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Characterization of novel myeloid differentiation antigen associated with acute myelogenous leukemia Askew, David Stephen 1985

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CHARACTERIZATION OF A NOVEL MYELOID DIFFERENTIATION ANTIGEN ASSOCIATED WITH ACUTE MYELOGENOUS LEUKEMIA By David Stephen Askew B. S c , U n i v e r s i t y of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1985 ®David S. Askew, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date December 08, 1985 i i ABSTRACT A monoclonal antibody has been developed that detects a unique c e l l surface antigen (NHL-30.5) with a molecular weight of 180,000 expressed on the human acute promyelocytic c e l l line HL-60. In addition to HL-60 and other AML c e l l lines, the antibody reacts with a significant proportion of hemopoietic cells from 40/48 patients with acute myeloid leukemia (AML), and on a variety of other hematologic disorders characterized by the presence of immature myeloid blast cells. In contrast i t does not react with normal mature hemopoietic cells, including lymphocytes, monocytes, granulocytes, erythrocytes, platelets, and splenocytes. Only one of 15 acute lymphoblastic leukemias has demonstrated reactivity (weakly) and a l l lymphoid c e l l lines tested have been uniformly negative. Reactivity with cells from patients in the chronic phase of chronic myeloid leukemia (CML) is also rare (7/26) and the number of NHL-30.5 positive cells is low (<20%). The acute phase of CML is strongly NHL-30.5-positive i f the blast c r i s i s is of the myeloid variant but is clearly negative in lymphoid blast c r i s i s . Analysis of normal differentiating bone marrow cells and mature peripheral blood mononuclear cells stained indirectly with the NHL-30.5 monoclonal antibody and FITC-second antibodies did not reveal a distinctly positive population. However, the cells with the highest fluorescence intensities (comprising 5% of the total population) sorted on a fluorescence activated c e l l sorter were highly enriched in both erythropoietic (CFU-E/BFU-E) and granulopoietic (CFU-C) progenitors. It therefore appears that the NHL-30.5 antigen is not an AML-associated marker but rather a normal myeloid differentiation antigen that is expressed on immature myeloid c e l l s . Consistent with this hypothesis is the observation that a number of AML-i i i derived c e l l lines that are blocked at an early stage of maturation lose NHL-30.5 expression when they are induced to terminally differentiate. These results support the concept that undifferentiated myeloid progenitors accumulate in AML patients due to a block in their capacity to differentiate into the stages characterized by loss of the NHL-30.5 antigen. The NHL-30.5 monoclonal antibody identifies a previously undescribed progenitor c e l l antigen and is potentially a useful reagent to differentiate myeloid leukemias from lymphoid leukemias, particularly in the acute phase of CML. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF ABBREVIATIONS v i LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENT S x i i Chapter I THE HEMOPOIETIC SYSTEM 1) Overview 1 2) Assays 3 (A) In V ivo : The Spleen Colony Assay 3 (B) In V i t r o : Assays for Clonogenic Progeni tors 5 3) Regulat ion of Stem C e l l D i f f e r e n t i a t i o n 11 (A) Regulatory Molecules 11 (B) Regulatory Role of the Microenvironment 15 (C) Theories of Stem C e l l Commitment 17 4) Neoplast ic Disorders of Myelopoiesis 19 (A) The Chronic M y e l o p r o l i f e r a t i v e Disorders and 19 the Acute leukemias (B) Chronic Myeloid Leukemia (CML) 21 (C) Acute Myeloid Leukemia (AML) 29 5) Ant igen ic Ana lys is of Leukemic Myelopoiesis 37 (A) Tumour Antigens 37 (B) Normal Myeloid D i f f e r e n t i a t i o n 44 (C) Leukemic Myeloid D i f f e r e n t i a t i o n 52 6) Thes is Object ive 57 References 59 Chapter II MATERIALS AND METHODS 88 1) Production of the NHL-30.5 Monoclonal Antibody 88 2) C e l l L a b e l l i n g Procedures 89 Binding Assay 89 Antigen Est imat ion 90 FACS ana lys is 90 3) Immunoprecipitation 94 4) Hemopoietic C e l l Lines 95 5) D i f f e r e n t i a t i o n of Myeloid Leukemia C e l l L ines 95 Granulocyt ic D i f f e r e n t i a t i o n 95 Monocytic D i f f e r e n t i a t i o n 97 6) Human C e l l Preparat ions 97 Pat ient Specimens 97 C e l l F rac t iona t ion 98 7) C e l l Sor t ing 100 8) Assays for Clonogenic Myeloid Progen i tors . 101 References 102 V Chapter III NHL-30.5: A MONOCLONAL ANTIBODY DEFINING AN ACUTE MYELOGENOUS LEUKEMIA (AML)-ASSOCIATED ANTIGEN. 1) In t roduct ion 104 2) Resul ts 105 R e a c t i v i t y with Normal and Leukemic c e l l s 105 D i f f e r e n t i a t i o n of HL-60 114 Immunoprecipitation 115 3) D iscuss ion 115 References 125 Chapter IV DIFFERENTIATION-LINKED EXPRESSION OF AN AML-ASSOCIATED ANTIGEN (NHL-30.5) ON MYELOID LEUKEMIA CELL LINES. 1) In t roduct ion 126 2) Resul ts 134 R e a c t i v i t y with Hemopoietic C e l l L ines 134 Induction of Granulocyt ic D i f f e r e n t i a t i o n 135 Induction of Monocytic D i f f e r e n t i a t i o n 139 3) D iscuss ion 145 References 151 Chapter V RESTRICTED EXPRESSION OF AN ACUTE MYELOGENOUS LEUKEMIA-ASSOCIATED ANTIGEN (NHL-30.5) ON NORMAL HEMOPOIETIC PROGENITOR CELLS. 1) In t roduct ion 154 2) Resul ts 156 FACS A n a l y s i s / C e l l Sor t ing 156 Funct iona l Studies 167 Immunoprecipitation of NHL-30.5 and My-10 167 3) D iscuss ion 173 References 176 Chapter VI SUMMARY AND CONCLUSION 178 LIST OF ABBREVIATIONS ALL - Acute lymphoblastic leukemia AML - Acute myelogenous leukemia BFU-E - Burst forming unit-erythroid BM - Bone marrow BPA - Burst promoting activity BSA - Bovine serum albumin CALLA - Common acute lymphoblastic leukemia antigen CFU-C - Colony forming unit-culture CFU-E - Colony forming unit-erythroid CFU-GM - Colony forming unit-granulocyte/macrophage CFU-GEMM - Colony forming unit-granulocyte erythrocyte magakaryocyte macrophage CFU-S - Colony forming unit-spleen CLL - Chronic lymphocytic leukemia CMML - Chronic myelomonocytic leukemia CML - Chronic myelogenous leukemia CR3 - Complement receptor 3 CSF - Colony stimulating factor CTL - Cytotoxic T lymphocyte DMEM - Dulbecco's modified Eagle's minimal essential medium DMSO - Dimethylsulfoxide DTH - Delayed type hypersensitivity EBSS - Earl's balanced salt solution EBV - Epstein Barr virus EDTA - Ethylenediaminetetraacetic acid Epo - Erythropoietin ET - Essential thrombocytosis FAB - French American British leukemia study group FACS - Fluorescent activated c e l l sorter FCS - Fetal calf serum FITC - Fluorescein isothiocyanate GaMIg - Goat anti-mouse immunoglobulin G-CSF - Granulocyte colony stimulating factor GM-CSF - Granulocyte macrophage colony stimulating factor G 6 P D - Glucose-6-phosphate dehydrogenase HAT - Hypoxanthine aminopterin and thymidine HBSS - Hanks balanced salt solution HBSS-Ca-Mg - HBSS minus calcium and magnesium 4-HC - 4-hydroperoxycyclophosphamide HLA - Human Leukocyte Antigen IL-3 - Interleukin 3 LAA - Leukemia associated antigen LCM - Leucocyte conditioned medium LFA-1 - Lymphocyte function antigen-1 LIA - Leukemia inhibitory activity Mac-1 - the mac-1 antigen MF - Myelofibrosis MLC - Mixed lymphocyte culture MPD - Myeloproliferative disease My-10 - the My-10 antigen NB-2 - monoclonal antibody against the transferrin receptor NBT - Nitrobluetetrazolium LIST OF ABBREVIATIONS CONTINUED NHL-62.14 - monoclonal antibody against the transferrin receptor NK - Natural k i l l e r PBS - Phosphate buffered saline PB - Peripheral blood Ph* - Philadelphia chromosome PHA - Phytohemagglutinin PV - Polycythemia vera RA - Retinoic acid RaMIg - Rabbit anti-mouse immunoglobulin Rh - Rhesus antigen SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis T200 - High molecular weight (200,000) leucocyte-common antigen TdT - Terminal deoxynucleotidyl transferase TPA - 12-0-tetradecanoylphorbol 13-acetate v i i i LIST OF TABLES Page TABLE I French-Amer ican-Br i t ish (FAB) c l a s i f i c a t i o n of acute myelogenous leukemia 34 TABLE II Cul ture condi t ions for hemopoietic c e l l l i n e s 96 TABLE III R e a c t i v i t y of the NHL-30.5 monoclonal antibody with the f i r s t 19 AML pat ients studied 110 TABLE IV A summary of a l l AML specimens conta in ing >10X NHL-30 .5 -pos i t ive c e l l s (NHL-30.5-posi t ive) I l l TABLE V A summary of a l l AML specimens conta in ing <10% NHL-30 .5 -pos i t ive c e l l s (NHL-30.5-negative) 112 TABLE VI Sequent ia l t es t ing of c e l l s from pat ients with AML and re la ted d isorders 113 TABLE VII Summary of the r e a c t i v i t y of NHL-30.5 with var ious pat ient categor ies 116 TABLE VIII R e a c t i v i t y of NHL-30.5 with c e l l s from pat ients i n the acute phase of CML 118 TABLE IX 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 es tab l ished myeloid leukemia c e l l l i n e s 128 TABLE X C h a r a c t e r i s t i c s of HL-60 c e l l s before and a f t e r induct ion of granu locy t ic d i f f e r e n t i a t i o n 130 TABLE XI C h a r a c t e r i s t i c s of HL-60 c e l l s before and a f t e r induct ion of monocytic d i f f e r e n t i a t i o n 131 TABLE XII A l t e r a t i o n s in oncogene expression assoc ia ted with induct ion of d i f f e r e n t i a t i o n in HL-60 c e l l s 133 TABLE XIII R e a c t i v i t y of NHL-30.5 monoclonal antibody with var ious leukemic c e l l l i n e s 136 TABLE XIV The number of progeni tors per 10 5 c e l l s i n NHL-30.5 sorted f r a c t i o n s from CML per iphera l blood 157 TABLE XV The number of progeni tors per 10 5 c e l l s i n NHL-30.5 sorted f r a c t i o n s from normal bone marrow 159 TABLE XVI The number of progeni tors per 10 5 c e l l s i n NHL-30.5 sorted f r a c t i o n s from normal per iphera l blood 160 TABLE XVII The d i s t r i b u t i o n of b las t progeni tors (b last co lon ies per 10^ c e l l s ) i n NHL-30.5 sorted f r a c t i o n s from the per iphera l blood and bone marrow of a pat ient with AML 166 ix TABLE XVIII The number of progenitors per 10^ bone marrow c e l l s plated i n the presence of p u r i f i e d NHL-30.5 monoclonal antibody 168 TABLE XIX The number of progenitors per 10 5 normal per iphera l blood c e l l s plated in the presence of p u r i f i e d NHL-30.5 monoclonal antibody 169 TABLE XX A comparison of the r e a c t i v i t y of the NHL-30.5 and My-10 monoclonal ant ibodies on the HL-60 and KG-1 AML c e l l l i n e s 171 TABLE XXI The a b i l i t y of the My-10 monoclonal antibody to block the binding of iodinated NHL-30.5 monoclonal antibody to HL-60 c e l l s . . . . 