of 2 d i f f e r e n t genotypes or with c e l l s from G6PD heterozygous . 115 females. L i t t l e i s known about the regulation of hemopoietic stem c e l l s . Although the output of highly d i f f e r e n t i a t e d elements i s obviously p r e c i s e l y c o n t r o l l e d , i t i s possible that regualtion at the stem c e l l l e v e l i s more lax. Three d i f f e r e n t theories suggest that the primary determinants are: (1) simply random events that d i c t a t e that with each c e l l d i v i s i o n , the stem c e l l has a defined chance of becoming a red c e l l precursor, a granulocyte precursor, 116 etc.; (2) l o c a l microenvironmental influences within the stroma of the bone marrow or other hematopoietic t i s s u e that program the d i r e c t i o n of stem c e l l development;^^ and (3) hormones operating at long range that i n t e r a c t with receptors at the surface of stem c e l l s to determine t h e i r commitment and 33 118 119 subsequent development. ' There are arguments favoring a l l three theories, and the mechanism of stem c e l l commitment to a given l i n e of d i f f e r -e n t i a t i o n remains one of the primary questions i n hematopoiesis today. CML — A STEM CELL DISORDER CML i s one of a group of diseases, known c o l l e c t i v e l y as the myeloprolif-e r a t i v e diseases (MPD), i n which transformation to neoplastic growth occurs i n a p l u r i p o t e n t stem c e l l . Resulting abnormalities i n the regulation of hemo-p o i e t i c c e l l p r o l i f e r a t i o n are not accompanied by s i g n i f i c a n t abnormalities i n terminal c e l l d i f f e r e n t i a t i o n . The concept of a group of myeloproliferative disorders with important common features was developed during the e a r l y part of t h i s century. Generally included are a group of c l i n i c a l disorders believed to represent v a r i e d manifestations of an underlying abnormality i n a p r i m i t i v e hemopoietic stem c e l l . This concept was based on the s i m i l a r i t i e s i n the h i s t o r i e s of these diseases, each characterized by predominant elevations of a sin g l e lineage, although the tendency f o r other c e l l l i n e s to be elevated was 120 a l s o noted. The term MPD was f i r s t proposed by Dameshek i n 1951, although conclusive evidence f o r the existence of a pluripotent stem c e l l was not obtained u n t i l 10 years l a t e r with the c h a r a c t e r i z a t i o n of the spleen colony-101 forming c e l l i n the mouse (discussed i n the previous section) and the demon-1 s t r a t i o n of the Ph chromosome i n multiple types of d i v i d i n g hemopoietic c e l l s . CML i s characterized by an elevation of the white blood c e l l (WBC) count, up to 100 times normal values, as a r e s u l t of the presence of increased numbers of a l l forms of mature and immature granulocytes. Studies on survivors of the atomic bombing of Hiroshima suggest that the f i r s t leukemic c e l l can appear 6 years before the wbc count reaches l e v e l s high enough to e l i c i t symptoms and 34 122 enable c l i n i c a l diagnosis. T y p i c a l presenting symptoms of patients with CML are fatigue, low-grade fever, weight l o s s , abnormal sweating and/or discomfort • 38 i n the upper abdomen due to an enlarged spleen and/or l i v e r . During the chronic phase of CML, wbc counts can usually be kept to accept-able l e v e l s by the use of therapeutic agents designed to k i l l p r o l i f e r a t i n g c e l l s e.g., the antimetabolite hydroxyurea and the a l k y l a t i n g agent busulfan (myleran). With therapy, t h i s stage of the disease can be r e l a t i v e l y symptom f r e e . CML usually terminates i n an acute phase which i s r e l a t i v e l y unresponsive to therapy. Median time before transformation to the acute phase i s 3 years. WBC counts r i s e to extreme l e v e l s and chromosomal changes i n add i t i o n to the Ph^ chromosome are often seen i n the bone marrow (see Figure 1). Median s u r v i v a l i s about 3 months with the cause of death usually being i n f e c t i o n or hemorrhage due to the f a i l u r e to form adequate numbers of fu n c t i o n a l 38 granulocytes and p l a t e l e t s . In almost a l l of the MPD (CML, e s s e n t i a l thrombocytosis (ET), PV, i d i o p a t h i c myelofibrosis (IMF)) that have been studied, more than one l i n e of mature myeloid c e l l s have been shown to be part of the neoplastic clone. Consequently, i t has been assumed that most myeloid neoplasms a r i s e i n one of 123 the multipotent stem c e l l compartments. The pluripotent stem c e l l i s generally believed to be the s i t e of onset for most cases of CML. Evidence for t h i s comes from cytogenetic studies, i n which the Ph^ chromosome has been a marker for the leukemic clone, and biochemical studies i n females with CML who are also heterozygous f o r G6PD isoenzymes i n whom a single isoenzyme type has been the marker of the leukemic clone. 35 (a) Cytogenetic Studies In 1 9 6 0 , a unique cytogenetic marker f o r the leukemic clone i n CML was 4 1 described. The Ph chromosome was found to be present i n the vast majority of bone marrow metaphases. I t was not present i n other d i v i d i n g c e l l s such as skin f i b r o b l a s t s , or PHA-stimulated T lymphocytes. In an attempt to determine the d i s t r i b u t i o n of the Ph^ chromosome i n various bone marrow c e l l types, Whang-Peng et a l estimated the percentages of e r y t h r o i d and granulocytic c e l l s i n stained d i r e c t marrow smears and corr e l a t e d 1 121 t h i s to the presence of the Ph chromosome i n d i v i d i n g bone marrow c e l l s . In 13 patients, the Ph^ chromosome was found i n 9 0 - 1 0 0 % of bone marrow metaphases. Since the h i s t o l o g i c a l study suggested that both e r y t h r o i d and granulocyte 1 precursors were p r o l i f e r a t i n g , i t was concluded that the Ph chromosome occurred 1 i n c e l l s of both the e r y t h r o i d and granulocytic lineages. In addition, Ph chromosomes were observed i n t e t r a p l o i d and oc t a p l o i d bone marrow c e l l s that were most l i k e l y of megakaryocytic lineage. More evidence f o r the involvement of er y t h r o i d c e l l s i n CML came with the 1 f i n d i n g of the Ph chromosome i n metaphases from c e l l s a c t i v e l y synthesizing 124 hemoglobin as measured by the uptake of radioactive i r o n . More recently, the 1 Ph chromosme has been c l e a r l y i d e n t i f i e d i n metaphases obtained from 114 125 i n d i v i d u a l e r y t h r o i d colonies cultured i n v i t r o . ' 1 In 1 9 7 1 , Chervenick et a l reported f i n d i n g the Ph chromosome i n granulo-126 p o i e t i c colonies grown i n v i t r o . In four patients with CML, analysis of i n d i v i d u a l colonies cultured i n methylcellulose from bone marrow and blood 1 mononuclear c e l l s revealed a Ph chromosome. Individual colonies were described as being of eosinophil, neutrophil, monocyte and macrophage o r i g i n . 1 The Ph chromosome has also been described i n granulocyte colonies cultured i n 1 v i t r o from patients with CML by others. Moore and Metcalf found the Ph 36 chromosome i n metaphases prepared from pooled granulocyte colonies cultured i n 127 agar from each of 6 patients with CML. This f i n d i n g was confirmed i n 128 125 i n d i v i d u a l granulocyte colonies cultured i n methylcellulose. ' Cultures of macrophages from patients with CML have a l s o been demonstrated to be Ph^ chromosome p o s i t i v e . Golde et a l cultured bone marrow c e l l s for 20-25 days under conditions that favour p r o l i f e r a t i o n and d i f f e r e n t i a t i o n along 129 the macrophage lineage. . E i g h t y - f i v e to n i n e t y - f i v e per cent of the c e l l s i n the d i f f u s i o n cultures were p o s i t i v e f o r alpha-naphthylbutyrase (granulocytes lack t h i s enzyme) and more than 80% of the observed metaphases contained the 1 1 Ph chromosome. McCarthy et a l have demonstrated the presence of the Ph 130 chromosome i n eosinophil colonies cultured i n v i t r o . 1 Recent data has suggested that the Ph chromosome may not be r e s t r i c t e d to c e l l s of the myeloid lineage i n CML, but may also be found i n c e l l s committed 131 to B-lymphoid d i f f e r e n t i a t i o n . In that report, cytogenetic analysis was performed on metaphase c e l l s from patients with chronic phase CML. C e l l s were given a mild hypotonic shock so that t h e i r membranes remained i n t a c t while s t i l l allowing metaphase c e l l s to be swollen s u f f i c i e n t l y f o r cytogenetic 1 a n a l y s i s . In addition to scoring f o r the presence of the Ph chromosome, c e l l membranes were simultaneously analyzed for the presence of surface immunoglobu-l i n M (a marker f o r c e l l s of the B-lymphocyte lin e a g e ) . In t h i s way, i t was 1 possible to determine i f c e l l s possessing the Ph chromosome were also immuno-gl o b u l i n bearing. Of 6 patients studied i n t h i s way, a l l m i t o t i c B-lymphocytes 1 expressed the Ph chromosomes. In summary, the cytogenetic data to date c l e a r l y suggest that i n CML transformation can occur i n a p l u r i p o t e n t stem c e l l capable of g i v i n g r i s e to a l l myeloid elements as well as c e l l s committed to the B-lymphoid pathway of d i f f e r e n t i a t i o n (see Figure 5). ^ T Cell (?) Lymphoid Stem Cell (5) B Cell (+) 131 Pluripotent Stem Cell Myeloid Stem Cell BFU-E (+) CFU-MEGA (+) Q Erythrocyte (+) 125 CFU-E (+) CFU-C (+) _ (g) Granulocyte W u« (S) Monocyte (+) 129 •* Eosinophil (+) 130 •\V/ Platelets (+) 121 Megakaryocyte (+) ure 5. S c h e m a t i c I l lustrat ion Showing the Involvement of the Ph i lade lph ia C h r o m o s o m e in the H e m o p o i e t i c L i n e a g e s . Numbers Refer to R e f e r e n c e s . 38 (b) Biochemical Studies Early support f o r the Lyon hypothesis of X chromosomal i n a c t i v a t i o n i n 132 mammalian females came i n 1963 from the electrophoretic study of lysates of f i b r o b l a s t cultures derived from females heterozygote for the A and B forms of 133 X-linked enzyme; glucose-6-phosphate dehydrogenase (G6PD). While mass cultures expressed both enzymes, clones o r i g i n a t i n g from si n g l e c e l l s expressed only type A or B, but not both. I t has since been reasoned that neoplasms a r i s i n g from a single c e l l i n such heterozygous females should e x h i b i t only one enzyme type. In 1967, Fialkow and co-workers applied t h i s system to the study of CML. Three heterozygous females with CML were studied and i n each case, a si n g l e enzyme type was demonstrated i n red blood c e l l s and granulocytes. In contrast, both type A and type B electrophoretic forms of the enzyme were present i n skin f i b r o b l a s t s . This r e s u l t was compatible with the c l o n a l o r i g i n of the tumor i n a stem c e l l common to the e r y t h r o i d and granulocytic lineages. During the l a s t 15 years, t h i s same group has confirmed t h i s f i n d i n g i n a t o t a l of 23 p a t i e n t s . In l a t e r studies, Fialkow and co-workers have also demonstrated the single isoenzyme type, c h a r a c t e r i s t i c of the leukemic clone i n heterozygous patients, i n p l a t e l e t s , macrophages, granulocytes cultured _in v i t r o and non-T-l y m p h o c y t e s . ' P l a t e l e t s were prepared form the p l a t e l e t r i c h plasma laye r , macrophages were obtained from the adherent layer of 8 day-old blood cultures, granulocytes were grown i n soft agar i n the present of leukocyte conditioned media, and non-T-cells were separated from T - c e l l s by the use of the E-rosette formation method. Recently, Fialkow's group have attempted to present unequivocal evidence fo r B lymphod involvement i n CML by combined use of the cytogenetic and bio-39 • 137 138 chemical markers of the leukemic clone. ' In order to obtain homogenous populations of c e l l s of the B lymphoid lineage f o r G6PD studies and karyotype an a l y s i s , a number of B lymphoid c e l l l i n e s were established using the Epstein-Barr v i r u s (EBV). (EBV i s a po l y c l o n a l a c t i v a t o r of B lymphocytes and studies of non-neoplastic t o n s i l derived B lymphoid c e l l l i n e s using G6PD and immuno-gl o b u l i n markers have shown that EBV transformed c e l l l i n e s are i n i t i a l l y p o l y c l o n a l , but become homogeneous i n culture by random s e l e c t i o n and over-139 growth of a single clone). In that study, 74 c e l l l i n e s were established from a sing l e patient with CML by seeding a small number of EBV-infected c e l l s i n each of a large number of m i c r o t i t r e wells. Of the 74 c e l l l i n e s , 72 were G6PD type A or B, but not both, and were therefore considered to be homogenous. Nine of the c e l l l i n e s 1 were Ph p o s i t i v e and expressed type B G6PD ( c h a r a c t e r i s t i c of the leukemic clone i n that p a t i e n t ) . I t was concluded that these 9 c e l l l i n e s were descendents of the leukemic clone and were therefore derived from a stem c e l l committed to the B lymphoid pathway of d i f f e r e n t i a t i o n (see below for a discussion of the remaining 65 c e l l l i n e s ) . This f i n d i n g represented the f i r s t d i r e c t demonstration of the involvement of the lymphoid series i n chronic phase CML. In summary, the biochemical data, l i k e the cytogenetic data, c l e a r l y suggest that i n CML transformation can occur i n a pluri p o t e n t stem c e l l capable of g i v i n g r i s e to a l l myeloid elements as well as c e l l s committed to the B lymphoid pathway of d i f f e r e n t i a t i o n . (c) C l i n i c a l Studies C l i n i c a l evidence a l s o suggests that CML arises i n a plu r i p o t e n t stem c e l l . As the disease progresses, i t often enters a terminal or acute b l a s t i c phase which i s characterized by a poor response to therapy and a predominance 40 of immature, poorly d i f f e r e n t i a t e d b l a s t c e l l s i n the blood and bone marrow. While i n most pat i e n t s , the b l a s t c e l l s show myeloid c h a r a c t e r i s t i c s , i n some the b l a s t c e l l s show lymphoid lineage s p e c i f i c features. In a few patients, a 140 s h i f t i n g pattern from one type to the other has also been observed. CYTOGENETIC CHANGES IN THE COURSE OF Ph 1-POSITIVE CML In 1980, Rowley reported the r e s u l t s of karyotypic analysis on 802 Ph^-p o s i t i v e patients with CML who had been examined with banding techniques by a 12 1 number of i n v e s t i g a t o r s . In 92% of the cases, the Ph -chromosome was formed by a t r a n s l o c a t i o n i n v o l v i n g chromosomes #9 and #22 with the breakpoints occurring i n i d e n t i c a l bands of chromosomes involved i n a l l cases. In the remaining cases, the r e c i p r o c a l t r a n s l o c a t i o n involved e i t h e r chromsome #22 and some chromosome other than #9, or was of a complex nature i n v o l v i n g three or more d i f f e r e n t chromosomes, two of which were #9 and #22 (with the possible exception of two cases). In a l l cases, except 2, the breakpoint i n chromosome #22 occurred i n the same chromosome band. The two exceptional cases both involve variant t r a n s l o c a t i o n s i n v o l v i n g chromosomes #12 and #22. I t appears that i n these cases, the breakpoint i n #22 occurred more d i s t a l to that observed i n the vast majority of cases. 1 The f a c t that i n 96% of patients with Ph - p o s i t i v e CML, the constant feature i s the t r a n s l o c a t i o n of genetic material from the end of chromosome #22 to the end of #9 has lead i n v e s t i g a t o r s to suggest that the c r i t i c a l event leading to neoplastic transformation appears to be r e l a t e d to t h i s s p e c i f i c t r a n s l o c a t i o n . However, one recent study has suggested that the a c q u i s i t i o n of the Ph^-chromosome i s a secondary event and that neoplastic transformation preceeds i t . Fialkow and co-workers studied 63 EBV transformed B lymphocyte 41 1 c e l l l i n e s from a single patient with Ph - p o s i t i v e CML who was also 137 138 heterozygous f o r types A and B of G6PD isoenzymes. ' A l l were negative f o r the Ph 1 chromosome and t h i s i n i t i a l l y suggested that they were derived from non-leukemic B lymphocytes. However, electrophoretic study of these c e l l s f o r G6PD phenotypes r a i s e d the p o s s i b i l i t y that at le a s t 1 some of the Ph negative c e l l l i n e s were a l s o derived from the leukemic clone. F o r t y - f i v e c e l l l i n e s displayed the B enzyme ( c h a r a c t e r i s t i c f o r the leukemic clone i n t h i s p a t i e n t ) , while 18 displayed the A enzyme. The difference betwen the observed r a t i o (45:18) and the expected r a t i o (1:1) was highly s i g n i f i c a n t 1 and suggested that the excess of Ph negative c e l l l i n e s expressing type B isoenzyme was due to the presence of B lymphocytes derived from the leukemic 1 clone but negative f o r the Ph chromosome. To inv e s t i g a t e t h i s f urther, 33 Ph 1 negative c e l l l i n e s that expressed the leukemic isoenzyme phenotype were cyto g e n e t i c a l l y studied. Detailed analysis revealed that 8/33 l i n e s were chromosomally abnormal, compared to 0/14 l i n e s expressing the other isoenzyme. This f i n d i n g of genetic i n s t a b i l i t y i n the Ph^ negative c e l l l i n e s that expressed the leukemic isoenzyme phenotype supported the hypothesis that the c e l l l i n e s were of the leukemic clone since genetic i n s t a b i l i t y i s a c h a r a c t e r i s t i c of malignant or premalignant c e l l s . The data discussed above c l e a r l y suggested that, i n that s i n g l e patient with CML, there were at l e a s t two steps involved i n the evolution of the neoplastic clone -r- one causing p r o l i f e r a t i o n of a pluri p o t e n t stem c e l l and the other inducing the Ph^ chromosome i n one or more descendents of t h i s progenitor clone. This p o s s i b i l i t y i s compatible with the reports of occasional patients with t y p i c a l CML whose marrow c e l l s are Ph^ negative at 1 presentation but l a t e r became Ph p o s i t i v e and the reported disappearance of 1 141-145 the Ph chromosome i n one case of CML b l a s t c r i s i s . 42 The p o s s i b i l i t y i s not, however, compatible with some of the cytogenetic studies suggesting a c l o n a l o r i g i n i n CML. In those rare patients where the p a t e r n a l l y derived chromosome #22 (or #9) can be distinguished from i t s maternally derived partner, the Ph^ chromosome has been shown to involve the 146—148 same #22 (or #9) chromosome i n a l l c e l l s studied. I f the a c q u i s i t i o n of 1 the Ph chromosome i s a secondary event, one may expect i t to involve maternal and paternal chromosomes on a random basis assuming that a t r a n s l o c a t i o n i n v o l v i n g e i t h e r one w i l l be of equal s i g n i f i c a n c e . Using a 149 150 s l i g h t l y d i f f e r e n t approach, F i t z g e r a l d et a l and Chaganti et a l showed that i n two i n d i v i d u a l s with a chromosomally mosaic c o n s t i t u t i o n , who also had Ph^ p o s i t i v e CML, the Ph^ chromosome was r e s t r i c t e d to only one c e l l l i n e . These observations suggest that the a c q u i s i t i o n of the Ph^ chromosome does not occur i n multiple c e l l s that have already gained a p r o l i f e r a t i v e advantage. I t i s c l e a r that more research i s needed before the r o l e of the Ph^ chromosome i n the pathogenesis of CML i s elucidated. The present understanding of the human genome and of the complex i n t e r a c t i o n s i n the control of gene function are too l i m i t e d to permit a v a l i d explanation of the r o l e of t h i s genetic event i n the development of CML. When patients with CML enter the terminal acute phase about 20% r e t a i n the 1 151 Ph -chromosome as the sole cytogenetic abnormality i n t h e i r leukemic c e l l s . In the remainder, however, other chromosomal abnormalities are superimposed on the c e l l l i n e marked with the Ph^ chromosome. In a number of cases, the changes i n the karyotype preceded the c l i n i c a l signs of b l a s t c r i s i s by 2 to 4 months and some authors now believe that the a d d i t i o n a l cytogenetic changes 152 herald b l a s t c r i s i s . Bone marrow chromosomes from 242 patients with Ph^-positive CML, who were i n the acute phase of t h e i r disease, have been analyzed with banding techniques 43 12 and reported by Rowley e_t a_l. Forty patients showed no change i n karyotype while 202 patients had a d d i t i o n a l chromosome abnormalities. The most common changes occurred i n combination to produce modal numbers of 47 to 52 chromosomes. Since v i r t u a l l y a l l patients who have been studied i n the acute phase of CML have already been exposed to chemotherapy during the chronic phase of t h e i r disease, i t i s usually impossible to determine to what extent the therapy a f f e c t s the pattern of abnormalities. However, the non-random nature of c e r t a i n changes (e.g., an a d d i t i o n a l chromosome #8) suggests that secondary or passive chromosome abnormalities may be r e f l e c t i n g the primary non-random cytogenetic event and subsequent c l o n a l s e l e c t i o n i n the i n c r e a s i n g l y more abnormal leukemic hematopoietic environment. 1 1 Ph -NEGATIVE CELLS IN Ph -POSITIVE CML The presence of a number of c e l l s without the Ph^-chromosome i n the bone 1 marrow of some patients with Ph - p o s i t i v e CML i s of considerable importance and c e r t a i n s i g n i f i c a n t findings w i l l now be discussed. In a recent study, c y t o g e n e t i c a l l y normal c e l l s were encountered i n 10 of 41 CML patients studied i n the early chronic phase of CML and ranged from 1.8% to 61% of the metaphases 153 analyzed. The percentage of normal c e l l s was found to gradually decrease during the course of the disease and i n the acute phase, no normal c e l l s were observed i n any of the p a t i e n t s . 1 Cytogenetically normal marrow c e l l s have also been observed i n Ph -p o s i t i v e bone marrows from CML patients treated with a p a r t i c u l a r l y intensive chemotherapy protocol evaluated at the Sloan-Kettering Cancer Center. Of 37 1 patients with Ph - p o s i t i v e CML who were treated with t h i s intensive therapy regimen (L-5 p r o t o c o l ) , 12 showed a temporary reduction i n the percentage of 1 Ph - p o s i t i v e marrow metaphases to l e s s than one t h i r d of the i n i t i a l values and 44 i n 7 of these patients, the frequency of Ph - p o s i t i v e p r o l i f e r a t i n g c e l l s was 154 reduced to zero. However, t h i s reduction was d i f f i c u l t to maintain and o v e r a l l s u r v i v a l of patients was not s i g n i f i c a n t l y extended. More recently, the same group have used a more intensive regimen (L-15 protocol) and have s i m i l a r l y observed a temporary reduction i n the percentage of Ph^-positive marrow metaphases to l e s s than one t h i r d of the pretreatment value i n 15 out of 155 28 patients studied. In order to determine whether the Ph^-negative c e l l s seen a f t e r intensive chemotherapy a c t u a l l y represent the re-emergence of t r u l y normal blood 1 elements, Singer et a l studied a patient with Ph - p o s i t i v e CML who was heterozygous f o r G6PD a l l e l e s and who obtained a p a r t i a l Ph 1-negative remission a f t e r the L-5 therapy p r o t o c o l . After 4 cycles of chemotherapy, 76% of marrow c e l l s were found to be Ph^-negative and 80% of the granulocytes were found to be non-clonal by G6PD ana l y s i s , suggesting that the Ph^-negative c e l l s were i n f a c t of the normal clone. These r e s u l t s suggest that r e s i d u a l normal stem c e l l s do p e r s i s t for some time i n the hematopoietic t i s s u e of CML patients i n the chronic phase of t h e i r disease. I t seems p l a u s i b l e that t h e i r p r o l i f e r a t i o n i s e i t h e r a c t i v e l y suppressed by the neoplastic clone or suppressed by as yet uncharacterized feedback i n h i b i t i o n mechanisms that function to i n h i b i t stem c e l l p r o l i f e r a t i o n as a normal part of the regulatory process. PRESENT OBJECTIVE Cytogenetic and biochemical studies demonstrate that the majority of human neoplasms consists of clones of mutant c e l l s . In CML, as well as i n c e r t a i n 4 5 r e l a t e d disorders such as PV, the bone marrow can often be shown to be populated by p r o l i f e r a t i n g neoplastic t i s s u e , the d i v i d i n g c e l l s of which a l l bear the same chromosome rearrangement. When CML i s brought i n t o true c l i n i c a l remission by intensive chemo-therapy, normal c e l l s recognized by t h e i r cytogenetic and/or biochemical c h a r a c t e r i s t i c s , may reappear i n the p r o l i f e r a t i n g marrow. Their presence among the d i v i d i n g c e l l s suggests that non-neoplastic hemopoietic stem c e l l s p e r s i s t beyond the time when t h e i r presence can no longer be detected i n standard marrow chromosome preparations, presumably because they are not a c t i v e l y p r o l i f e r a t i n g . Their presence i n the hemopoietic system signals hope f o r recovery of a normal marrow i n these patients provided the s i t u a t i o n can be manipulated s u i t a b l y . The p r i n c i p a l objective of t h i s work was to t r y to develop more s e n s i t i v e methods that would allow c y t o g e n e t i c a l l y normal hemopoietic stem c e l l s , with the capacity f o r normal p r o l i f e r a t i o n and d i f f e r e n t i a t i o n , i f present to be demonstrated i n patients with Ph^-positive CML and to use such methods to evaluate t h e i r frequency during the course of the disease. By u t i l i z i n g i n  v i t r o colony assays f o r the more p r i m i t i v e precursor c e l l s of the hemopoietic system and long-term bone marrow cultures, a system was developed i n which mechanisms that appear to suppress normal c e l l s i n vivo were apparently reduced. By using chromosomal markers for the malignant clone and performing karyotypic analysis on hemopoietic colonies, i t was possible to d i f f e r e n t i a t e between progeny of cyt o g e n e t i c a l l y a l t e r e d and cy t o g e n e t i c a l l y normal stem c e l l s . I f c y t o g e n e t i c a l l y normal and presumably non-malignant, r e s i d u a l stem c e l l s can be demonstrated in v i t r o , then factors that influence t h e i r p r o l i f e r -ation may be further investigated. Indeed, the fundamental difference between normal and transformed c e l l populations might be made accessible to study under 46 i n v i t r o c o n d i t i o n s . 47 REFERENCES 1. 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Proc N a t l Acad S c i USA 58: 1468-1471, 1967. 135. Fialkow PJ, Jacobson RJ, Papayannopoulou T: Chronic myelocytic leukemia: c l o n a l o r i g i n i n a stem c e l l common to the granulocyte, erythrocyte, p l a t e l e t and monocyte/macrophage. Am J Med 63: 125-130, 1977. 136. Fialkow PJ, Denman AM, Jacobson RJ, Lowenthal MN: Chronic myelocytic leukemia. O r i g i n of some lymphocytes from leukemic stem c e l l s . J C l i n Invest 62: 815-823, 1978. 137. Martin PJ, N a j f e l d V, Hansen JA, Penfold GK, Jacobson RJ, Fialkow PJ: Involvement of the B-lymphoid system i n chronic myelogenous leukemia. Nature 287: 49-50, 1980. 138. Fialkow PJ, Martin PJ, N a j f e l d V, Penfold GK, Jacobson RJ, Hansen JA: Evidence f o r a multistep pathogenesis of chronic myelogenous leukemia. Blood 58: 158-163, 1981. 139. Nilsson K: The nature of lymphoid c e l l l i n e s and t h e i r r e l a t i o n s h i p to the v i r u s . 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L i s k e r R, Casas L, Mutchinick O, Lopez-Ariza B, Labardini J : Patient with chronic myelogenous leukemia and l a t e appearing Philadelphia chromosome. Cancer Genet Cytogenet 6: 275-277, 1982. 145. Hagemeijer A, Smit EME, Lowenberg B, Abels J : Chronic myeloid leukemia with permanent disappearance of the Ph chromosome and development of new c l o n a l subpopulations. Blood 53: 1-13, 1979. 