172 X LIST OF FIGURES FIGURE I Schematic representat ion of the hierarachy of hemopoietic progenitor compartments cur ren t l y i d e n t i f i e d by colony assay procedures 10 FIGURE II Schematic representat ion of the f luorescence ac t iva ted c e l l sor te r (FACS) 93 FIGURE III FACS p r o f i l e s of the binding of NHL-30.5 to per iphera l blood c e l l s from a normal, AML, and CML donor 106 FIGURE IV FACS p r o f i l e s of the binding of NHL-30.5 to bone marrow c e l l s from a normal, AML, and CML pat ient 107 FIGURE V FACS p r o f i l e s of the binding of NHL-30.5 to populat ions of granulocytes , monocytes, lymphocytes, PHA-stimulated lymphocytes, e ry throcytes , sp lenocytes , and p l a t e l e t s from normal donors 109 FIGURE VI FACS p r o f i l e s of the binding of NHL-30.5 to the HL-60 c e l l l i n e before and a f te r induct ion of d i f f e r e n t i a t i o n with DMSO 117 FIGURE VII Immunoprecipitation of the NHL-30.5 antigen from 1 2 5 I - l a b e l l e d HL-60 c e l l s 119 FIGURE VIII Immunoprecipitation of the NHL-30.5 antigen from an ^ 2 5 j _ i a D e n e ( j AML per iphera l blood specimen conta in ing 77% b las t c e l l s 120 FIGURE IX FACS ana lys is of the r e a c t i v i t y of NHL-30.5 with HL-60 c e l l s at var ious time i n t e r v a l s fo l lowing the induct ion of g ranu locy t ic d i f f e r e n t i a t i o n with DMSO 137 FIGURE X FACS ana lys is of the r e a c t i v i t y of NHL-30.5 with HL-60 c e l l s at var ious time i n t e r v a l s fo l lowing the induct ion of granu locy t ic d i f f e r e n t i a t i o n with r e t i n o i c ac id 138 FIGURE XI Immunoprecipitation of the NHL-30.5 antigen and t r a n s f e r r i n receptor from HL-60 c e l l s before and a f t e r induct ion of d i f f e r e n t i a t i o n with DMSO 140 FIGURE XII Immunoprecipitation of the NHL-30.5 antigen and t r a n s f e r r i n receptor from HL-60 c e l l s l a b e l l e d with 32 P 141 FIGURE XIII FACS ana lys is of the r e a c t i v i t y of NHL-30.5 with HL-60 c e l l s at var ious time i n t e r v a l s fo l lowing induct ion of monocytic d i f f e r e n t i a t i o n with TPA. 142 x i FIGURE XIV FACS ana lys is of the r e a c t i v i t y of NHL-30.5 with KG-1 c e l l s at var ious time i n t e r v a l s fo l lowing induct ion of monocytic d i f f e r e n t i a t i o n with TPA 144 FIGURE XV Morphological appearance of HEL cul tures before and a f t e r induct ion of d i f f e r e n t i a t i o n with TPA 146 FIGURE XVI FACS ana lys is of the r e a c t i v i t y of NHL-30.5 with adherent HEL c e l l s at var ious time i n t e r v a l s fo l lowing induct ion of monocytic d i f f e r e n t i a t i o n with TPA 147 FIGURE XVII FACS ana lys is of the r e a c t i v i t y of NHL-30.5 with the nonadherent HEL c e l l s at var ious time i n t e r v a l s fo l lowing induct ion of monocytic d i f f e r e n t i a t i o n with TPA 148 FIGURE XVIII Saturat ion curve for the binding of 1 2 5 I - l a b e l l e d NHL-30.5 monoclonal antibody to HL-60 c e l l s . 149 FIGURE XIX Graphica l representat ion of the frequency of myeloid progeni tors in NHL-30.5 sorted f r a c t i o n s from CML per iphera l blood 161 FIGURE XX Graphica l representat ion of the frequency of myeloid progenitors in NHL-30.5 sorted f r a c t i o n s from normal bone marrow 162 FIGURE XXI Graphica l representat ion of the frequency of myeloid progenitors in NHL-30.5 sorted f r a c t i o n s from normal per iphera l blood 163 FIGURE XXII Fluorescence p r o f i l e s of the r e a c t i v i t y of NHL-30.5 with per iphera l blood and bone marrow c e l l s from a pat ient with AML. C e l l s were sorted on the basis of the i r r e a c t i v i t y with NHL-30.5 at the ind ica ted gates and plated in standard methy lce l lu lose assays 165 FIGURE XXIII A comparison of the NHL-30.5 and My-10 antigens by immunoprecipitation 170 x i i ACKNOWLEDGEMENTS Throughout the course of this work I have had the pleasure of associating with a number of individuals whose contribution to the completion of this thesis I would like to acknowledge. I wish to thank: My Supervisor Dr. Fumio Takei, and other Senior Investigators of the Terry Fox Laboratory for stimulating my interest in the many facets of experimental hematology, and for providing the f a c i l i t i e s and training necessary for the completion of this work. Members of my supervisory committee, Drs. D. Brooks and J. Levy for their helpful suggestions. Cam Smith, Darlene Nipius, Visia Dragowska and other research technologists in this laboratory for their patience in teaching me their s k i l l s and for assisting with this project. Dr. W. Gibson, for the enthusiasm in his teaching. Fellow graduate students for encouragement and companionship. Pam Quick and HDC. My family, for their untiring support. Darien and Ken: "Think where man's glory most begins and ends, and say my glory was I had such friends" W.B. Yeats (1865-1939) x i i i What am I , L i f e ? A thi n g of watery s a l t Held i n cohesion by un r e s t i n g c e l l s , Which work they know not why, which never h a l t , Myself u n w i t t i n g where t h e i r Master dwells? John Masef i e l d (1878-1967), Sonnets, 14 1 C H A P T E R I THE HEMOPOIETIC SYSTEM 1) OVERVIEW OF THE HEMOPOIETIC SYSTEM The f u l l y d i f f e r e n t i a t e d hemopoietic c e l l s in the per iphera l blood have a f i n i t e l i f e s p a n and are incapable of s e l f - r e n e w a l . Since hemopoiesis must occur throughout the l i f e s p a n of an i n d i v i d u a l , cont inua l replacement of these r e l a t i v e l y short l i v e d mature c e l l s i s maintained by a populat ion of l e s s d i f f e r e n t i a t e d hemopoietic c e l l s . The hemopoietic system i s thus comprised of a h ierarchy of a d iverse range of c e l l s at d i f f e r e n t stages of d i f f e r e n t i a t i o n . The developmental sequence of these c e l l s has been known for some time and i s based l a r g e l y on the fact that the major i ty are in the l a t e r stages of maturation and are therefore morphological ly recognizable as precursors of the g r a n u l o c y t i c , megakaryocyte , or e ry thro id l i n e a g e s . In normal a d u l t s , the primary s i t e s for ac t ive hemopoiesis are r e s t r i c t e d to bone marrow in the ver tebrae, r i b s , sternum, p e l v i s , scapulae, s k u l l and extreme proximal port ions of the long bones. The hemopoietic t i ssue w i th in the marrow i s compartmentalized by p lates of bony trabeculae protruding in to the marrow c a v i t y , although under condi t ions of severe hematologic s t r e s s expanded output can be accomplished by increas ing the proport ion of hemopoietic t i ssue at the expense of the fat conta in ing areas. In order to maintain homeostasis the hemopoietic system must have the capac i ty to se l f - renew, and th is a b i l i t y res ides in a populat ion of hemopoietic stem c e l l s . This stem c e l l pool provides c e l l s for d i f f e r e n t i a t i o n at va r iab le rates according to the demand for f u n c t i o n a l blood c e l l s , and at the same time maintains a r e l a t i v e l y constant reserve of 2 stem ce l l s . Stem cells thus possess two important features: a) an unrestricted differentiation potential, i.e. the capacity to produce cells representative of a l l blood c e l l lineages (1), and b) an extensive self renewal capacity, i.e. the a b i l i t y to give rise to new stem cells that are also pluripotent (2). Early evidence for the existence of these primitive stem cells came from studies showing that mouse bone marrow contained a class of cells capable of repopulating lethally irradiated mice with a functional hemopoietic system (3,4). Furthermore, Barnes et al (5) reported that in some mice recovering from sublethal doses of irradiation, virtually a l l dividing cells in hemopoietic tissue (including lymphoid tissue) possessed the same unique radiation-induced chromosomal marker. Since these chromosomal abnormalities are generated randomly in single cells, the identification of the same marker in differentiated cells of a number of lineages suggested that the markers arose in a pluripotent stem c e l l . Further evidence that these stem ce l l s , or at least a subpopulation of stem cells, possess both myeloid and lymphoid (T and B) differentiation potential has been inferred from several studies in the mouse system (6). The existence of a stem c e l l common to both the myeloid and lymphoid lineages in man is supported from studies of clonal neoplastic disorders, particularly chronic myelogenous leukemia (CML) (7). Approximately 90% of patients with CML contain the Philadelphia (Ph^) chromosome in their dividing marrow cell s . The identification of this marker in red c e l l precursors, granulocytes, platelets and some B-lymphocytes (8,9) suggested that these cells were descendants of a pluripotent stem c e l l . This is further supported by studies with the X-linked glucose-6-phosphate dehydrogenase (G6PD) marker (see section on CML). Female patients with CML who are heterozygous at this 3 locus show only a s i n g l e enzyme type in the i r hemopoietic c e l l s (10,11) , an observat ion that fur ther documents the monoclonali ty of th is d i s o r d e r . S i m i l a r l y , one pat ient with s i d e r o b l a s t i c anemia was demonstrated to express a s i n g l e G6PD isoenzyme in both T and B lymphocytes in add i t ion to myeloid c e l l s (12). Since no morphological , cytochemical , or ant igen ic markers are known that are e x c l u s i v e l y r e s t r i c t e d to hemopoietic stem c e l l s , the i d e n t i f i c a t i o n of these c e l l s has r e l i e d heav i ly upon recogni t ion of the i r progeny. Progress in the development of c l o n a l assays for these p r im i t i ve c e l l s over the past 20 years has grea t ly f a c i l i t a t e d ana lys is of the organ iza t ion of the hemopoietic system and the fac tors that regulate hemopoietic stem c e l l d i f f e r e n t i a t i o n . These assays are reviewed in the fo l lowing two sec t ions and the i r con t r ibu t ion to our current understanding of d i f f e r e n t i a t i o n in the var ious hemopoietic l ineages i s summarized in Figure I (13). 2) ASSAYS (A) In V ivo : The Spleen Colony Assay In 1961 T i l l and McCulloch (14) descr ibed the f i r s t assay for the q u a n t i t a t i v e detect ion of p r im i t i ve hemopoietic c e l l s . This assay i s based on the observat ion that when hemopoietic c e l l s are in jec ted in t ravenously in to l e t h a l l y i r r a d i a t e d syngeneic mice, a populat ion of c e l l s wi th in the i n i t i a l inoculum i s capable of forming macroscopical ly v i s i b l e nodules on the r e c i p i e n t sp leen . The c e l l i n i t i a t i n g these spleen co lon ies was termed CFU-S (colony forming u n i t - s p l e e n ) . The widespread use of th is assay as a method of measuring the s i z e of the stem c e l l compartment i s based on observat ions that the CFU-S appears to f u l f i l l the c r i t e r i a by which a c e l l i s c l a s s i f i e d as a hemopoietic stem c e l l : a) P r o l i f e r a t i v e capac i ty : The s i z e of the A i n d i v i d u a l co lon ies (approximately 10^ to 10?) r e f l e c t s the extensive p r o l i f e r a t i v e capaci ty of the c e l l that gave r i s e to the colony, b) D i f f e r e n t i a t i o n p o t e n t i a l : H i s t o l o g i c a l examination of i n d i v i d u a l spleen co lon ies revealed the presence of e r y t h r o i d , g r a n u l o c y t i c , and megakaryocytic l i n e s of d i f f e r e n t i a t i o n suggesting that the CFU-S was indeed a mult ipotent c e l l (1) . Ear ly s tudies ind icated that some CFU-S may have lymphoid in a d d i t i o n to myeloid d i f f e r e n t i a t i v e p o t e n t i a l (15-18), although th is has never been c l e a r l y e s t a b l i s h e d . The CFU-S i s cur rent ly viewed as a stem c e l l r e s t r i c t e d to myelopoies is . c) C l o n a l i t y : Evidence for the monoclonal o r i g i n of these co lonies was obtained by generat ing spleen co lon ies with c e l l s conta in ing rad ia t ion- induced chromosomal markers. Unique karyotypes were observed in a number of co lonies and the marker was found in a very high propor t ion of the metaphases obtained from each colony (19). These r e s u l t s were a l s o confirmed using stem c e l l d e f i c i e n t (W/Wv) mice as r e c i p i e n t s (1) . d) Se l f renewal capac i ty : Ana lys is of CFU-S by s e r i a l t ransp lanta t ion s tud ies has demonstrated that some of the c e l l s wi th in spleen co lon ies are themselves capable of spleen colony formation (2). There are some l i m i t a t i o n s associated with the use of the CFU-S assay as a q u a n t i t a t i v e measure of stem c e l l s however. Only a f r a c t i o n of the colony- forming c e l l s in jec ted into an i r r a d i a t e d r e c i p i e n t w i l l a c t u a l l y s e t t l e in the spleen (2) , the remaining c e l l s e i ther dying or growing in the bone marrow. This problem i s fur ther compounded with the observat ion that more than one spleen colony may o c c a s i o n a l l y a r i s e from a s i n g l e CFU-S (5) . There i s a l s o accumulating evidence that the CFU-S popula t ion , as def ined by the spleen colony assay, i s a heterogeneous populat ion that can vary in both se l f - renewa l capaci ty and d i f f e r e n t i a t i o n p o t e n t i a l (20). 