146. Gahrton G, Lindsten J , Zech L: Clonal o r i g i n of the Philadelphia chromo-some from e i t h e r the paternal or the maternal chromosome number 22. Blood 43: 837-840, 1974. 57 147. Hossfeld DK: A d d i t i o n a l chromosomal i n d i c a t i o n f o r the u n i c e l l u l a r o r i g i n of chronic myelocytic leukemia. Z Krebsforsch 83: 269-273, 1975. 148. Hayata I, Kakati S, Sandberg AA: On the monoclonal o r i g i n of chronic myelocytic leukemia. Proc Japan Acad 50: 381-385, 1974. 149. F i t z g e r a l d PH, P i c k e r i n g AF, Eiby JR: Clonal o r i g i n of the Philadelphia chromosome and chronic myeloid leukemia: evidence from a sex chromosome mosaic. Br J Haematol 21: 473-480, 1971. 150. Chaganti RSK, Bailey RB, Jhanwar SC, A r l i n ZA, Clarkson BD: Chronic myelogenous leukemia i n the monosomic c e l l l i n e of a f e r t i l e Turner syndrome mosaic (45,X/46,XX). Cancer Genet Cytogenet 5: 215-221, 1982. 151. Rowley JD: chromosome abnormalities i n human leukemia. Ann Rev Genet 14: 17-39, 1980. 152. S i l v e r RT, Schleider MA, S e n t e r f i t LB, Rothe DJ: Early recognition of terminal phase CML by s e r i a l chromosome a n a l y s i s . Proc Am Assoc Cancer Res 18: 220, 1977. 153. Sonia SI, Sandberg AA: Chromosomes and causation of human cancer and leukemia. XXIX. Further studies on karyotypic progression i n CML. Cancer 41: 153-163, 1978. 154. Cunningham I, Gee T, Dowling M, Chaganti R, Bailey R, Hopfan ^, Bowden L, Turnbull A, Knapper W, Clarkson B: Results of treatment of Ph + chronic myelogenous leukemia with an intensive treatment regimen (L-5 p r o t o c o l ) . Blood 53: 375-395, 1979. 155. Goto T, N i s h i k o r i M, A r l i n Z, Gee T, Kempin S, Burchenal J , S t r i f e A, Wisniewski D, Lambek C, L i t t l e C, Jhanwar S, Chaganti R, Clarkson B. Growth c h a r a c t e r i s t i c s of leukemic and normal hematopoietic c e l l s i n Ph + chronic meylogenous leukemia and e f f e c t s of intensive treatment. Blood 59: 793-808, 1982. 156. Singer JW, A r l i n ZA, N a j f e l d V, Adamson JW, Kempin SJ, Clarkson BD, Fialkow PJ: Restoration of nonclonal hematopoiesis i n chronic myelogenous leukemia (CML) following a chemotherapy-induced los s of the Ph chromo-some. Blood 56: 356-360, 1980. 58 C H A P T E R T W O A METHOD FOR OBTAINING HIGH QUALITY CHROMOSOME PREPARATIONS FROM SINGLE HEMOPOIETIC COLONIES ON A ROUTINE BASIS INTRODUCTION Early chromosomal data, and more recent studies of G6PD heterozygotic females, a l l i n d i c a t e that the chronic myeloproliferative disorders 1 2 3 (polycythemia vera (PV), chronic myeloid leukemia (CML), ' and i d i o p a t h i c 4 myeloid metaplasia, ) generally a r i s e from the transformation of a s i n g l e c e l l . Such studies have furt h e r shown that the i n i t i a l l y transformed c e l l i s capable not only of extensive self-renewal, but a l s o of d i f f e r e n t i a t i o n along a l l of the myeloid pathways and, i n some instances, at l e a s t some of the lymphoid pathways.^ Thus, during the early stages of these diseases, members of the transformed clone outgrow and replace to varying degrees the normal hemopoietic c e l l elements i n the marrow, although d i f f e r e n t i a t i o n of these abnormal progenitors i n t o r e l a t i v e l y normal non-dividing mature blood c e l l s may proceed i s a more or l e s s normal fashion. A recurrent problem i n assessing the status of patients with such stem c e l l disorders l i e s i n the d i f f i c u l t y of obtaining information about changes at the stem c e l l l e v e l . Unfortunately, such information i s u n l i k e l y to be provided by cytogenetic analysis of fresh marrow aspirates since the most prevelant d i v i d i n g c e l l s i n the marrow are usually the most d i f f e r e n t i a t e d and, at the present time, methodology for the d i r e c t i d e n t i f i c a t i o n of metaphases s p e c i f i c a l l y from stem c e l l s does not e x i s t . However, clonogenic assays f o r p r i m i t i v e hemopoietic c e l l s do e x i s t and progenitors can be stimulated to p r o l i f e r a t e and d i f f e r e n t i a t e _in v i t r o . Under appropriate culture conditions, 59 i n d i v i d u a l committed myeloid progenitor c e l l s can give r i s e to i d e n t i f i a b l e colonies.^ (See previous chapter f o r a more complete discussion of the i n v i t r o assays for hemopoietic progenitors.) Pure e r y t h r o i d and pure granulopoietic colonies can be r e a d i l y distinguished from each other i n the l i v i n g state by the color and d i s t i n c t i v e arrangement of t h e i r d i f f e r e n t component c e l l types. The erythroblasts i n maturing e r y t h r o i d colonies synthesize s u f f i c i e n t hemoglobin to become v i s i b l y red and the c e l l s are grouped i n small subcolonies or c l u s t e r s . In contrast, the c e l l s i n granulopoietic colonies remain c l e a r and are generally more evenly dispersed. Mixed colonies containing both erythroblasts and c e l l s of the granulocyte s e r i e s , and hence thought to derive from plur i p o t e n t progenitors, have also been observed, although at much lower frequencies.^ I t i s thus possible to obtain information about a spectrum of r e l a t i v e l y rare hemopoietic c e l l types simply by the analysis of t h e i r c l o n a l progeny generated i n v i t r o (see Figure 6). The importance of such an approach i s exemplified by recent studies of two female G6PD heterozygotes with PV and another with CML. In these 3 patients, a l l of the c i r c u l a t i n g red c e l l s and granulocytes were shown to be derived from the abnormal clone, whereas the more p r i m i t i v e colony-forming c e l l populations i n t h e i r marrows included normal as 1 8 9 well as abnormal representatives. ' ' Chromosomal studies of i n d i v i d u a l hemopoietic colonies o f f e r a powerful approach to the study of a c e l l compartment not i d e n t i f i a b l e i n d i r e c t bone marrow chromosome preparations. This i s of p a r t i c u l a r relevance to the myeloproliferative diseases where chromosomal markers are frequently 2 encountered, e s p e c i a l l y i n CML. I t i s , therefore, not s u r p r i s i n g that attempts along these l i n e s have been made for almost as long as human 10-13 hemopoietic colony assays have been a v a i l a b l e . However, most colonies i n BONE MARROW ASPIRATE • • < Stem cells Differentiated cells Banded karyotypes from direct preparations are predominantly from highly differentiated proliferating hemopoietic cells. Figure 6. A Schematic Illustration Demonstrating For Primitive Precursor Cells. HEMOPOIETIC COLONY IN VITRO Banded karyotypes from individual colonies are exclusively from progeny of single hemopoietic progenitor cells committed to a particular path-way of differentiation (erythroid or granulocytic) The Rationale Of Using Clonogenic A s s a y s O 61 which c e l l d i v i s i o n i s s t i l l o ccurring contain l e s s than 1,000 c e l l s , and the t e c h n i c a l problem of processing such small numbers of c e l l s has severely l i m i t e d the a c q u i s i t i o n of extensive data. Since the main objective of t h i s t h e s i s was to t r y to demonstrate the extent to which c y t o g e n e t i c a l l y normal stem c e l l s , with the capacity f o r normal p r o l i f e r a t i o n and d i f f e r e n t i a t i o n 1 p e r s i s t i n patients with Ph - p o s i t i v e CML, a r e l i a b l e method f o r obtaining high q u a l i t y chromosome preparations from i n d i v i d u a l hemopoietic colonies was of prime importance. In t h i s chapter, the development of a methodology that r o u t i n e l y allows most selected colonies to be karyotyped i s described. MATERIALS AND METHODS A number of pe r i p h e r a l blood and bone marrow samples were obtained through the courtesy of physicians i n the Province of B r i t i s h Columbia. Some peri p h e r a l blood samples were a l s o obtained from normal healthy volunteers. A l l samples from patients were l e f t o v e r specimens taken with informed consent f o r the purpose of c l i n i c a l diagnosis, staging, or follow-up. Peripheral blood mononuclear c e l l s and bone marrow buffy coat c e l l s were prepared and cultured i n 35 x 10 mm standard nontissue culture P e t r i dishes i n 1.1 ml of culture 14 medium as described i n Appendix I. (Also, see Figure 4, Chapter One.) Washed c e l l s were added to a f i n a l medium that consisted of 0.8% methylcellulose, 30% f e t a l c a l f serum (FCS), 0.1% deionized bovine serum -4 albumin, 9% human leukocyte conditioned medium, 10 2-mercaptoethanol, 2.5 units/ml of e r y t h r o p o i e t i n (Epo, Step I I I , Connaught Laboratories, Toronto, Ontario), and alpha-medium (see Appendix II for precise d e t a i l s ) . P eripheral blood preparations were usually plated at a f i n a l c e l l 5 concentration of 4 x 10 c e l l s per dish except for specimens from patients with PV or CML where t h i s c e l l concentration was expected to give r i s e to a larger 15 than normal number of colonies. In these cases, the p l a t i n g concentration 62 was lowered appropriately t o give 30-50 large colonies per dish. Bone marrow 5 preparations were p l a t e d at a f i n a l concentration of 2 x 10 c e l l s per dish. A l l reagents were prescreened and selected f o r optimal e r y t h r o i d and 16 granulocyte colony growth-supporting capacity against laboratory standards. Cultures were incubated at 37°C i n a 5% CC^-air environment and maintained at high humidity. Procedure for Harvesting and Processing Colonies Dishes were f i r s t examined using an inverted microscope a f t e r 8-9 days and at d a i l y i n t e r v a l s t hereafter. The a c t u a l day of colony harvest was chosen to maximize the number of c e l l s recovered i n metaphase. Preliminary r e s u l t s i n d i c a t e d that, on average, a f t e r 14 days colonies contained few d i v i d i n g c e l l s , although they were more mature and hence easier to recognize and micro-17 manipulate. These observations are i n keeping with previous studies of the growth c h a r a c t e r i s t i c s of e r y t h r o p o i e t i c colonies which showed p r o l i f e r a t i o n and maturation to be c l o s e l y associated, cessation of colony growth c o i n c i d i n g 14 c l o s e l y with the onset of hemoglobin production. Analyzable metaphases could be recovered from most large (>500 c e l l ) colonies with c a r e f u l s e l e c t i o n of recognizable but immature colonies. Colonies were scored as e r y t h r o i d only i f a s i g n i f i c a n t proportion of c e l l s i n them contained s u f f i c i e n t hemoglobin to give them a d i s t i n c t l y reddish hue. Colonies were c l a s s i f i e d as granulocytic by t h e i r c h a r a c t e r i s t i c morphology containing small, c l e a r c e l l s d i s p e r s i n g r e l a t i v e l y homogenously from a more concentrated c e n t r a l core of c e l l s . The presence of lobulated n u c l e i was also confirmed on the f i n a l s l i d e s . Colonies that d i d not f a l l c l e a r l y i n t o e i t h e r of these two categories were not used. One hour p r i o r to the end of the incubation period, c e l l d i v i s i o n s were 63 arrested at metaphase by the ad d i t i o n of 0.1 ml of colcemid (Gibco) to each 1.1 ml c u l t u r e . The colcemid was d i l u t e d to 1 mg/ml i n Hank's BSS and then applied using a 26-gauge syringe needle to ensure even a p p l i c a t i o n over the surface of the methylcellulose. Single colonies were removed i n d i v i d u a l l y i n t o a f i n e l y drawn out Pasteur p i p e t t e ( i n t e r n a l diameter of approx. 0.5 mm) using an inverted microscope at a f i n a l magnification of 75X. The colony, suspended i n approximately 0.01 ml of the methylcellulose culture surrounding i t , was then t r a n s f e r r e d i n t o a m i c r o t i t e r well (Linbro) containing 0.1 ml of 0.075 M hypotonic KC1 and dispersed by gently p i p e t t i n g up and down. After 20 minutes at room temperature, the e n t i r e contents of the m i c r o t i t e r well were tra n s f e r r e d onto a microscope s l i d e that had been pretreated with 0.01% p o l y l y s i n e (w/v) (Sigma #P1886, St. Louis, Mo.) i n d i s t i l l e d water f o r 90 18 19 minutes. ' The pretreatment was c a r r i e d out by p l a c i n g one drop of the p o l y l y s i n e s o l u t i o n onto the s l i d e and then coverslipping. I t thus f a c i l i t a t e d the a p p l i c a t i o n of an adhesive coat of p o l y l y s i n e on the glass surface. Immediately p r i o r to use, the c o v e r s l i p was washed o f f and the s l i d e b l o t t e d dry. The colony, s t i l l suspended i n the hypotonic s o l u t i o n , was allowed to s i t on the p o l y l y s i n e t reated area of the s l i d e i n a humid environment f o r 10 minutes. Then the excess hypotonic s o l u t i o n was gently removed using an absorbent paper, and 2 drops of 3:1 methanol a c e t i c a c i d f i x a t i v e was gently dropped with a Pasteur pipette onto the area of the s l i d e where the colony had been placed. A f t e r 15-30 seconds, 2 more drops of the same f i x a t i v e was added. Afte r about 1 minute, the s l i d e was quickly dried high over an open flame. The s l i d e , with the colony now f i r m l y f i x e d to i t , was then immersed i n fresh f i x a t i v e f o r 15 minutes before a i r drying and scanning. C e l l recovery using t h i s procedure was found to be 80-90% (Table I ) . 64 TABLE I Percentage of c e l l s recovered when hemopoietic colonies grown i n standard methylcellulose cultures were harvested f o r chromosomes using s l i d e s pretreated with p o l y l y s i n e ^ Experiment Percent C e l l s Recovered +SE A B C 85 + 3 82 + 16 90 + 5 In order to determine the c e l l recovery attainable using t h i s method, a peri p h e r a l blood sample from a healthy donor was set up f o r the growth of hemopoietic colonies. On the 11th day of incubation, 10 large e r y t h r o i d colonies were plucked following the normal procedure and pooled i n t o a volume of hypotonic s o l u t i o n required to give a f i n a l c e l l concentration of about 500-1000 c e l l s per ml. Twenty aliquots of 0.1 ml were tra n s f e r r e d to i n d i v i d u a l m i c r o t i t e r wells f o r remainder of the 20-min hypotonic treatment. Ten al i q u o t s were then t r a n s f e r r e d to a p o l y l y s i n e - t r e a t e d microscope s l i d e and the remaining 10 to an untreated microscope s l i d e . The c e l l s on the p o l y l y s i n e - t r e a t e d s l i d e s were treated according to the procedure f o r harvesting colonies f o r chromosome a n a l y s i s . The aliquots on the untreated s l i d e were quickly dried with the a i d of heat lamp. A l l s l i d e s were f i x e d i n 3:1 methanol:acetic a c i d and stained with Giemsa. The procedure was repeated using two other donors. The average number of c e l l s on the untreated s l i d e s was taken to represent 10 0% recovery, and when t h i s value was compared to the average number of c e l l s on the p o l y l y s i n e - t r e a t e d s l i d e , the average c e l l recovery using t h i s technique was determined. 65 Metaphases suitable for analysis were sequentially Q and G-banded 20 21 according to routine methods as described i n Appendix I I I . ' Selected metaphases were fluorescent-reverse banded using chromomycin A3 and methylgreen 22 23 (see Appendix I I I ) . ' SCE Studies The s i s t e r chromatid exchange (SCE) assay for the presence of chromosomal breaking agents was used as part of the development of the present methodol-24 -3 ogy. For SCE analysis of e r y t h r o i d colonies, 0.05 ml of 10 M BrdU i n H^ O was added to each 1.1 ml culture 48 hours p r i o r to harvest. For the SCE -3 analysis of epo-stimulated fresh marrow, 0.25 ml of 10 M BrdU was added to each 5.5 ml c u l t u r e containing 0.5 ml fresh bone marrow aspirate and 5 ml of alpha-medium with 15% FCS, and incubated i n a loosely capped t e s t tube at 37°C i n a 5% CO^-air environment f o r 48 hours. Chromosomes were prepared by routine 25 methods as described i n Appendix VI. For PHA-stimulated per i p h e r a l blood, -3 0.25 ml of 10 BrdU was added to each 5.5 ml culture containing 0.5 ml fresh blood, 0.05 ml PHA (M) (Gibco) and 5 ml of alpha-medium with 15% FCS. Cultures o were incubated i n a loosely capped t e s t tube at 37 C i n a 5% CC^-air environment f o r 72 hours. Chromosomes were prepared by routine methods (see ' 2 6 Appendix V). For s k i n f i b r o b l a s t s , a l i n e was established from a punch biopsy performed on a healthy male volunteer. A f t e r 6 passages, c e l l s were grown on s l i d e s f o r chromosomes according to established methods (see Appendix 27 VI). F o r t y - f i v e hours p r i o r to harvest, BrdU, at a f i n a l concentration of 50 JUM, was added to each c u l t u r e . A l l cultures containing BrdU were protected from d i r e c t l i g h t throughout t h e i r incubation. D i f f e r e n t i a l l y stained metaphases were obtained using the fluorescence-plus-Giemsa methodology (see 28 Appendix VII). 66 RESULTS Lack of Overlap Between I n d i v i d u a l l y Removed Colonies In order to show that a single colony of c e l l s c o n s i s t i n g of several hundred c e l l s could be removed from a 35 mm dish containing up to 30 s i m i l a r colonies, the following mixing experiment was performed. Peripheral blood samples were taken from a healthy male and a healthy female volunteer, and prepared i n the usual manner. Equal number of c e l l s from the two i n d i v i d u a l s 5 were mixed and plated at 2 x 10 c e l l s of each type per dish. F i f t y e r y t h r o i d colonies were harvested from 10 dishes on days 10 and 11 of growth and, from these, 35 preparations y i e l d e d metaphases. These were then Q banded and scored f o r the number of metaphases that could be p o s i t i v e l y i d e n t i f i e d as male or female. The r e s u l t s are shown i n Table I I , experiment A. Twenty-eight of the 35 colonies with metaphases contained two or more that could be analyzed. Of these, 19 colonies y i e l d e d only male metaphases and the remaining nine contained only female metaphases. Figure 7 shows a G-banded metaphase obtained from a single e r y t h r o i d colony of female o r i g i n . On average, 630 +60 c e l l s and 12+1 analyzable metaphases were recovered from a si n g l e colony. When the experiment was repeated, using c e l l s from a d i f f e r e n t p a i r of male and female donors, s i m i l a r r e s u l t s were obtained (Table I I , experiment B). SCE Assay f o r Clastogens In V i t r o Various authors have suggested that some c e l l s may be prone to the development of chromosomal breakage a f t e r periods of more than 1 or 2 days i n cul t u r e . In order to assess the possible importance of t h i s phenomenon for hemopoietic c e l l s p r o l i f e r a t i n g and d i f f e r e n t i a t i n g i n methylcellulose cultures, the incidence of SCE i n 9- to 11-day-old e r y t h r o i d colony c e l l s was measured and compared to the value obtained with the incidence of SCE f o r epo-stimulated fresh marrow, PHA-stimulated p e r i p h e r a l blood or skin f i b r o b l a s t s . 67 TABLE II Results of karyotype analysis performed on metaphases obtained from single e r y t h r o i d colonies harvested from cultures containing colonies of both male and female o r i g i n . Male - Female Mixing Experiment Expt. A Expt. B No. of Colonies Containing 2 or More Metaphases 28 30 Colonies Containing Only Male Karyotypes 19 16 Colonies Containing Only Female Karyotypes 9 14. C e l l s Recovered Per Colony 630 ±60* Not done Analyzable Metaphases Per Colony 12 ± 1* Not done Maximum Analyzable Metaphases Per Colony 19 62 * Average ± SE for 28 colonies used for cytogenetic analysis. Figure 7. A G-Banded Metaphase And Karyotype Obtained From A Single Erythroid Colony. 69 Figure 8. A Differentially Stained Metaphase From A Single Erythroid Colony. 70 Figure 9 . A Chromomycin A 3 - Methyl Green Reverse Banded Metaphase From A Granulocyte Colony From A Male With P h 1 - P o s i t i v e CML. 71 The r e s u l t s are shown i n Table I I I . The l e v e l of SCE was not increased i n cultured hemopoietic c e l l s and was s i m i l a r to previously reported normal 29 30 values. ' Figure 8 shows an example of a d i f f e r e n t i a l l y stained metaphase from an er y t h r o i d colony. Peripheral blood and bone marrow c e l l s from an i n d i v i d u a l with Bloom syndrome were a l s o studied. These showed the 3 1 c h a r a c t e r i s t i c marked increase i n SCE frequency and served as a p o s i t i v e c o n t r o l (Table I I I ) . Karyotypes from Single Colonies from Assays of PV and CML As a preliminary t e s t of the a p p l i c a b i l i t y of t h i s method to studies of patients with myeloproliferative disease, three such i n d i v i d u a l s were studied: one with PV and two with P t J - p o s i t i v e CML. Both e r y t h r o i d and granulocyte colonies were obtained i n a l l cases from peripheral blood cultures. Table IV shows the number and type of colonies plucked per i n d i v i d u a l , the average number of analyzable metaphases per colony, and the outcome of karyotype analysis using Q and G-banding f o r the two more extensively studied p a t i e n t s . In the patient with PV, each of the 6 e r y t h r o i d colonies analyzed showed a 1 normal male karyotype. In the female patient with Ph - p o s i t i v e CML, each of the f i v e e r y t h r o i d and two granulocytic colonies analyzed was 46,XX,Ph^ (see Appendix IX f o r cytogenetic nomenclature). Figure 9 i l l u s t r a t e s the usefulness of the chromomycin A3-methyl green reverse banding method f o r the i d e n t i f i c a -t i o n of the Ph^ chromosome. The metaphase shown was obtained from a sin g l e granulocyte colony from a 40-year-old male with Ph^-positive CML. DISCUSSION The technique presented here permits high q u a l i t y chromosome preparations, suitable f o r banding, to be r o u t i n e l y obtained from single e r y t h r o i d or granulopoietic colonies. Previous problems due to c e l l loss were overcome by TABLE III SCE frequency i n metaphases from erythroid colonies harvested from 1- to 2-week-old methylcellulose cultures. Also shown for comparison are SCE frequencies i n epo-stimulated fresh marrow, PHA-stimu-lated peripheral blood, and skin fibroblasts. Bloom syndrome values serve as a positive control. Expt. Diagnosis C e l l Type No. Cells Counted SCE/Metaphase ± SE QL * Plasmacytoma Erythroid colonies from marrow 30 3.6 ± 0.7 WK Secondary Erythrocytosis Erythroid colonies from marrow 30 4.8 ± 0.5 OL * Plasmacytoma Erythroid colonies from marrow 30 3.1 + 0.6 GK Normal Erythroid colonies from peripheral blood 30 5.3±0.5 HS * Lung Cancer Epo-stimulated fresh marrow 30 3.8 ± 0.4 WK Secondary Erythrocytosis Epo-stimulated fresh marrow 15 4.510.6 HT 12 Normal PHA-stimulated peripheral blood 30 3.1 + 0.4 HT 21 Normal PHA-stimulated peripheral blood 50 4.6 ± 0.3 HT 31 Normal PHA-stimulated peripheral blood 46 3.9 + 0.3 HT 41 Normal PHA-stimulated peripheral blood 79 3.7 + 0.2 FT 1 Normal Skin fibroblasts - passage six 30 4.2+0.4 JB Bloom Syndrome Epo-stimulated fresh marrow 5 115 + 26 JB Bloom Syndrome PHA-stimulated peripheral blood 10 97 + 22 * Untreated patients in which there was no evidence of marrow involvement. 73 TABLE IV Outcome of karyotype analysis performed on single hemopoietic colonies grown from: (a) a peripheral blood sample from a 40-year-old male with the c l i n i c a l diagnosis of polycythemia vera; (b) a peripheral blood sample from a 47-year-old female with the c l i n i c a l diagnosis of chronic myelogenous leukemia.^ (a) PATIENT: RW DIAGNOSIS: Polycythemia vera SOURCE OF SAMPLE: Blood Colony Type No. of Karyotypes Per Colony Result Erythroid 2 46, XY Erythroid 3 46, XY Erythroid 3 46, XY Erythroid 2 46, XY Erythroid 3 46, XY Erythroid 3 46, XY PATIENT: SC DIAGNOSIS: Chronic myelogenous leukemia SOURCE OF SAMPLE: Blood Colony Type No. of Karyotypes Per Colony Result Erythroid 6 46, XX, Ph 1 Erythroid 3 46, XX, Ph 1 Erythroid 3 46, XX, Ph 1 Erythroid 2 46, XX, Ph 1 Erythroid 1 46, XX, Ph 1 Granulocyte 12 46, XX, Ph 1 Granulocyte 3 46, XX, Ph 1 Diagnosis based on the recommendations of the Polycythemia Vera Study Group (32). Cultured at the time of diagnosis when peripheral white count was 166,000 per cu. mm and d i f f e r e n t i a l compatible with CML. 74 the use of p o l y l y s i n e coated s l i d e s and s e l e c t i o n of colonies at a stage when they could be recognized but when c e l l d i v i s i o n had not yet ceased. The o r i g i n a l impetus f o r t h i s work was the need for methology to analyze stem c e l l populations i n patients with hemopoietic disorders. Technically, i t was therefore important to show that i n cultures containing a r t i f i c i a l ' mixtures of c y t o g e n e t i c a l l y d i s t i n c t progenitors (achieved by using male and female donors), the method yi e l d e d k a r y o t y p i c a l l y pure colonies. This i s consistent with previous studies i n d i c a t i n g such colonies o r i g i n a t e from si n g l e 33 progenitors and suggests that i t should be possible to i d e n t i f y chromosomally unique colonies i n assays of n a t u r a l l y occurring heterogeneous populations. Cytogenetic analysis of i n d i v i d u a l e r y t h r o i d or granulopoietic colonies o f f e r s several advantages over the conventional method of obtaining metaphases from fresh bone marrow aspirates. F i r s t , i t permits the assignment of karyotypes to known progenitor populations. This i s possible because the s i z e and content of d i f f e r e n t i a t e d c e l l s i n each colony analyzed can be separately determined and these c h a r a c t e r i s t i c s serve to i d e n t i f y the type and stage of development of the o r i g i n a l progenitor. Second, since the l a r g e s t colonies are believed to derive from the most 34 p r i m i t i v e c e l l types, karyotype analysis of such colonies should provide a view that c l o s e l y approximates the cytogenetic composition of the stem c e l l compartment. In hemopoietic disease states where cytogenetic changes accompany transformation, analysis of the predominant d i v i d i n g c e l l types i n the marrow would be expected to provide quite a d i f f e r e n t p i c t u r e , highly skewed i n favour of those subpopulations that had gained a s e l e c t i v e growth advantage. Thus r e s i d u a l , normal, quiescent stem c e l l s would escape detection by d i r e c t marrow studies although t h e i r presence might be demonstrable a f t e r stimulation i n v i t r o . Recent studies of G6PD heterozygotes suggest that t h i s i s indeed the 75 8 9 case i n at l e a s t some patients with PV and CML. ' S i m i l a r l y , cytogenetic 35 changes heralding progression from a chronic stage to acute leukemia might be apparent i n colony-forming c e l l s before becoming detectable i n d i r e c t marrow preparations. T h i r d , since numerous karyotypes may be obtained from a single colony, the chance of a cy t o g e n e t i c a l l y unusual progenitor being an a r t i f a c t i s remote. F i n a l l y , i t should be noted that since p r i m i t i v e hemopoietic progenitor c e l l s c i r c u l a t e i n the per i p h e r a l blood of patients with myeloproliferative 3 6 disorders, h o r i z o n t a l studies of such i n d i v i d u a l s are possible without the necessity of repeated bone marrow a s p i r a t i o n . Although the present method has been s p e c i f i c a l l y developed for the an a l y s i s of myeloid colonies i n which normal erythroid and granulocytic d i f f e r e n t i a t i o n i s proceeding, i t could be r e a d i l y adapted for the analysis of other types of colonies, both hemopoietic and nonhemopoietic, where assessment of progenitor c e l l s may be of s i m i l a r importance. However, as i n the present a p p l i c a t i o n , adaptation of the method to other types of colonies i s l i k e l y to depend on optimization of the culture conditions used, methods f o r i d e n t i f y i n g the type of c e l l s produced, and a knowledge of the colony growth k i n e t i c s . SUMMARY The need f o r improved methodology to f a c i l i t a t e cytogenetic analysis of hemopoietic stem c e l l populations, p a r t i c u l a r l y i n studies of the hemopoietic malignancies, has been recognized f o r a number of years. Since p r i m i t i v e hemopoietic c e l l s can be stimulated to form colonies of i d e n t i f i a b l e progeny under appropriate conditions i n v i t r o , i t should i n theory, be possible to obtain such information. However, hemopoietic colonies of human o r i g i n r a r e l y contain more than 1000 c e l l s , and i n handling such small samples, c e l l l o s s has h i s t o r i c a l l y been a major problem. 76 A method has been developed that allows from two up to more than 50 metaphases per colony to be obtained from most er y t h r o i d and granulopoietic colonies harvested i n d i v i d u a l l y from standard methylcellulose assay cultures. Of key importance i s the s e l e c t i o n of large but s t i l l immature colonies and the use of p o l y l y s i n e coated s l i d e s , which ensures recovery of 80-90% of the sample. The method y i e l d s reproducibly high q u a l i t y metaphases su i t a b l e for analysis a f t e r G, Q, and fluorescent-reverse banding. Cytogenetic analysis of 58 colonies removed i n d i v i d u a l l y from cultures i n i t i a t e d with a mixture of male and female c e l l s showed both male and female colonies to be present as expected. In a l l instances, only one type of metaphase was ever found i n a si n g l e colony. A procedure f o r measuring the incidence of s i s t e r chromatid exchange (SCE) i n the progeny of p r i m i t i v e hemopoietic progenitors has also been established. SCE values obtained f o r 1- to 2-week-old colonies derived from normal progenitors were s i m i l a r to previously published normal SCE values. Preliminary a n a l y s i s of e r y t h r o i d and granulocytic colonies from one patient with polycythemia vera (PV) and two patients with chronic myelogenous leukemia (CML) i n d i c a t e that preparations of equal q u a l i t y are obtained from such i n d i v i d u a l s and thus, for the f i r s t time, the Ph^-chromosome could be r e a d i l y demonstrated i n e r y t h r o i d colonies. 77 REFERENCES 1. Adamson JW, Fialkow PJ, Murphy S, Prchal JF, Steinmann L: Polycythemia vera: Stem c e l l and probable c l o n a l o r i g i n of the disease. N Engl J Med 295: 913-916, 1976. 2. Nowell PC, Hungerford DA: A minute chromosome i n human chronic granulo-c y t i c leukemia. Science 132: 1497, 1960. 3. Fialkow PJ, G a r t l e r SM, Yoshida A: Clonal o r i g i n of chronic myelocytic leukemia i n man. Proc Natl Acad S c i USA 58: 1468-1471, 1967. 4. Jacobson RJ, Salo A, Fialkow PJ: Agnogenic myeloid metaplasia: A c l o n a l p r o l i f e r a t i o n of hematopoietic stem c e l l s with secondary myelofibrosis. Blood 51: 189-194, 1978. 5. Martin PJ, N a j f e l d V, Hansen JA, Penfold GK, Jacobson RJ, Fialkow PJ: Involvement of the B-lymphoid system i n chronic myelogenous leukemia. Nature 287: 49-50, 1980. 6. Metcalf D: Hemopoietic Colonies. In V i t r o Cloning of Normal and Leukemic C e l l s , Recent Results i n Cancer Research. Springer-Verlag, New York, pp 1-227, 1977. 7. Fauser AA, Messner HA: Granulo-erythropoietic colonies i n human bone marrow, pe r i p h e r a l blood, and cord blood. Blood 52: 1243-1247, 1978. 8. Prchal JF, Adamson JW, Murphy S, Steinmann L, Fialkow PJ: Polycythemia vera: The i n v i t r o response of normal and abnormal stem c e l l l i n e s to e r y t h r o p o i e t i n . J C l i n Invest 61: 1044, 1978. 9. Singer JS, A r l i n AZ, Najfeld V, Adamson JW, Kempin SJ, Clarkson BD, Fialkow PJ: Restoration of nonclonal hematopoiesis i n chronic myelogen-ous leukemia (CML) following a chemotherapy-induced loss of the Ph chromosome. Blood 56: 356-360, 1980. 10. Chervenick PA, E l l i s CD, Pan SF, Lawson AL: Human ^Leukemic c e l l s : In v i t r o growth of colonies containing the Philadelphia (Ph ) chromosome. Science 174: 1134-1136, 1971. 11. Aye MT, T i l l JE, McCulloch EA: C y t o l o g i c a l studies of granulopoietic colonies from two patients with chronic myelogenous leukemia. Exp Hematol 1: 115-118, 1973. 12. Moore MAS, Metcalf D: Cytogenetic analysis of human acute and chronic myeloid leukemic c e l l s cloned i n agar culture. Int J Cancer 11: 143-152, 1973. 13. B u l l J : Cytogenetic studies of marrow and p e r i p h e r a l blood granulocyte colonies i n treated chronic myelogenous leukemia. Blood C e l l s 1: 161-162, 1975. 14. Gregory CJ, Eaves AC: Human marrow c e l l s capable of e r y t h r o p o i e t i c d i f f e r -e n t i a t i o n i n v i t r o . D e f i n i t i o n of three e r y t h r o i d colony responses. Blood 78 49: 855-864, 1977. 15. Eaves AC, Eaves CJ: Abnormalities i n the e r y t h r o i d progenitor compartments i n patients with chronic myelogenous leukemia (CML). Exp Hematol 7 (Suppl 5): 65-75, 1979. 16. Eaves CJ, Eaves AC: E r y t h r o i d progenitor c e l l numbers i n human marrow— Implications f o r r e g u l a t i o n . Exp Hematol 7 (Suppl 5): 54-64, 1979. 17. Dube ID. Cytogenetic analysis of single hemopoietic colonies. M.Sc. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1980. 18. Mazia D, Schatten G, Sale W: Adhesion of c e l l s to surfaces coated with P o l y l y s i n e . J C e l l B i o l 66: 198-200, 1975. 19. Rajendra BR, S c i o r r a L J , Lee M: A new and simple technique f o r chromosomal preparations from per i p h e r a l blood lymphocytes, amniotic c e l l c ultures, skin f i b r o b l a s t s , bone marrow and single c e l l clones when the y i e l d s from harvest are low. Hum Genet 55: 363-366, 1980. 20. Caspersson T, Zech L, Johanssen C: D i f f e r e n t i a l binding of a l k y l a t i n g flurorchromes i n human chromosomes. Exp C e l l Res 60: 315-319, 1970. 21. Seabright M: A rapid banding technique for human chromosomes. Lancet 2: 971-972, 1971. 22. Sahar E, L a t t SA: Enhancement of banding patterns i n human metaphase chromosomes by energy t r a n s f e r . Proc Natl Acad S c i USA 75: 5650-5654, 1978. 23. Chamberlin J , Magenis PE: Fluorescent s t a l k s . Am J Hum Genet 31: 91A, 1979. (and personal communications) 24. Latt SA, Schreck RR: S i s t e r chromatid exchange a n a l y s i s . Am J Hum Genet 32: 297-313, 1980. 25. Sandberg A: The Chromosomes i n Human Cancer and Leukemia. E l s e v i e r , New York, p. 102, 1980. 26. Moorhead PS, Nowell PC, Mellman, WJ, Batipps DM, Hungerford DA: Chromosome preparations of leucocytes cultured from human pe r i p h e r a l blood. Exp C e l l Res 20: 613-616, 1960. 27. Schneider EL, Tice RR, Kram D: Bromodeoxyuridine-differential s t a i n i n g technique: a new approach to examining s i s t e r chromatid exchange and c e l l r e p l i c a t i o n k i n e t i c s . In: Methods i n C e l l Biology, V o l . 25 (D Prescott, ed.), Academic Press, New York, pp. 379-409, 1978. 28. Perry P, Wolff S: New Giemsa method f o r the d i f f e r e n t i a l s t a i n i n g of s i s t e r chromatids. Nature 251: 156-158, 1974. 29. Beck B, Obe G: The human leucocyte t e s t system. Hum Genet 29: 127-134, 1975. 79 30. Dube ID: The incidence of s i s t e r chromatid exchanges i n cultured human lymphocytes. B.Sc. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1977. 31. Chaganti RSK, Schonberg S, German J : A many-fold increase i n s i s t e r chromatid exchanges i n Bloom's syndrome lymphocytes. Proc Natl Acad S c i USA 71: 4508-4512, 1974. 32. Wasserman LR: The treatment of polycythemia vera. Semin Hematol 13: 57-78, 1976. 33. Prchal JR, Adamson JW, Steinmann L, Fialkow PJ: Human erythroid colony formation i n v i t r o : Evidence f o r c l o n a l o r i g i n . J C e l l Physiol 89: 489-492, 1976. 34. Eaves CJ, Humphries RK, Eaves AC: In v i t r o c h a r a c t e r i z a t i o n of e r y t h r o i d precursor c e l l s and the e r y t h r o p o i e t i c d i f f e r e n t i a t i o n process. In: C e l l u l a r and Molecular Regulation of Hemoglobin Switching (G. Stamatoyann-opoulos, AW Nienhuis, eds.), Grune & Stratton, New York, pp 251-273, 1979. 35. Michaux J-L, Van Den Berghe H, Rodhain J , Sokal G, David G, Hulhoven R: Etude simultanee du caryotype et de 1'histo^.ogie medullaire dans l a leucemie myeloide chronique a chromosome Ph . Nouv Rev F r Hematol 15: 575-583, 1975. 36. Eaves AC, Henkelman DH, Eaves CJ: Abnormal erythropoiesis i n the myelo-p r o l i f e r a t i v e disorders: an analysis of underlying c e l l u l a r and humoral mechanisms. Proceedings of the NIH Conference on "Erythropoietin i n the Regulation of Er y t h r o p o i e s i s " . Exp Hematol 8(Suppl 8): 235-247, 1980. 80 C H A P T E R T H R E E CYTOGENETIC STUDIES OF EARLY MYELOID PROGENITOR COMPARTMENTS IN CHRONIC PHASE 1 1 Ph -POSITIVE CML. METHYLCELLULOSE ASSAYS REVEAL THE PERSISTENCE OF Ph -NEGATIVE COMMITTED PROGENITORS THAT ARE SUPPRESSED FROM DIFFERENTIATING IN VIVO INTRODUCTION Chronic myeloid leukemia (CML) i s a myeloproliferative disease t y p i c a l l y characterized by an increased rate of r e l a t i v e l y normal granulopoiesis. During the e a r l y or chronic phase of CML, white blood c e l l (wbc) counts can be reduced to acceptable l e v e l s by non-specific a l k y l a t i n g agents, such as busulfan (myleran) or antimetabolic agents, such as hydroxyurea. With therapy, the chronic phase can thus be r e l a t i v e l y symptom-free and l a s t s f o r about three years from the time of presentation. The more terminal or acute phase i s characterized by an acceleration i n the rate of granulopoiesis with concomit-tant f a i l u r e of hemopoietic precursors to complete normal d i f f e r e n t i a t i o n , r e s u l t i n g i n a preponderance of immature c e l l s i n the marrow and blood. This stage i s r e l a t i v e l y unresponsive to currently a v a i l a b l e therapy and usually terminates f a t a l l y i n about three months due to the i n a b i l i t y of normal blood elements to function, or as a r e s u l t of therapeutic complications. In s p i t e of the advent of new therapeutic protocols, o v e r a l l s u r v i v a l i n CML has not improved s u b s t a n t i a l l y i n 60 years.^ ^- 2-11 „ . ,12-16 ^ Cytogenetic and, more recently, biochemical studies have shown that CML a r i s e s from the transformation to neoplastic growth of a single c e l l capable of extensive self-renewal as well as d i f f e r e n t i a t i o n along the major hematopoietic lineages ( i . e . , i n a p l u r i p o t e n t stem c e l l ) . In 85-90% of cases, a l l c e l l s of the transformed clone are marked by a unique chromosomal tr a n s l o c a t i o n that r e s u l t s i n an abnormally small chromosome #22; the 81 Philadelphia chromosome (Ph ). ' The Ph p e r s i s t s throughout the course of CML and i n the acute phase, a d d i t i o n a l non-random chromosome abnormalities are 19 1 often seen i n the leukemic c e l l s . The Ph i s thus a u s e f u l marker, not only c l i n i c a l l y , but a l s o i n experimental oncology where, as i s the case here, i t can be used to study the dynamic i n t e r a c t i o n s between leukemic and non-leukemic c e l l populations i n v i t r o . An important question i n CML i s the f a t e of those p l u r i p o t e n t stem c e l l s that are not involved i n the o r i g i n a l transformation. Since the vast majority of bone marrow metaphases from most patients bear the Ph^, i t has been postulated that the normal p l u r i p o t e n t stem c e l l pool has ei t h e r been destroyed or e f f e c t i v e l y suppressed from p r o l i f e r a t i n g . The question of t h e i r existence i s not only of academic importance, since i t s r e s o l u t i o n w i l l provide i n s i g h t s i n t o the very early events that occur i n the course of transformation, but of c l i n i c a l s i g n i f i c a n c e since a t o t a l destruction of the normal p l u r i p o t e n t stem c e l l pool would make a complete cure impossible using current approaches. On the other hand, i f r e s i d u a l normal stem c e l l s e x i s t i n most patients, then i t may be possible to make c o r r e l a t i o n s between t h e i r presence and prognosis and response to therapy. Indeed, the existence of normal stem c e l l s may play a major r o l e i n future treatment s t r a t e g i e s . For example, German has suggested r e s t o r a t i o n of normal hematopoiesis may be accomplished by autologous trans-p l a n t a t i o n with an enriched population of i n v i t r o - s e l e c t e d normal stem c e l l s 20 following a b l a t i o n of diseased marrow by standard measures. A l t e r n a t i v e l y , i f normal stem c e l l s e x i s t i n a quiescent state then normal hematopoiesis may be restored i f t r u l y leukemia-cell s p e c i f i c cytotoxic agents can be employed. There i s some a p r i o r i evidence f o r the existence of normal hemopoietic c e l l s i n the diseased marrow of CML patients. A transient reappearance of chromosomally normal bone marrow c e l l s has been observed i n some patients 82 21-24 1 following c e r t a i n therapeutic regimens and i n at l e a s t one case, these Ph -25 1 negative c e l l s were shown to be non-clonal. In addition, occasional Ph -negative metaphases have been detected i n d i r e c t marrow preparations when 26 27 s u f f i c i e n t numbers of metaphases have been analyzed. ' For example, Sokal studied 195 patients and detected 1 or more Ph^-negative c e l l s i n preparations from 22 untreated and 34 treated p a t i e n t s . In 25% of the cases where Ph^-27 negative c e l l s were detected, only one normal metaphase was seen. However, since the c e l l types responsible f o r these Ph^-negative metaphases are unknown, t h e i r demonstration could ind i c a t e e i t h e r the presence of a minor population of normal myeloid c e l l s , or the p r o l i f e r a t i o n of other marrow c e l l types develop-mentally outside the p o t e n t i a l of the abnormal clone (e.g., f i b r o b l a s t s ) . Several groups have attempted to address t h i s issue by performing cytogenetic studies on recognizable hemopoietic colonies generated i n assays of c e l l s from 1 24 28—33 patients with Ph - p o s i t i v e CML. ' These reports w i l l now be discussed. In 1971, Shadduck and Nankin published the f i r s t report of cytogenetic 28 studies on committed hemopoietic c e l l s . Granulocyte colonies were cultured from precursors i n the p e r i p h e r a l blood of two patients with known Ph^-positive CML. C e l l s were harvested en masse and chromosome analysis revealed the presence of the Ph^-chromosome i n a l l 19 metaphases from one p a t i e n t and i n a l l 5 from the other. I t i s not clear from that b r i e f report whether or not the patients were treated and no hematological data were given. 29 Chervenick et a l were the f i r s t to study i n d i v i d u a l colonies. In that report, granulocyte progenitors from the marrow of 4 patients were cytogeneti-1 c a l l y examined. In one patient, 1 out of 8 colonies was Ph -negative and i n 1 1 another, 3 out of 6 were Ph -negative, the remainder being Ph - p o s i t i v e . In the t h i r d p a t i ent, the two colonies f o r which r e s u l t s were obtained were Ph^-p o s i t i v e . In a fourth patient, rendered pancytopenic by busulfan, 10 meta-83 1 phases from pooled granulocyte colonies were a l l Ph -negative. This then was the f i r s t report i n which cy t o g e n e t i c a l l y normal myeloid precursors, committed to granulocyte d i f f e r e n t i a t i o n , were detected. Unfortunately, very l i t t l e c l i n i c a l and hematological data was made av a i l a b l e i n that report. Moore and Metcalf used the i n v i t r o agar culture t e c h n i q u e , ^ i n i t i a l l y developed for mouse bone marrow c e l l s , to study c e l l s committed to granulocyte 30 d i f f e r e n t i a t i o n . Granulocyte aggregates, c l u s t e r s and colonies were cytog e n e t i c a l l y analyzed from 2 untreated and 2 treated (with busulfan) 1 patients i n the chronic phase of Ph - p o s i t i v e CML. A t o t a l of 57 metaphases were studied and a l l were Ph^-positive. The authors noted that t h e i r r e s u l t s 29 were i n contrast to those of Chervenick et a l and suggested that perhaps i n 1 some patients, but not i n others, a minor population of Ph -negative granulo-c y t i c colony-forming c e l l s p e r s i s t i n the bone marrow. Aye et a l generated granulocyte colonies i n methylcellulose cultures using 3 1 a method s i m i l a r to that used i n t h i s work. T h i r t y marrow granulocyte progenitors (CFU-C) and 10 blood CFU-C from one untreated patient, with a 3 3 3 3 leukocyte count of 38.6 x 10 /mm (normal range = 4-11 x 10 /mm ) and 13% myeloblasts (normal = <4%), were a l l Ph^-positive. The other patient i n that report was treated (with busulfan) and, at 5 years since the i n i t i a l diagnosis 3 3 was made, had only 4.4 x 10 leukocyte/mm and 3% myeloblasts. A l l 30 marrow 1 CFU-C were found to be Ph - p o s i t i v e . The authors made no attempt to account f o r 29 the discrepancy between t h e i r findings and those of Chervenick et a l . Singer ^ t a l used the X-linked G6PD isoenzyme marker for leukemic clone i n 1 t h e i r study of 5 treated, heterozygous patients with Ph - p o s i t i v e CML (see 32 Chapter One f o r a discussion of t h i s methodological approach). Granulocytic colonies were grown from precursors i n marrow and pe r i p h e r a l blood using the methylcellulose assay and granulopoiesis stimulated by eit h e r a feeder l a y e r of 84 peripheral blood c e l l s or a more p u r i f i e d source of colony stimulating f a c t o r s . A t o t a l of 1,308 colonies were analyzed and only 2 d i d not bear the isoenzyme c h a r a c t e r i s t i c of the leukemic clone. The authors attempted to explain t h i s exclusion (99%) of normal progenitors by the f a c t that a l l patients studied were more than 8 months post-diagnosis and, presumably, the disease was at an advanced stage i n which nonleukemic stem c e l l s are d i f f i c u l t to detect. A l t e r n a t i v e l y , they noted the p o s s i b i l i t y that only some patients have Ph^-negative CFU-C. More than a year a f t e r t h i s t h e s i s was i n i t i a t e d , G r i l l i et a l published t h e i r findings on the use of the methylcellulose assay to c u l t i v a t e e r y t h r o i d colonies (BFU-E) and CFU-C from precursors i n the peripheral blood of 4 1 33 patients i n the chronic phase of Ph - p o s i t i v e CML. In 3 patients, a l l 1 colonies analyzed were Ph - p o s i t i v e but i n the fourth, 2 out of 6 CFU-C and 1 out of 10 BFU-E were cy t o g e n e t i c a l l y normal. Unfortunately, neither c l i n i c a l nor hematological data are a v a i l a b l e on any of the patients i n that report. The f i n d i n g of the Ph^-chromosome i n BFU-E confirms the observation reported i n Chapter Two and previously published by t h i s a u t h o r . ^ 1 F i n a l l y , Goto et a l recently observed Ph -negative BFU-E and CFU-C, cultured from marrow and/or blood precursors, i n 5 out of 8 patients studied i n 24 the chronic phase. The main emphasis i n that report was the e f f e c t of intensive therapy on growth c h a r a c t e r i s t i c s of normal and leukemic c e l l popula-ti o n s and, as a r e s u l t , d e t a i l s of the data are sketchy and very l i t t l e c l i n i c a l and hematological information were given on pretreatment p a t i e n t s . 1 In summary, although Ph -negative hemopoietic precursor c e l l s have 1 occasionally been observed i n patients i n the chronic phase of Ph - p o s i t i v e CML, the r e s u l t s of the reports are c o n f l i c t i n g . Their i n t e r p r e t a t i o n i s d i f f i c u l t due to the small number of cases i n each report, the d i f f e r e n t 85 culture conditions used, and the lack of relevant c l i n i c a l data i n most cases. The work described i n t h i s chapter was designed to systematically study a series of patients i n the chronic phase of CML on whom relevant c l i n i c a l and hematological data were a v a i l a b l e . The r e s u l t s suggest that Ph^-negative hemopoietic stem c e l l s p e r s i s t during the early stages of cl o n a l expansion i n CML, even though t h e i r a b i l i t y to d i f f e r e n t i a t e further has already been suppressed. MATERIALS AND METHODS C l i n i c a l and hematological data for the 19 patients included i n t h i s survey are shown i n Table V, VI and VII. A l l patients were considered to be i n the chronic phase of CML when studied. They represent an unselected group accrued over a 3 year period (1979-1983) c o n s i s t i n g of 9 males and 10 females. Ages range from 12 to 61 years with a mean of 39 years. Nine marrow and 10 blood specimens were from patients who were newly diagnosed and studied p r i o r to chemotherapy. Seven marrow and 12 blood specimens were from patients who had been previously diagnosed and treated. For those patients who had under-gone therapy, t h i s consisted of the a l k y l a t i n g agent busulfan and/or the a n t i -metabolite hydroxyurea. Marrow and blood c e l l s used for culture were part of aspirates taken for diagnostic purposes and were obtained with informed consent. Cytogenetic analysis of G-banded d i r e c t marrow preparations were performed on a l l patients at the time of diagnosis as a part of the routine laboratory work-up. For patients studied a f t e r the i n i t i a l diagnosis was made, repeat d i r e c t cytogenetic analyses were s i m i l a r l y performed on bl a s t s i n the same marrow and/or blood specimen used to set-up the methylcellulose cultures. (For d e t a i l s , see Results below.) At l e a s t 15 metaphases from each d i r e c t specimen 86 were analyzed and i n only 3 instances were l e s s than 20 metaphases analyzed. To study the cytogenetic composition of p r i m i t i v e hemopoietic progenitor c e l l compartments, marrow buffy coat c e l l s or l i g h t density peripheral blood mononuclear c e l l s were separated, washed and plated i n methylcellulose cultures as described i n Appendices I and I I . In such cultures, large e r y t h r o i d and 37 granulocyte colonies arose from committed precursors. Mixed granulocyte/erythroid (CFU-G/E) colonies were also o c c a s i o n a l l y observed and 38 are thought to a r i s e from more p r i m i t i v e precursors. The f i n a l composition of each 1.1 ml culture was 0.8% methylcellulose (Dow Chemicals, Vancouver), 30% FCS, 0.1% deionized BSA (Sigma Chemicals, St. Louis, Missouri), 9% human -4 leukocyte conditioned medium, 10 M 2-mercaptoethanol (Sigma), c e l l s , alpha medium, and 2-5 units/ml of human urinary e r y t h r o i p o i e t i n ( p a r t i a l l y p u r i f i e d 39 i n our laboratory to a s p e c i f i c a c t i v i t y of >100 units/mg protein ). Routinely, 2 x 10^ c e l l s were plated i n each 1.1 ml culture but a d d i t i o n a l 4 4 cultures were also set-up at lower c e l l concentrations (8 x 10 and 2 x 10 c e l l s per culture) to accommodate possible elevated progenitor concentrations frequently encountered i n CML.^ Incubation of cultures was at 37°C i n a s t r i c t l y c o n t r o l l e d 5% CO^-air environment maintained at high humidity. Numbers of p r i m i t i v e e r y t h r o i d progenitors (BFU-E, >8 erythroblast c l u s t e r s per colony, see Figure 3), and granulocyte progenitors (CFU-C), per set number of marrrow c e l l s or per ml of blood were determined as a part of the routine laboratory workup. (Note: Blood progenitors can be scored per ml of blood since the t o t a l blood volume remains r e l a t i v e l y unchanged i n CML. However, since the marrow compartment can increase i n s i z e by extension i n t o the long bones, progenitor numbers are reported per set number of c e l l s . ) Control values shown i n Table V are those obtained under the same conditions used 4 1 here. 87 To demonstrate the existence of chromosomally normal progenitor c e l l s , large, w e l l - i s o l a t e d and r e a d i l y i d e n t i f i a b l e erythroid, granulocyte and mixed grnulocyte/erythroid colonies were selected, plucked and processed i n d i v i d u a l -l y . A d e t a i l e d d e s c r i p t i o n of the method used was described i n Chapter Two and Reference 10. A minimum of two G-banded metaphases were analyzed per colony and i n most cases, karyotypes prepared. The genotypes of the progenitors from which the colonies arose were thus i n f e r r e d by cytogenetic analysis of t h e i r d i f f e r e n t i a t i n g descendants. This approach was based on the assumption that 1 1 colonies were derived from si n g l e progenitor c e l l s , e i t h e r Ph - p o s i t i v e or Ph -negative. This was supported by the f i n d i n g that f o r 941 colonies analyzed i n a recent report from our laboratory, i n no instance were 2 d i f f e r e n t karyotypes 42 obtained from a s i n g l e colony. RESULTS Sixteen marrow and 22 blood specimens from nineteen patients with Ph^-1 p o s i t i v e CML were evaluated f o r the presence of Ph -negative progenitors (BFU-E, CFU-C and CFU-G/E) by cytogenetic analysis of i n d i v i d u a l colonies generated 1 i n methylcellulose assays. To compare the proportion of Ph -negative progenitors with the proportion of Ph^-negative c e l l s i n l a t e r compartments, simultaneous d i r e c t marrow or unstimulated blood preparation were a l s o c y t o g e n e t i c a l l y analyzed (with 3 exceptions, Cases 17b, 23b, 26c). Direct 1 preparations revealed the presence of the standard Ph t r a n s l o c a t i o n , t(9;22), i n 17 cases and v a r i a n t Ph^ t r a n s l o c a t i o n i n 2 cases [t(12;22) i n Case 7, t(16;22) i n Case 13] (see Appendix IX for cytogenetic nomenclature). In addi-t i o n , d i r e c t preparations showed no evidence of chromosomal mosaicism i n any of the specimens. For assessment of progenitors, at l e a s t 14 colonies were analyzed per specimen whenever pos s i b l e since such a sample s i z e permits detection of Ph^-88 negative colonies with 95% confidence i f they comprise 20% or more of the 43 population. Fourteen or more colonies were analyzed i n 21 of the 38 specimens. Chromosomally normal progenitors were not detected i n specimens from 7 of the 10 untreated patients (Table V) or i n specimens from 10 of the 11 treated patients (Table V I ) . Chromosomally normal progenitors were detected i n 4 patients (Table V I I ) . Three were newly diagnosed, untreated and had WBC counts of les s than 31,000 3 per mm at presentation. The fourth was studied 5 months a f t e r diagnosis and i n i t i a t i o n of chemotherapy with hydroxyurea. These cases are described i n d i v i d u a l l y below. Case 16. At presentation, a l l 30 bone marrow metaphases analyzed were 1 1 Ph - p o s i t i v e . Of 10 colonies analyzed from marrow assays, 3 were Ph -negative 1 (33%), the remainder being Ph - p o s i t i v e . One month l a t e r , a f t e r splenectomy 1 but before other therapy, 7 of 24 marrow colonies were again Ph -negative (30%). 1 Case 17. Like the previous case, a l l d i r e c t marrow metaphases were Ph -p o s i t i v e (25 analyzed). However, 10 of 15 blood progenitors (67%) and 1 of 2 marrow progenitors were Ph^-negative at the time of study. Case 18. This patient was f i r s t studied 5 months a f t e r presentation and i n i t i a t i o n of chemotherapy with hydroxyurea. At that time, a l l 21 bone marrow metaphases analyzed were Ph^-positive while 2 of 14 marrow progenitors (14%) and 4 of 13 blood progenitors (31%) were chromosomally normal. Case 19. At presentation, a l l 31 d i r e c t marrow metaphases were Ph^-p o s i t i v e . No colonies of s u f f i c i e n t s i z e f o r cytogenetic analysis were produced i n marrow assays set up at the same time. However, a l l 12 colonies 1 obtained from the corresponding blood assys were Ph -negative. Metaphases from these colonies a l l contained the same polymorphic heterochromatic region i n one chromosome #9 [var(9) (cen, QFQ43)] that was seen i n the d i r e c t preparation, TABLE V Results of cytogenetic studies of hemopoietic progenitors in 7 patients with untreated, newly diagnosed Ph^-positive CML in whom no normal progenitors were found Months Cytogenetic Studies „ „ Since WBC , Hg Platelets „ . Progenitor Numbers Normal : Ph'-Positive Case # Age Sex „ . - 3 , ' . 