5 In summary, although there i s evidence to support the existence of a common lympho-myelopoietic stem c e l l , the p r e c i s e l o c a t i o n of the CFU-S w i t h i n the hi e r a r c h y of hemopoietic c e l l d i f f e r e n t i a t i o n has not been e s t a b l i s h e d . The CFU-S i s c l e a r l y a c e l l with multipotent myeloid d i f f e r e n t i a t i o n p o t e n t i a l , but there i s as yet no d e f i n i t i v e evidence i m p l i c a t i n g t h i s c e l l i n lymphopoiesis. (B) In V i t r o ; Assays f o r Clonogenic Progenitors The advent of i n v i t r o colony assays has provided much informat i o n about a population of c e l l s that are intermediate between the stem c e l l and maturing e f f e c t o r c e l l compartments. Members of t h i s i n t e r v e n i n g p o p u l a t i o n are termed progenitors and they appear to d i f f e r from stem c e l l s i n that they l a c k s e l f - r e n e w a l capacity and are r e s t r i c t e d i n t h e i r d i f f e r e n t i a t i o n p o t e n t i a l . Due to the considerable heterogeneity of a l l hemopoietic c e l l p o p u l a t i o n s , the i n i t i a l development of an agar c l o n i n g system capable of supporting the growth of g r a n u l o c y t i c or macrophage co l o n i e s ( 2 1 , 2 2 ) was a major t e c h n i c a l achievement that provided an i n v i t r o system i n which i n d i v i d u a l c e l l populations could be analyzed under c o n t r o l l e d c o n d i t i o n s . The assay i n v o l v e s the im m o b i l i z a t i o n of hemopoietic c e l l s i n s e m i - s o l i d medium c o n t a i n i n g appropriate n u t r i e n t s , serum and growth f a c t o r s . The p r o l i f e r a t i o n of a s i n g l e progenitor c e l l i s therefore able to g i v e r i s e to a clone of mature descendants s u f f i c i e n t l y l o c a l i z e d to be i d e n t i f i e d as a colony. The progenitors g i v i n g r i s e to col o n i e s of granulocytes and macrophages were i n i t i a l l y termed CFU-C (colony forming u n i t i n c u l t u r e ) and more r e c e n t l y CFU-granulocyte/macrophage (CFU-GM). Subsequently the c u l t u r e c o n d i t i o n s that allow the p r o l i f e r a t i o n of what appear to be the e a r l i e s t committed c e l l s i n each myeloid d i f f e r e n t i a t i o n lineage have s i n c e been 6 descr ibed (23). These studies have demonstrated considerable heterogeneity in each pathway of d i f f e r e n t i a t i o n . Colonies of e ry thro id c e l l s were f i r s t documented using the plasma c l o t system (24). Subsequently the f i b r i n c l o t (25), methy lce l lu lose (26), and agar systems have been used, although agar does not appear to support the l a t e r phases of e ry thro id d i f f e r e n t i a t i o n very wel l (27). When an appropr ia te combination of nut r ients and s t i m u l i are provided, these s e m i - s o l i d media are able to support the growth of co lon ies of hemoglobin syn thes iz ing e r y t h r o b l a s t s . One of the features c h a r a c t e r i s t i c of the co lon ies der ived from the more p r imi t i ve of these ery thro id progeni tors i s the i r o rgan iza t ion into d i s c r e t e c l u s t e r s . The migratory a b i l i t i e s of these progeni tors dur ing ear ly c e l l d i v i s i o n s i s l o s t when they enter the terminal stages of e ry thro id d i f f e r e n t i a t i o n and th is contr ibutes to the formation of a mu l t i c lus te red colony. The term BFU-E (burst - forming u n i t - e r y t h r o i d ) i s used to designate the c e l l of o r i g i n . The d e f i n i t i o n of sequent ia l stages 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 in the progenitor compartment i s based on the decreasing p r o l i f e r a t i v e capaci ty and the loss of migratory a b i l i t y that accompanies the d i f f e r e n t i a t i v e d i v i s i o n s . D i f fe rent s ized e ry th ro id co lon ies have therefore been shown to detect progeni tors in at l eas t three stages of d i f f e r e n t i a t i o n along the e ry thro id pathway (28,29). The most p r i m i t i v e BFU-E i s the precursor of the large mul t i c lus te red co lon ies (>8 c l u s t e r s ) that cons is t of many ery throb las ts (>1000). The more mature BFU-E g ive r i s e to smal ler bursts conta in ing 3-8 c l u s t e r s of e ry throb las ts (30), and s i n g l e or paired c l u s t e r s of e ry throb las ts o r i g i n a t e from a more d i f f e r e n t i a t e d c e l l type designated as the colony forming u n i t - e r y t h r o i d (CFU-E) . These CFU-E were the f i r s t e ry thro id co lonies to be cu l tured i n v i t r o and they are e a s i l y recognized as 1-2 t ight c l u s t e r s conta in ing 8-50 c e l l s . 7 A l l e r y t h r o i d progenitors assayable i n these c l o n a l c u l t u r e s appear to represent a continuum of d i f f e r e n t stages of d i f f e r e n t i a t i o n . Further support f o r t h i s concept of an e r y t h r o p o i e t i c h i e r a r c h y comes from s t u d i e s demonstrating d i f f e r e n c e s i n the p r o p e r t i e s of c e l l s that generate these d i f f e r e n t s i z e d e r y t h r o i d c o l o n i e s . These include: d i f f e r e n c e s i n p r o g e n i t o r c e l l s i z e , d e n s i t y , c e l l surface antigen expression, s e n s i t i v i t y to e r y t h r o p o i e t i n and leukocyte-derived f a c t o r s , and normal c y c l i n g s t a t u s (31). The granulocyte progenitor, known as the CFU-C, i s a l s o the precursor f o r macrophages and i s commonly r e f e r r e d to as the CFU-granulocyte/macrophage (CFU-GM) (23). Heterogeneity i n t h i s precursor population i s w e l l e s t a b l i s h e d and major subpopulations have been i d e n t i f i e d that d i f f e r i n t h e i r buoyant d e n s i t y , p r o l i f e r a t i v e c a p a c i t y , c e l l c y c l e s t a t u s , s e n s i t i v i t y to pathway-specific regulatory molecules (32), and c e l l surface a n t i g e n i c phenotype (33). Since the p r o l i f e r a t i v e a c t i v i t y of CFU-GM r e f l e c t s the c o n c e n t r a t i o n and type of colony s t i m u l a t i n g f a c t o r ( s ) present (34), d i f f i c u l t i e s are sometimes encountered when t r y i n g to s u b c l a s s i f y these progenitors i n t o d i f f e r e n t stages of maturation. S i m i l a r progressions of c e l l types of decreasing p r o l i f e r a t i v e capacity have been i d e n t i f i e d f o r the megakaryocyte progenitor c e l l compartment (35). Several of the more d i r e c t l i n e s of evidence i n d i c a t i n g that the myeloid c o l o n i e s described above are derived from s i n g l e colony-forming c e l l s i n c l u d e : 1. m i c r o c u l t u r e of s i n g l e c e l l s l e a d i n g to colony formation (36). 2. male/female mixing experiments that produce co l o n i e s c o n t a i n i n g e i t h e r a male or female karyotype (37). 3. Demonstration of a s i n g l e G6PD isoenzyme i n bone marrow c o l o n i e s from a G6PD heterozygote (38). 8 The c o l o n i e s described above detect progenitors that are r e s t r i c t e d to a p a r t i c u l a r myeloid l i n e a g e . Culture techniques that permit the c l o n a l growth of hemopoietic progenitors with m u l t i l i n e a g e d i f f e r e n t i a t i o n p o t e n t i a l have s i n c e been described by s e v e r a l groups of i n v e s t i g a t o r s (39-42). Colonies c o n t a i n i n g hemopoietic c e l l s of s e v e r a l lineages were f i r s t described using c u l t u r e s of f e t a l l i v e r c e l l s i n agar (39). Notably these mixed e r y t h r o i d c o l o n i e s grew i n the absence of detectable e r y t h r o p o i e t i n , although i t i s now known that the conditioned medium from the pokeweed mitogen-activated spleen c e l l s used i n these c u l t u r e s contained i n t e r l e u k i n - 3 ( I L - 3 ) . Since most s t u d i e s report these c o l o n i e s to contain granulocytes, e r y t h r o c y t e s , macrophages and megakaryocytes the c e l l of o r i g i n has been termed a CFU-GEMM. The presence of mast c e l l s and e o s i n o p h i l s i n c l o n a l l y derived mixed c o l o n i e s have a l s o been reported (43). The lymphopoietic p o t e n t i a l of CFU-GEMM i s supported by a number of studies i n d i c a t i n g the presence of lymphoid c e l l s bearing e i t h e r B or T c e l l markers i n mixed co l o n i e s (44-48). G-6PD a n a l y s i s of the myeloid and T lymphoid c e l l s w i t h i n these c o l o n i e s has e s t a b l i s h e d that the c o l o n i e s o r i g i n a t e from a s i n g l e lympho-myelopoietic progenitor ( r a t h e r than more than one clonogenic c e l l ) (46). Recently, Nakahata et a l (49) documented the s i n g l e c e l l o r i g i n of human mixed c o l o n i e s that express va r i o u s combinations of myeloid c e l l lineages ( n e u t r o p h i l , e r y t h r o c y t e , macrophage, megakaryocyte, e o s i n o p h i l , and b a s o p h i l ) . These c o l o n i e s contained combinations of c e l l s i n 2-5 l i n e a g e s , demonstrating that there i s considerable heterogeneity i n the progenitor c e l l compartment that gives r i s e to human mixed c o l o n i e s . These r e s u l t s are i n agreement with s t u d i e s of the s i n g l e - c e l l o r i g i n of c o l o n i e s expressing various combinations of c e l l l i n eages i n the mouse system (50). 9 The i d e n t i f i c a t i o n of a progenitor capable of mixed colony formation i n v i t r o ra ised the quest ion of the prec ise r e l a t i o n s h i p between th is CFU-GEMM and the CFU-S in the h ierarchy of hemopoietic d i f f e r e n t i a t i o n (Figure I ) . A number of s tudies now suggest that the CFU-GEMM and CFU-S may represent p a r t i a l l y over lapping populat ions: both possess s i m i l a r buoyant d e n s i t i e s and each acquire the c h a r a c t e r i s t i c of increas ing densi ty and smal ler s i z e assoc ia ted with development (51-54). S i m i l a r i t i e s a lso ex is t in the p r o l i f e r a t i v e s ta te of CFU-S and CFU-GEMM (54-56) and there i s a s i g n i f i c a n t c o r r e l a t i o n between the number of spleen co lonies and the number of mixed co lon ies obtained from i n d i v i d u a l spleen co lonies (54). D i rect evidence for th is over lap has come from c e l l separat ion experiments where f e t a l l i v e r CFU-S have been f rac t ionated into two populat ions and the major i ty of the mixed colony forming c e l l s segregated into only one of these f r a c t i o n s (57). The a b i l i t y of CFU-GEMM to undergo se l f - renewal has a lso been documented (58). Evidence for a more d i r e c t r e l a t i o n s h i p between CFU-GEMM and CFU-S was obtained by the demonstration that mixed co lonies in v i t r o from both f e t a l l i v e r (59) and adult bone marrow (60) contain CFU-S. The existence of an in v i t r o colony forming c e l l more p r i m i t i v e than the CFU-GEMM has been suggested from the i d e n t i f i c a t i o n of unique co lon ies composed p r imar i l y of und i f fe ren t ia ted b last c e l l s in the mouse (61). The progeni tors of these co lonies were designated S - c e l l s ( 'stem' c e l l s ) and appeared to be more p r im i t i ve than CFU-GEMM s ince r e p l a t i n g of primary stem c e l l co lon ies revealed a high incidence of secondary stem c e l l co lon ies in a d d i t i o n to GEMM c o l o n i e s . The human equivalent of the S - c e l l has been reported to e x i s t in u m b i l i c a l cord blood (62) but i t s presence in adult hemopoietic t i ssue has not been documented. This may r e f l e c t the i n a b i l i t y of present cu l tu re condi t ions to st imulate the growth of the human S - c e l l . 