3. Specimen* Presenta- (per mm ) g/dl (per mm ) tion BFU-E+ CFU-C BFU-E+ CFU-C CFU-G/E Total 1 39 F 0 15,000 13.4 249,000 M 17 34 0: :11 0:3 0 :8 0: :22 2(a) 42 M 0 29,000 10.3 1,700,000 B 320 373 0; :12 0:3 0: :3 0: :18 M 6 10 0: :14 - 0: :14 3(a) 32 M 0 56,000 11.7 1,300,000 B 96,000 77,500 0: :18 0:6 0: :3 0: :27 M 15 22 0: :5 0:1 0: :6 4 37 F 0 130,000 10.7 670,000 B 34,000 40,000 0: :12 _ 0: : 12 M 20 77 0: i l l - 0: i l l 5 48 F 0 156,000 10.8 490,000 B 12,000 15,400 0: :14 _ 0: : 14 M 18 24 0: :13 0:2 0: : 15 6 52 M 3 290,000 10.6 250,000 B 60,000 120,000 0: :18 - 0: ; 18 7 12 M 0 490,000 7.8 260,000 B 31,000 1,000,000 0: 15 - 0: 15 M 10 500 0: 17 - 0: 1 0: 18 Controls (n = 18) B x = 53 x = 26 (range 1CH-284) (range 4->-147) 6 _ Controls (n = 29) M x = 28 x = 64 (range 1+284) (range 19+218) CO •Specimens: B = Blood; M = Marrow ^ ^Blood values are per ml. Marrow values are per 2 x 10^ buffy coat c e l l s . See text for d e t a i l s . +>8 cl u s t e r s . ^Values from Eaves and Eaves (reference 41). Range shown i s that delimited by the geometric mean +_ 2 standard deviations. TABLE VI R e s u l t s o f c y t o g e n e t i c s t u d i e s o f hemopo i e t i c p r o g e n i t o r * i n 10 p a t i e n t s w i t h t r e a t e d , p r e v i o u s l y - d i a g n o s e d , P h ^ - p o s i t i v e CML i n whom no normal p r o g e n i t o r s were found Case H A g e Sex Months S i n c e P r e s e n t a -t i o n WBC (per mnr) Hg g /d l P l a t e l e t s (pe r mm3) Specimen* P r o g e n i t o r Numberst BFU-E* CFU-C C y t o g e n e t i c S t u d i e s Normal : P h ^ - P o s i t i v e BFU-E* CFU-C CFU-G/E T o t a l 8 ( a ) 38 (b) ( c ) 21 39 43 14,900 64 ,700 29 ,300 17.2 14.9 15.4 652,000 1,038,000 1,080,000 170 18 15,000 10 63 90 24 ,250 180 0 :28 0:14 0:41 0:1 0 :2 0:251 0:8 0:38 0:14 0:41 0:1 35 29 39,800 9 .5 384,000 31 ,000 60 90 ,430 390 0 :13 0:6 0:1 0 :3 0:1 0 :15 0:9 10 11 12(a) (b) 2 ( b ) 13 14 3 (b ) 15 61 33 35 42 48 56 33 25 F M M M M F M 21 96 37 41 13 94 2 16 50 ,000 52,800 53 ,100 27 ,600 55,000 69 ,400 75,000 85 ,000 326,000 10.0 14.7 15.2 16.9 10 .3 10.4 12.2 10.9 7.4 227,000 338,000 398,000 169,000 1,700,000 387 ,000 775,000 887 ,000 902,000 33,000 8 4 ,800 7 10,000 1,500 4 ,000 140 24 12,000 60 77 ,000 58 15,795 41 28 ,092 2 ,021 9 ,263 30 12 393,120 254 0:5 0:8 0:10 0:25 0 :9 0:12 0 :5 0:30 0:8 0 :3 0:6 0:1 0:8 0:2 0:2 0:1 0:11 0:2 0:1 0:14 0:10 0:10 0:27 0:11 0 :13 0:16 0:30 0:8 0:4 0:6 o * Spec imens: B = B l o od ; M = Marrow. + B l ood v a l u e s a r e per m l . Marrow va l ues a r e per 2 x 1 0 5 b u f f y c o a t c e l l s . * >8 C l u s t e r s . § Poo l ed c o l o n i e s . TABLE VII R e s u l t s o f c y t o g e n e t i c s t u d i e s o f hemopo ie t i c p r o g e n i t o r s i n 4 p a t i e n t s w i t h P h ^ p o s i t i v e CML i n whom normal p r o g e n i t o r s were d e t e c t e d Case H Age Sex Months S i n c e P r e s e n t a -t i o n WBC (per mm3) Hg g/d i P l a t e l e t s (pe r mnr) Specimen* P r o g e n i t o r Numberst BFU-E* CFU-C D i r e c t C y t o g e n e t i c Normal : P h 1 -BFU-E* CFU-C S t ud i e s P o s i t i v e CFU-G/E To t a l 16(a) 38 F 0 18,300 16.3 590,000 M 24 26 0:30 3:7 - 3:7 l b ) 1 13,100 14.9 580,000 M 20 40 - 7:17 - 7:17 17 59 F 0 26,300 14.4 660,000 B 240 381 10:5 _ 10:5 M 2 102 0:25 1:1 - 1:1 18 34 M 5§ 26,400 12.4 224,000 B 820 666 0:21 4:7 0:2 4 :9 M 52 147 2:12 - 2:12 19(a) 24 F 0 30,600 12.6 1,200,000 B 630 887 0:31 10:0 2:0 - 12:0 (b) 2 77,000 10.8 667,000 B 440 3 ,410 0:21 0:25 - 0:25 ( c ) 8§ 88 ,800 11.2 980,000 B 6 ,800 5,522 - 0:9 - 0:9 * Spec imens: B - B l o o d ; M = Marrow. t B lood v a l u e s are per m l . Marrow va l ues a re per 2 x 10-> bu f f y coa t c e l l s . * >8 C l u s t e r s . 5 T r ea t ed w i t h hyd roxyu rea . 92 thus excluding the p o s s i b i l i t y of a specimen error (see Appendix IX for cytogenetic nomenclature). Two months l a t e r , and p r i o r to any treatment, a second blood specimen was obtained. This time, a l l 25 colonies analyzed were 1 Ph - p o s i t i v e . A l l 21 unstimulated peripheral blood metaphases obtained from t h i s specimen were also Ph^-positive. Eight months a f t e r presentation and two months a f t e r the s t a r t of chemotherapy with hydroxyurea, a repeat analysis again revealed the exclusive presence of Ph^-positive progenitors i n the p e r i -pheral blood of t h i s p a t i e n t . DISCUSSION Chronic myeloid leukemia i s a disease of c l o n a l o r i g i n . In the majority of cases, karyotypic studies of marrow at the time of diagnosis show only leukemic metaphases characterized by the Ph^-chromosome. This marker i s extremely u s e f u l as i t permits one to d i s t i n g u i s h between cyt o g e n e t i c a l l y normal and abnormal hemopoietic populations. This marker has been used here to study the dynamic i n t e r r e l a t i o n s h i p s of these two populations at the l e v e l of the fresh bone marrow aspirate, i n d i c a t i v e p r i m a r i l y of p r o l i f e r a t i n g hemopoietic c e l l s close to t h e i r t erminally d i f f e r e n t i a t e d state, and at the l e v e l of cultured hemopoietic colonies, i n d i c a t i v e of a more p r i m i t i v e precursor c e l l population (see Figure 6). In the present s e r i e s of 19 p a t i e n t s i n the chronic phase of CML, a l l had almost complete replacement of the cytogenetically normal hemopoietic c e l l s i n t h e i r bone marrow by the Ph^—positive clone as determined by the d i r e c t cytogenetic studies. This i s i n keeping with the findings i n most s e r i e s of patients and suggests that once CML has become s u f f i c i e n t l y advanced to permit diagnosis the Ph^-positive clone appears to have a d e f i n i t e p r o l i f e r a t i v e advantage over the normal c e l l s . 9 3 No chromosomally normal myeloid precursors were detected i n blood or marrow specimens from 7 out of 10 untreated patients or from 10 of 11 treated p a t i e n t s . This f i n d i n g i s consistent with the r e s u l t s of most previous studies -2Q 30—32 where analysis was, however, r e s t r i c t e d to granulopoietic progenitors. ' I t thus appears that by the time of diagnosis, the proportion of normal hemo-p o i e t i c progenitor c e l l s i s often so low, i . e . , < 1 0 - 2 0 % , as to preclude t h e i r detection by the analysis of r e a l i s t i c numbers of colonies. The presence of chromosomally normal progenitors i n newly diagnosed or 24 29 3 3 conventionally treated patients has only been r a r e l y observed. ' ' In the present s e r i e s , such c e l l s were r e a d i l y detected i n 4 patients and included members of the p l u r i p o t e n t stem c e l l compartment (CFU-G/E) as well as p r i m i t i v e but committed e r y t h r o p o i e t i c and granulopoietic progenitor c e l l types. For 2 of the 3 untreated patients i n whom chromosomally normal progenitors were found, repeat analyses were performed during the f i r s t few months following presentation and before i n i t i a t i o n of chemotherapy. The r e s u l t s of these follow-up studies suggest that the proportion of chromosomally normal progenitors decreases with time and continued c l o n a l expansion, as i n d i c a t e d by an i n c r e a s i n g number of c i r c u l a t i n g progenitors and a r i s i n g WBC count. Since the elevation i n early progenitor numbers i s , on average, more pronounced than 40 , 4 4 the elevation i n the WBC count (see Figure 10 (a) and (b)), i t would be a n t i c i p a t e d that normal progenitors, even i f t h e i r numbers remained constant, would i n most patients already have been d i l u t e d to undetectable l e v e l s by the time of diagnosis. Conversely, i t would also be a n t i c i p a t e d that i n patients with low WBC counts normal progenitors, i f present at normal l e v e l s , would be detectable. To further t e s t t h i s hypothesis, the proportion of a l l p r i m i t i v e BFU-E present (per unit volume of blood) that should have been Ph^-negative, assuming 94 10C 10' ID O DO o 1 CL 10 D LU I Z> L L CQ 5 10 2 10' 10^  I I I I • Total BFU-•E • vs • — WBC . . • . • •••• , . x - • — • • • • • • • • ••• • • • _ • .•• • • • o • . ° / / • • • ••' o • o m * o • • — • • • • o Normal — • • • CML I I I 1 10-Figure 10 (a). 10L 10; 10 10' 10 V WBC per ml of Blood A Graph Showing The E levat ion In The Number Ot Erythro id Progeni tors As A Funct ion Ot The White Blood Cell Count. ( A . E a v e s , Unpublished Data.) 10 95 10l 10' 10' 10' 10' 10^  CFU-C vs WBC • • • to£b6 o • o ° Normal • CML i 10C 10: 10 10 10° 10° 10' WBC per ml of Blood Figure 10 (b). A Graph Showing The Elevation In The Number Of Granulocyte Progenitors As A Function Of The White Blood Cell Count. (A.Eaves, Unpublished Data.) 96 the persistence of a normal population, was determined f or each patient. These values were then used to determine whether or not the number of large e r y t h r o i d colonies a c t u a l l y analyzed was s u f f i c i e n t to permit detection of chromosomally 43 normal p r i m i t i v e BFU-E (see Table V I I I ) . Three of the untreated patients (Cases 2a, 17 and 19) had s u f f i c i e n t l y low l e v e l s of BFU-E that the number analyzed was enough to i d e n t i f y a normal population with 95% confidence. In 2 of these (Cases 17 and 19) Ph^-negative colonies were found. Similar findings 42 were made i n a recent report from t h i s group. In that study, 3 more patients with s u f f i c i e n t l y low l e v e l s of BFU-E were studied and i n a l l cases Ph^-negative colonies were detected. Thus i n untreated CML, i t appears that chromosomally normal, p r i m i t i v e hemopoietic progenitor c e l l s with the capacity f o r normal p r o l i f e r a t i o n and d i f f e r e n t i a t i o n commonly p e r s i s t f o r s i g n i f i c a n t periods of time at normal or near normal l e v e l s . The d i f f i c u l t y i n demonstrating t h e i r existence as time goes on i s apparently due to t h e i r many-fold d i l u t i o n by the P h 1 - p o s i t i v e clone. In the group of treated p a t i e n t s , Ph 1-negative colonies were detected i n only one case (#18) of the 4 (Cases 36, 8a, 126, 18) where s u f f i c i e n t numbers of colonies were analyzed, according to the assumptions d e t a i l e d above. This r e s u l t i s not s u r p r i s i n g since chemotherapy would be expected to reduce both normal and abnormal populations. I t seems that i f conventional therapy has a s e l e c t i v e e f f e c t against the abnormal clone, i t i s not usually s u f f i c i e n t to be detectable at the progenitor c e l l l e v e l . The f i n d i n g i n Case 19 of 100% chromosomally normal progenitors i n blood during the course of c l o n a l expansion, suggests that domination of the marrow may tend to precede entry of abnormal progenitors i n t o the pe r i p h e r a l c i r c u l a -1 t i o n . The f a c t that i n t h i s case, the blood progenitors went from 100% Ph -97 TABLE VIII Expected and observed Ph^-negative BFU-E* assuming persistence of normal progenitors at normal levelst Case # BFU-E Percentage Expected To Be % Mosaicism % Ph -Negative per ml blood Normal Excluded Progenitors Observed + min x max+ Untreated Patients 2(a) 320 3 17 89 16 0 § 3(a) 96,000 0.01 0.06 0.3 11 0 4 34,000 0.03 0.2 0.8 23 0 5 12,000 0.09 0.5 2.4 20 0 6 60,000 0.02 0.09 0.5 18 0 7 31,000 0.03 0.2 0.9 19 0 67 S 100! 0 § 17 240 4 22 100 80 19(a) (b) 630 440 2 2 8 12 45 65 23 12 Treated Patients 2(b) 11,000 0.1 0.5 2 24 0 0 § 3(b) 140 7 39 100 10 8(a) 170 6 32 100 8 o 5 8(b) 15,000 0.07 0.4 1.9 8 0 9 31,000 0.03 0.2 0.9 20 0 10 33,000 0.03 0.2 0.9 20 0 12(a) 4,900 0.2 1 6 26 0 12(b) 7 100 100 100 11 0 § 13 1,600 0.6 3 18 21 0 14 4,000 0.2 1 7 18 0 15 112,000 0.01 0.04 0. 2 53 0 18 820 1.2 6.4 35 24 36§ * >8 c lus ters . + See Table V for normal values. +Maximum and minimum values calculated (>8 c lusters) per ml blood. using normal values for mean number of BFU-E § Cases where suf f i c ient numbers of colonies were analyzed to permit detection of P h J -negative BFU-fT., assuming maximum persistence of normal progenitors. 98 negative to 10 0% Ph^-positive within 2 months gives some i n d i c a t i o n of the r a p i d i t y with which t h i s process can occur. This f i n d i n g , plus the r a p i d repopulation of marrow by Ph^-positive c e l l s observed i n patients rendered 1 24 p a r t i a l l y Ph -negative by intensive therapy may help to explain why most 1 patients have 10 0% Ph - p o s i t i v e blood progenitors by the time of diagnosis. Cytogenetically normal progenitors were detected i n 3 untreated patients 1 (Cases 16, 17 and 19), whose d i r e c t bone marrow preparations showed only Ph -p o s i t i v e metaphases, even though i n each case enough c e l l s were analyzed to 1 permit the detection of Ph -negative metaphases had they been present at the l e v e l observed among progenitors. Mechanisms that suppress normal hemopoiesis i n CML thus appear to be more e f f e c t i v e at the l e v e l of terminally d i f f e r e n t i a t e d c e l l s than at the l e v e l of early p l u r i p o t e n t and committed progenitor c e l l compartments. A s i m i l a r phenomenon has recently been observed i n polycythemia vera, a r e l a t e d myeloproliferative disorder i n which the e r y t h r o i d lineage i s predominantly involved. In that study, the authors used the X-linked G6PD isoenzyme system as a marker for the neoplastic clone i n 2 heterozygous 45 females. While only one isoenzyme type was detectable i n c i r c u l a t i n g erythocytes, granulocytes and p l a t e l e t s , cultures of blood and marrow revealed a s u b s t a n t i a l number of BFU-E and CFU-E that were marked by the other isoenzyme. In an attempt to account for t h e i r f i n d i n g s , the authors suggested that i n h i b i t i o n of normal e r y t h r o i d d i f f e r e n t i a t i o n occurred between the BFU-E and the more mature e r y t h r o i d colony-forming u n i t . I t i s possible that i n CML, the p r o l i f e r a t i o n of p r i m i t i v e normal progeni-tors i s suppressed by normal feedback i n h i b i t i o n mechanisms that f a i l to l i m i t the growth of t h e i r leukemic counterparts. The recently described d i f f e r e n t i a l s e n s i t i v i t y of CFU-C from normal and CML donors to the i n h i b i t o r y e f f e c t s of 99 46 prostaglandin-E^ could exemplify such a mechanism. Since a s i g n i f i c a n t proportion of the most p r i m i t i v e hemopoietic progenitor compartments are normally q u i e s c e n t , ^ t h e i r numbers would, according to such a hypothesis, be expected to decline slowly. In contrast, l a t e r compartments where turnover 47 i s normally high would be expected to be depleted r a p i d l y . Regardless of the mechanism, our present findings i n d i c a t e that the factors that prevent p r i m i t i v e normal progenitors from generating mature progeny i n vivo are of reduced effectiveness _in v i t r o . Consistent with t h i s p r e d i c t i o n are the r e s u l t s discussed i n Chapter Four. SUMMARY Individual hemopoietic colonies generated i n methylcellulose cultures from progenitors i n blood and marrow were cyt o g e n e t i c a l l y analyzed to investigate the l e v e l and extent of normal stem c e l l suppression that occurs i n patients 1 with Ph - p o s i t i v e CML. Ten patients were studied at diagnosis p r i o r to the i n i t i a t i o n of chemotherapy and 4 of these were studied again 1 to 16 months l a t e r . Another 9 patients were studied f o r the f i r s t time 2 to 96 months a f t e r diagnosis and i n i t i a t i o n of chemotherapy. No chromosomally normal metaphases were found i n e i t h e r d i r e c t marrow preparations or i n hemopoietic colonies obtained from simultaneous assays of marrow and/or blood samples from 15 of the 19 patients studied. In the other 4, chromosomally normal hemopoietic progeni-tors were r e a d i l y demonstrable even though a l l d i v i d i n g c e l l s i n the bone marrow belonged to the Ph^-positive clone at the time of study. These r e s u l t s i n d i c a t e that the suppressive e f f e c t s of c l o n a l expansion on normal hemopoiesis are more pronounced, and apparent sooner, i n the more d i f f e r e n t i a t e d compart-ments. In addition, they support the view that the o r i g i n a l population of normal stem c e l l s does not disappear r a p i d l y , although t h e i r numbers may be 100 d i l u t e d to undetectable l e v e l s depending upon the extent of c l o n a l expansion at the stem c e l l l e v e l by the time of diagnosis. 101 REFERENCES 1. 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Singer JW, Fialkow PJ, Steinmann L, N a j f e l d V, Stein S, Robinson WA: Chronic myelocytic leukemia (CML): f a i l u r e to detect r e s i d u a l normal committed stem c e l l s jLn v i t r o . Blood 53: 264-268, 1979. 33. G r i l l i G, Carbonell F, Fliedner TM: Studi c i t o g e n e t i c i s i u p r o g e n i t o r i e r i t r o p o i e t i c i e m i e l o p o i e t i c i i n v i t r o n e l l a leucemia mieloide cronica. Haematol 66: 733-739, 1981. 34. Pike BL, Robinson WA: Human bone marrow colony growth i n agar-gel. J C e l l P h ysiol 76: 77-84, 1970. 35. Greenberg PL, Nichols W, Schrier SL: Granulopoiesis i n acute myeloid leukemia and preleukemia. New Engl J Med 284: 1225-1232, 1971. 36. Iscove NN, Senn JS, T i l l JE, McCulloch EA: Colony formation by normal and leukemic human marrow c e l l s i n c u l t u r e . E f f e c t of conditioned medium from human leukocytes. Blood 37: 1-5, 1971. 37. Gregory CJ, Eaves AC: Human marrow c e l l s capable of e r y t h r o p o i e t i c d i f f e r -e n t i a t i o n i n v i t r o . D e f i n i t i o n of three e r y t h r o i d colony responses. Blood 49: 855-864, 1977. 38. Fauser AA, Messner HA: Granuloerythropoietic colonies i n human bone marrow periph e r a l blood and cord blood. Blood 52: 1243-1248, 1978. 39. K r y s t a l G, Eaves C, Eaves A: F i f t y - f o l d p u r i f i c a t i o n of human erythropoie-t i n using CM A f f i - g e l blue. Blood 60(Suppl 1): 87a, 1982. 40. Eaves AC, Eaves CJ: Abnormalities i n the e r y t h r o i d progenitor compartments i n p a tients with chronic myelogenous leukemia (CML). Exp Hematol 7 (Suppl 5): 65-75, 1979. 41. Eaves CJ, Eaves AC: Erythroid progenitor c e l l numbers i n human marrow — Implication f o r regulation. Exp Hematol 7 (Suppl 5): 54-64, 1979. 42. Dube ID, Gupta CM, Kalousek DK, Eaves CJ, Eaves AC: Cytogenetic studies of early myeloid progenitor compartments i n Ph - p o s i t i v e chronic myeloid leukemia (CML). I. Persistence of Ph 1- negative committed progenitors that are suppressed from d i f f e r e n t i a t i n g i n vivo. Br J Haematol ( i n press) 43. Hook EB: Exclusion of chromosomal mosaicism. Tables of 90%, 95% and 99% confidence l i m i t s and comments on use. Am J Human Genet 29: 94-97, 1977. 104 44. Goldman JM, Shiota F, Th'ng KH, Orchard KH: C i r c u l a t i n g granulocyte and eryt h r o i d progenitor c e l l s i n chronic granulocytic leukemia. Br J Haematol 46: 7-13, 1980. 45. Adamson SW, Singer JW, Catalano P, Murphy S, L i n N, Steinman L, Ernst C, Fialkow P: Polycythemia vera. Further i n v i t r o studies of haemato-p o i e t i c r e g u l a t i o n . J C l i n Invest 66: 1363-1368, 1980. 46. A g l i e t t a M, Piacibellow W, Gauosto F: I n s e n s i t i v i t y of chronic myeloid leukemia c e l l s to i n h i b i t i o n of growth by prostaglandin E . Cancer Res 40: 2507-2511, 1980. 47. Eaves CJ, Humphries RK, Eaves AC: _In v i t r o c h a r a c t e r i z a t i o n of er y t h r o i d precursor c e l l s and the er y t h r o p o i e t i c d i f f e r e n t i a t i o n process. In: C e l l u l a r and Molecular Regulation of Hemoglobin Switching (G. Stamatoyan-nopoulos, AW Nienhuis, eds), Grune & Stratton, pp. 251-278, 1979. 48. Fauser AA, Messner HA: P r o l i f e r a t i v e state of human pl u r i p o t e n t hemopoitic progenitors (CFU-GEMM) i n normal i n d i v i d u a l s and under regeneration conditions a f t e r bone marrow transplantation. Blood 54: 1197-1200, 1979. 105 C H A P T E R F O U R CYTOGENETIC STUDIES OF EARLY MYELOID PROGENITOR COMPARTMENTS IN 1 1 CHRONIC PHASE Ph -POSITIVE CML. LONG-TERM CULTURE REVEALS Ph -NEGATIVE COMMITTED PROGENITORS IN A MAJORITY OF PATIENTS INTRODUCTION Chronic myeloid leukemia (CML) i s a c l o n a l disorder that a r i s e s i n the -1 2 p l u r i p o t e n t hemopoietic stem c e l l compartment. ' T y p i c a l l y , a l l p r i m i t i v e progenitor compartments are enlarged by the abnormally expanding clone so that by the time of diagnosis c i r c u l a t i n g BFU-E and CFU-C progenitor numbers may be 3 4 increased more than 1000-fold. ' A dramatic expansion of the terminally d i f f e r e n t i a t i n g compartments i s usually l i m i t e d to the granulocyte-monocyte lineages, although s i g n i f i c a n t increases i n p l a t e l e t production frequently also occur. In 85-90% of cases, the clone i s marked by the presence of the 1 Philadelphia (Ph ) chromosome, a r e a d i l y v i s i b l e and uniquely a l t e r e d 5-7 chromosome 22 r e s u l t i n g from a balanced chromosomal t r a n s l o c a t i o n . Thus at 1 presentation, the Ph chromosome i s usually seen i n a l l marrow metaphases and 1 8 9 Ph - p o s i t i v e e r y t h r o i d and granulocyte progenitors also predominate. ' Nevertheless, r e s t o r a t i o n of Ph^-negative hemopoiesis from r e s i d u a l normal stem c e l l s can be obtained with aggressive treatment p r o t o c o l s ^ a n d as was 1 demonstrated i n the previous Chapter Ph -negative progenitors may be found i f 9 s u f f i c i e n t numbers of hemopoietic colonies are examined. In t h i s Chapter, the r o l e of i n v i t r o growth i n f a c i l i t a t i n g the detection of Ph^-negative hemopoietic stem c e l l s has been further investigated by u t i l i z i n g long-term marrow cul t u r e s . In long-term marrow cultures, an a r t i f i c i a l environment can be attained 106 i n which hemopoiesis occurs, i n c l u d i n g d i f f e r e n t i a t i o n of precursors under appropriate conditions and, at l e a s t i n murine systems, self-renewal of c e l l s i n the i n i t i a l inoculum. In murine cu l t u r e s , self-renewal of the e a r l i e s t detectable hemopoietic stem c e l l and consequent production of granulocytic, 12-15 megakaryocytic and e r y t h r o i d progenitor c e l l s occurs f o r many months. The maintenance of hemopoiesis i n v i t r o i s apparently dependent upon the p r i o r development of a bone marrow derived adherent c e l l layer containing a v a r i e t y of c e l l types, i n c l u d i n g c e l l s with the features of adipocytes, endothelial c e l l s and macrophages and thought to be equivalent to the marrow stroma i n vivo . The murine system has been s u c c e s s f u l l y modified by several groups for . ^ ^—21 the long-term culture of human marrow. I f normal hematopoietic stem c e l l s e x i s t i n at l e a s t some CML patients but are suppressed from p r o l i f e r a t i n g in vivo, as previous observations suggest (Chapter Three and reference 9), then the long-term marrow culture system might enhance the chances of t h e i r detection f o r the following reasons: ( i ) p r i m i t i v e hemopoietic progenitor c e l l types detectable by colony assays are r o u t i n e l y maintained i n the adherent layers of long-term cultures, and ( i i ) long-term marrow culture permits the study of any observable e f f e c t that the i n t e r a c t i o n between the d i f f e r e n t c e l l populations i n the culture may have on the quiescent normal stem c e l l pool. In t h i s Chapter, the f i n d i n g of a dramatic increase i n the proportion of Ph^-negative e r y t h r o i d (BFU-E), granulocyte (CFU-C) and mixed granulo-cyte/erythroid (CFU-G/E) hemopoietic precursors detected a f t e r long-term culture of marrow from treated as well as untreated patients with t y p i c a l Ph^-p o s i t i v e CML i s reported. These findings extend the observation recently 1 reported by our group of Ph -negative progenitors i n long-term marrow cultures 22 from 3 patients with newly diagnosed untreated CML and, further, provide 107 evidence f o r t h e i r existence i n patients with established, treated disease. These observations support the conclusion that i n both treated and untreated patients, a s i g n i f i c a n t proportion of Ph^-negative progenitors can be detected i n v i t r o i n s p i t e of the absence of t h e i r progeny i n d i r e c t marrow prepara-t i o n s . This shows that differences e x i s t between normal and leukemic stem c e l l response to microenvironmental influences i n v i t r o and suggests that such differences may be exploited to therapeutic advantage. MATERIALS AND METHODS C l i n i c a l and hematological data f o r the 9 patients included i n t h i s study are shown i n Table IX. There were 5 males and 4 females. The average age of the patients was 37 years (range 20 to 39). A l l patients were i n the chronic phase of CML. Three were studied within one month of diagnosis and before the i n i t i a t i o n of any therapy. The remaining 6 were studied 5 to 83 months a f t e r presentation and i n i t i a t i o n of conventional treatment with busulfan and/or hydroxyurea. Cytogenetic analysis of uncultured bone marrow metaphases revealed the presence of the Ph^ i n a l l cases at the time of study. In no pat i e n t was there evidence f o r the p r o l i f e r a t i o n of cy t o g e n e t i c a l l y normal bone marrow c e l l s . Marrow c e l l s used f o r culture were part of aspirates taken f o r diagnostic purposes and were obtained with informed consent. Culture Procedures A d e t a i l e d d e s c r i p t i o n of the procedures used to i n i t i a t e and maintain 23 long-term marrow cultures has been previously reported. (See Appendix VIII) 7 B r i e f l y , an a l i q u o t of marrow aspirate containing 2-2.5 x 10 nucleated c e l l s was incubated i n a 60 mm x 15 mm Falcon t i s s u e culture dish containing 8 ml of growth medium. Up to ten cultures were i n i t i a t e d from each specimen. In one TABLE IX C l i n i c a l and hematological data for the nine cases included i n the long-term culture study Months WBC (per mm^ ) Progenitor Numbers* Case 1 Age Sex Since Presenta-tion Hg g/dl Platelets (per mm^ ) Marrow BFU-E'*' CFU-C Blood BFU-E1" CFU-C Untreated Patients K a ) 38 F 0 18,000 16.3 590,000 24 26 1,444 241 K b ) 1 13,000 14 .9 580,000 20 40 - -2 60 F 0 27,000 14.4 660,000 2 102 381 381 3 30 M 0 142,000 14.8 205,000 14 100 45,787 78,120 Treated Patients 4 34 M 5 25,000 12.4 224,000 52 147 1,958 666 5 39 M 9 16,000 13.4 249,000 17 74 93 36 6 20 F 14 40,000 13.0 600,000 6 105 3,507 2,963 7 39 M 43 32,000 15.4 1,080,000 10 180 18,995 14 ,175 8 38 F 14 4,600 13.5 198,000 4 27 14 7 9 33 M 83 53,000 14.7 338,000 8 58 2,369 12,110 Controls^ x - 28 range: 1-284 x » 64 range: 19-218 x - 53 range: 10-284 x =• 26 range: 4-147 o CO * Per 200,000 marrow c e l l s plated or per ml blood, t >8 clusters. § Values from Reference 29. Range shown i s that delimited by the geometric mean ± S.E.M. 109 case (Case 1), excess red c e l l s were removed at t h i s time by c e n t r i f u g a t i o n of the nonadherent c e l l s over Ficoll-Hypaque and subsequent return of the washed l i g h t density c e l l s to the cultures i n new growth medium. Cultures were o o incubated f o r the f i r s t 4 days at 37 C and then t r a n s f e r r e d to 33. C. Cultures were fed on the seventh day a f t e r i n i t i a t i o n by removal of the nonadherent c e l l s followed by c e n t r i f u g a t i o n and resuspension i n new medium i n the o r i g i n a l t i s s u e culture dish. Thereafter, cultures were fed at weekly i n t e r v a l s by demi-depopulation of the nonadherent c e l l f r a c t i o n and complete replacement of the growth medium. Myeloid progenitor c e l l s i n the adherent c e l l f r a c t i o n were assayed a f t e r 2 to 8 weeks of long-term culture by removal of the adherent layer with collagenase and subsequent p l a t i n g of the nucleated c e l l s i n 0.8% methylcellu-lose i n Iscove's medium using 5.0 units/ml of human urinary erythropoietin 24 ( p u r i f i e d i n our laboratory to a s p e c i f i c a c t i v i t y of 100-200 units per mg ) 23 as previously described (see Appendix I I ) . Myeloid progenitor c e l l s i n s t a r t i n g marrow were s i m i l a r l y assayed (reported i n Table IX). Cytogenetic Studies An a l i q u o t of marrow from a l l patients studied was used to obtain simultaneous d i r e c t preparations of Giemsa banded bone marrow metaphases (see Appendix IV). Cytogenetic analysis of these preparations revealed the presence