10 ULTIMATE STEM CELL • — * Lymphopoiesis ERYTHROID PROGENITORS MEGAKARYOCYTE PROGENITORS GRANULOCYTE PROGENITORS FIGURE I Diagrammatic r e p r e s e n t a t i o n of the hiera r c h y of hemopoietic progenitor compartments c u r r e n t l y i d e n t i f i e d by colony assay procedures. From r e f (13). 11 Further evidence supporting the existence of a stem c e l l more immature than the CFU-GEMM comes from s t u d i e s with the long term bone marrow c u l t u r e system. Successful long-term c u l t u r e s were generated from marrow suspensions depleted of l a - p o s i t i v e c e l l s suggesting that, i n contrast to CFU-GEMM, the c e l l s i n i t i a t i n g long-term c u l t u r e s are la-ne g a t i v e (63-65). Successful long-term c u l t u r e s can a l s o be generated from marrow c e l l s treated w i t h the a l k y l a t i n g agent 4-hydroperoxy-cyclophosphamide (4-HC). Since CFU-GEMM are known to be h i g h l y s e n s i t i v e to t h i s drug (66,67), i t appears that another stem c e l l (not represented by CFU-GEMM) i s r e s i s t a n t to 4-HC, presumably because i t i s not i n a c t i v e c e l l c y c l e (66). This drug has a l s o been used i n the i n v i t r o purging of bone marrow during autologous bone marrow t r a n s p l a n t a t i o n . Bone marrow treated with 4-HC (at l e v e l s that are l e t h a l to CFU-GEMM) can r e c o n s t i t u t e f u l l hemopoietic f u n c t i o n i n s u p r a l e t h a l l y i r r a d i a t e d p a t i e n t s (68,69) suggesting that the stem c e l l r e s p o n s i b l e f o r hemopoietic r e c o n s t i t u t i o n i s 4-HC-resistant. 3) REGULATION OF STEM CELL DIFFERENTIATION A) Regulatory Molecules The development of i n v i t r o assays f o r hemopoietic progenitor c e l l s has provided a unique system f o r the i n v e s t i g a t i o n of the r e g u l a t i o n of hemopoiesis. The importance of such regulatory mechanisms i s p a r t i c u l a r l y evident when one considers the short h a l f l i v e s of the c i r c u l a t i n g p e r i p h e r a l blood c e l l s . The hemopoietic system i s a c e l l renewal system which balances c e l l 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 at rates p r o p o r t i o n a l to the demand f o r these f u l l y d i f f e r e n t i a t e d c e l l s and i s therefore r e s p o n s i b l e f o r maintaining the number of c i r c u l a t i n g c e l l s w i t h i n a narrowly defined range. The p r e c i s i o n and f l e x i b i l i t y of the marrow i n meeting ongoing blood c e l l 12 requirements and in responding to s p e c i f i c hemostatic s t ress impl ies that the p r o l i f e r a t i o n of th is t i ssue i s modulated by s e n s i t i v e b i o l o g i c a l c o n t r o l mechanisms. The regula t ion of th is developmental process i s thought to res ide in a group of hemopoietic growth fac tors and some i l l - d e f i n e d i n t e r a c t i o n s with marrow stromal c e l l s . Those growth fac tors involved in the regu la t ion of the ea r l y stages of hemopoiesis st imulate the p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of c e l l s capable of forming more than one blood c e l l type and have been termed mul t i l ineage growth f a c t o r s . Those c o n t r o l l i n g the l a t e r stages act on c e l l s with a more r e s t r i c t e d d i f f e r e n t i a t i o n program and are therefore considered to be l i n e a g e - s p e c i f i c growth fac tors (70). The Colony St imulat ing Factors The i d e n t i f i c a t i o n of a fac tor required for the p r o l i f e r a t i o n of granulocyte/macrophage progeni tors in s e m i - s o l i d medium provided the f i r s t candidate for a pathway-speci f ic regulator of hemopoiesis. The p r o l i f e r a t i o n of CFU-GM in v i t r o was shown to be dependent on a s u f f i c i e n t concentrat ion of a p ro te in re leased by feeder layer c e l l s (21,71) or condi t ioned medium from cu l tu res of these feeder layers (72,73). Further ana lys is of th is condi t ioned medium showed that i t s b i o l o g i c a l a c t i v i t y resided in a family of g lycopro te ins termed colony s t imula t ing fac tors or CSF's (74). The use of condi t ioned media to f a c i l i t a t e the in v i t r o growth of hemopoietic c e l l s has s ince provided the condi t ions in which other myeloid progeni tor c e l l types can be st imulated to form colonies (23). P u r i f i c a t i o n of the d i f f e r e n t b i o l o g i c a l a c t i v i t i e s present in th is condit ioned medium has revealed a s e r i e s of growth f a c t o r s which are progress ive ly r e s t r i c t e d in the i r b i o l o g i c a l a c t i v i t y and target c e l l s (75). I n t e r e s t i n g l y , the CSF's can be produced by most t i ssues but they are d is t ingu ished from each other in the i r unique s p e c i f i c i t y for subsets of hemopoietic c e l l s . GM-CSF, for example i s a mul t i l ineage growth 13 f a c t o r capable of s t imula t ing the p r o l i f e r a t i o n and development of granulocyte and macrophage progeni tor c e l l s . This fac tor has been i s o l a t e d from both lung t i s s u e (76) and pokeweed mitogen-stimulated spleen c e l l s (77), although i t s p rec ise r o l e in v ivo i s not c l e a r l y e s t a b l i s h e d . For rout ine human c e l l c u l t u r e purposes the usual source of th is fac tor i s leukocyte -condi t ioned medium (76) or p lacenta l -cond i t ioned medium (79). The growth fac tors G-CSF and M-CSF are more r e s t r i c t e d in the i r a c t i v i t i e s , s t imu la t ing predominantly subsets of granulocyte (80) or macrophage co lon ies (81) r e s p e c t i v e l y . The concept of l ineage and s t a g e - r e l a t e d growth fac tors i s not absolute however, s ince both GM-CSF (82) and G-CSF (83) have been shown to support the ear ly d i v i s i o n s of p lu r ipo ten t p recursors , although they lack the a b i l i t y to support the terminal p r o l i f e r a t i v e and d i f f e r e n t i a t i v e events in l ineages other than the granulocyte/macrophage pathway. In a d d i t i o n , G-CSF i s considered unique among the CSF c lasses in i t s a b i l i t y to induce terminal d i f f e r e n t i a t i o n in murine myeloid leukemia c e l l l i n e s (84). It i s now poss ib le to c l e a r l y d i s t i n g u i s h on both molecular and f u n c t i o n a l grounds, four major c lasses of CSF in the mouse: GM-CSF, G-CSF, M-CSF, and IL-3 (74,85). These fac tors have been p u r i f i e d to homogeneity (74,86-88) and in the case of GM-CSF and IL -3 , molecular ly cloned (89-91). Factors s t imu la t ing both eos inoph i l and megakaryocyte colony formation i n agar cu l tu res of bone marrow have a lso been descr ibed (92). In contrast to the murine system, human CSF's are not as wel l charac te r i zed , although human analogues with b i o l o g i c a l a c t i v i t y s i m i l a r to mouse GM-CSF (93), M-CSF (94), G-CSF (95) and IL-3 (96) have now been i d e n t i f i e d and molecular c lon ing of these fac to rs i s expected in the near fu ture . 14 E r y t h r o p o i e t i n and IL-3 Since CFU-E show a marked dependence on the presence of e r y t h r o p o i e t i n (Epo) in the cu l ture medium, i t has long been assumed that c e l l s at th is l e v e l 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 are responsive to Epo (31). In contrast to the C S F ' s , however, th is hormone has c l e a r l y been demonstrated to have a p h y s i o l o g i c a l ro le in s t imula t ing ery thro id d i f f e r e n t i a t i o n in v ivo (97). The major e f fec t of Epo i s genera l ly considered to be from the mature BFU-E stage to the nondiv id ing ery throblast stage, although i t i s not c e r t a i n whether i t ac ts as a mitogenic st imulus or a s u r v i v a l fac tor (31). The search for fac tors other than Epo which might in f luence the ea r l y stages of e r y t r o p o i e s i s has i d e n t i f i e d a molecule ( IL-3) that may act at the l e v e l of p lur ipotent progenitor c e l l s (98) and ear ly committed e r y t h r o c y t i c precursors (p r im i t i ve BFU-E) (29). The e f fec ts of th is fac tor appear to be a d d i t i v e with those of M-CSF and ery thropo ie t in in that IL -3 , in combination with Epo or CSF-1 (M-CSF) y i e l d s much la rger co lonies conta in ing e r y t h r o i d or monocytic c e l l s than Epo or CSF-1 alone (70). A number of i n v e s t i g a t o r s have i d e n t i f i e d fac to rs with s i m i l a r b i o l o g i c a l a c t i v i t i e s and current evidence suggests that the a c t i v i t y of these fac tors res ides in the same macromolecule. These f a c t o r s inc lude : IL-3 (99), burs t -p romot ing -ac t iv i t y (BPA) (100), hematopoiet ic growth fac tor (HCGF) (101), P - c e l l growth fac tor (102), and mul t i -CSF (103). The mechanism of ac t ion of th is molecule has not been i d e n t i f i e d but i s an area cur rent ly under ac t ive i n v e s t i g a t i o n (104). For i n  v i t r o c lonogenic assays the source of IL-3 i s usua l ly condi t ioned medium from agar or l e c t i n - s t i m u l a t e d l e u k o c y t e - r i c h f rac t ions of human b lood, or from pokeweed mitogen-st imulated mouse spleen c e l l s for mouse progen i tors . A v a i l a b l e evidence suggests that i t i s the T-lymphocytes in these t i ssues that produce th is fac tor in response to e i ther mitogens or s p e c i f i c antigens (105,106). 