of the standard Ph^ t r a n s l o c a t i o n t(9;22)(q34;q11) i n a l l cases at the time of study, and i n no p a t i e n t was there any evidence f o r the p r o l i f e r a t i o n of cy t o g e n e t i c a l l y normal bone marrow c e l l s (Table X). To demonstrate the existence of chromosomally normal progenitor c e l l s , large, w e l l - i s o l a t e d and r e a d i l y i d e n t i f i a b l e erythroid, granulocyte and mixed granulocyte/erythroid colonies a r i s i n g i n methylcellulose cultures from progen-i t o r s i n fresh marrow or i n the adherent layers of long-term cultures were Methodology: O Metaphases from Fresh Marrow Fresh Marrow 9-12 Days J O Colony Assays (BFU-E, CFU-C, CFU-G/E) Metaphases from Individual Colonies 2-8 Weeks Long-term Culture Adherent Cells 9-12 Days O Colony Assays (BFU-E, CFU-C, CFU-G/E) Metaphases from Individual Colonies Figure 1 1 . Schematic Illustration ot the Methodological Approach used in Chapter Four. 111 selected, plucked and processed i n d i v i d u a l l y . This i s schematically i l l u s t r a t e d i n Figure 11. A d e t a i l e d d e s c r i p t i o n of the cytogenetic technique used has been previously described i n d e t a i l i n Chapter Two and reference 8. A minimum of two G-banded metaphases were analyzed per colony and i n most cases, karyotypes prepared. The genotypes of the progenitors from which the colonies arose were thus i n f e r r e d by cytogenetic analysis of t h e i r d i f f e r e n t i a t i n g descendents. This approach was based on the assumption that colonies were 1 1 derived from single progenitor c e l l s , e i t h e r Ph - p o s i t i v e or Ph -negative. Evidence supporting t h i s assumption has been presented i n Chapter Three and reference 9. Whenever po s s i b l e , at l e a s t 14 colonies were analyzed per s p e c i -1 men since such a sample s i z e permits detection of Ph -negative colonies with 25 95% confidence i f they comprise 20% or more of the population. Occasionally, 2 or more colonies were pooled f o r a n a l y s i s . These are i n d i c a t e d as pooled colonies i n Table IX. RESULTS The d i f f e r e n t i a l e f f e c t s of long-term marrow culture on the maintenance of 1 1 Ph - p o s i t i v e and Ph -negative progenitors from the 9 d i f f e r e n t chronic phase CML patients included i n t h i s study are summarized i n Table 2. The i n i t i a l progenitor data f o r 2 of the 3 untreated patients and 2 of the 6 treated patients have been presented i n Chapter Three. They are shown again here to f a c i l i t a t e comparison with the r e s u l t s obtained a f t e r 1 to 6 weeks of long-term c u l t u r e . Untreated Patients Two of the 3 newly diagnosed patients studied had low peripheral WBC counts at presentation, and assessment of t h e i r blood l e v e l s of granulocyte progenitors (CFU-C) and p r i m i t i v e e r y t h r o i d progenitors (BFU-E) suggested that 112 these compartments had not yet expanded markedly (see Table IX). Consistent 1 with t h i s f i n d i n g was the demonstration of a s i g n i f i c a n t degree of Ph -1 negative/Ph - p o s i t i v e mosaicism amongst t h e i r numbers, i n s p i t e of the absence 1 of Ph -negative metaphases i n d i r e c t marrow preparations from these same 2 p a t i e n t s . The t h i r d untreated patient (Case 3) presented with a peripheral WBC count of 142,000 and blood progenitor l e v e l s that were increased approximately 200-fold above normal. In t h i s p a t i ent, a Ph^-negative population was not detected i n i n i t i a l methylcellulose assays. Long-term marrow cultures from a l l 3 patients y i e l d e d r e s u l t s s i m i l a r to 22 those previously reported for untreated CML. (See Figures 12(a)-(h).) This included a tendency f o r supranormal numbers of nucleated c e l l s to be present i n the nonadherent f r a c t i o n during the f i r s t 3 weeks, although normal or reduced numbers were measured the r e a f t e r . At a l l times the progenitor content of both adherent and nonadherent f r a c t i o n s was also e i t h e r within normal l i m i t s or reduced. A previous study i n t h i s laboratory had shown that most of the p r i m i t i v e 1 22 progenitors i n the adherent layer were usually Ph -negative by 4 weeks. In the present study, cytogenetic analysis of colonies from long-term culture assays was therefore performed i n i t i a l l y a f t e r 3 or 4 weeks. For the 3 newly diagnosed patients studied here, a l l progenitors analyzed at t h i s time proved 1 to be Ph -negative (Table X). 1 An increase i n the proportion of Ph -negative progenitors present a f t e r 3 to 4 weeks implies that there must have been a s i g n i f i c a n t difference i n the 1 1 maintenance of Ph -negative and Ph - p o s i t i v e progenitors during t h i s i n t e r v a l . Table XI shows the number of p r i m i t i v e Ph^-positive BFU-E present i n i t i a l l y and the maximum number that could have been present i n the adherent layer 3 to 4 1 weeks l a t e r and s t i l l have escaped detection. Changes i n p r i m i t i v e Ph -TABLE X Ratios of cytogenetically normal : Ph 1-positive metaphases in d i r e c t bone marrow aspirates and ratios of cytogenetically normal : Ph 1-positive colonies in methylcellulose assays before and after long-term culture of progenitors Normal : Ph 1-Positive Case * TJ..<I~I Long-Term Cultures _. Direct Bone I n i t i a l Colony Time of Marrow Assay BFU-E* CFU-C CFU-G/E Total Assay Untreated Patients 1 (a) 0 : 30 3 : 7 11 : 0 1 : 0 - 12 : 0 Wk 4,6 Kb) 0 : 13 7 : 17 10 : 0 - 10 : 0 Wk 1 2 0 : 25 1 ! ; 1 5 : 0 4 : 0 5 : 0 14 : 0 Wk 3,6 3 0 : 16 0 i 14 7 : 0 6 i 0 1 : 0 14 : 0 Wk 3 Treated Patients 4 0 :  21 6 : 21 13 : 0 1 : 0 - 14 : 0 Wk 4 5 0 : 25 0 : 10 10 : 0 24 : 1+ 2 : 0 28 : 0+ 12 : 0 Wk 2 , 3 , 6 6 0 :  9 0 : 16 1 : 0 13 i 0 - 14 : 0 Wk 3 7 0 :  25 0 : i 25 0 : 13 0 : 46+ 0 : 1 0 : i 14 Wk 2,3,6 e 0 : 31 0 : 11 0 : 1 0 i 0 : ; 5 ,22+ - 0 : 6 Wk 3 9 0 : 10 0 : 10 . * - -* >8 clusters. t Metaphases from pooled colonies. t No colonies that could be cytogenetically analyzed were derived from these long-term cultures at any time. 114 negative BFU-E numbers between 0 and 3-4 weeks are also shown. The dramatic decline i n Ph^-positive BFU-E i n a l l 3 experiments i s r e a d i l y seen and 1 contrasts with the much slower decline of Ph -negative BFU-E. The k i n e t i c s of the Ph^-negative BFU-E i n these experiments c l o s e l y resembles that of BFU-E i n 23 normal long-term marrow cul t u r e s . To investigate the p o s s i b i l i t y that t h i s abnormal behaviour of the Ph^-p o s i t i v e progenitors might be r e l a t e d to t h e i r exclusion from or i n a c t i v a t i o n i n the adherent layer during the i n i t i a l phases of i t s formation, an experiment to evaluate one week-old cultures was undertaken. The marrow used was a repeat aspirate from Case 1 obtained 1 month a f t e r the f i r s t . As can be seen i n Table X, Case 1(b) only Ph^-negative p r i m i t i v e BFU-E were detected i n the adherent f r a c t i o n of these 1-week-old cu l t u r e s . Treated Patients To evaluate the e f f e c t of conventional treatment on the subsequent behavior of Ph^-positive progenitors i n long-term marrow cultures, and to determine how long a f t e r diagnosis PtJ-negative stem c e l l s might s t i l l be detected i n such cu l t u r e s , 6 patients with established treated disease were studied. In a l l 6 cases treatment was i n i t i a t e d soon a f t e r diagnosis. The i n t e r v a l from diagnosis to the time of study varied from 5 to 83 months (Table IX). Cytogenetic analysis of hemopoietic colonies generated i n methylcellulose assays of the same marrow specimens used to i n i t i a t e long-term cultures f a i l e d to detect the presence of Ph^-negative progenitors i n 5 of the 6 treated patients (Table X). In the remaining treated patient (Case 4) 22% of marrow 1 progenitors were Ph -negative at the time of study, and a s i m i l a r d i s t r i b u t i o n of progenitor genotypes was found i n the perip h e r a l blood. Long-term cultures established from marrow aspirates from a l l 6 patients were monitored f o r up to 6 to 8 weeks, and the number of p r i m i t i v e hemopoietic 115 progenitors detected was s u f f i c i e n t to permit cytogenetic data to be c o l l e c t e d i n a l l but 1 experiment. Long-term cultures from t h i s patient's marrow (Case 9) f a i l e d to develop a t y p i c a l adherent layer, remained hypocellular and y i e l d e d few colonies, none of which could be cytogenetically analyzed. Long-term cultures from the other 5 treated patients were s i m i l a r i n appearance and behaviour to cultures of untreated CML marrow. Cytogenetic analyses were performed on colonies generated i n assays of adherent layers harvested i n i t i a l l y a f t e r 3 to 4 weeks and i n some instances again a f t e r 6 weeks. For 3 of the treated patients (Cases 4, 5 and 6), the r e s u l t s were s i m i l a r to those obtained f o r the 3 untreated patients, i . e . , 1 v i r t u a l l y a l l progenitors present a f t e r 3 weeks were Ph -negative. In the 1 other 2 experiments, only Ph - p o s i t i v e progenitors were detected (Table X). As shown above for cultures established with marrow from untreated p a t i e n t s , conversion to Ph^-negativity i n long-term cultures of treated CML marrow was associated with an abnormally r a p i d decline of the o r i g i n a l l y dominant Ph^-positive population (see Table XI). In one of the 2 treated CML experiments where Ph^-negative progenitors were not detected (Case 8), t h i s abnormal behaviour of the Ph^-positive population was also observed. However, i n the other (Case 7), a d i f f e r e n t pattern was seen. In t h i s experiment, the 1 number of p r i m i t i v e BFU-E ( a l l of which were co n s i s t e n t l y Ph - p o s i t i v e ) present i n the adherent layer was maintained i n the range previously determined for a 23 ser i e s of normal marrow cultures. Long-term Cultures A comparison was made between cultures from those patients that converted 1 1 to Ph -negative hemopoietic progenitor p r o l i f e r a t i o n to those i n which Ph -p o s i t i v e progenitor p r o l i f e r a t i o n p e r s i s t e d throughout the long-term culture 116 TABLE XI Number of Ph^-positive and Ph^-negative primitive BFU-E* in the adherent layer of long-term CML cultures^ Duration of Long-Term Culture (wks) 0 Ph^-Positive 3-4 6 Ph 0 1 -Negative 3-4 6 Case 1 2100 <17 _ 900 71 2 125 <15 - 125 36 3 1750 <4 - <403 11 4 5571 <91 - 929 432 5 2125 <48 - <617 184 6 750 1 - <120 <1 8 500 1 - <130 <1 A* 7750 <17 <2S <930 17 25 B § 1250 <25 - <225 25 c|| 1500 6 <36 <390 109 331 D ' 1500 55 - <615 <13 7 1250 452 37 <12S0 <186 <16 >8 cl u s t e r s . ^Values were estimated by multiplying the t o t a l number of primitive BFU-E per adherent layer by the percent determined to be Ph 1-positive (or negative). When none of a particular genotype were detected, maximum values were calculated. In these instances, the percent value used was the maximum that could be excluded with 95* confidence according to the number of colonies analyzed. t § *"A = Case 1, Expt. 2 in Reference 22 : B - Case 2 in Reference 22. ||C = Case 3 in Reference 22; = Case 4 in Reference 22-117 period. Growth k i n e t i c parameters s p e c i f i c a l l y examined as a function of time i n culture were: the t o t a l number of nucleated c e l l s per nonadherent layer (Figure 12a), the t o t a l number of granulopoietic progenitors (CFU-C) per non-adherent and per adherent l a y e r (Figures 12b & 12c), the t o t a l number of mature e r y t h r o i d progenitors (CFU-E) per nonadherent and per adherent layer (Figures 12d & 12e), the t o t a l number of p r i m i t i v e e r y t h r o i d progenitors (BFU-E) per nonadherent and per adherent layer (Figures 12f & 12g), and the t o t a l number of e r y t h r o i d progenitors g i v i n g r i s e to colonies of greater than 8 c l u s t e r s per adherent layer (corresponding to those BFU-E selected f o r cytogenetic analysis) (Figure 12h). No s i g n i f i c a n t differences were observed between cultures that 1 1 remained c o n s i s t e n t l y Ph - p o s i t i v e and those i n which Ph -negative progenitors were detected. DISCUSSION Although i t i s now c l e a r that many patients with CML may s t i l l have a s i g n i f i c a n t population of chromosomally normal progenitors i n t h e i r marrows at the time when t h e i r disease i s f i r s t recognized, very l i t t l e i s known about what happens to these c e l l s as the disease progresses. In the previous Chapter, i t was shown that i n d i r e c t marrow preparations, where the majority of the d i v i d i n g c e l l s can be assumed to be terminally d i f f e r e n t i a t i n g granulocytic 1 and e r y t h r o i d c e l l s , the proportion of Ph -negative metaphases i s lower than that predicted by the degree of mosaicism i n the p r i m i t i v e progenitor compart-ments from which these two lineages derive. This i s consistent with the concept that the presence of an expanded neoplastic clone may have a suppressive e f f e c t i n vivo on the d i f f e r e n t i a t i v e capacity of normal progenitor 9 26 c e l l s . ' i f t h i s suppression also a f f e c t s the self-maintaining a b i l i t y of r e s i d u a l normal stem c e l l s , then with time, t h i s population also would be expected to disappear, although t h i s might occur r e l a t i v e l y slowly. 118 10 F i g u r e 12(a). N u c l e a t e d C e l l s per N o n - A d h e r e n t L a y e r a s a F u n c t i o n of the N u m b e r of W e e k s in L o n g - T e r m C u l t u r e . N u m b e r s R e f e r to P a t i e n t s . 119 W e e k s In L o n g - T e r m Cul ture F igure ( 1 2 b ) . G r a n u l o c y t e P r o g e n i t o r s p e r N o n - A d h e r e n t L a y e r a s a F u n c t i o n of the Number of W e e k s In L o n g - T e r m C u l t u r e . N u m b e r s R e f e r to P a t i e n t * . 120 4 10 W e e k s in L o n g - T e r m C u l t u r e F i g u r e 12 ( c ) . G r a n u l o c y t e P r o g e n i t o r s per A d h e r e n t L a y e r a s a F u n c t i o n of the Number of W e e k s in L o n g - T e r m C u l t u r e . Numbers Re fer to P a t i e n t s . 1 2 1 F i g u r e 12 (d) . M a t u r e E r y t h r o i d P r o g e n i t o r s per N o n - A d h e r e n t L a y e r as a F u n c t i o n of the Number of W e e k s in L o n g - T e r m C u l t u r e . N u m b e r s R e f e r to P a t i e n t s . 122 10 F igure 12 ( e ) . Mature E r y t h r o i d P r o g e n i t o r s per Adherent L a y e r as a Funct ion of the Number of W e e k s in L o n g - T e r m Cu l ture . Numbers Re fer to P a t i e n t s . 123 W e e k s In L o n g - T e r m Culture Figure 12 (f). Primitive Erythro id Progenitors per N o n - A d h e r e n t L a y e r as a Funct ion of the Number of W e e k s in L o n g - T e r m Culture. Numbers Refer to P a t i e n t s , 124 1 2 3 4 6 6 7 8 W e e k s In Long - T e r m Culture F igure 12(g) . Primit ive E r y t h r o i d P rogen i to rs per Adherent L a y e r a s a Funct ion of the Number of W e e k s In L o n g - T e r m Cu l ture . Numbers R e f e r to P a t i e n t s . 125 Figure 12 (h). Primit ive E ry thro id Progeni tors (Giving R ise to More Than 8 C lus te rs ) per Adherent Layer a s a Funct ion of the Number of W e e k s In L o n g - T e r m Culture. Numbers Refer to Pat ients. 126 In t h i s study, the long-term culture system was u t i l i z e d to explore t h i s 1 question. The r e s u l t s show that a r e a d i l y detectable Ph -negative hemopoietic progenitor population, capable of normal growth and d i f f e r e n t i a t i o n i n v i t r o , may frequently p e r s i s t i n the marrow of CML patients even a f t e r more than a year of conventional treatment. Such c e l l s become detectable i n long-term marrow cultures because the Ph^-positive population usually declines r a p i d l y (Table XI). To date, a t o t a l of 13 patients have been studied i n t h i s laboratory — 9 22 i n t h i s report and 4 i n a previous one. In only 1 case (an untreated 1 patient) was there evidence f o r the p r o l i f e r a t i o n of Ph -negative c e l l s i n the d i r e c t marrow preparation (Case 1, experiment 1 of reference 22). Analysis of hemopoietic colonies produced i n assays of fresh marrow revealed the presence of Ph^-negative progenitors i n 4 cases (Cases 1,2 and 4 i n t h i s report and Case 1 i n reference 22). Three of these were cases who presented with low WBC counts (<27,000) and were studied at diagnosis. The fourth was a patient who at diagnosis 5 months previously was also i n t h i s category (WBC was 23,000 at that time.) Analysis of hemopoietic colonies produced i n assays of long-term 1 culture adherent layers showed complete conversion to Ph -ne g a t i v i t y i n 6 of the 7 newly diagnosed patients studied (Cases 1-3 in t h i s report and Cases 1-3 i n reference 22), and i n 3 of the 6 treated patients studied (Cases 4-6 i n t h i s r e p o r t ) . No Ph^-negative progenitors were detected i n long-term cultures from 3 of the remaining 4 patients. One of these was an untreated patient (Case 4 i n reference 22). Two were treated patients whose disease had been diagnosed and treated the longest (Cases 7 & 8 i n t h i s r e p o r t ) . In the remaining experi-ment (Case 9), the number of progenitors recovered was very low and none of these could be cy t o g e n e t i c a l l y analyzed. No examples of p e r s i s t i n g progenitor mosaicism i n long-term culture have yet been found. 127 I f marrows from 9 patients were able to maintain a Ph^-negative population i n the long-term culture system, why were such c e l l s not detected i n cultures from the other 4 patients studied? The simplest explanation i s that i n each of 1 these 4 cases, the i n i t i a l aspirate was devoid of Ph -negative progenitors. However, i t could also be argued that the composition of the adherent layers obtained might have been i n s u f f i c i e n t to support Ph^-negative progenitors or have l e d to t h e i r suppression as occurs i n vivo. In 2 cases where Ph^-negative progenitors were not detected (Case 8 i n t h i s report and Case 4 i n reference 22), the number of progenitors recovered from both adherent and nonadherent f r a c t i o n s was low, a l b e i t s t i l l detectable. The actual values measured were not, however, lower than maximum numbers of 1 Ph - p o s i t i v e progenitors that could have been present i n the other long-term CML marrow cultures where most of the p e r s i s t i n g progenitors were Ph^-negative (Table XI). There i s thus no reason to assume that i n these 2 cases Ph^-negative progenitors would not have been maintained had they been present at the outset. In the 3rd case, no progenitors were recovered. This patient had been treated f o r approximately 8 years and because of t h i s , i t seems l i k e l y that h i s marrow was no longer able to e s t a b l i s h a competent adherent l a y e r . The 4th case where Ph^-negative progenitors were not detected (Case 7) i s perhaps the most i n t e r e s t i n g . High numbers of p r i m i t i v e Ph^-positive progenitors were detected i n the adherent layer of long-term cultures established from t h i s patient's marrow, i n contrast to the very poor recoveries of P h 1 - p o s i t i v e progenitors i n a l l other long-term CML marrow culture experi-ments (Table XI). At present, the p o s s i b i l i t y cannot be excluded that i n t h i s case, s i g n i f i c a n t numbers of Ph^-negative c e l l s were a l s o present, but were not detected because they f a i l e d to achieve a s u f f i c i e n t s u r v i v a l advantage over the Ph^-positive population due to the unusual establishment i n v i t r o of condi-128 tions s i m i l a r to those p r e v a i l i n g i n vivo. Nevertheless, from t h i s l i m i t e d s e r i e s , i t would appear that the frequency of patients with detectable Ph^-negative progenitors tends to decrease as the time since diagnosis and i n i t i a -t i o n of therapy increases. To e s t a b l i s h whether t h i s trend has general v a l i d -i t y w i l l c l e a r l y require assessment of a much larger number of patients and/or repeated studies of the same patients a f t e r varying periods of time. Previous studies of long-term marrow cultures of both human and murine o r i g i n have i n d i c a t e d that progenitor maintenance i s a function of the p r i m i -21 23 27 t i v e c e l l s that early on become a part of the adherent l a y e r . ' ' The 1 abnormally low numbers of p r i m i t i v e Ph - p o s i t i v e progenitors t y p i c a l l y found i n the adherent layer of 3 to 4 week-old cultures may be accounted f o r , at l e a s t p a r t i a l l y , by a r e l a t i v e l y decreased a b i l i t y of P h 1 - p o s i t i v e c e l l s to adhere to the bottom of the culture f l a s k e i t h e r d i r e c t l y or i n d i r e c t l y v i a other adherent c e l l types. A l t e r n a t i v e l y , they may adhere but then be unable to survive or p r o l i f e r a t e . The r e s u l t s of the second experiment set up with 1 marrow c e l l s from Case 1 in d i c a t e d that Ph - p o s i t i v e progenitors were not part of the adherent f r a c t i o n recovered a f t e r 7 days. This i s consistent with both hypotheses. However, t h i s abnormal behaviour i s not a u n i v e r s a l property of 1 a l l Ph - p o s i t i v e progenitors, since exceptions can be found (e.g., Case 7). 1 This abnormal behaviour i s also not unique to Ph - p o s i t i v e c e l l s . A s i m i l a r conversion to cytogenetic and f u n c t i o n a l normalcy i n some, but not, a l l long-term cultures established with c e l l s from newly diagnosed AML patients has been 28 recently documented. The usefulness of the long-term culture method i n f a c i l i t a t i n g the detection of Ph^-negative progenitors i s thus not always p r e d i c t a b l e . At l e a s t 1 two c o n t r i b u t i n g factors are involved. On the one hand, the number of Ph -negative progenitors i n the i n i t i a l inoculum i s of prime importance. On the 129 other hand, the composition of the adherent layer a l s o seems to play a major r o l e . I t appears that heterogeneity may e x i s t among CML patients with respect to both of these f a c t o r s and, i n the f i n a l a n a lysis, i t i s t h e i r i n t e r a c t i o n that determines whether or not Ph^-negative progenitors can be demonstrated i n a given pati e n t . SUMMARY 1 In the previous Chapter, evidence was presented f o r the existence of Ph -negative hemopoietic progenitors i n marrows from patients with newly diagnosed, untreated ph^-positive CML. In t h i s Chapter, the observations were extended to patients with treated as well as untreated CML. Ten marrows from 9 patients i n 1 the chronic phase of Ph - p o s i t i v e CML were evaluated f o r t h e i r content of chromosomally normal hemopoietic progenitors. Hemopoietic colonies cultured i n methylcellulose from progenitors i n the i n i t i a l marrow aspirate were cyto-g e n e t i c a l l y analyzed. In addition, progenitors were analyzed from the adherent layers of long-term l i q u i d marrow cultures established from the i n i t i a l marrow sample. A l l p a tients were e x c l u s i v e l y Ph^-positive upon examination of fresh marrow. In 3 cases, from 20-50% of progenitors i n the uncultured marrow were Ph^-negative. In 6 cases, 3 with well-established, treated CML, progenitors 1 derived from the adherent layer of long-term cultures were e x c l u s i v e l y Ph -negative. These r e s u l t s suggest that hemopoietic stem c e l l s that do not belong to the cytogenetically marked leukemic clone e x i s t i n the marrows of most patients with CML f o r a considerable period of time a f t e r presentation. In addi t i o n , the observation of a conversion to e x c l u s i v e l y Ph^-negative progenitor c e l l p r o l i f e r a t i o n i n most long-term cultures suggest that important differences e x i s t between normal and leukemic stem c e l l responses to the microenvironment of the long-term cultures. 130 REFERENCES 1. Fialkow PJ, Jacobsen RJ, Papayannopoulos T: Chronic myelocytic leukemia: Clonal o r i g i n i n a stem c e l l common to the granulocyte, erythrocyte, p l a t e l e t and monocyte/macrophage. Am J Med 63: 125-130, 1977 2. Bernheim A, Berger R, Preud'homme JL, Labaume S, Bussel A, Barot-Ciorbara R: Ph i l a d e l p h i a chromosome p o s i t i v e blood B lymphocytes i n chronic myelo-c y t i c leukemia. Leuk Res 5: 331-339, 1981 3. Eaves AC, Eaves CJ: Abnormalities i n the e r y t h r o i d progenitor compartments i n patients with chronic myelogenous leukemia (CML). Exp Hematol 7 (Suppl 5): 65-75, 1979 4. Goldman JM, Shiota F, Th'ng KH, Orchard KH: C i r c u l a t i n g granulocyte and e r y t h r o i d progenitor c e l l s i n chronic granulocytic leukemia. Br J Haematol 46: 7-13, 1980 5. Tough IM, Jacobs PA, Courtbrown W: Cytogenetic studies on bone marow i n chronic myeloid leukemia. Lancet 1: 844-846, 1963 6. Whang J , F r e i E, T j i o JM, Carbone PP, Brecher G: The d i s t r i b u t i o n of the Phi l a d e l p h i a chromosome i n patients with chronic myelogenous leukemia. Blood 22: 664-673, 1963 7. Rowley JD: A new consistent chromosomal abnormality i n chronic myelogenous leukemia i d e n t i f i e d by quinacrine fluorescence and Giemsa s t a i n i n g . Nature 243: 290-293, 1973 8. DubS ID, Eaves CJ, Kalousek DK, Eaves AC: A method for obtaining high q u a l i t y chromosome preparations from single hemopoietic colonies on a routine b a s i s . Cancer Genet Cytogenet 4: 157-168, 1981 9. Dub€ ID, Gupta CM, Kalousek DK, Eaves CJ, Eaves AC: Cytogenetic studies of early myeloid progenitor compartments i n Ph - p o s i t i v e chronic myeloid leukemia (CML): I. Persistence of Ph -negative committed progenitors that are suppressed from d i f f e r e n t i a t i n g i n vivo. Br J Haematol ( i n press) 10. Goto T, N i s h i k o r i M, A r l i n Z, Gee T, Kempin S, Burchenal J , S t r i f e A, Wisniewski D, Lambek C, L i t t l e C, Jhanwar S, Chaganti R, Clarkson B: Growth c h a r a c t e r i s t i c s of leukemic and normal hemopoietic c e l l s i n Ph + chronic myelogenous leukemia and e f f e c t s of intensive treatment. Blood 59: 793-808, 1982 11. Smalley RV, Vogel J , Huguley CM, M i l l e r D: Chronic granulocytic leukemia: cytogenetic conversion of bone marrow with c y c l e - s p e c i f i c chemotherapy. Blood 50: 107-113, 1977 12. Dexter TM, A l l e n TD, Lajtha LG, S c h o f i e l d R, Lord BI: Stimulation of d i f f e r e n t i a t i o n and p r o l i f e r a t i o n of haemopoietic c e l l s i n v i t r o . J C e l l P h y s i o l 82: 461-473, 1973. 131 13. Testa NG, Dexter TM: Long-term production of e r y t h r o i d precursor c e l l s (BFU) i n bone marrow culture. D i f f e r e n t i a t i o n 9: 193-195, 1977. 14. Dexter TM, Moore MAS, Sheridan APC: Maintenance of hemopoietic stem c e l l s and production of d i f f e r e n t i a t e d progeny i n allogeneic and semi-allogeneic bone marrow chimeras i n v i t r o . J Exp Med 145: 1612-1616, 1977. 15. Dexter TM, A l l e n TD, Lajtha LG: Conditions c o n t r o l l i n g the p r o l i f e r a t i o n of hemopoietic stem c e l l s i n v i t r o . J C e l l P hysiol 91: 335-344, 1977. 16. Gartner S, Kaplan H: Longterm culture of human bone marrow c e l l s . Proc N a t l Acad S c i USA 77: 4756-4759, 1980. 17. Hocking W, Golde DW: Long-term human bone marrow cultures. Blood 56: 118-124, 1980. 18. Greenberger JS, Sakakeeny M, Parker L: In v i t r o p r o l i f e r a t i o n of hemopoie-t i c stem c e l l s i n long marrow cultures: p r i n c i p l e s i n mouse applied to man. Exp Hematol (Suppl 5): 135-148, 1979. 19. Moore MS, Broxmeyer HE, Sheridan AP, Meyers PA, Jacobsen N, Winchester RJ: Continuous human bone marrow culture: Ia antigen c h a r a c t e r i z a t i o n of probable p l u r i p o t e n t i a l stem c e l l . Blood 55: 682-680, 1980. 20. Toogood IRG, Dexter TM, A l l e n TS, Lajtha LG: The development of a l i q u i d culture system f o r the growth of human bone marrow. Leuk Res 4: 449-461, 1980 . 21. Eaves C, Coulombel L, Eaves A: Analysis of hemopoiesis i n long-term human marrow cul t u r e s , i n Killraann Sv-Aa, Cronkite EP, Muller-Berat CN (eds): Haemopoietic Stem C e l l s . Copenhagen, Munksgaard, 1983, p 287-298. 22. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC: Long-term marrow culture reveals chromosomally normal hematopoietic progenitor c e l l s i n patients with P h i l a d e l p h i a chromosome-positive chronic myelogenous leukemia. N Engl J Med 308: 1493-1498, 1983. 23. Coulombel L, Eaves AC, Eaves CJ: Enzymatic treatment of longterm human marrow cultures reveals the p r e f e r e n t i a l l o c a t i o n of p r i m i t i v e hemopoietic progenitors i n the adherent la y e r . Blood 62: 291-297, 1983. 24. K r y s t a l G, Eaves AC, Eaves CJ: F i f t y - f o l d p u r i f i c a t i o n of human erythro-p o i e t i n using CM A f f i - g e l Blue. Blood 60(Suppl 1): 87a, 1982 (abstr) 25. Hook EB: Exclusion of chromosomal mosaicism. Tables of 90%, 95% and 99% confidence l i m i t s and comments on use. Am J Human Genet 29: 94-97, 1977. 26. Adamson SW, Singer JW, Catalano P, Murphy S, L i n N, Steinman L, Ernst C, Fialkow P: Polycythemia vera. Further i n v i t r o studies of hematopoietic regulation. J C l i n Invest 66: 1363-1368, 1980. 27. Mauch P, Greenberger JS, Botnik LE, Hannon EC, Hellman S: Evidence f o r structured v a r i a t i o n i n the self-renewal capacity of hemopoietic stem c e l l s within long-term bone marrow cultures. Proc Natl Acad S c i USA 77: 132 2927-2930, 1980. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC: Variable persistence of normal and leukemic c e l l s i n long-term AML marrow cultures (manuscript i n preparation) Eaves CJ, Eaves AC: E r y t h r o i d progenitor c e l l numbers i n human marrow— Implication f o r regulation. Exp Hematol 7 (Suppl 5): 54-64, 1979. 133 C H A P T E R F I V E CYTOGENETIC STUDIES OF EARLY MYELOID PROGENITOR COMPARTMENTS IN ACUTE PHASE Ph 1-POSITIVE CML. METHYLCELLULOSE ASSAYS REVEAL 1 PERSISTENCE OF Ph -POSITIVE COMMITTED PROGENITORS INTRODUCTION In the preceeding two chapters, attention was focused on the question of the existence of chromosomally normal hemopoietic stem c e l l s i n the chronic phase of Ph^-positive CML. In t h i s chapter, the i n v e s t i g a t i o n i s extended to patients i n the acute phase of CML. Within a mean of three years a f t e r diagnosis, the r e l a t i v e l y benign chronic phase of CML evolves i n t o a terminal acute phase that i s unresponsive to a v a i l a b l e therapy. In the acute phase, hemopoietic progenitor c e l l maturation becomes progressively impaired and eventually terminates i n the severe maturational block that i s c h a r a c t e r i s t i c of acute myelogenous leukemia (AML).^ The development of maturationally defective progenitor c e l l s i s usually the r e s u l t of c l o n a l evolution with concomitant changes i n the 1 karyotypes of the leukemic clone. When patients with Ph - p o s i t i v e CML enter the terminal acute phase, a d d i t i o n a l chromosomal abnormalities are superimposed 1 2 3 on the Ph - p o s i t i v e stem l i n e i n about 80% of patients. ' A change i n the karyotype of the leukemic c e l l s i s considered to be a grave prognostic sign; the median s u r v i v a l from the time of change u n t i l death being l e s s than f i v e 4 months. The cytogenetic changes seen i n the acute phase are of a nonrandom 2 5 nature (see Figure 1) ' and probably f a l l i n t o the category of secondary or passive changes, as discussed i n Chapter One. ( I t should be noted, however, that since v i r t u a l l y a l l of the patients studied i n the acute phase have been 134 treated with an a l k y l a t i n g agent, such as busulphan or an antimetabolite, such as hydroxyurea, the p o s s i b i l i t y e x i s t s that t h i s therapy a f f e c t s the pattern of abnormalities seen.) In about 80% of acute phase pat i e n t s , the c e l l s of the malignant clone e x h i b i t morphological and biochemical c h a r a c t e r i s t i c s of non-terminally d i f f e r e n t i a t e d myeloid c e l l s (myeloblasts). In the remaining cases, the malignant c e l l s e x h i b i t c h a r a c t e r i s t i c s of non-terminally d i f f e r e n t i a t e d ' 6 7 lymphoid c e l l s (lymphoblasts). ' ' A recent study of immunoglobulin gene arrangement i n lymphoblasts from patients i n lymphoid b l a s t c r i s i s of CML has l e d to the suggestion that b l a s t c r i s i s expansions represent unique subclones at d i f f e r e n t stages of B - c e l l - Q precursor d i f f e r e n t i a t i o n . S e r i a l i n v e s t i g a t i o n s of a si n g l e patient i n d i c a t e d that the separate c e l l u l a r expansions during d i f f e r e n t b l a s t c r i s i s can o r i g i n a t e from d i s t i n c t B - c e l l precursors at d i s c r e t e , g e n e t i c a l l y d i s t i n -" 8 guishable stages of maturation. These findings support the concept that while the i n i t i a l transformation event(s) i n CML probably occurs i n a p l u r i -potent stem c e l l , the "second" event(s) that p r e c i p i t a t e the acute phase of t h i s disease can occur i n any one of a host of more d i f f e r e n t i a t e d c e l l types and the acute phase subclone exhibits many of the phenotypic c h a r a c t e r i s t i c s of the c e l l involved i n t h i s second event. Indirect evidence suggesting that the a d d i t i o n a l chromosome changes seen i n the acute phase are markers of the b l a s t c e l l populations comes from the frequent observation of a reversion to the o r i g i n a l Ph^-positive stem l i n e when 9 1 0 1 1 1 2 " 1 b l a s t c e l l s are temporarily ablated by therapy. ' ' ' . However, Ph -negative marrow metaphases have only r a r e l y been observed among patients i n the acute phase of CML. Sokal e_t a l reported Ph 1-negative metaphases i n 5 out of 101 patients i n the b l a s t i c s t a g e ^ and Zaccaria et a l found Ph^-negative 14 metaphases i n 3 out of 12 patients presenting i n the b l a s t i c stage. There 135 has been only one reported attempt to use clonogenic assays for hemopoietic precursors to c y t o g e n e t i c a l l y study the stem c e l l population during the acute phase. In that report, the si n g l e patient studied showed c l i n i c a l and morpho-l o g i c a l signs of transformation to b l a s t c e l l c r i s i s but no a d d i t i o n a l chromo-1 somal abnormalities were superimposed on the Ph - p o s i t i v e stem l i n e . Cytogenetic analysis of small and poorly d i f f e r e n t i a t e d c e l l c l u s t e r s revealed 1 15 23 metaphases a l l bearing the Ph as the sole abnormality. 1 In t h i s Chapter, the d i f f e r e n t i a t i o n p o t e n t i a l of the Ph - p o s i t i v e clone 1 and the a d d i t i o n a l subclones that characterize the acute phase of Ph - p o s i t i v e CML i s investigated. Further, the e f f e c t s of karyotypic evolution on the a b i l i t y of progenitor c e l l s to d i f f e r e n t i a t e normally i s examined. MATERIALS AND METHODS C l i n i c a l and hematological data on the 6 patients studied i n the acute phase are summarized i n Table XII (see Results f o r a more d e t a i l e d c l i n i c a l summary of each p a t i e n t ) . A l l patients had been treated with conventional therapy (busulfan, hydroxyurea). Cases 1,2 and 3 were diagnosed as being i n 1 the acute phase of Ph - p o s i t i v e CML at the times of study. Cases 4,5 and 6 1 were i n i t i a l l y diagnosed as cases of Ph - p o s i t i v e acute lymphocytic leukemia but, as w i l l be demonstrated, at l e a s t two of these were more l i k e l y cases of Ph^-positive CML that were f i r s t diagnosed a f t e r transformation to the acute lymphoblastic phase had occurred. Cases 1, 2 and 5 were studied i n the chronic phase of t h e i r disease and these data were reported i n Chapter Three (Cases 13, 9 and 14 i n Table VI). Cases 1, 2 and 5 were studied only once a f t e r acute transformation. Case 4 was studied twice a f t e r transformation and Case 3 was studied 8 times from the f i r s t signs of acute transformation up u n t i l the time of death 11 months l a t e r . Case 6 was studied 3 times. Three patients (Cases 136 1,2 and 3) died of t h e i r disease during the course of t h i s study. Case 4 underwent bone marrow transplantation and has had no relapse since (7 months at the time of w r i t i n g ) . Case 5 i s s t i l l i n remission. Prognosis of Case 6 was poor at the time of w r i t i n g . Cytogenetic analysis of G-banded d i r e c t marrow preparations were performed on a l l patients at the time of diagnosis, i n most instances as a part of the routine laboratory work-up. In the acute phase, repeat d i r e c t cytogenetic analyses were usually performed on b l a s t s i n the same marrow and/or blood specimens used to set-up the methylcellulose cultures (see Table XII for these data). Marrow and blood specimens were part of samples taken f or diagnostic purposes and were obtained with informed consent. To study the cytogenetic composition of p r i m i t i v e hemopoietic progenitor c e l l compartments, procedures i d e n t i c a l to those described i n previous chapters were used. Marrow buffy coat c e l l s or l i g h t density peripheral blood mono-nuclear c e l l s were separated, washed and plated i n methylcellulose cultures as described i n Appendices I and I I . In such cultures, large erythroid and granu-locyte colonies arose from committed precursors. Mixed granulocyte/erythroid (CFU-G/E) colonies were also occasionally observed. Numbers of p r i m i t i v e e r y t h r o i d progenitors (BFU-E, >8 erythroblast c l u s t e r s per colony) and granulocyte progenitors (CFU-C), per set number of marrow c e l l s or per ml of blood were determined as a part of the routine laboratory workup. (Note: Blood progenitors can be scored per ml of blood since the t o t a l blood volume remains r e l a t i v e l y unchanged i n CML. However, since the marrow compartment can increase i n s i z e by extension i n t o the long bones, progenitor numbers are reported per set number of c e l l s . ) Control values shown i n Table V are'those reported previously from t h i s l a b o r a t o r y . ^ In an attempt to demonstrate the existence of chromosomally normal progen-1 3 7 i t o r c e l l s , large, w e l l - i s o l a t e d and r e a d i l y i d e n t i f i a b l e erythroid, granulo-cyte and mixed granulocyte/erythroid colonies were selected, plucked and processed i n d i v i d u a l l y . A d e t a i l e d d e s c r i p t i o n of the method used i s described i n Chapter Two and Reference 17. A minimum of two G-banded metaphases were analyzed per colony and i n most cases, karyotypes prepared. The genotypes of the progenitors from which the colonies arose were thus i n f e r r e d by cytogenetic analysis of t h e i r d i f f e r e n t i a t i n g descendants. This approach was based on the assumption that colonies were derived from single progenitor c e l l s , e i t h e r Ph^-p o s i t i v e or Ph^-negative. This was supported by the f i n d i n g that f o r 941 colonies analyzed i n a recent report from t h i s laboratory, i n no instance were 18 2 d i f f e r e n t karyotypes obtained from a single colony. Occasionally, two or more colonies were pooled for an a l y s i s . (These are in d i c a t e d as "pooled col o n i e s " i n Table XIII.) RESULTS  Case _1_ This 51 year-old man was diagnosed i n 1973 as having CML. He was treated i n t e r m i t t e n t l y with busulfan and i n A p r i l 1981, cytogenetic studies of bone marrow were performed as a part of routine staging. At that time, a l l 23 metaphases analyzed revealed the presence of an unusual Ph^ translocation i n an otherwise normal male karyotype. The tr a n s l o c a t i o n involved chromosomes 16 and 22 [46,XY,t(16;22)(q24;q11)]. Hemopoietic colonies cultured from blood 1 precursors at that time were a l l Ph - p o s i t i v e (13 colonies analyzed, reported i n Table VI, Case 13). By May 1982, the patient had undergone a myeloblastic transformation. Cytogenetic studies were performed on b l a s t c e l l s i n peripheral blood as a part of t h i s work. The analysis of 50 metaphases revealed 4 c e l l l i n e s : 46,XY,+C, TA8LE XII C l i n i ca l and hematological data on the 6 patients studied in the acute phase Months . - *» (HTL-I A ™ »•«-• prTtor t ion BFU-E* CFU-C 1 49 M 107 38,000 11.2 251,000 8 2.115 2.655 2 35 F 34 148.000 9.7 31,000 B M 32 18 192 74 3(a) 58 F 7 155,000 8.7 105,000 B 8.953 160 (b) 8 125,000 11.1 96,000 8 M 500 S.5 24 90 (0 9 14,600 12.1 393,000 M 3.5 24.5 (d) 10 115,000 10.4 160,000 B H 3.25 3.40 9.75 4.13 (e) 11 1,200 10.5 118.000 M 3.5 19.0 (f) 12 150,000 12.5 116,000 B 17.5 31.5 (9) 13 112,000 11.0 31,000 B M 8.0 1.75 38.0 7.0 (h) 16 71,000 14.6 38,000 B 13.5 18.0 4(a) 29 M 2 5,400 9.8 212,000 B M 121 19.5 184 152 (b) 5 6,400 10.6 333,000 8 M ISO 28 126 284 5 46 M 22 10,600 16.4 174.000 B M 38 94 278 92 6(a) 62 F 1 1.100 11.5 86,000 B M 2 0.5 2 5 (b) 1.5 200 11.1 69,000 B M 3 0.28 0 15 (c) 2 5,400 10.9 502,000 B M 174 51 132 162 * Specimens: 8 • Blood; M « Marrow s t Blood values are per ml. Marrow values are per 2 x 10 buffy coat c e l l s . (See Table V for control values) X >8 c lus ters . 139 -17,i(17q),t(16;22)/46,XY,t(16;22)/46,XY,+C,-F,i(17q),t(16;22)/47,XY,+C,+C, -17,i(17q),t(16;22). One possible sequence of c l o n a l evolution accounting f o r t h i s r e s u l t i s 46,XY,t(16;22) — * 47,XY,+C,t(16;22) — * 47,XY,+C,i(17q),t(16;22). This l a t t e r l i n e p ossibly gave r i s e to two c e l l l i n e s with a d d i t i o n a l abnormalities; one with a missing 17 and the other with a missing chromosome of the F group. The 46,XY,+C,-17,i(17q),t(16;22) c e l l l i n e then p o s s i b l y gave r i s e to the 47,XY,+C,+C,-17,i(17q),t(16;22). (See Appendix IX for explanation of cytogenetic nomenclature.) Hemopoietic colonies were cultured from blood precursors i n the same sample used f o r cytogenetic a n a l y s i s . Colonies a r i s i n g i n methylcellulose cultures were of abnormal morphology i n some cases and d i f f i c u l t to c l a s s i f y . The l e a s t abnormal colonies were large, hemoglobin containing colonies that resembled large, e r y t h r o i d colonies except f o r the actual morphology of the colony. Instead of c e l l s being arranged i n 8 to 16 spaced c l u s t e r s of about 50 c e l l s , each around a c e n t r a l point, colonies apparently consisted of about 800 c e l l s scattered densely around a c e n t r a l o r i g i n . Four such colonies y i e l d e d 1 two or more metaphases each and a l l were characterized by the Ph as the sole abnormality. Another abnormal colony type i d e n t i f i e d i n cultures from blood progenitors were of unusual morphology and lacked the c h a r a c t e r i s t i c red color i n d i c a t i v e of hemoglobin synthesis i n e r y t h r o i d colonies. Karyotypes from three such colonies were 46,XY,t(16;22) while 7 were 47,XY,+C,+C, -17,i(17q),t(16;22). Diffuse colonies containing approximately 500 c e l l s each were also observed. Karyotypes from three such colonies were 47,XY,+C,+C,-17,i(17q), t(16;22) while 6 were 46,XY,t(16;22). Two macrophage-like colonies containing about 200 c e l l s each were s u c c e s s f u l l y karyotyped as 47,XY,+C,+C,-17,i(17q), 140 t(16;22) and 46,XY,t(16;22). Normal appearing granulocyte colones were derived from two c e l l l i n e s : 47,XY,+C,+C,-17,i(17q),t(16;22) and 46,XY,t(16;22) (6 colonies analyzed). Two granulocyte colonies that had unusually d i f f u s e arrangements of constituent c e l l s were of the 47 chromosome c e l l l i n e described above. A further 6 colonies were completely u n i d e n t i f i a b l e . A l l were of the 47 chromosome c e l l lineage. The p a t i e n t died s h o r t l y a f t e r the l a s t study and a repeat t r i a l was not p o s s i b l e . At best, i t can be s a i d that the most abnormal stem l i n e detected i n the unstimulated p e r i p h e r a l blood [47,XY,+C,+C,-17,i(17q),t(16;22)] gave r i s e to a host of abnormal colonies i n v i t r o . I t appears that the o r i g i n a l Ph^-p o s i t i v e c e l l l i n e was the only c e l l l i n e capable of g i v i n g r i s e to recogniz-able, a l b e i t morphologically abnormal, e r y t h r o i d colonies. Case 2 This 36 year-old woman was diagnosed as having t y p i c a l Ph^-positive CML i n February 1978 and treated with busulfan. In December 1980, she was evaluated f o r chromosomally normal progenitors while s t i l l i n chronic phase. At that time, a l l 9 marrow progenitors and 15 blood progenitors were Ph^-positive (Case #9, Table VI). By June 1981, she had undergone myeloblastic transformation. At that time, chromosomal analysis of a l l 28 marrow and blood d i r e c t metaphases revealed a 57 chromosome c e l l l i n e with 5 marker chromosomes; 1 57,XX,+ 1 ,+2,+ 10 ,+ 11 ,+ 14 ,- 15 ,-17 ,+21 ,+ 22 ,+Ph ,+Mar1 ,+Mar2 ,+Mar3 ,+Mar4 ,+Mar5 . These cytogenetic findings were rather unusual for b l a s t c e l l c r i s i s i n CML where isochromosome 17, d u p l i c a t i o n of chromosome 8, and d u p l i c a t i o n of Ph^ are generally found. The degree of abnormality was, however, consistent with the terminal phase of CML and the patient died s h o r t l y a f t e r . Hemopoietic colonies f a i l e d to grow from buffy coat marrow c e l l s . TABLE XIII Results of cytogenetic studies on hemopoietic progeni tors In 6 pa t ients in the acute Phase Case I Specimen Blast Cel l Karyotypes* Karyotypes from Hemopoietic Progeni tors Number of Colonies Analyzed 3(a) lt>) (O (d) (e) ( f ) (9) W 4(a) <t» 5 6(a) lb) (c) 46,XY. tC .-17, l (17q) . t (16 ;22)/46.XY, t (16 ;22)/ 4 6 , X T . « C , - F . l ( 1 7 q ) . t ( 1 6 ; 2 2 ) / 4 7 . X » , » C , » C , - 1 7 . 5 7 , X X . » l . * 2 , t l 0 . t l l , » 1 4 , - 1 5 . - 1 7 . * 2 1 , « 2 2 , + P h 1 , •Har1,•Mart,+Mar3,*Mar4,»Mar5 5 7 , X X , t l , « 2 , » l O , * l l , « 1 4 , - 1 5 , - 1 7 . « 2 1 , « 2 2 . « P h 1 •Mar1,•Mart,•Mar3,*Mar4,•Mar5 46,XX,Ph 1 /44.XX.-17,-18,-19.-Ph , .+del (6q) , •Ha r.der(9)t(9p;17q),der(J)t(9q;22q) ND ND 46,XX.Ph'/46,XX 44,XX,-17 , -18 ,-Ph 1 ,*deU6q),der(9)t(9p;17q), der(2)t(9q;llq)/46,XX,Ph' ND 46,XX,Ph 1 /44,XX,-17,-18,-Ph 1 .+del (6q) , der(9)t(9p;17q).der(2)t(9q;22q) 46,XX.Ph 1 /44,XX,-17,-18,-Ph 1 ,+del (6q) , der(9 ) t (9p;17q).der(9 ) t (9q;22q) NO 44,XX.-17,-18,-Ph 1 .^del (6q) ,der (9 ) t (9p;17q) . der(f)t(9q;22q) /46 .XX.Ph' 43,XX,•del (6q) .-10,-14.-17,-18,-19.-20,-Ph ' , •Marl.•Mart,+Mar3.der(9 ) t (9p;17q), der(9Jt (9q;22q1/44,XX,•del(6q1,-17,-18.-1?, W , ^ r l . de r ( ? J t ( 9p ;17q ) ,de r ( o ) t ( 9q ;22q ) ND NO 46,XY/46,XY,Ph 1 ND 46,Xy/46,XY,Ph' 46,XY,Ph' 46,XX NO 46,XX/46,XX,Ph' ND 46, XX 46,XY. t (16;22)/ 4 7 ,XY.^C.^C,- I7 , i (17q) , t (16;22) 4 6 , X X , P h 1 / 5 7 , X X , M . r t . M 0 . M l , • H . . - 1 5 . - 1 7 . * 2 1 , » 2 2 , » P h ' . « M « r l . •Mart.•Mart, •Ma^.+MarS 46,XX;Ph ' 46,XX.Ph ' 46 .XX.Ph 1 46.XX.Ph ' 46 ,XX ,Ph l 46,XX,Ph ' 46 .XX.Ph 1 46,XX,Ph ' 46.XX.Ph 1  46.XX.Ph 1 ND 46.XX.Ph 1 46,XY/46.XY,Ph 1 46,XY/46,XY,Ph 1 46,XY/46,XY,Ph 1 46,XY/46.XY,Ph 1 46.XY.Ph 1 46,XX/46,XX,Ph 1 46,XX 46,XX 46,XX 46,XX/46,XX,Ph 1 37 52 19 15 12 17 13 17 20 9 18 11 78 f 28 2(30*) •>t 32(80') 16(61+) * See Appendix IX for cytogenet ic nomenclature, t Number of metaphases from pooled co lon i es . 142 However, a marrow suspension culture containing medium and f e t a l c a l f serum was ca r r i e d f o r 4 days and the c e l l s p r o l i f e r a t i n g at that time harvested f o r 1 cytogenetic a n a l y s i s . A mosaic p i c t u r e with 5 c e l l s bearing the Ph as the sole abnormality and 8 characterized by the 57 chromosome c e l l l i n e was revealed from t h i s suspension culture study. Hemopoietic progenitors i n peripheral blood gave r i s e e x c l u s i v e l y to abnormally small colonies, each containing approximately 50 c e l l s . Fifty-two metaphases were analyzed from 35 pooled colonies. Forty-one were marked with the Ph 1 as the sole abnormality while 11 were of the 57 chromosome number c e l l l i n e . Case 3 This 56 year-old woman was diagnosed i n A p r i l 1980 with t y p i c a l Ph 1-p o s i t i v e CML. In J u l y , 16 blood progenitors were analyzed and a l l bore the Ph 1 as the sole abnormality (Case #4, Table V I ) . She was treated with hydroxyurea. In November 1980, she underwent lymphoblastic transformation. At that time, cytogenetic analysis of unstimulated blood c e l l s revealed two c e l l 1 l i n e s — o n e characterized by the Ph as the sole abnormality and the other containing 44 chromosomes and numerous abnormalities: 44,XX,-17,-18,-19 , -Ph 1,+del(6q),+Mar,der(9)t(9p;17q),der(9)t(9q;22q). Hemopoietic colonies cultured from blood progenitors were ind i s t i n g u i s h a b l e from normal controls and 10 colonies were 46,XX,Ph1. In December, the patient was i n remission as a r e s u l t of therapy. At that time, a l l 19 blood and 15 marrow progenitors were 46,XX,Ph1. The patient was s t i l l i n remission i n January of 1981 and d i r e c t bone marrow analysis revealed one normal d i p l o i d c e l l and 55 c e l l s c a r r y i n g the Ph 1 as the sole abnormality. 1 Twelve marrow progenitors were studied and a l l were 46,XX,Ph . In February 1981, the patient had relapsed again and the 44 chromosome 143 c e l l l i n e that was seen i n November predominated i n the d i r e c t bone marrow (25 c e l l s analyzed). Seventeen blood and 13 marrow progenitors were cytogenetical-l y studied and a l l c a r r i e d the Ph^ as the sole abnormality. The patient underwent two remissions and two relapses before her death i n September 1981. In remission, the 46,XX,Ph^ c e l l l i n e reappeared i n d i r e c t marrow and/or unstimulated p e r i p h e r a l blood preparations. In relapse, the 44 chromosome c e l l l i n e predominated, often with superimposed abnormalities. At the time of death, the following marrow c e l l l i n e predominated: 43,XX,+del(6q),-10,-14,-17,-18,-19,-20,-Ph1,+Mar1,+Mar2,+Mar3,der(9)t(9p;17q), der(9)t(9q/22q). Hemopoietic colonies were s u c c e s s f u l l y cultured from blood and marrow progenitors throughout the f i n a l stages of the disease. In a l l 1 cases, the 46,XX,Ph c e l l l i n e was the only c e l l l i n e that gave r i s e to hemopoietic colonies (152 colonies analyzed). I t appeared that the other c e l l l i n e s were incapable of g i v i n g r i s e to colonies i n v i t r o under the conditions used (which are not optimized even f o r normal lymphoid c e l l colony formation). Case 4 This 31 year-old man f i r s t presented i n August of 1982 with the c l i n i c a l and hematological signs of acute u n d i f f e r e n t i a t e d leukemia. Analysis of bone marrow revealed the Ph^ as the sole abnormality i n a l l 64 c e l l s studied. I t was suggested that the patient probably had Ph^-positive CML that had evolved in t o the acute phase before becoming c l i n i c a l l y evident. The patient was treated with chemotherapy designed to reduce the number of u n d i f f e r e n t i a t e d b l a s t s i n the hemopoietic system. In l a t e September, the patient was i n a chemotherapy-induced remission 1 with the Ph being detected i n only 4 out of 172 marrow metaphases. In November, the b l a s t c e l l population reappeared i n the marrow and 50% of marrow 144 karyotypes were Ph^-positive (15 metaphases studied). Hemopoietic colonies were cultured from blood and marrow precursors at that time. Of the blood 1 progenitors, 2 out of 16 e r y t h r o i d colonies (BFU-E) studied were Ph - p o s i t i v e , the remainder being normal d i p l o i d . One granulocyte colony (CFU-C) studied was 1 1 Ph - p o s i t i v e . Thirteen of 37 metaphases from pooled CFU-C were Ph - p o s i t i v e and a l l 7 from pooled BFU-E were Ph^-positive. A s i m i l a r mosaic p i c t u r e was seen among marrow progenitors: 2 CFU-C studied were Ph^-positive, 1 out of 4 metaphases from pooled CFU-C were Ph^-positive, and 3 out of 23 metaphases from pooled mixed CFU-C and BFU-E were Ph^-positive. The p a t i e n t was treated i n t o a second remission i n December 1982. At that time, 100% bone marrow metaphases were Ph^-negative. In early January, 2 out of 50 marrow metaphases were Ph^-positive. At that time, 78 metaphases from pooled hemopoietic colonies cultured from blood progenitors were analyzed. Seventy-five were normal d i p l o i d while 3 metaphases from pooled granulocyte colonies were Ph^-positive. Seven BFU-E from marrow progenitors were normal d i p l o i d while one was 46,XY,PhJ. In mid-January 1983, the patient was transplanted with h i s s i s t e r ' s marrow and i s reportedly well and free of disease at the present time (10 months at the time of w r i t i n g ) . Case 5 This 45 year-old man presented i n November 1980 with an abnormally high proportion of lymphoid b l a s t c e l l s i n h i s marrow and blood. Cytogenetic analysis of d i v i d i n g blood c e l l s revealed a t y p i c a l Ph^-positive p i c t u r e (15 c e l l s analyzed). One hundred marrow metaphases were analyzed. Eighty were of the following karyotype: 55,XY,Ph1,+2,+4,+6,+10,+11,+13,+17,+21,+Ph1. F i f t e e n were: 54,XY,Ph1 ,+ 2,+5 ,+6,+ 10 ,+ 13 ,+ 17 ,+ 21 ,+Ph1 . The remaining 5 were 46,XY,Ph 1. The patient was treated with chemotherapy designed to reduce the 145 numbers of lymphoblasts. By December 1980, he was diagnosed as being i n complete hematological remission of acute lymphocytic leukemia (ALL). At that time, 3 marrow metaphases were 46,XY and 3 were 46,XY,Ph^. In November 1981, the pa t i e n t was s t i l l i n complete remission even though 1 11 out of 22 marrow metaphases were Ph - p o s i t i v e . The patient remained i n remission up u n t i l December 1982 at which time, a l l of 7 marrow metaphases studied were Ph^-positive. In November 1982, the patient showed signs of c e n t r a l nervous system involvement and, at the time of w r i t i n g , i s gravely i l l . Cytogenetic a n a l y s i s was performed on hemopoietic colonies cultured from marrow precursors i n a marrow sample that became a v a i l a b l e i n October 1982. A l l 27 BFU-E studied were 46,XY,Ph1. One mixed colony (CFU-G/E) was also 1 karyotyped as 46,XY,Ph . Case 6 This 62 year-old woman presented i n mid-January 1983 with c l i n i c a l and hematological signs of acute lymphocytic leukemia. Chromosome analysis of d i r e c t bone marrow metaphases revealed the presence of 3 c e l l l i n e s . The major 1 c e l l l i n e c a r r i e d 59 chromosomes i n c l u d i n g the Ph . The other c e l l l i n e s were 1 46,XX,Ph and 46,XX. The patient was treated with therapy designed to reduce b l a s t c e l l p r o l i f e r a t i o n and by early February, the marrow karyotype was normal d i p l o i d (7 c e l l s studied). At that time, however, P h 1 - p o s i t i v e hemopoietic colonies were cultured from marrow progenitors. Two BFU-E were karyotyped as 46,XX,Ph1 and 46,XX re s p e c t i v e l y . In addition, 24 karyotypes from pooled CFU-C were 46,XX while 6 were 46,XX,Ph1. In mid-February, the patient showed signs of relapse and the Ph 1 was seen i n 2 out of 36 metaphases; the remainder being normal d i p l o i d . Growth of hemopoietic colonies from blood and marrow progenitors was poor. Two karyo-146 types were obtained from pooled blood BFU-E and 4 from pooled marrow BFU-E. A l l were 46,XX. In early March 1983, the bone marrow appeared to be a normal remission bone marrow. Seventy-three bone marrow metaphases analyzed were 46,XX. However, hemopoietic colonies cultured from marrow progenitors again revealed a mosaic p i c t u r e . Fourteen BFU-E were 46,XX but 4 of 59 karyotypes from pooled BFU-E 1 1 were 46,XX,Ph , the remainder being normal d i p l o i d . One CFU-C was 46,XX,Ph while 2 karyotypes from pooled CFU-C were 46,XX. One mixed CFU-G/E colony was 46,XX. Blood progenitors were cytogenetically studied simultaneously; 22 BFU-E, 9 CFU-C and 1 CFU-G/E were a l l k a r y o t y p i c a l l y normal. Likewise f o r 80 karyotypes from pooled CFU-C and BFU-E. The patient has relapsed since March and her prognosis i s now poor. A d d i t i o n a l karyotypes have been obtained from hemopoietic colonies cultured from blood and marrow progenitors as part of a separate ongoing project i n our laboratory. These r e s u l t s revealed predominantly normal d i p l o i d hemopoietic colonies i n s p i t e of chromosomally abnormal lymphoblastic stem c e l l l i n e s i n vivo. Rarely, P h 1 - p o s i t i v e hemopoietic colonies were observed. DISCUSSION The methylcellulose assay f o r hemopoietic progenitor c e l l s was used to inves t i g a t e precursor c e l l involvement i n 6 patients with P h 1 - p o s i t i v e leukemia. Two issues were addressed simultaneously: (a) the question of the existence of cytogenetically normal precursor c e l l s i n the acute phase of CML was studied, and (b) the a b i l i t y of the a d d i t i o n a l malignant stem l i n e s to complete normal d i f f e r e n t i a t i o n programs. The findings are summarized i n Table XIV. TABLE XIV Percentage of c e l l s bearing the Ph^chromosome, with or without addi t iona l chromosomal abnormal i t ies , among marrow metaphases and myeloid progenitors for the 6 pat ients studied in the acute phase Case # Phenotype of Percentage of Marrow Metaphases* Percentage of Myeloid Progeni tors* Blast Ce l l s Bearing the Ph 1 Bearing the Ph 1  Relapse Remission Relapse Remission 1 Myeloid >94 NA >88 NA 2 Myeloid >89 NA t NA 3 Lymphoid >88 >90 >98 >91 4 Undi f ferent ia ted 50 4 21 12 5 Lymphoid >96 50 NA >89 6 Lymphoid 5 95 NA i •t *When none of a pa r t i cu l a r genotype were detected, maximum values were ca l cu l a t ed . In these cases , the percentage used was the maximum that could be excluded with 95% confidence according to the number of co lonies a n a l y z e d . J 9 +No colonies grew that could be cy togenet i ca l l y analyzed. ^Estimate based on resu l ts of the analys is of pooled co lon ies . 