15 Detect ion of another mul t i l ineage growth fac tor has recent ly been repor ted . I n i t i a l s tudies i d e n t i f i e d a fac tor in condi t ioned medium that conferred upon very p r imi t i ve c e l l s the a b i l i t y to respond to CSF-1 (M-CSF) (107). Th is f a c t o r , termed hemopoietin 1, has recent ly been p u r i f i e d and shown to be d i s t i n c t from IL-3 (108). The e f fec t of th is fac tor i s s y n e r g i s t i c , r equ i r ing the presence of other growth fac tors and having no c o l o n y - s t i m u l a t i n g a c t i v i t y by i t s e l f . It seems l i k e l y that the cont ro l of hemopoiesis i s accomplished by appropr ia te combinations of p o s i t i v e and negative i n f l u e n c e s . The p o s s i b l e involvement of a va r i e ty of i n h i b i t o r y fac tors that may regulate hemopoiesis i s a c o n t r o v e r s i a l issue and c o n f l i c t i n g reports have been obta ined. A number of p o t e n t i a l phys io log ic i n h i b i t o r s have been i d e n t i f i e d that are thought to act e i t h e r d i r e c t l y on progenitor populat ions or i n d i r e c t l y by modulating CSF r e l e a s e . D e f i n i t i v e evidence that such i n h i b i t o r s play a r o l e in the regu la t ion of hemopoiesis in v ivo i s s t i l l l ack ing however. There can be no doubt that there are a number of d i s t i n c t molecular spec ies that are necessary for the growth and d i f f e r e n t i a t i o n of hemopoietic c e l l s in c u l t u r e . With the exception of Epo however, the na tura l p h y s i o l o g i c a l ro le of these f a c t o r s , both s t imula tory , and i n h i b i t o r y , in the r e g u l a t i o n of the c e l l types that they appear to in f luence in v i t r o has yet to be e s t a b l i s h e d . Whether inappropr iate expression of these growth f a c t o r s plays a ro le in leukemogenesis i s a quest ion that should be answered i n the near fu tu re . (B) Regulatory Role of the Microenvironment The int imate s t r u c t u r a l a s s o c i a t i o n between hemopoietic and stromal c e l l s in the marrow suggests that the stromal c e l l s may in f luence hemopoietic 16 development poss ib ly by supplying the necessary c e l l matrix and d i f f u s i b l e growth fac tors required for hemopoiesis. The prec ise nature of these i n t e r a c t i o n s i s unknown and experimental evidence for such a regulatory r o l e i s l a r g e l y c i r c u m s t a n t i a l . The i n i t i a l observat ions that CFU-S developing in the spleen d i s p l a y a predominance of e r y t h r o p o i e s i s , while those developing in the bone marrow d i s p l a y a predominance of granulopoies is suggested that CFU-S d i f f e r e n t i a t i o n i s in f luenced by i t s microenvironment (109). Transplanta t ion of marrow stroma in to the spleen or the t ransplanta t ion of spleen stroma subcutaneously, demonstrated that each type of organ stroma regenerated i t s d i s t i n c t i v e a b i l i t y to support hemopoiesis and cont ro l the d i f f e r e n t i a t i o n of p lu r ipo ten t stem c e l l s . The most c i t e d example of a defect in the i n t e r a c t i o n between stroma and hemopoiesis i s the g e n e t i c a l l y determined macrocytic anemia in mice c a r r y i n g the S tee l mutation ( S l / S l ^ ) . The hemopoietic abnormality in these mice appears to r e f l e c t a stromal defect rather than a stem c e l l defect s ince the anemia may be cured by the t ransplanta t ion of a normal spleen but not by t ransp lanta t ion of marrow stem c e l l s (110). An approach to the ana lys is of stromal and hemopoietic c e l l i n t e r a c t i o n s has been the development of a l i q u i d cu l ture system that supports the p r o l i f e r a t i o n of CFU-S for severa l weeks. The a b i l i t y of these long-term bone marrow cu l tures to maintain the se l f - renewal and 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 CFU-S i s thought to r e f l e c t the presence of a complex adherent layer that presumably serves as the in v i t r o equivalent of the marrow stroma (111). In conf i rmat ion of the in v ivo studies on the S l / S l d mouse, Dexter and Moore (112) demonstrated that Steel -hemopoiet ic c e l l s could be supported by a normal marrow-derived adherent l a y e r , but the S tee l -der ived adherent - layer was 17 incapable of support ing long term hemopoiesis. Recent s tudies of CFU-S s t imula tory and i n h i b i t o r y a c t i v i t i e s in the hemopoietic t i ssues of S i mice have suggested that the S i defect may res ide in the production of a p r o l i f e r a t i o n st imulator from an as yet u n i d e n t i f i e d regula tor -produc ing c e l l (113). For the purpose of th is thesis i t i s s u f f i c i e n t to say that there i s now an accumulating body of evidence to support the regulatory ro le of the microenvironment on hemopoiesis. Although the nature of these i n t e r a c t i o n s i s l a r g e l y unknown, i t i s be l ieved that c lose range c e l l u l a r i n t e r a c t i o n s , the e labora t ion of the prev ious ly discussed growth f a c t o r s , and perhaps a host of short range stromal c e l l - d e r i v e d fac tors that are as yet u n i d e n t i f i e d , may a l l be invo lved . Charac te r i za t ion of the c e l l s comprising the stroma and i t s in v i t r o analogue (114) w i l l be important in he lp ing to e luc ida te the mechanism of these i n t e r a c t i o n s . (C) Theor ies of Stem C e l l Commitment Two theor ies are commonly c i t e d to expla in how stem c e l l se l f - renewa l and d i f f e r e n t i a t i o n i s regulated. The f i r s t model, proposed by Curry and T r e n t i n (109,115) i s based on h i s t o l o g i c a l s tudies of the composition of spleen co lon ies and proposes that commitment of p lur ipotent stem c e l l s i s a r e s u l t of s p e c i f i c induct ive microenvironmental s igna ls surrounding the stem c e l l s . Th is theory f inds support in s tudies demonstrating a regulatory r o l e of the microenvironment on hemopoiesis (see previous sec t ion on the microenvironment). It i s a lso poss ib le that d i f f u s i b l e growth fac tors such as the CSF 's may in f luence these commitment d e c i s i o n s . In experiments us ing paired daughter c e l l s of i n d i v i d u a l granulocyte-macrophage progen i to rs , Metcalf showed that GM-CSF can d i r e c t l y in f luence both the rate of 18 proliferation and the differentiation pathway entered by the progeny of these granulocyte/macrophage precursor cells (116). The second theory, developed by T i l l et al (117) proposes that the decision between self-renewal and commitment to differentiate is a stochastic event, and that only the probabilities of such events can be influenced by environmental factors. This model was i n i t i a l l y developed to explain the marked variation in the distribution of CFU-S, CFU-C, BFU-E, and CFU-E in spleen colonies (2). Such variation is consistent with various determination events occurring at random. An extension of this model proposes that stem c e l l commitment is governed by progressive and stochastic restriction in the differentiation potential of stem cells (118). This concept is supported by the identification of bipotent progenitors such as erythroid-eosinophil (119), granulocyte-macrophage (22), erythroid-megakaryocyte (60,120), neutrophil-erythroid (121), and oligopotent progenitors containing two or three lines of differentiation (43,50,122). Further support for the stochastic concept comes from analysis of the differentiation of hemopoietic colonies derived from the two progeny of a single parent progenitor c e l l . The single progenitors used in these studies were isolated using a micromanipulation technique from blast c e l l colonies cultured from the spleens of 5-fluorouracil-treated mice (61). Eighteen to 24 hours later the paired progenitors were separated, replated, and the two colonies derived from each paired progenitor were analyzed for the presence of various combinations of lineages. The results clearly documented dissimilar patterns of differentiation in the two daughter cells, providing strong evidence for the stochastic model of stem c e l l differentiation (123). An extension of these studies also revealed disparate differentiation in colonies derived from paired progenitors that were replated sequentially (124). 19 Although these r e s u l t s are consistent with the basic p r i n c i p l e of a s t o c h a s t i c process of d i f f e r e n t i a t i o n , some skewing in l ineage expression was noted and th is might r e f l e c t the in f luence of some e x t r i n s i c f a c t o r . In the human system, paired daughter c e l l s from My-10 a n t i g e n - p o s i t i v e cord blood c e l l s were a lso recent ly shown to produce co lonies c o n s i s t i n g of d i s s i m i l a r l ineage combinations, thus support ing the studies in the mouse (125). Of course these two models are not n e c e s s a r i l y mutually e x c l u s i v e . One can combine elements of both models and suggest that the d e c i s i o n between stem c e l l se l f - renewa l and d i f f e r e n t i a t i o n i s b a s i c a l l y a s t o c h a s t i c process that can be modulated by the hemopoietic microenvironment. 4) NEOPLASTIC DISORDERS OF MYELOPOIESIS (A) The Chronic M y e l o p r o l i f e r a t i v e Diseases and the Acute Leukemias As ou t l ined in the previous sect ions the hemopoietic system can be represented by a l ineage diagram i l l u s t r a t i n g the d i f f e r e n t i a t i o n pathways that begin at the p lur ipotent stem c e l l l e v e l and end at the l e v e l of the mature e f f e c t o r c e l l (F igure I ) . A var ie ty of neop las t i c d isorders a f f e c t the hemopoietic system and they manifest themselves by var ious degrees of impairment of th is d i f f e r e n t i a t i o n scheme. The broadest c l a s s i f i c a t i o n of the leukemias contains two groups: the acute and chronic leukemias. Each of these i s i n turn categor ized as e i ther lymphoid or myeloid and fur ther s u b d i v i s i o n involves a more de ta i l ed ana lys is of the leukemic c e l l phenotypes. The chronic mye lopro l i f e ra t i ve d isorders (MPD's) are a group of hematologic neoplasms character ized by expansion of a l l three myeloid compartments in the bone marrow. In the per iphera l blood however there i s u s u a l l y a predominance of only one l ineage and th is contr ibutes to the c l i n i c a l p resenta t ion . Granulocytes are increased in chronic myelogenous 20 leukemia (CML), red c e l l s are increased i n polycythemia vera (PV), and p l a t e l e t s are increased i n e s s e n t i a l thrombocytosis (ET). The use of G6PD isoenzyme a n a l y s i s has demonstrated that a l l of the MPD's i n v o l v e the c l o n a l expansion of a n e o p l a s t i c stem c e l l which has retained a v a r i a b l e c a p a c i t y f o r g r a n u l o c y t i c , monocytic, e r y t h r o c y t i c , and megakaryocytic d i f f e r e n t i a t i o n (126). Accumulating evidence a l s o suggests that the n e o p l a s t i c stem c e l l i n CML, PV, and ET i s capable of d i f f e r e n t i a t i o n along the B c e l l l i n e a g e (127). Although a l l myeloid elements at the time of diagnosis are of the abnormal clone, the a b i l i t y of these c e l l s to continue to d i f f e r e n t i a t e i n t o f u n c t i o n a l end c e l l s e x p l a i n s why the chronic course of these d i s o r d e r s can be maintained f o r s e v e r a l years with minimal chemotherapy. The n a t u r a l progression of CML i s somewhat more aggressive than the other MPD's i n that t r a n s i t i o n to an acute phase occurs on average three years f o l l o w i n g i n i t i a l d i a g n o s i s . This conversion i s thought to inv o l v e the c l o n a l e v o l u t i o n of a c e l l w i t h i n the malignant clone that u l t i m a t e l y leads to the rapid accumulation of n o n d i f f e r e n t i a t i n g b l a s t c e l l s that possess c h a r a c t e r i s t i c s of e i t h e r acute myelogenous leukemia (AML) (60% of cases) or acute lymphoblastic leukemia (ALL) (30% of cases) (128). I t i s important to d i s t i n g u i s h between myeloid and lymphoid b l a s t c r i s i s s ince the l a t t e r often responds to chemotherapy with v i n c r i s t i n e and prednisone and the former i s l a r g e l y r e f r a c t o r y to treatment (129). T r a n s i t i o n to an acute phase a l s o occurs i n PV, ET, and MF although l e s s f r e q u e n t l y ( l e s s than 10% of cases) (124). In c ontrast to the ch r o n i c leukemias, the acute leukemias encompass a group of r e l a t e d d i s o r d e r s that are a l l associated with replacement of normal bone marrow by n o n d i f f e r e n t i a t i n g , and consequently n o n f u n c t i o n a l , c e l l s that appear blocked at a p a r t i c u l a r l e v e l of maturation. F a i l u r e of normal hemopoiesis i s thus the most serious pathophysiologic consequence of acute 21 leukemia. The i d e n t i f i c a t i o n of a 'preleukemia phase has been i d e n t i f i e d i n some o l d e r p a t i e n t s and t h i s i s c h a