148 Persistence of Ph. 1-positive Progenitors In Case 1, Ph 1-negative progenitors were not detected. However, i n view of the large increase over normal i n the number of blood progenitors (see Table XI), Ph 1-negative progenitors would not have been detected, even i f they p e r s i s t e d at normal l e v e l s per ml blood, unless very large numbers of colonies 20 were studied (>150). 1 In Cases 2 and 3, Ph -negative progenitors were not detected i n s p i t e of reductions of progenitor numbers to normal and subnormal l e v e l s by 1 chemotherapy. Assuming that Ph -negative progenitors p e r s i s t e d at normal l e v e l s i n these two p a t i e n t s , then they should have been detected. However, the chemotherapy presently used i n the acute phase of CML (busulfan, hydroxyurea, cytosine arabinoside, daunorubicin, adriamycin, 6-thioguanine, 6-mercaptopurine, v i n c r i s t i n e prednisone, etc.) i s cytotoxic to p r o l i f e r a t i n g 20 normal as well as malignant c e l l s . I t may therefore be argued that f a i l u r e 1 to detect Ph -negative progenitors i n treated patients can not be taken as proof of t h e i r suppression by the leukemic clone. The f i n d i n g of a high degree mosaicism at the progenitor l e v e l i n Case 4 suggests that large numbers of Ph 1-negative progenitors p e r s i s t e d i n the hematopoietic system of t h i s p a t i e n t . While t h i s f i n d i n g i n i t s e l f i s s i g n i f i c a n t , t h i s Case i s unusual and even though the c l i n i c a l diagnosis was 1 Ph - p o s i t i v e CML presenting i n acute u n d i f f e r e n t i a t e d b l a s t c r i s i s , i t may not be possible to extrapolate the findings to other cases of CML. The f a c t that a f t e r conventionally induced remissions greater than 50% of marrow metaphases 1 1 were Ph -negative i s most unusual for CML where the Ph - p o s i t i v e clone usually 5 dominates a l l l e v e l s of hemopoiesis a f t e r s i m i l a r therapy. The r a p i d a c c e l e r a t i o n of t h i s patient's disease and the predominance of u n d i f f e r e n t i a t e d b l a s t s i n relapse support the cytogenetic diagnosis of P h 1 - p o s i t i v e acute 149 un d i f f e r e n t i a t e d leukemia. The f i n d i n g i n t h i s case demonstrates the usefulness of performing cytogenetic studies on hemopoietic colonies as an a i d i n the establishment of the degree and extent of involvement of the transformed clone at the stem c e l l l e v e l . In t h i s instance, i t i s possible that the transformation event associated with malignant growth occurred i n a neoplastic c e l l long before the neoplastic clone had gained dominance over the hemopoietic system, as i s usually the case by the time of diagnosis. This suggestion i s compatible with 21 22 previous notions about c l o n a l evolution and tumorigenesis. ' 1 Case 5 was i n i t i a l l y diagnosed as having Ph - p o s i t i v e ALL. However, a l l myeloid progenitors studied i n remission were P h 1 - p o s i t i v e . According to conventional concepts, i n ALL the i n i t i a l transformation occurs i n a c e l l r e s t r i c t e d to lymphoid d i f f e r e n t i a t i o n . The f i n d i n g of the Ph 1 i n c e l l s committed to the myeloid lineage, as well as the observation of lymphoblasts i n 1 marrow and blood suggest that t h i s patient was more l i k e l y a case of Ph -p o s i t i v e CML that presented only a f t e r lymphoblastic transformation had occurred. Case 5 thus belongs i n the same group as Cases 2,3 and 4 i l l u s t r a t e the persistence of the P h 1 - p o s i t i v e clone even a f t e r acute transformation has occurred. Case 6 i s possibly s i m i l a r to Case 2 i n which the second event occurred much e a r l i e r than usual i n the course of c l o n a l expansion. The exclusive f i n d i n g of P h 1 - p o s i t i v e e r y t h r o i d and granulopoietic progenitors i n f i r s t remission suggests that the malignant clone was p l u r i p o t e n t i a l ( i . e . , capable of both lymphoid and myeloid d i f f e r e n t i a t i o n ) as i s the case i n CML 1 presenting i n lymphoblastic c r i s i s . I f t h i s i s i n f a c t the case, then the Ph -p o s i t i v e clone should eventually predominate a l l l e v e l s of hemopoiesis. At the time of w r i t i n g , t h i s had not yet occurred and future r e s u l t s are anxiously 150 awaited. The observations suggest that, i n at l e a s t c e r t a i n cases of CML, the transformed stem c e l l l i n e can give r i s e to progeny that undergo b l a s t i c trans-formation before c l o n a l expansion has progressed very f a r . As a r e s u l t , the maturational block that characterizes b l a s t c r i s i s occurs i n a minor population of leukemic c e l l s at a much e a r l i e r than usual stage of disease. I t i s conceivable that i n these cases, the f i r s t round of chemotherapy would e s s e n t i a l l y eradicate the premature b l a s t c e l l population so that the patient would be c l i n i c a l l y returned to a stage of very early CML, i . e . , before the 1 Ph - p o s i t i v e clone was able to dominate the hemopoietic system. Under such a circumstance, the proportion of P h 1 - p o s i t i v e progenitors should slowly increase with disease progression. F a i l u r e of B l a s t C e l l Progenitors to D i f f e r e n t i a t e Normally In V i t r o The findings i n t h i s Chapter demonstrate the a p p l i c a b i l i t y of the human hemopoietic stem c e l l culture system to study the l e v e l at which the chromosomal mutations commonly seen i n the acute phase of CML a f f e c t d i f f e r e n t i a t i o n . I t i s now possible to address c e r t a i n basic questions: Are a l l progenitor c e l l s that p r o l i f e r a t e i n vivo capable of d i f f e r e n t i a t i o n i n culture? In Cases 1,2 and 3, cytogenetic analysis of d i r e c t bone marrow c l e a r l y showed the presence of p r o l i f e r a t i n g c e l l l i n e s i n addition to the one bearing the Ph 1. In Case 1, only one of the 3 a d d i t i o n a l c e l l l i n e s gave r i s e to colonies i n c u l t u r e . In Case 2, the c e l l l i n e bearing chromosomal abnormalities i n addition to the Ph 1 gave r i s e to grossly abnormal colonies. In Case 3, numerous a d d i t i o n a l c e l l l i n e s were observed i n d i r e c t marrow preparations as the disease progressed, however, i n no instance did any of 151 these give r i s e to any type of recognizable erythroid or granulocyte colonies. I t could be argued that i n t h i s l a t t e r case, the c e l l l i n e s d i d not form colonies _in v i t r o because they were committed to lymphoid or some other pathway of d i f f e r e n t i a t i o n . The f a c t that t h i s patient underwent a lymphoblastic transformation supports t h i s hypothesis. The f i n d i n g suggests that c e r t a i n mutant progenitor c e l l s , while f u l l y competent of p r o l i f e r a t i o n i n vivo, are unable to d i f f e r e n t i a t e in v i t r o . Is there any phenotypic difference between colonies derived from normal and abnormal c e l l s ? In Cases 1 and 2, progenitor c e l l s bearing various degrees of chromosomal mutation gave r i s e to e r y t h r o i d and granulocyte colonies that were c l e a r l y abnormal. The data suggest that c e r t a i n mutations are compatible with normal d i f f e r e n t i a t i o n while others are not. SUMMARY Individual hemopoietic colonies generated i n methylcellulose cultures from blood and marrow progenitors were cyt o g e n e t i c a l l y analyzed i n an attempt to determine i f stem c e l l s not involved i n the transformed clone p e r s i s t i n the 1 acute phase of CML. Four p a t i e n t s i n the acute phase of Ph - p o s i t i v e CML were studied and i n a l l cases, there was exclusive persistence of the P h 1 - p o s i t i v e stem l i n e during the acute phase. Because of the extensive chemotherapy that these patients had undergone p r i o r to study, i t was impossible to determine i f normal progenitors were present and d i l u t e d or suppressed by the leukemic clone. Two patients with a t y p i c a l P h 1 - p o s i t i v e acute leukemia were also studied. In both cases, s i g n i f i c a n t numbers of Ph^-negative progenitors were detected. This r e s u l t demonstrates the a p p l i c a b i l i t y of the hemopoietic colony assay to 152 the delin e a t i o n of the extent to which the transformed clone i s involved i n the hemopoietic system i n cases of a t y p i c a l acute leukemia. In the course of t h i s work, three patients were studied i n the acute phase of t h e i r disease. At such times, bone marrow c e l l l i n e s were characterized by chromosomal abnormalities i n addition to the Ph . I t was thus possible to use these gross chromosomal abnormalities as a re p e r t o i r e of genetic mutations. I t was possible to demonstrate mutations that (a) have no e f f e c t on myeloid d i f f e r e n t i a t i o n , (b) have d r a s t i c e f f e c t on myeloid d i f f e r e n t i a t i o n , and (c) are completely incompatible with myeloid d i f f e r e n t i a t i o n . This observation demonstrates that the i n v i t r o clonogenic assays may be further used to study the maturational blocks that frequently accompany acute transformation. I t may indeed be possible to delineate myeloid b l a s t c r i s i s i n a manner analogous to lymphoid b l a s t c r i s e s , using well characterized steps i n i n v i t r o d i f f e r e n t i a t i o n as markers of myeloid precursors. 153 REFERENCES 1. K o e f f l e r PH, Golde DW: Chronic myelogenous leukemia—new concepts. New Eng J Med 304: 1201-1209 & 1269-1274, 1981. 2. Mitelman F, Leuan G: C l u s t e r i n g of aberrations to s p e c i f i c chromosomes i n human neoplasms. I I I . A survey of 287 neoplasms. Hereditas 82: 162-174, 1976. 3. Rowley JD: Chromosomes i n leukemia and lymphoma. Sem Hematol 15: 301-319, 1978. 4. Whang-Peng J , Canellos GP, Carbone PP, T j i o JH: C l i n i c a l implications of cytogenetic variants i n chronic myelocytic leukemia (CML). Blood 32: 755-766, 1968. 5. Rowley JD: Ph^-positive leukemia, i n c l u d i n g chronic myelogenous leukemia. C l i n i c s i n Hematol 9: 55-86, 1980. 6. Boggs DR: Hematopoietic stem c e l l theory i n r e l a t i o n to possible lympho-b l a s t i c conversion of chronic myeloid leukemia. Blood 44: 449-453, 1974. 7. Janossy G, Woodruff RK, Paxton A, Greaves MF, Capellaro D, Kirk B, Innes EM, Eden OB, Lewis C, Ca^ovsky D, Hoffbrand AV: Membrane marker and c e l l separation studies i n Ph - p o s i t i v e leukemia. Blood 51: 861-877, 1978. 8. Bakhshi A, Minowada J , Arnold A, Crossman J , Jensen JP, Whang-Peng J , Waldman TA, Korsmeyer SJ: Lymphoid b l a s t c r i s i s of chronic leukemia represent stages i n the development of B - c e l l precursors. N Eng J Med 309: 826-831, 1983. 1 9. Pedersen B: Influence of hyperdiploidy on Ph prevalence response to therapy i n chronic myelogenous leukaemia. Br J Haematol 14: 507-512, 1968. 10. Cannellos GP, DeVita VT, Whang-Peng J , Carbone PP: Hematologic and cyto-genetic remission of b l a s t i c transformation of chronic granulocytic leukemia. Blood 38: 671-679, 1971. 11. Stern R, Sorenson J , Wurster-Hill DH, Cornwell GG, Mclntyre OR: Chromosome changes i n a patient achieving complete remission i n the acute phase of chronic myelogenous leukemia. Am J Hematol 6: 155-161, 1979. 12. Sandberg AA: The Chromosomes i n Human Cancer and Leukemia. E l s e v i e r North Holland Inc., New York, p. 748, 1980. 1 1 13. Sokal JE: S i g n i f i c a n c e of Ph -negative marrow c e l l s i n Ph - p o s i t i v e chronic granulocytic leukemia. Blood 56: 1072-1076, 1980. 14. Zaccaria A,^Ruggero D, Guolo ML, Guarini A, Frezza R, Baccarani M, Tura S. Ph - p o s i t i v e chronic myeloid leukemia presenting i n b l a s t i c 154 c r i s i s : Report of 12 cases. B o l l Inst Sieroter Milan 57: 383-391, 1978. 15. Moore MAS, Metcalf D: Cytogenetic analysis of human acute and chronic myeloid leukemic c e l l s cloned i n agar culture. Int J Cancer 11: 143-152, 1973 . 16. Eaves CJ, Eaves AC: Erythroid progenitor c e l l numbers i n human marrow— Implication f o r regula t i o n . Exp Hematol 7 (Suppl 5): 54-64, 1979. 17. Dube' ID, Eaves CJ, Kalousek DK, Eaves AC: A method f o r obtaining high q u a l i t y chromosome preparations from single hemopoietic colonies on a routine b a s i s . Cancer Genet Cytogenet 4: 157-168, 1981. 18. Dube ID, Gupta CM, Kalousek DK, Eaves CJ, Eaves AC: Cytogenetic studies of e arly myeloid progenitor compartments i n Ph - p o s i t i v e chronic myeloid leukemia (CML). I. Persistence of Ph -negative committed progenitors that are suppressed from d i f f e r e n t i a t i n g i n vivo. Br J Haematol (in press). 19. Hook EB: Exclusion of chromosomal mosaicism. Tables of 90%, 95% and 99% confidence l i m i t s and comments on use. Am J Hum Genet 29: 94-97, 1977. 20. Goldman JM, Lu DP: New approaches i n chronic granulocytic leukemia— o r i g i n , prognosis and treatment. Sem Hematol 19: 241-256, 1982. 21. Nowell PC: The c l o n a l evolution of tumor c e l l populations. Science 194: 23-28, 1976. 22. Fialkow PJ: Clonal and stem c e l l o r i g i n of blood c e l l neoplasms. In: Contemporary Hematology/Oncology (J Lobue, AS Gordon, R S i l b e r , FM Muggia, eds), Plenum, New York, p 1, 1980. 155 C H A P T E R S I X SUMMARY AND CONCLUSIONS Chronic myeloid leukemia (CML), l i k e most malignancies, i s a c l o n a l 1 neoplasm, t r a c i n g i t s o r i g i n to a sing l e transformed c e l l . In i t i s exemplified the h i e r a r c h i c a l structure t y p i c a l of many malignancies i n which c e l l s of l i m i t e d p r o l i f e r a t i v e p o t e n t i a l are produced by an expanding stem 2 c e l l population. Considerable evidence indicates that i n CML, the c e l l that i s i n i t i a l l y transformed i s a member of the normal pluripotent stem c e l l compartment of the blood-forming system. During the chronic phase of the disease, t h i s c e l l p r o l i f e r a t e s extensively, while r e t a i n i n g i t s capacity f o r d i f f e r e n t i a t i o n i n t o f u n c t i o n a l l y and morphologically diverse blood c e l l s . As a r e s u l t , by the time of diagnosis, a l l of the c i r c u l a t i n g red c e l l s , granulocytes, monocytes and p l a t e l e t s are progeny of the neoplastic clone, 1 as may also be 3 the case f o r some B lymphocytes. The morphologically unmarked neoplastic members of the stem c e l l compartment, l i k e t h e i r normal counterparts, are thus greatly d i l u t e d by t h e i r terminally d i f f e r e n t i a t i n g progeny. Information about changes i n the s i z e and composition of the stem c e l l compartment i s important, both f o r the development of new treatment s t r a t e g i e s , and as an approach to the analysis of mechanisms leading to c l o n a l dominance. However, the a c q u i s i t i o n of such information requires the use of methods that allow both normal and neoplastic stem c e l l s to be detected and distinguished. Methods f or quantitating normal p r i m i t i v e hemopoietic c e l l s based on t h e i r a b i l i t y to generate colonies of recognizable blood c e l l s are 4 av a i l a b l e , and a long-term marrow culture system i n which such p r i m i t i v e 156 c e l l s can be maintained f o r many weeks has a l s o been recently developed.^ The Philadelphia (Ph 1) chromosome provides a consistent marker of the neoplasic clone i n most patients.^ In t h i s t h e s i s , e f f o r t s were focused on the use of cytogenetic analysis of hemopoietic colonies to evaluate the stem c e l l compartment i n t h i s disease. A technique for obtaining analyzable metaphases i n high y i e l d from i n d i v i d u a l colonies was developed, and then used to 'determine the cytogenetic composition of colony-forming progenitors present i n fresh marrow or blood samples, or i n long-term marrow cultures maintained for various periods of time. Chronic Phase Studies In the present studies, hemopoietic progenitors from 19 P h 1 - p o s i t i v e patients i n t y p i c a l chronic phase were cultured i n methylcellulose and t h e i r c l o n a l progeny c y t o g e n e t i c a l l y analyzed. Four of these patients were studied twice and 2 were studied three times, giving a t o t a l of 27 separate inves-t i g a t i o n s . Of these, 10 were c a r r i e d out using specimens from patients studied at diagnosis and p r i o r to the i n i t i a t i o n of chemotherapy. In 5 cases, hemopoietic colonies analyzed c y t o g e n e t i c a l l y were derived from progenitors i n marrow only, i n 11 cases they were cultured from blood progenitors only, and i n 11 cases both marrow and blood derived progenitors were assayed. Ten marrow aspirates c o l l e c t e d on d i f f e r e n t occasions from 9 chronic phase patients were used to i n i t i a t e long-term cultures that were maintained with weekly feeding for 2-8 weeks before hemopoietic progenitors i n the adherent c e l l f r a c t i o n were removed and stimulated to form colonies i n methylcellulose assays. These were, i n turn, cytogenetically analyzed. Six of the 10 specimens were from patients with previously diagnosed, treated CML. In a l l cases, only colonies y i e l d i n g at l e a s t two G-banded metaphases were included i n the data. 157 A l l patients studied i n the chronic phase of t h e i r disease were exc l u s i v e l y P h 1 - p o s i t i v e i n d i r e c t preparations. Ph 1-negative progenitor c e l l s were detected among blood and marrow progenitors i n 7 patients. A l l were newly diagnosed, untreated p a t i e n t s . The incidence of Ph^-negative c e l l s ranged from 5% to 100%. As a group, these patients were among the most recently diagnosed with lower than average white blood c e l l counts (<31,000 cells/mm^). Chromosomally normal hemopoietic progenitors were r e a d i l y detected i n the adherent layers of long-term marrow cultures established from 7 of the 10 specimens. In 2 of the remaining 3, only Ph^-positive progenitors were detected. In the remaining case, s u f f i c i e n t hemopoietic colonies f o r cytogenetic analysis were not obtained. Four of the 7 specimens showing chromosomally normal progenitors were from patients with newly diagnosed CML, but 3 were from patients studied 5-14 months postdiagnosis and i n i t i a t i o n of chemotherapy. In 4 of the 7 cases, chromosomally normal progenitors were not detected i n methylcellulose assays of the marrow used to i n i t i a t e the long-term cultures. In long-term cultures established from a l l of the 7 marrow specimens, the Ph^-positive clone declined r a p i d l y so that by 4-6 weeks, 100% of progenitors were chromosomally normal. In 1 of the 2 long-term cultures where Ph 1-negative progenitors were not detected, the Ph^-positive clone also declined r a p i d l y . However, i n the other c u l t u r e , the number of P h 1 - p o s i t i v e progenitors present a f t e r 3 and 6 weeks remained high, showing k i n e t i c s s i m i l a r to that exclusive to the Ph^-negative population i n other cultures. The combined i n vitr o - c y t o g e n e t i c approach used here reveals a number of i n t e r e s t i n g findings: ( 1) Chromosomally normal progenitors were detected i n methylcellulose 158 assays only when the specimens were obtained from patients at or soon a f t e r diagnosis with r e l a t i v e l y low white blood c e l l counts. These were probably cases i n which the leukemic clone had not yet expanded to such a degree so as to preclude the detection of a normal-sized population of non-neoplastic progenitors, based on the analysis of r e a l i s t i c numbers of colonies. This suggests that i n most patients i n the chronic phase of t h e i r disease, the leukemic clone greatly d i l u t e s the r e s i d u a l normal hemopoietic c e l l population, not only at the l e v e l of the terminally d i f f e r e n t i a t e d c e l l s , but also among the c e l l s of the more p r i m i t i v e myeloid progenitor c e l l compartments. (2) The long-term culture assay revealed the presence of s i g n i f i c a n t numbers of chromosomally normal progenitors i n the majority of specimens from both newly diagnosed patients and patients with well-established, treated CML. This observation suppports the hypothesis that non-neoplastic hemopoietic stem c e l l s p e r s i s t i n the marrows of most patients with CML for considerable periods of time. (3) The r a p i d decline of the Ph^-positive clone i n most long-term CML marrow cultures suggests that differences e x i s t between Ph^-positive and 1 Ph -negative stem c e l l responses during the development of the adherent layer. I t i s also p o s s i b l e that, as a r e s u l t of c e r t a i n as yet undefined properties, Ph 1-negative stem c e l l s may p r e f e r e n t i a l l y become incorporated i n t o the adherent c e l l layer. Nevertheless, the f a i l u r e of the P h 1 - p o s i t i v e progenitor population to decline r a p i d l y i n one long-term culture a l s o suggests that the d i f f e r e n t i a l behaviour usually exhibited by P h 1 - p o s i t i v e c e l l s i s not an 1 i n t r i n s i c a l l y f i x e d property of the Ph - p o s i t i v e genotype. (4) The f a c t that Ph 1-negative progenitors were not detected i n long-term cultures from 1 patient even though i n t h i s patient, the 159 P h 1 - p o s i t i v e population d i d r a p i d l y decline, suggests that heterogeneity e x i s t s among patients with respect to the a b i l i t y of t h e i r r e s i d u a l 1 Ph -negative stem c e l l s to p e r s i s t i n vivo. On the other hand, the p o s s i b i l i t y that such c e l l s were simply d i l u t e d to undetectable l e v e l s i n the p a r t i c u l a r marrow samples used to i n i t i a t e t h i s culture cannot be r u l e d out. (5) The r e s u l t s of the short-term methylcellulose assays for progenitors i n blood and marrow provide evidence f o r the suppression of normal stem c e l l p r o l i f e r a t i o n i n vivo. In 9 cases, chromosomally normal c e l l s were detected at s i g n i f i c a n t l y higher l e v e l s among myeloid progenitors than among marrow metaphases. In a l l cases, s u f f i c i e n t numbers of marrow metaphases were analyzed to permit the detection of the progeny of these chromosomally normal progenitors i f they had been p r o l i f e r a t i n g i n vivo. Acute Phase Studies To in v e s t i g a t e cytogenetic changes that accompany acute transformation i n CML and the e f f e c t s they have at the myeloid progenitor c e l l l e v e l , 4 patients i n the acute phase of CML were studied as well as 2 patients with a t y p i c a l 1 Ph - p o s i t i v e acute leukemia. A t o t a l of 13 marrow and 12 blood specimens were inv e s t i g a t e d on 16 d i f f e r e n t occasions. Four of the 6 patients studied i n the acute phase showed only Ph^-positive progenitors. In the other cases, s i g n i f i c a n t numbers of Ph 1-negative progenitors were detected. I t i s possible that these l a t t e r patients entered the b l a s t i c transformation stage before the neoplastic clone had expanded to the l e v e l normally seen. Three of the patients were studied at times when marrow c e l l l i n e s 1 existed with multiple chromosome abnormalities i n addition to the Ph . Using gross chromosomal abnormalities as a re p e r t o i r e of genetic mutations, i t was possible to demonstrate mutations that (a) have no observable e f f e c t on 160 myeloid d i f f e r e n t i a t i o n , (b) have d r a s t i c e f f e c t on myeloid d i f f e r e n t i a t i o n , and (c) are completely incompatible with myeloid d i f f e r e n t i a t i o n assayed i n  v i t r o . CONCLUSION Perhaps the most s i g n i f i c a n t f i n d i n g here i s the demonstration of chromosomally normal myeloid progenitors i n most patients i n the chronic phase of CML. To what extent t h i s information may be u t i l i z e d i n the future manage-ment of t h i s disease remains to be seen. The long-term culture r e s u l t s suggest that normal and leukemic stem c e l l s may display d i f f e r e n t s u r v i v a l patterns under p a r t i c u l a r conditions. The r e s u l t s obtained i n the course of t h i s work r a i s e many new questions and suggest further experiments designed to investigate tumorigenesis i n CML. For example, although the findings are consistent with the persistence of r e s i d u a l normal stem c e l l s at normal or near normal l e v e l s i n patients i n the chronic phase of t h e i r disease, the p o s s i b i l i t y e x i s t s that some of the Ph^-negative progenitors detected were not b i o l o g i c a l l y normal. Malignant 7 8 transformation i s thought to involve at l e a s t 2 changes i n the genome ' and evidence f o r Ph 1-negative but c l o n a l B lymphocytes i n a si n g l e patient has 9 1 been reported. On the other hand, persistence of Ph -negative, nonclonal myeloid stem c e l l s i n at l e a s t once CML patient has also been documented. 1^ C l e a r l y , information from patients with CML where nonclonality can be confirmed by cytogenetic or biochemical studies w i l l be required to e s t a b l i s h whether c l o n a l expansion i n the myeloid stem c e l l compartment can precede 1 a c q u i s i t i o n of the Ph -chromosome. Future experiments designed to analyze the basis of the growth k i n e t i c differences between P h 1 - p o s i t i v e and Ph 1-negative stem c e l l s i n long-term 161 marrow cultures may f a c i l i t a t e the development of new therapeutic protocols designed to eradicate the leukemic clone more s p e c i f i c a l l y and permit re-establishment of normal hemopoiesis. I f t h i s can be done, a disease that has remained incurable may f i n a l l y be conquered. 162 REFERENCES 1 . Fialkow PJ: C e l l lineages i n hematopoietic neoplasia studied with glucose-6-phosphate dehydrogenase c e l l markers. J C e l l P hysiol (Suppl 1) : 37-43, 1982. 2. Salmon SE. Cloning of Human Tumor Stem C e l l s . Alan R L i s s Inc., New York, 357 pages, 1980. 3. Bernheim A, Berger R, Preud'homme JL, Labaume S, Bussel A, Barot-Ciorbaru R: Philadelphia chromosome p o s i t i v e blood B lymphocytes i n chronic myelocytic leukemia. Leuk Res 5: 331-339, 1981. 4. Eaves CJ, Eaves AC: Erythropoiesis. In: Hematopoietic Stem C e l l s , (DW Golde & F Takaku, eds), M Dekker Inc, New York (in press). 5. Coulombel L, Eaves AC, Eaves CJ: Enzymatic treatment of longterm human marrow cultures reveals the p r e f e r e n t i a l l o c a t i o n of p r i m i t i v e hemopoietic progenitors i n the adherent layer. Blood 62: 291-297, 1983. 6. Shaw MT. Chronic Granulocytic Leukemia, Praeger S c i e n t i f i c , New York, 251 pages, 1981. 7. Nowell P: The c l o n a l evolution of tumor c e l l populations. Science 194: 23-28, 1976. 8. Neiman PE, Payne LN, Jordon L, Weiss RA: Malignant lymphoma of the bursa of f a b r i c i u s - a n a l y s i s of early transformation. Cold Spring Harbor Conference on C e l l P r o l i f e r a t i o n 7: 519-528, 1980. 