r a c t e r i z e d by chronic marrow i n s u f f i c i e n c y e v o l v i n g over time i n t o acute leukemia (131). The p r e c i s e frequency of the preleukemic phase i s unknown since many p a t i e n t s that present with an acute leukemia may have p r e v i o u s l y experienced asymptomatic hematologic a b n o r m a l i t i e s . The excessive production of nonfunctional immature c e l l s i n AML i s i n d i r e c t contrast to CML where a large population of f u l l y d i f f e r e n t i a t e d c e l l s i s produced. Since CML u l t i m a t e l y evolves i n t o an AML-like leukemia, the abnormal myelopoiesis c h a r a c t e r i s t i c of these two di s o r d e r s w i l l be considered i n f u r t h e r d e t a i l . (B) Chronic Myelogenous Leukemia (CML)  Leve l of Stem C e l l involvement The most compelling evidence that CML i s a c l o n a l d i s o r d e r of hemopoeitic stem c e l l s comes from the a n a l y s i s of G6PD heterozygotes with t h i s disease. The genetic locus f o r G6PD i s on the p o r t i o n of the X-chromosome that i s randomly i n a c t i v a t e d e a r l y on i n embryonic development (132). The G6PD gene has s e v e r a l a l l e l e s that s p e c i f y isoenzymes that can be e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h e d . Since only one G6PD gene i s a c t i v e i n each somatic c e l l , normal c e l l s i n females who are heterozygous f o r the A and B a l l e l e s c o n s i s t of two populations, one s y n t h e s i z i n g isoenzyme A and the other isoenzyme B. On the other hand, tumours that o r i g i n a t e i n a s i n g l e c e l l would express only a s i n g l e enzyme phenotype. The c l o n a l theory of CML i s s t r o n g l y supported by s t u d i e s w i t h t h i s enzyme marker. Females who are heterozygous f o r G6PD and who have CML express double-enzyme phenotypes i n nonhemopoietic t i s s u e s but only one type of enzyme i n CML granulocytes (133). Although i t i s conceivable 22 that many c e l l s were a l te red i n i t i a l l y , only one clone i s evident by the time the d isease i s c l i n i c a l l y over t . This leukemic populat ion thus o r i g i n a t e d from a s i n g l e c e l l that possessed a p r o l i f e r a t i v e advantage. Cytogenet ic s tudies have a lso been used to i n f e r the monoclonal i ty of CML. The i d e n t i f i c a t i o n of the Ph^ chromosome in 90-100% of d i v i d i n g marrow c e l l s has been taken as evidence for a s i n g l e c e l l o r i g i n . Th is type of evidence i s l e s s convincing than isoenzyme studies because i t r e l i e s on the i d e n t i f i c a t i o n of a chromosomal abnormality that i s very s p e c i f i c for CML. Indeed, there i s evidence to suggest that the ear ly events in the pathogenesis of CML predispose to the development of the Phi chromosome and that the a c q u i s i t i o n of the Phi leads to a fur ther growth advantage (134). Isoenzyme and cytogenet ic s tudies have been used to determine 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 c e l l that i n i t i a l l y acquired the s e l e c t i v e growth advantage. I d e n t i f i c a t i o n of the Ph^ chromosome or a monoclonal pat tern of G6PD isoenzyme expression in granulocytes , monocytes, macrophages, e r y t h r o c y t e s , megakaryocytes, e o s i n o p h i l s , and basophi ls has demonstrated that CML i s a c l o n a l d isorder of a p lur ipotent hemopoietic stem c e l l common to these pathways (8,10,135-137). Notably, the Ph^ chromosome i s absent from bone marrow f i b r o b l a s t s and other mesenchymal t issues (138) and these c e l l s express a double isoenzyme phenotype (133). This type of ana lys is has been combined with in v i t r o assays for c lonogenic progeni tors and has demonstrated that CFU-C, BFU-E, CFU-E and CFU-GEMM-derived co lon ies in CML pat ients possess both the P h 1 chromosome (139-141) and a s i n g l e G6PD enzyme i d e n t i c a l to the i r c i r c u l a t i n g myeloid c e l l s (142-144). CML thus appears to involve a multipotent myeloid stem c e l l . That th is stem c e l l i s capable of lymphoid as wel l as myeloid d i f f e r e n t i a t i o n i s 23 c o n s i s t e n t with s e v e r a l c l i n i c a l observations. The i d e n t i f i c a t i o n of the Phi-chromosome (9) and the d e t e c t i o n of a s i n g l e isoenzyme patte r n (145) i n the B-lymphocytes of p a t i e n t s with CML provides strong support f o r the involvement of a s u b s t a n t i a l proportion of the B-lineage, although not a l l B c e l l s a r i s e from the CML clone (134). In confirmation of the involvement of the B-lineage, Epstein-Barr v i r u s transformed B-lymphoblastoid l i n e s were e s t a b l i s h e d from a s i n g l e patient with CML and a l l c e l l l i n e s that were Phi chromosome p o s i t i v e a l s o expressed a monoclonal patt e r n of the G6PD enzyme type present i n the leukemic myeloid c e l l s (134). The extent to which T-lymphocytes are involved i n CML has h i s t o r i c a l l y been more d i f f i c u l t to a s c e r t a i n . Attempts to demonstrate the Phi chromosome i n p e r i p h e r a l blood T - c e l l s from CML p a t i e n t s have g e n e r a l l y been unsu c c e s s f u l and the m a j o r i t y of the T - c e l l s appear to be p o l y c l o n a l by G6PD a n a l y s i s , although T - c e l l s i n p a t i e n t s with poorly c o n t r o l l e d disease may be members of the n e o p l a s t i c clone (145). I t i s p o s s i b l e that small numbers of P h i - p o s i t i v e T - c e l l s are present but they are undetectable due to the predominance of normal l o n g - l i v e d T - c e l l s or T - c e l l r e s t r i c t e d stem c e l l s that antedated the development of the disease. Consistent with the hypothesis that CML r e s u l t s from the transformation of a stem c e l l with both myeloid and lymphoid d i f f e r e n t i a t i o n p o t e n t i a l i s the existence of myeloid and lymphoid b l a s t c r i s i s i n the terminal phase of CML. Although the vast m a j o r i t y of lymphoid b l a s t c r i s e s possess a preB phenotype ( i n c l u d i n g immunoglobulin gene rearrangement) (146,147), s e v e r a l cases of T-lymphoid b l a s t c r i s e s have r e c e n t l y been reported (142-144) suggesting that, at l e a s t i n some cases the disease may i n v o l v e a stem c e l l capable of T-lymphocyte d i f f e r e n t i a t i o n . 24 E t i o l o g y and Pathogenesis That CML a r i s e s from a p r i m i t i v e progenitor that r e t a i n s the c a p a c i t y to produce and replace d i f f e r e n t i a t e d myeloid and p o s s i b l y lymphoid c e l l s i s now w e l l e s t a b l i s h e d . The p r e c i s e nature of the i n i t i a l transformation event that has such a marked e f f e c t on myelopoiesis remains obscure. I t i s known that CML may develop f o l l o w i n g exposure to r a d i a t i o n (151) although i n the vast m a j o r i t y of cases no environmental f a c t o r s have d e f i n i t e l y been i m p l i c a t e d . Recently i t was reported that the Ph^ chromosome recurred i n donor c e l l s f o l l o w i n g an a l l o g e n e i c bone marrow t r a n s p l a n t a t i o n (152). This would suggest p e r s i s t e n c e of the f a c t o r that i n i t i a l l y induced the disease, thus r e i n d u c i n g leukemia i n the donor c e l l s p o s s i b l y by the t r a n s f e c t i o n of oncogenic m a t e r i a l from r e s i d u a l leukemic c e l l s to the donor c e l l s . D onor-cell relapse f o l l o w i n g a l l o g e n e i c bone marrow t r a n s p l a n t a t i o n i s a rare event however (153), suggesting that i f t r a n s f e c t i o n i s indeed the mechanism by which leukemogenesis i s i n i t i a t e d i n donor c e l l s , the e f f i c i e n c y of t h i s process must be very low. Genetic I n s t a b i l i t y and the P h i l a d e l p h i a chromosome The l i k e l i h o o d that tumour-associated chromosomal changes play an important r o l e i n leukemogenesis i s h i g h l y suggested by t h e i r nonrandom nature. CML was the f i r s t human neoplasm to be associated with a s p e c i f i c and r e p r o d u c i b l e chromosomal abnormality, the P h i l a d e l p h i a (Ph^-) chromosome. O r i g i n a l l y described by Nowell and Hungerford (154), the Ph 1 chromosome i s now known to t y p i c a l l y r e s u l t from 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 (155). The presence of t h i s marker i n at l e a s t 90% of p a t i e n t s w i t h CML (156) suggests that the a c q u i s i t i o n of the Phi may represent an important event i n the pathogenesis of CML. This r e c i p r o c a l t r a n s l o c a t i o n has been shown to i n v o l v e two oncogenes. C - s i s , the c e l l u l a r homologue of the Simian sarcoma 25 v i r u s transforming gene i s normally located on chromosome 22, and c - a b l , the c e l l u l a r homologue of the murine Abelson leukemia v i r u s oncogene i s l o c a t e d on chromosome 9. The Ph 1 t r a n s l o c a t i o n moves the c-abl locus from chromosome 9 to chromosome 22 and the c - s i s locus moves i n the opposite d i r e c t i o n (157,158). The most consi s t e n t f i n d i n g i n CML, i n c l u d i n g those cases that i n v o l v e complex t r a n s l o c a t i o n s , i s the movement of m a t e r i a l from 9 to 22 (159). In a d d i t i o n , t h i s c - a b l - c o n t a i n i n g region always appears to j o i n chromosome 22 at a h i g h l y s p e c i f i c breakpoint c l u s t e r region (bcr) (160), suggesting that the c-abl locus may play the most important r o l e i n the development of the disease. The t r a n s l o c a t e d c-abl gene i s rearranged and a m p l i f i e d i n the CML c e l l l i n e K562 (161), and an anomalous 8kb t r a n s c r i p t has been detected i n both K562 c e l l s and hemopoietic c e l l s from a l l P h i - p o s i t i v e CML p a t i e n t s thus f a r examined (162,163). This CML-specific 8kb RNA has been shown to be a fused t r a n s c r i p t of the a b l and bcr genes (164). The c-abl gene product produced by these Ph 1 - p o s i t i v e c e l l s a l s o appears to be abnormal i n that i t possesses a t y r o s i n e kinase a c t i v i t y s i m i l a r to the v-abl p r o t e i n (the normal c e l l u l a r gene product has no detectable t y r o s i n e kinase a c t i v i t y ) (165). Presumably t h i s p r o t e i n i s a h y b r i d p r o t e i n t r a n s l a t e d from the anomalous RNA f u s i o n product. Together these r e s u l t s suggest that t r a n s l o c a t i o n of the c-abl locus may lead to rearrangement and a m p l i f i c a t i o n of the gene r e s u l t i n g i n the production of a hybrid p r o t e i n that has kinase a c t i v i t y c h a r a c t e r i s t i c of i t s transforming counterpart i n the Abelson leukemia v i r u s . The abnormal a b l t r a n s c r i p t has been reported to be absent i n Ph^-negative CML (166), although one p a t i e n t w i t h apparently Ph^-negative CML r e c e n t l y showed rearrangement of the bcr locus r e s u l t i n g from a j o i n t t r a n s l o c a t i o n of bcr and c-abl to chromosome 12 (167). I t would therefore appear that the proximity of the 26 c-abl gene to the bcr locus i s an important step i n the pathogenesis of CML, although presumably other mechanisms are involved i n cases of Phi-negative CML where no rearrangements of the a b l and bcr genes are detected. I n t e r e s t i n g l y , a p r e l i m i n a r y report has suggested that the Phi chromosome of childhood ALL may a c t u a l l y be d i s t i n g u i s h e d from that of CML by the absence of breakpoints w i t h i n the bcr region (168). Since normal maturing myeloid c e l l s express c - a b l , the presence of an a l t e r e d c-abl oncogene product i n CML c e l l s i s an i n t r i g u i n g observation, although why t h i s a l t e r a t i o n has such a d r a s t i c e f f e c t on myelopoiesis i s not yet known. The t r a n s f e r of m a t e r i a l i n the reverse d i r e c t i o n from chromosome 22 to 9 i s not associated with the t r a n s c r i p t i o n a l a c t i v a t i o n of the c - s i s gene, and therefore i t i s u n l i k e l y that t h i s gene i s invo l v e d i n CML (158) An important question regarding the e t i o l o g y of CML i s whether the a c q u i s i t i o n of the Phi chromosome i s a primary or secondary event. Compatible with the view that i t i s secondary are reports of o c c a s i o n a l p a t i e n t s w i t h CML whose marrow c e l l s are Phi-negative at pr e s e n t a t i o n but l a t e r become P h i - p o s i t i v e (169-171). Fialkow et a l (134) provided more d i r e c t evidence by demonstrating the existence of Phi-negative c e l l s d e rived from the leukemic clone using a combination of G6PD and cytogenetic a n a l y s i s . In these s t u d i e s the B-lymphocytes from a patient with CML were transformed w i t h the Eps t e i n - B a r r v i r u s (EBV) and a number of B - c e l l l i n e s , some P h i - p o s i t i v e and some Phi-negative, were e s t a b l i s h e d . Of the Phi-negative l i n e s only those expressing the type B isoenzyme c h a r a c t e r i s t i c of the p a t i e n t s leukemia were g e n e t i c a l l y unstable. Phi-negative c e l l l i n e s that expressed the type A enzyme d i d not have k a r y o t y p i c a b n o r m a l i t i e s . These r e s u l t s suggest that the formation of the Phi chromosome i s not the s o l e event i n the pathogenesis of CML. I t may w e l l be that at l e a s t two steps are required i n the e v o l u t i o n of 27 CML; the f i r s t i n v o l v i n g a h e r i t a b l e change at the p l u r i p o t e n t stem c e l l l e v e l and the second inducing the Ph* chromosome i n the descendants of t h i s c e l l (134). The i n i t i a l transformation event may i n f a c t predispose a c e l l to the subsequent a c q u i s i t i o n of the Ph^ - chromosome and t h i s may i n turn confer a p r o l i f e r a t i v e advantage over both normal stem c e l l s and n e o p l a s t i c stem c e l l s that are Ph^-negative. The chronic phase of CML i s unstable and at some point the disease progresses to an aggressive acute leukemia that i s r a p i d l y f a t a l . Transformation to the acute phase i s u s u a l l y accompanied by the development of a d d i t i o n a l chromosomal abnormalities superimposed on the Ph^ chromosome. The mechanism underlying t h i s progressive genetic i n s t a b i l i t y i s unknown but i t has been suggested that a c t i v a t i o n of a genetic t r a n s p o s i t i o n system may account f o r the a c c e l e r a t i n g chromosomal i n s t a b i l i t y c h a r a c t e r i s t i c of t h i s and many other n e o p l a s t i c d i s o r d e r s (172). D i f f e r e n t i a t i o n of the Ne o p l a s t i c Clone Leukemic myeloid c e l l s appear to mature normally during the ch r o n i c phase of CML, although s u b t l e abnormalities i n granulocyte (173,174) and p l a t e l e t (175) f u n c t i o n have been reported. Normal d i f f e r e n t i a t i o n i s a l s o observed i n v i t r o where c l o n a l l y - d e r i v e d P h ^ - p o s i t i v e progenitors are capable of d i f f e r e n t i a t i n g i n t o c o l o n i e s that are morphologically i n d i s t i n g u i s h a b l e from c o l o n i e s derived from nonclonal progenitors (176). Despite the normal morphological appearance of CML c o l o n i e s a number of ab n o r m a l i t i e s are evident. Studies have demonstrated that the frequency of a l l c l a s s e s of c i r c u l a t i n g progenitors i s g r e a t l y elevated i n CML (177-180) and t h i s increase represents expansion of the leukemic clone. Although the c a r d i n a l f e a t u r e of CML i s an increase i n the number of c i r c u l a t i n g granulocytes, e r y t h r o c y t i c and megakaryocytic progenitor compartments are 28 enlarged to the same extent as the granulocyte progenitor compartment ( s e v e r a l thousand f o l d ) suggesting that there i s no p r e f e r e n t i a l commitment to the granulocyte l i n e a g e i n CML (177). S i m i l a r l y , there appears to be no li n e a g e s p e c i f i c i t y i n the c y c l i n g s t a tus of CML progenitors (181). I t has been reported that 70% of the CFU-C from CML pa t i e n t s have a den s i t y < 1.062 g/cm2 whereas only 5% of c o n t r o l CFU-C have t h i s d ensity (176). I t i s not known i f t h i s low d e n s i t y i s i n t r i n s i c to the CML progenitors (126). The a b i l i t y of CML progenitors to execute t h e i r normal d i f f e r e n t i a t i o n programs i s not complete however, since a number of CML p a t i e n t s have been shown to e x h i b i t a v a r i a b l e capacity f o r Epo-independent e r y t h r o i d d i f f e r e n t i a t i o n i n v i t r o (182). Terminal 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 may a l s o be adversely a f f e c t e d since many pa t i e n t s are anemic i n s p i t e of the elevated l e v e l s of e r y t h r o i d progenitors (181). This might r e f l e c t a defect i n t r i n s i c to the progenitors or a secondary e f f e c t of the leukemia. Suppression of Normal Hemopoiesis Ph 1 -negative c e l l s have been found to p e r s i s t i n the marrow of p a t i e n t s w i t h CML but t h e i r growth i s apparently suppressed by the malignant clone i n v i v o (183,184). Several i n v e s t i g a t o r s have explored the p o s s i b i l i t y that leukemic c e l l s may suppress the p r o l i f e r a t i o n of normal stem c e l l s and thus confer a growth advantage upon the leukemic progenitors. Thymidine s u i c i d e experiments have demonstrated that the c i r c u l a t i n g progenitors i n CML are i n a c t i v e c e l l c y c l e whereas i n normal i n d i v i d u a l s very few are c y c l i n g (181,185). This suggests a defect i n CML that prevents the progenitors from l e a v i n g the c e l l c y c l e and ent e r i n g G Q - I t i s l i k e l y that the o v e r a l l p r o l i f e r a t i v e advantage of the leukemic clone r e f l e c t s a combination of the expanded progenitor compartment, the c e l l c y c l e a c t i v i t y of these p r o g e n i t o r s , and suppression of normal hemopoiesis by leukemia-derived i n h i b i t o r y f a c t o r s . 29 The r o l e of known i n h i b i t o r s i n the observed suppression of normal hemopoiesis i s unclear, although defects i n a v a r i e t y of regulatory networks that may i n v o l v e chalones, c e l l - a s s o c i a t e d i n h i b i t o r s , i s o f e r r i t i n s and prostaglandins have been proposed (173,186-188). (C) Acute Myelogenous Leukemia  Lev e l of Stem C e l l Involvement In AML the n e o p l a s t i c c e l l s are unable to f o l l o w normal d i f f e r e n t i a t i o n pathways although some features of g r a n u l o p o i e t i c d i f f e r e n t i a t i o n are u s u a l l y evident i n the leukemic b l a s t c e l l s . A number of nonrandom chromosomal ab n o r m a l i t i e s have been i d e n t i f i e d i n AML c e l l s and although these changes are not as s p e c i f i c as the Phi i s f o r CML, the data w i t h i n a given p a t i e n t i n d i c a t e that the disease i s c l o n a l at the time of study. Studies of G6PD heterozygotes with AML confirm these observations of c l o n a l i t y (189). Moreover, the i d e n t i f i c a t i o n of a s i n g l e G6PD isoenzyme i n p l a t e l e t s , e r y t h r o c y t e s , and leukemic b l a s t s suggests that the b l a s t population i n AML o r i g i n a t e s from a multipotent stem c e l l common to these lineages (189). In younger p a t i e n t s the disease appears to be expressed i n c e l l s w i t h d i f f e r e n t i a t i o n r e s t r i c t e d to the granulocyte-macrophage pathway. On the bas i s of these f i n d i n g s Fialkow et a l have suggested that there i s heterogeneity i n the stem c e l l o r i g i n of AML: involvement of a multipotent stem c e l l i n e l d e r l y p a t i e n t s and a granulocyte-macrophage r e s t r i c t e d stem c e l l i n c h i l d r e n . This was based on a small number of p a t i e n t s however and may not n e c e s s a r i l y r e f l e c t true age-related d i f f e r e n c e s i n the disease. Further evidence was r e c e n t l y obtained f o r the heterogeneity of AML i n two a d u l t AML p a t i e n t s who expressed a s i n g l e G6PD isoenzyme i n t h e i r monocytic b l a s t s (190). A l l other myeloid c e l l populations expressed a double 30 enzyme phenotype, suggesting that in these pat ients transformation occurred at the l e v e l of a progenitor with d i f f e r e n t i a t i o n r e s t r i c t e d to monocytic d i f f e r e n t i a t i o n . Immunoglobulin heavy chain gene rearrangements were recen t ly reported in the b las t c e l l s from two of fourteen c h i l d r e n with AML, suggest ing that , at l a s t in some cases, AML may a lso involve stem c e l l s capable of B lymphoid d i f f e r e n t i a t i o n (191). More convincing evidence for B - c e l l involvement i s a recent report of two AML pa t ien ts , one with r e s t r i c t e d (g ranu locy t ic ) and the other with mult ipotent ( e r y t h r o c y t i c , g r a n u l o c y t i c , and megakaryocyte) d i f f e r e n t i a t i v e expression of the involved stem c e l l s (192). G-6PD a n a l y s i s of EBV-transformed B - c e l l s from the pat ient whose abnormal clone showed mult ipotent expression showed involvement of the B - l ineage . In c o n t r a s t , evidence for B c e l l involvement was not detected in the pat ient whose abnormal clone showed r e s t r i c t e d express ion. These f ind ings underscore the heterogeneity of stem c e l l involvement in AML. An a l t e r n a t i v e explanat ion i s that the disease always o r ig ina tes in a mult ipotent stem c e l l , and that in some cases i t i s not capable of d i f f e r e n t i a t i n g down one or more l i n e a g e s . Never the less , the d i s c r i m i n a t i o n between AML with r e s t r i c t e d or mult ipotent d i f f e r e n t i a t i v e expression may r e f l e c t d i f fe rences in e t i o l o g y and pathogenesis, and may therefore have future prognost ic and therapeut ic i m p l i c a t i o n s . E t i o l o g y and Pathogenesis The abnormal b last c e l l populat ion that character izes AML i s unable to complete normal myeloid d i f f e r e n t i a t i o n programs, and i t s accumulation r a p i d l y leads to f a i l u r e of normal hemopoiesis. As in the case of CML the nature of the transforming event(s) that i n i t i a t e s the disease remains obscure, although i t s a s s o c i a t i o n with agents known to cause DNA damage such as radiotherapy and chemotherapy used in the treatment of other malignancies (193,194) and carcinogens such as benzene (195) i s wel l documented. 