9. Fialkow PJ, Martin PJ, N a j f e l d V, Penfold GK, Jacobsen RJ, Hansen JA: Evidence for a multi-step pathogenesis of chronic myelogenous leukemia. Blood 58: 158-163, 1981. 10. Singer JW, A r l i n ZA, Najfeld V, Adamson JW, Kempin SJ, Clarkson BD, Fialkow PJ: Restoration of non-clonal hematopoiesis i n chronic myelogenous leukemia (CML) following a chemotherapy-induced loss of the 1 Ph chromosome. Blood 56: 356-360, -1980, 163 Appendix IA Procedure f o r the Preparation of Hemopoietic Stem C e l l s f o r P l a t i n g from Fresh  Marrow Aspirate A l l manipulations of human c e l l s were c a r r i e d out i n a laminar flow Biogard hood under s t e r i l e conditions. A l l apparatus that came i n t o d i r e c t contact with human c e l l s were autoclaved before they were discarded. Procedure: 1. Measure the t o t a l volume of marrow using a s t e r i l e c a l i b r a t e d p i p e t t e . 2. Remove 0.1 ml of aspirate and use t h i s to do a s t a r t i n g nucleated c e l l count on the hemocytometer (magnification: 100X). 3. Using a s t e r i l e Pasteur pipette, t r a n s f e r the e n t i r e sample to a 17 x 100 mm p l a s t i c tube (Falcon 2057) and spin the specimen at 1 ,00 0 rpm for 4 minutes at room temperature (LEC HN-S centrifuge). 4. Remove the buffy coat with a s t e r i l e Pasteur p i p e t t e and t r a n s f e r to another Falcon 2057 tube. 5. Let the tube stand at room temperature f o r 10-20 minutes to f a c i l i t a t e sedimentation of r e s i d u a l red c e l l s . 6. Transfer the e n t i r e contents of the tube, except f o r the red c e l l button, to another Falcon 2057 tube, and add enough 2% f e t a l c a l f serum i n alpha medium (2% FCS) to bring the volume up to 10 ml. 7. Spin the specimen at 950 rpm for 10 minutes at 4 -C (S o r v a l l RC-3 automatic, r e f r i g e r a t e d c e n t r i f u g e ) . 8. Pour o f f the supernatant and resuspend i n 2% FCS to give a volume of 10 ml. 9. Centrifuge as i n step 7. 10. Pour o f f the supernatant and resuspend the specimen i n 2% FCS to give a f i n a l volume of 3 ml. 11. Remove 0.1 ml of the c e l l suspension and use t h i s to do a f i n a l c e l l count on the hemocytometer. 6 12. D i l u t e the c e l l s i n 2% FCS ( f i n a l concentration of 2 x 10 c e l l s / m l ) . 164 Appendix IB Procedure f o r the Preparation of Hemopoietic Stem C e l l s f o r P l a t i n g from  Peripheral Blood. A l l manipulations of human c e l l s were c a r r i e d out i n a laminar flow Biogard hood under s t e r i l e conditions. A l l apparatus that came i n t o d i r e c t contact with human c e l l s were autoclaved before they were discarded. Procedure: 1. Measure the t o t a l volume of blood using a s t e r i l e c a l i b r a t e d p i p e t t e . 2. Remove 0.1 ml of blood and use t h i s to do a s t a r t i n g nucleated c e l l count on the hemocytometer (magnification: 100X). 3. Gently layer 10 ml of blood on top of 15 ml of lymphocyte separation medium (Bionetics 8410-01) i n a s t e r i l e 50 ml co n i c a l tube (Falcon 2070). 4. Centrifuge the specimen at 1,700 rpm for 30 minutes at room temperature (LEC HN-S c e n t r i f u g e ) . 5. Using a Pasteur p i p e t t e , c a r e f u l l y aspirate o f f and discard the plasma layer down to 2mm above the lymphocyte l a t e r . 6. Transfer the lymphocyte layer (3-5 ml) to a Falcon 2057 tube and add 2% FCS to give a f i n a l volume of 10 ml. 7. Spin the specimen at 950 rpm for 10 minutes at 4*C (So r v a l l RC-3 automatic, r e f r i g e r a t e d c e n t r i f u g e ) . 8. Pour o f f the supernatant and resuspend the p e l l e t i n 10 ml 2% FCS. 9. Spin as i n step 7. 10. Pour o f f the supernatant and resuspend the p e l l e t i n 1 ml of 2% FCS. 11. Remove 0.1 ml of the c e l l suspension and use t h i s to do a f i n a l c e l l count on the hemocytometer. 12. D i l u t e the c e l l s i n 2% FCS to give a f i n a l c e l l concentration of 4 x 10^ c e l l s / m l . 165 Appendix IIA Procedure for the Preparation of the Culture Medium f o r Hemopoietic Stem C e l l s Medium was made up i n batches of 102 ml. Medium was frozen i n 17 x 100 mm Falcon 2057 s t e r i l e , disposable p l a s t i c tubes. 2.7 ml of medium was aliquoted per tube. For p l a t i n g , the tubes were thawed and 0.3 ml of the f i n a l c e l l suspension was added to each tube. A f t e r mixing by vortex, 1.1 ml of the medium containing c e l l s were plated per 35 x 10 mm standard dish (Lux 5221-R) using p l a t i n g needles (Monojeck 202, 15 gauge, 1.5", blunt, on standard 3 cc syringes). To prepare 102 ml of culture medium: * 40 ml 2.2 ml methylcellulose (Dow Chemicals) 1 ml L-g^utamine, 29.2 mg/ml (General Biochemicals 10510) 1 ml 10 M mercaptoethanol (Baker Chemical Co. 08865) 30 ml f e t a l c a l f serum (one l o t used throughout) 10 ml bovine serum albumin (Sigma A 4503) 10 ml human leukocyte conditioned medium 10 ml erythropoietin at 27.5 units/ml (epo, Step I I I , Connaught Laboratories) * 2.2% methylcellulose was made i n batches of 2 l i t e r s according to the routine protocol used i n the laboratory of Dr. C.J. Eaves (See Appendix Prepared according to the procedures ou t l i n e d i n Appendix IIC. Procedure: 1. Mix 22 grams of Dow standard grade methylcellulose with 22 grams of Dow premium grade methylcellulose. Autoclave. 2. Add the mixture of methylcellulose grades to 1 l i t e r of autoclaved double d i s t i l l e d water at 80"c i n a 2 - l i t e r Erlenmeyer f l a s k . Use continuous s t i r r i n g by magnetic s t i r r e r while adding the powder. 3. B o i l the mixture f r o 1 minute and l e t cool to room temperature. 4. Add 1 l i t e r of 2 x alpha-medium at 37*C (Connaught Laboratories). 5. S t i r at 4*C overnight to complete the c l a r i f i c a t i o n process. 6. Dispense 40 ml aliquots and l e t stand at 20"c f o r 2 weeks as a s t e r i l i t y check. 7. Freeze the a l i q u o t s f or 24 hours, then thaw i n the r e f r i g e r a t o r . Store at 4*C. 166 Appendix IIB Preparation of Methylcellulose Methylcellulose 4000 cps (Dow Chemicals) 2.3%, 2 l i t e r s . 1. Weigh out 23 grams each of Dow standard grade and Dow premium grade. 2. Mix, autoclave i n a nalgene beaker covered with f o i l f o r 15 min on dry time. Store i n drying cabinet. 3. Pour 1 l i t e r double d i s t i l l e d water i n t o s p e c i a l double arm 2 l i t e r Erlenmeyer f l a s k containing a large s t i r bar. Close side arms with small stoppers and cover the mouth of the f l a s k with f o i l . Autoclave on l i q u i d c ycle. 4. When water i s s t i l l hot from autoclave, place f l a s k on s t i r r e r / h o t p late and add methylcellulose through funnel. 5. S t i r continuously and slow increase the heat so that f l a s k i s brought to a low b o i l for 1 minute . 6. S t i r u n t i l methylcellulose has cooled (usually about 1 hour). Add 1 l i t e r of prewarmed 2 x alpha medium and s t i r f o r 10-20 minutes. Stopper f l a s k . 7. S t i r at 4*C overnight. 8. Dispense i n t o 100 ml Brockway b o t t l e s i n 40 ml a l i q u o t s . 9. Let b o t t l e s stand at room temperature f o r about 2 weeks as a s t e r i l i t y check. 10. Freeze e n t i r e batch to c l a r i f y f u r ther. 11. Store at 4 " c . 167 Appendix IIC Preparation of Standard PHA Conditioned Media. 1. C o l l e c t s t e r i l e blood with preservative-free heparin at a f i n a l concentration of 50 u/ml blood, i . e . , 2500 u/50ml blood. 2. Remove al i q u o t f or nucleated c e l l count. 3. Add 5 ml of 1% metyhylcellulose ( i . e . , 2X d i l u t i o n of standard 2% stock i n alpha medium per 50 ml blood) and mix by continuous rocking for 5 minutes. Use 18 guage needle to take up methylcellulose; change to 21 guage to put i n t o syringe. 4. Stand syringe upright on plunger and sediment (rocket method) u n t i l j ust before buffy coat s t a r t s to form (usually about 20-30 min, but can be longer). Record the actual time of sedimentation. 5. Using a b u t t e r f l y needle and keeping syringe upright, push out white c e l l r i c h plasma i n t o a s t e r i l e tube. Take f i r s t few drops o f f i n t o another tube (waste). 6. Spin at 1000 rpm for 10 minutes at room temperature. Pour o f f plasma and resuspend c e l l s i n 2% FCS (4 ml per 50 ml of s t a r t i n g volume of whole blood). For serum free media, wash c e l l s twice with alpha medium and resuspend i n alpha medium. 6 7. Count and d i l u t e c e l l s to 5 x 10 c e l l s / m l . 8. Make up a mixture of PHA, FCS, 2-mercaptoethanol so that these ingredients are 5X the f i n a l desired concentration, e.g., i n the following r a t i o s : 10 ml FCS, 1 ml 10 M 2-mercaptoethanol, 0.5 ml re c o n s t i t u t e d PHA and 8.5 ml of alpha media. 9. Mix c e l l s with the PHA mixture i n a r a t i o of 4 parts c e l l s : 1 part mixture. 2 10. Put i n t o t i s s u e culture f l a s k s , 30 ml t o t a l volume per 75 cm f l a s k . (24 ml c e l l s + 6 ml PHA mixture.) 11. Incubate at 37*C i n 5% CO for 1 week. 2 12. Harvest by p i p e t t i n g o f f mixture and put i n t o s t e r i l e centrifuge tubes. 13. Centrifuge at 300Xg and pour or p i p e t t e supernatant i n t o s t e r i l e b o t t l e s for storage. 168 Appendix I II Chromosome Banding Procedures. A l l s l i d e s used f o r banding were a i r dryed and aged f o r at l e a s t 2 days. A l l procedures were c a r r i e d out at room temperature. A) Q-Banding. 1 1. Immerse s l i d e i n 100% ethanol f o r 2 minutes. 2. Immerse s l i d e i n 70% ehtanol f o r 2 minutes. 3. Immerse s l i d e i n 50 % ethanol f o r 2 minutes. 4. Immerse s l i d e i n 20% ethanol f o r 2 minutes. 5. Immerse s l i d e i n Sorensen's buffer pH 6.8 f o r 5 minutes. 6. Immerse s l i d e i n 0.5% (w/v) quinacrine dihydrochloride (Sigma Q-0250) f o r 2 0 minutes. 7. Dip s l i d e i n Sorensen's buffer pH 6.8. 8. Repeat step 7 i n fresh buffer. 9. Immerse s l i d e i n fresh buffer f o r 5 minutes. 10. Remove s l i d e and b l o t dry. 11. Mount s l i d e i n buffer. 12. Seal edges of c o v e r s l i p with rubber cement and allow to dry i n subdued l i g h t . 1 Caspersson, T •, Zech, L., Johanssen, C. D i f f e r e n t i a l binding of a l k y l a t i n g fluorochromes i n human chromosomes. Exp. C e l l . Res. 60_, 315-319, 1970. 169 B) G Banding Trypsin solution: +Rehydrate stock t y r p s i n (Bacto 0153-61) with 10 ml Hanks Ca and Mg -free balanced s a l t s o l u t i o n . Dilute 1-5 ml i n 60 ml of 0.9% (w/v) NaCl. 1. Dip s l i d e i n t r y p s i n s o l u t i o n f o r 5-90 seconds. 2. Dip s l i d e i n 1% C a C l 2 (w/v) b r i e f l y . 3. Immerse s l i d e i n 1% caCl^ f or 60 seconds. 4. Rinse s l i d e b r i e f l y i n d i s t i l l e d water. 5. Repeat step 4 i n f r e s h d i s t i l l e d water. 6. Stain s l i d e i n 4% Giemsa (Gurr 35086) i n Sorensen's bu f f e r pH 6.8 f o r 3 minutes. 7. Rinse s l i d e b r i e f l y i n d i s t i l l e d water. 8. Repeat step 7. 9. Blot s l i d e dry. 10. Coverslip. Note: Times i n t r y p s i n and Giemsa are variable and t r i a l and error should be used to determine the optimum times for best banding. 1 Seabright, M. A r a p i d banding technique for human chromosomes. Lancet i i , 971-972, 1971. 170 Reverse-fluorescent Banding. ' Stock methyl green s o l u t i o n : 0.11 grams methyl green (BDH Chemicals 34052) i n Sorensen's buffer pH 6.8. Chromomycin-A3 b u f f e r : 0.15 M NaCl, 0.0025 M MgCl , 0.03 M KCl, 0.01M Na 2HP0 4. Adjust to pH 7.0 _ 4 Chromomycin-A3 s t a i n i n g s o l u t i o n : Dissolve 5.0 X 10 grams chromomycin-A3 (Sigma C 1637) i n above buffer. 1. Mount s l i d e i n chromomycin-A3 s t a i n i n g s o l u t i o n and leave i n dark f or 15 minutes to 2 hours. 2. Rinse o f f c o v e r s l i p i n d i s t i l l e d water. 3. Immerse s l i d e i n 4% methyl green i n Sorensen's buffer pH 6.8 f o r 10 minutes. 4. Rinse s l i d e i n d i s t i l l e d water. 5. Mount s l i d e i n 100% g l y c e r o l . Sahar, E., L a t t , S.A. Enhancement of banding patterns i n human metaphase chromosomes by energy t r a n s f e r . Proc. N a t l . Acad. S c i . USA 75: 5650-5654, 1978. Chamberlain, J . Personal communication. 171 Appendix IV Procedures f o r obtaining bone-marrow metaphases: A) THC method. 1 1. Add 0.25 ml of fresh marrow to a s t e r i l e test-tube containing 9 ml of 0.075 ml KCl, 0.08 ml Colcemid (Gibco 120-52110), 1 ml t r y p s i n EDTA (Gibco 610-5300). 2. Incubate for 20 minutes at 37*C. 3. Centrifuge at 200Xg f o r 8 minutes. 4. Discard supernatant. 5. F i x with 8 ml 3:1 :: Methanol:glacial a c e t i c a c i d . 6. Centrifuge as i n step 3. 7. Wash with 8 ml of above f i x a t i v e . 8. Centrifuge as above. 9. Repeat steps 7 and 8. 10. Resuspend p e l l e t of c e l l s i n fresh f i x a t i v e and drop onto clean, wet s l i d e s . 1 Hozier, J . C , Lindquist, L.L. Banded karyotypes from bone marrow: A c l i n i c a l u s eful approach. Hum. Genet. S3, 205-209, 1980. 172 B) 24-hour unstimulated marrow method. 1. Add 0.25 ml of fr e s h marrow to 10 ml RPMI culture medium with 15% f e t a l c a l f serum i n a s t e r i l e tube. 2. Incubate overnight at 37*C. 3. Aspirate o f f supernatant. 4. Add p e l l e t of c e l l s to a s t e r i l e test-tube containing 9 ml of 0.075 M KCL, 0.08 ml Colcemid (Gibco 120-5201) and 1 ml t r y p s i n EDTA (Gibco 610-5300). 5. Harvest as i n steps 2-10 i n previous procedure (A). T i j o . J.H., Whang, J . Chromosome preparations of bone marrow c e l l s without p r i o r i n v i t r o culture or i n vivo c o l c h i c i n e administration. Stain Technol. 37, 17-20, 1962. 173 Appendix V 1 Procedure f o r Obtaining Metaphase Chromosomes from Peripheral blood. Chromosome culture medium: 100 ml Dulbecco's Modified Eagle medium (Gibco 188G), 11 ml of f e t a l c a l f serum, 2 ml heparin sodium (1000 usp units/ml) and 1 ml of p e n i c i l l i n - s t r e p t o m y c i n (1000 units p e n i c i l l i n per ml and 10,000 un i t s streptomycin per ml. Difco 5854-59) 1. Add 0.5 ml fresh blood to 5 ml of above medium i n a s t e r i l e tube with a screw cap. 2. Add 0.1 ml of PHA-M form (Gibco R15-0576). 3. Incubate at 37"c f o r for 72 hours. 4. Add 0.1 ml colcemid (Gibco 120-5210). 5. Incubate f o r 1 hour. 6. Centrifuge at 200X g f o r 8 minutes. 7. Discard supernatant. 8. F i x with 8 ml of 3:1 :: Methanol:glacial a c e t i c a c i d . 9. Centrifuge as above. 10. Wash with 8 ml fresh f i x a t i v e . 11. Repeat steps 8 and 9 above two or three times. 12. Resuspend p e l l e t of c e l l s i n fresh f i x a t i v e and drop onto clean, wet, s l i d e s . 1Moorhead, P.S., Nowell, P.C., Mellman, W.J., Battips, D.M., Hungerford, D.A. Chromosome preparations of leukocytes cultured from human peri p h e r a l blood. Exp. C e l l Res. 20, 613-616, 1960. 174 Appendix VI Procedure f o r Obtaining Metaphase Chromosomes from F i b r o b l a s t s . 1. Remove a l l medium from f i b r o b l a s t cultures i n T25 f l a s k s . 2. Rinse c e l l s with 2 ml Hank's balanced s a l t s o l u t i o n . 3. Add 2 ml t r y p s i n s o l u t i o n (1 ml t r y p s i n (Bacto 0155361) to 20 ml Hank's balanced s a l t s o l u t i o n ) . 4. Let s i t for 2 minutes at room temperature. 5. Pour o f f t r y p s i n and incubate culture at 37"c for 15 minutes. 6. Add 10 ml minimum e s s e n t i a l medium with 2% f e t a l c a l f serum. Pipet up and down to break up clumps. 7. Place 1.5 ml of the c e l l suspension on s t e r i l e s l i d e s i n a square p e t r i dish (Falcon 10 12). 8. Incubate c e l l s on s l i d e s f o r 30 minutes. 9. Add 15 ml warm medium and incubate for 24 hours. 10. Change medium and incubate f o r 16 hours. 11. Add 0.2 ug/ml colcemid (Gibco 120-5210) f o r 3 hours. 12. Aspirate o f f medium. Add 15 ml 1% (w/v) sodium c i t r a t e f o r 5 minutes. 13. Add 15 ml cold f i x a t i v e (3:1 :: methanol:glacial a c e t i c acid) f o r 5 minutes. 14. Place s l i d e s i n c o p l i n j a r of fresh f i x a t i v e f o r 5 minutes. 15. Repeat step 14 i n fresh f i x a t i v e . 16. Gently remove s l i d e s , blow gently, flame gently. 17. Slowly dip s l i d e s i n 75% g l a c i a l a c e t i c a c i d i n d i s t i l l e d water for 10-15 seconds. 18. Flame s l i d e s gently. Tap once, flame dry. 175 Appendix VII 1 Procedure f o r D i f f e r e n t i a l Staining of Metaphase Chromosomes. 1. B r i e f l y r i n s e s l i d e s i n d i s t i l l e d water. 2. Immerse s l i d e s i n 0.5 mg/ml Hoechst 33258 i n d i s t i l l e d water f o r 10 minutes. 3. Rinse s l i d e s i n d i s t i l l e d water. 4. Mount s l i d e s i n 0.16 M sodium phosphate/ 0.04 M sodium c i t r a t e buffer pH 7 .0 . 5. Place s l i d e s 35 cm from a 150 watt grow lamp f o r 24 hours. 6. Remove c o v e r s l i p s . 7. Stain i n Giemsa (5% i n Sorensen's buffer pH 6.8). ^Perry, P., Wolff, S. New giemsa method for the d i f f e r e n t i a l s t a i n i n g of s i s t e r chromatids. Nature 251, 156-158, 1974. 176 Appendix VIII Procedures f o r ^ I n i t i a t i o n , Maintenance"and Analysis of Long-term Marrow  Cultures. A) I n i t i a t i o n . Culture medium: Alpha medium (supplemented with 40 mg glutamine/100 ml, 4 mg inositol/100 ml and 1 mg f o l i c ^cid/100 ml) containing ^2.5% f e t a l c a l f serum, 12.5% horse serum, 10 M mercaptoethanol, 10 M hydrocortisone-21 hemisuccinate sodium s a l t . 7 1. Add an al i q u o t of fresh bone marrow c e l l s containing 2.5-3 X 10 c e l l s to the culture medium to give a f i n a l volume of 8 ml i n each 60mm X 15mm ti s s u e culture dish. 2. Mix gently by p i p e t t i n g . 3. Incubate at 37"c i n a humid 5% CO^ i n a i r environment f o r 3-4 days. B) Maintenance. 1. Remove 2-3 ml of media from the dish. 2. Swirl the dish gently to detach red c e l l s from the bottom. 3. Remove the remaining media and the non-adherent c e l l s . 4. Add 7.5 ml new medium to the t i s s u e culture dish. 5. Centrifuge c e l l s at 200X g for 8 minutes. 6. Resuspend c e l l s i n 0.5 ml fresh medium. 7. Add 0.5 ml of c e l l suspension to o r i g i n a l t i s s u e culture dishes. 8. Incubate at 33"C i n a humid 5% CO^ i n a i r environment f o r 3-4 days. C) Weekly Feeding. 1. Remove 2-3 ml of media from the dish. 2. Swirl the dish gently to detach red c e l l s from the bottom. 3. Take out a l l the medium and c e l l s . 4. Return one h a l f of the medium and c e l l s to the dish. 5. Add 4 ml of fresh medium to each dish. 6. Incubate at 33*C as above f o r 7 days. 177 D)Processing of Adherent Layers. 1. Remove a l l the non-adherent c e l l s and a l l the medium. 2. Wash the adherent layer twice with 2 ml of Hank's balanced s a l t s o l u t i o n (HBSS) without calcium and magnesium. 3. Add to the dish 2 ml f e t a l c a l f serum and 8 ml of Collagenase s o l u t i o n (Sigma C-0130, 13mg/10ml HBSS). 4. Incubate at 37*C for 3 hours. 5. Remove a l l the medium and detached c e l l s . 6. Wash the dish with 2-3 ml of HBSS and pool c e l l s with those obtained i n step 5. 7. Centrifuge c e l l s at 200X g f o r 8 minutes. 8. Resuspend p e l l e t and d i l u t e to 1 X 10 c e l l s / m l . 9. Plate c e l l s i n methylcellulose culture media (see Appendix I I ) . 1Coulombel, L., Eaves, A.C., Eaves, C.J. Enzymatic treatment of longterm , human marrow cultures reveals the p r e f e r e n t i a l l o c a t i o n of p r i m i t i v e hemopoietic progenitors i n the adherent l a y e r . Blood 62, 291-297, 1983. 178 Appendix IX Cytogenetic Nomenclature; The cytogenetic nomenclature used throughout t h i s work was the International System f o r Human Cytogenetic Nomenclature (1978) . In the construction of the karyotype the autosomes are numbered from 1 to 22 as nearly as pos s i b l e i n descending order of length. The sex chromosomes are r e f e r r e d to as X and Y. The symbols p and q are used to designate, r e s p e c t i v e l y , the short and long arms of each chromosome. A) Normal Karyotypes. Normal human karyotypes are designated as follows: 46,XX Normal female. 46,XY Normal male. B) Numerical Chromosome Aberrations. The + and - signs are placed before the appropriate symbol to in d i c a t e a d d i t i o n a l or missing whole chromosomes, e.g., 47,XX,+8 47 chromosomes, XX sex chromosomes, an a d d i t i o n a l chromosome #8. 45,XY,-12 45 chromosomes, XY sex chromosomes, a missing chromosome #12. C) Chromosome Mosaics. The chromosome c o n s t i t u t i o n of the d i f f e r e n t c e l l l i n e s i n a mosaic are l i s t e d i n order of the predominant clones. 45, X/46,XY A chromosome mosaic with two c e l l l i n e s , the major one with 45 chromosomes and a s i n g l e X, the other with a normal male karyotype. 45,XX/47,XX,+7 A chromosome mosaic with two c e l l l i n e s , one with normal female karyotype, the other with 47 chromosomes, XX sex chromosomes, and an a d d i t i o n a l chromosome #7. D) Structural Chromosome Aberrations. (a) The + or - signs are placed a f t e r the appropriate symbol to in d i c a t e an increase or decrease i n the length of a chromosome, e.g., 46, XX,11p+ 46 chromosomes, XX sex chromosomes, one chromosome #11 with a d d i t i o n a l chromatin on the short arm. 47, XX,+6q- 47 chromosomes, XX sex chromosomes, an a d d i t i o n a l chromosome #6 that i s missing a part of i t s long arm. (b) Isochromosomes are designated as follows; 46,X,i(Xq) Female karyotype with 46 chromosomes, in c l u d i n g one normal X chromosome and one chromosome represented by an isochromosome for the long arm of the X. 179 (c) Reciprocal translocations are designated as follows; 46,XY,t(9;22) Breakage and reunion have occurred i n chromosomes #9 and #22. 46,XY,t(9q; 22q) Breakage and reunion have occurred i n the long arm of chromosome #9 and the long arm of chromosome #22. 46 ,XY,t(9;22) (q34;rq11) Breakage and reunion have occurred at bands q34 i n chromosome #9 and q11 i n chromosome #22. 46,XY,Ph Abbreviated nomenclature f o r a normal male karyotype with a standard Philadelphia chromosome r e s u l t i n g from a r e c i p r o c a l t(9;22)(q34;q11). (d) Marker chromosomes. Any morphologically distinguishable abnormal chromosome that cannot be f u l l y characterized may be designated by the symbol mar. I f more that one marker chromosome occurs i n a karyotype they are num-bered seq u e n t i a l l y . e.g., 48,XX,+mar1,+mar2 48 chromosomes, XX sex chromosomes and 2 marker chromosomes. (e) Derivative chromosomes. A derivative chromosome i s one of the st r u c -t u r a l l y rearranged chromosomes generated by a rearrangement i n v o l v i n g two or more chromosomes. I f both chromosomes of the same p a i r are involved i n the rearrangements they are distinguished by p l a c i n g one and two dashes under the respective chromosomes, e.g., 46,XX,der(9)t(9p; 117q),der(J>)t(9q;22q) 46 chromosomes, XX sex chromosomes, one 9;17 tra n s l o c a t i o n r e s u l t i n g i n a chromosome #9 broken i n the long arm and reunited with material from the long arm of chromosome #17, and one 9;22 tr a n s l o c a t i o n r e s u l t i n g i n the other chromosome #9 being broken i n the long arm and reunited with material from the long arm of chromosome #22. 1 Report of the Standing Committee on Human Cytogenetic Nomenclature. Cytogenetics and C e l l Genetics 2±, 309-404,1978. Appendix X Ode to the Colony Plucker The tumor b i o l o g i s t glowed with excitement, Just to think of the basic advancement A r i s i n g from the experiment. The reward was hard to r e s i s t , But, i n h i s mind d i d the words p e r s i s t Of the tumor c y t o g e n e t i c i s t : On the very best day, No matter what way, Ten per-cent, I say, W i l l be l o s t at the s t a r t ; I t ' s j u s t one small part Of t h i s wonderful a r t . Of the r e s t , ten per-cent w i l l be D i v i d i n g , you see Leaving l e s s f o r me. And that's at best! So l e t ' s not j e s t , We're t a l k i n g 'bout le s s and l e s s . Now, y o u ' l l be pleased to know, That i n ten per-cent, or so, A metaphase w i l l show! But, before you smile with d e l i g h t , Recall that most of these are too t i g h t : Less than ten per-cent just r i g h t . And i n a very experienced hand, Only h a l f of these w i l l band. And they won't a l l be so grand. Some w i l l s t a i n i n one t i n y patch. And, of course, some w i l l scratch; Generally making chromosomes impossible to match. Perhaps t h i s seems rather hopeless, But, point zero four f i v e per-cent success, At the end of t h i s elegant process, Can u s u a l l y be quite s u f f i c i e n t , I f you'd only be l e s s p r o f i c i e n t At g i v i n g me colonies with c e l l s so d e f i c i e n t ! So, before you say any more, Go back to your cultures and score A l l colonies of a thousand c e l l s or more. And i f you're s t i l l of the view That h a l f of these'11 do; Then I ' l l gladly pluck up a few! P U B L I C A T I O N S Ian D.Dube 1. Dube, I.D., Eaves, A.C, and Eaves, C J . A new technique for the cytogenetic analysis of cells from single hemopoietic colonies of bone marrow or peripheral blood origin. Am. J. Hum. Genet. _32/ 68A ( 1980). 2. Dube", I.D., Eaves, C.J., Kalousek, D.K., and Eaves, A.C. A method for obtaining high quality chromosome preparations from single hemopoietic colonies on a routine basis. Cancer Genet. Cytogenet. 4: 157-168 (1981). 3. Dube, I.D., Kalousek, D.K., Gupta, CM. , Eaves, C J . and Eaves, A.C Cytogenetic analysis of single hemopoietic colonies in leukemia can reveal different c e l l populations from those identified in direct preparations. Am. J. Hum. Genet. 3i3, 62A (1981). 4. Gupta, CM., Coulombel, L., Eaves, Ay Eaves, C , Dube, I.D. and Kalousek, D.K. Unexpected proliferation of Ph -negative cells in CML marrow cultures under conditions that favour myelopoiesis. Am. J. Hum. Genet. 34, 70A ( 1982) . 5. Kalousek, D.K., Dube, I.D., Eaves, C J . and Eaves, A.C Cytogenetic analysis of myeloid progenitors in Ph -positive ALL. J. C e l l . Biochem. (Suppl. 7B), 53 (1983). 6. Eaves, A.C, Dube. I.D., Gup1j.a, CM., Eaves, C J . and Kalousek, D.K. Persistence of functional Ph -negative stem cells in a patient with established Ph -positive CML using long-term marrow culture. Exp. Hematol. JH (Suppl JU), 82 (1983). 7. Dube, I.D., Coulombel, L., Gupta, C , Kalousek, D.K., Eaves, C J . and Eaves, A.C. Chromosomally normal myeloid progenitors in Philadelpia-positive CML. Am. J. Hum. Genet. 3_5, 62A (1983). 8. Dube, I.D., Gupta, CM., Kalousek, D.K., Eaves, C J . , and Eaves, A.C Cytogenetic studies of early myeloid progenitor compartments in Ph -positive chronic myeloid leukaemia (CML). I. Persistence of Ph -negative committed progenitors that are suppressed from differentiating in vivo. Br. J. Haematol, (in press 1984). 9. Dube, I.D., Kalousek, D.K., Coulombel, L., Gupta, CM., Eaves, C J . and Eaves^ A.C. Cytogenetic studies of early myeloid progenitor compartments in Ph -positive chronic myeloid leukemia (CML). II. Long-term culture reveals the persistence of Ph -negative progenitors in_treated as well as newly diagnosed patients. Blood (in press 1984). 10. Dube, I.D., Kalousek, D.K., Coulombel, L., Eaves, C J . and Eaves, A.C In vitro cytogenetic studies in chronic myeloid leukemia (CML) (Abstr), in Human Tumor Cloning (editors Salmon, S.E. and Trent, J.M.) Grune & Stratton: New York (in press 1984). 11. Dube, I.D., Kalousek, D.K., Coulombel, L., Eaves, C J . and Eaves, A.C. In vitro cytogenetic studies in chronic myeloid leukemia (CML), in Human Tumor Cloning (editors Salmon, S.E. and Trent, J.M.) Grune & Stratton: New York (in press 1984). PUBLICATIONS Ian D.Dube' 12. Dube, I.D., Kalousek, D.K., Eaves, C.J. and Eaves, A.C. V a r i a t i o n i n Ph - p o s i t i v e progenitor maintenance i n long-term CML marrow c u l t u r e s . J . C e l l . Biochem. (Suppl. 8A), 85 (1984). 13. Dube, I.D., Kalousek, D.K., Coulombel, L., Eaves, C.J. and Eaves, A.C. V a r i a t i o n i n Ph - p o s i t i v e progenitor maintenance i n long-term CML marrow c u l t u r e s , i n Genes and Cancer, UCLA Symposium on Molecular and C e l l u l a r Biology, New Series, Volume 17 ( e d i t o r s Bishop, J.M., Greaves,M., and Rowley, J.D.) Alan R. L i s s , Inc: New York (in press 1984). 14. Coulombel, L., Eaves, C.J., Dube, I.D., Kalousek, D.K. and Eaves, A.C. Var i a b l e persistence of leukemic progenitor c e l l s i n long-term CML and AML marrow cul t u r e s , i n Long-Term Bone Marrow Culture: Present Experience and  Future P o s s i b i l i t i e s (editors Wright, D.G. and Greenberger J.S.) Alan R. L i s s , Inc: New York (in press 1984). 

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