31 The disease may a r i s e de novo or i t may be preceeded by syndromes of hemopoietic d y s f u n c t i o n c o l l e c t i v e l y r e f e r r e d to as preleukemia. The preleukemic phase g e n e r a l l y occurs i n older i n d i v i d u a l s , and AML e v o l v i n g from t h i s phase has a poorer prognosis than AML a r i s i n g with no recognized preleukemic phase (131). Treatment of the disease i s designed to reduce the leukemic population by courses of chemotherapy u n t i l the leukemic c e l l s are e s s e n t i a l l y undetectable. In contrast to CML, G6PD stu d i e s have shown that c y t o t o x i c therapy allows normal g r a n u l o p o i e s i s to re-emerge (189), although not i n a l l cases (196). The leukemic clone u l t i m a t e l y regains dominance during r e l a p s e . Chromosomal Abnormalities A number of nonrandom chromosomal changes have been a s s o c i a t e d w i t h AML although none of them are as cons i s t e n t as the Phi chromosome i n CML. Abnormal karyotypes have g e n e r a l l y been reported i n 50% of AML p a t i e n t s using conventional banding techniques (156), although recent s t u d i e s using h i g h - r e s o l u t i o n chromosome techniques suggest that abnormalities may be present i n c e l l s from a l l AML p a t i e n t s (197). Nonrandom changes i n c l u d e trisomy 8, monosomy 7, and other abnormalities i n chromosomes 5, 17, and 21. In aggressive disease these nonrandom changes may occur i n a background of m u l t i p l e chromosome rearrangements suggesting that they are the primary change and other abnormalities r e f l e c t c l o n a l e v o l u t i o n (198). Probably of gr e a t e r b i o l o g i c a l importance are rearrangements that c o n s i s t e n t l y a s s o c i a t e w i t h a p a r t i c u l a r s u b c l a s s i f i c a t i o n of AML. These would include the t r a n s l o c a t i o n s i n v o l v i n g chromosomes 8 and 21 i n M2-AML and chromosomes 15 and 17 i n M3-AML. Although the meaning of these c o n s i s t e n t t r a n s l o c a t i o n s i s yet to be e l u c i d a t e d , t h e i r a s s o c i a t i o n with c e l l s that are blocked at the myeloblast (M2-AML) or promyelocyte (M3-AML) stage of d i f f e r e n t i a t i o n i s i n t r i g u i n g . An 32 interesting report of an association between an inversion of chromosome 16 and abnormal eosinophils in M4-AML has also been documented (199). The Ph^ translocation has been demonstrated in a number of patients with AML (200). Some of these cases probably represent CML in blast c r i s i s , although in other cases i t appears to be distinct from CML in that complete remission is associated with loss of the Phi chromosome (200,201). In some cases a genetic predisposition to DNA damage or chromosomal instability has been implicated in the genesis of AML. Such conditions include Down's syndrome (202), ataxia-telangiectasia (203), congenital agranulocytosis (204), celiac disease (205), Bloom's syndrome (206), Fanconi's anemia (207), Wiscott-Aldrich syndrome (208) and von Recklingshausen's neurofibromatosis (209). Associations with immune deficiency states (210) and immunosuppressed transplant recipients (211) have also been reported. As with most cancers, AML probably arises from a complex interplay between host and environmental risk factors. Role of Cellular Oncogenes Identification of the constant chromosome regions that are involved in myeloid hemopoietic malignancies is of considerable importance in view of the increasing evidence implicating cellular oncogenes in normal myeloid differentiation. Chromosomal aberrations have the potential to cause changes in normal oncogene regulation and thus contribute to leukemic myelopoiesis. The myb gene is the cellular homologue of the transforming gene of a virus that is know to cause AML in chickens (212). The c-myb gene is specifically expressed in hemopoietic cells and appears to be tightly regulated during myeloid c e l l differentiation (213). This oncogene was recently reported to be amplified in a case of AML (214). Interestingly, the c-myb gene codes for a protein that has some degree of structural homology to 33 the c-myc gene product (215). The c-myc gene has a l s o been reported to be a m p l i f i e d i n AML (216,217). The t r a n s c r i p t i o n of c-myb and c-myc i n myelogenous leukemia c e l l l i n e s i s l o s t when these l i n e s are induced to d i f f e r e n t i a t e (218,219), suggesting a p o s s i b l e r o l e i n the normal d i f f e r e n t i a t i o n process. The f p s / f e s oncogene may a l s o prove important i n t h i s regard s i n c e i t s expression has been shown to be l a r g e l y confined to normal hemopoietic t i s s u e (220) and hematologic malignancies (221). This gene becomes t r a n s c r i p t i o n a l l y l e s s a c t i v e i n myeloid leukemia c e l l l i n e s only when these c e l l s are induced to undergo monocytic d i f f e r e n t i a t i o n (222). Also notable i s the demonstration that the N-ras gene i s a l t e r e d i n some cases of AML and that t h i s a l t e r e d gene i s capable of transforming NIH 3T3 c e l l s or r a t - 1 f i b r o b l a s t s (223-226). An amino a c i d s u b s t i t u t i o n at codon 13 of the N-ras gene has r e c e n t l y been i m p l i c a t e d i n the conversion of t h i s gene i n t o one w i t h transforming a c t i v i t y (227). D i f f e r e n t i a t i o n of the N e o p l a s t i c Clone In the case of AML the leukemic b l a s t c e l l s are i d e n t i f i e d by t h e i r l a c k of d i f f e r e n t i a t e d c h a r a c t e r i s t i c s . The most common method f o r the c l a s s i f i c a t i o n of AML involv e s the use of morphological and cytochemical c r i t e r i a to c a t e g o r i z e the leukemic b l a s t c e l l population i n t o subgroups that appear to c o r r e l a t e with d i f f e r e n t stages of normal hemopoietic c e l l d i f f e r e n t i a t i o n (228). S p e c i f i c subtypes are therefore defined by the d i r e c t i o n of d i f f e r e n t i a t i o n and the degree of c e l l u l a r maturation. S i x subgroups are c u r r e n t l y recognized i n the FAB c l a s s i f i c a t i o n (Table I ) . Two main models have been advanced to e x p l a i n the existence of the predominating b l a s t population. The f i r s t model proposes e i t h e r a block i n a normal myeloid d i f f e r e n t i a t i o n pathway or an uncoupling between p r o l i f e r a t i o n 34 TABLE I F rench-Amer ican-Br i t ish (FAB) c l a s s i f i c a t i o n of acute myelogenous leukemia. A c l a s s i f i c a t i o n scheme based on morphological and cytochemical c r i t e r i a (228). Ml Mye lob last ic M2 Myelob last ic with evidence of maturation M3 Promyelocytic M4 Myelomonocytic M5 Monocytic M6 Erythroleukemia 35 and differentiation (229,230). The second model is based on the observation that despite their general adherence to normal myeloid differentiation programs, leukemic blasts may occasionally express markers of differentiation that are out of context for a normal c e l l at the equivalent stage of differentiation. The latter model proposes that the blast population represents a novel lineage consisting of components of normal differentiation assembled abnormally (231). This hypothesis finds support in the identification in single cells of markers of more than one lineage that are not normally expressed coincidentally. These would include the coexpression of lymphoid and myeloid markers (232-239), as well as the coexpression of markers characteristic of different myeloid lineages (233,240). Such observations support the concept of aberrant gene expression in the leukemic cells, although an alternate explanation is that in some of the cases the leukemic cells are arrested at a developmental stage characterized by multilineage marker expression. Autoradiographic studies have shown that the majority of AML blast cells are nondividing cells (241). It is interesting to note however, that the differentiation potential of many of these abnormal end-cells does not appear to be irreversibly blocked (229,242). It is now evident that fresh AML cells or c e l l lines derived from patients with AML w i l l undergo various degrees of differentiation in response to a number of physiological (229,243,244) and nonphysiological (222,245) inducers of differentiation (see Chapter IV). Blast Cell Progenitors Several assays have been described that support the growth of blast c e l l progenitors in AML. Culture conditions that favour the growth of these cells include the addition of PHA-stimulated leukocyte-conditioned medium as a stimulatory factor (246) and the removal of T cells from the c e l l suspension 3 6 to be assayed (247). In these assay systems the majority of the leukemic blasts f a i l to proliferate but their progenitors are able to undergo 1-5 cycles of c e l l division to form clusters of poorly differentiated c e l l s . The cells within these colonies are morphologically similar to, and possess the same chromosomal aberrations (248) and G6PD isoenzyme (7), as blast cells in direct marrow preparations from the patient. Analysis of the replating efficiency of these colonies demonstrated that some of the blast progenitors possessed the stem c e l l characteristic of self-renewal (249). Presumably the capacity for self-renewal in these cells is biologically relevant since this property would allow clonogenic blast progenitors to maintain an independent leukemic population in vivo. Considerable heterogeneity in self-renewal capacity is observed between patients but this property appears to be a stable characteristic in individual patients (250) and may in fact be negatively correlated with prognosis (251). The c l i n i c a l significance of these blast progenitors is further exemplified by the finding of a significant correlation between their numbers and the blast count (252) and the observation that, in contrast to the blast cells, they are actively cycling (253,254). A distinction between the blast progenitor population and the total blast population resides in their respective sedimentation and c e l l surface antigen profiles (255,256). The a b i l i t y of the leukemic clone to suppress normal hemopoiesis is evident in AML patients that present with anemia, granulocytopenia, and thrombocytopenia (257). Whether or not this reflects physical exclusion by the expanding mass of leukemic cells or leukemia-derived inhibitory ac t i v i t i e s is uncertain. An acidic i s o f e r r i t i n termed leukemic inhibitory activity (LIA) has been reported in the serum of AML patients and was shown to inhibit normal granulopoiesis in vitro (188). Unfortunately, the lack of 37 c u l t u r e c o n d i t i o n s capable of supporting e a r l y stem c e l l growth prevents the a n a l y s i s of i n h i b i t i o n at stages p r i o r to the progenitor l e v e l . In c o n t r a s t to CML, complete remission i n AML appears to represent the reestablishment of normal hemopoiesis. In an AML marrow that i s i n c l i n i c a l r e mission only normal karyotypes are found (140), a l l lineages express both forms of G6PD (258), the progenitor l e v e l s approach normal dimensions (23), and LIA can not be detected (188). An exception to t h i s was r e c e n t l y reported i n an AML p a t i e n t who entered a complete remission c h a r a c t e r i z e d by m o r p h o l o g i c a l l y and k a r y o t y p i c a l l y normal hemopoietic c e l l s but marrow progenitors were nonetheless c l o n a l l y derived (196). 5) ANTIGENIC ANALYSIS OF LEUKEMIC MYEL0P0IESIS (A) Tumour Antigens A n a l y s i s of c e l l u l a r d i f f e r e n t i a t i o n r e l i e s on the i d e n t i f i c a t i o n of s p e c i f i c gene products that are associated with various stages of maturation. W i t h i n the f i e l d of experimental hematology extensive use has been made of morphological and cytochemical techniques to i d e n t i f y the developmental sequence of the terminal stages of hemopoiesis. Many of these features have been shown to e x i s t i n leukemic as w e l l as normal c e l l s and t h i s provides a basis f o r current c l a s s i f i c a t i o n schemes. Heterogeneity i n the c l i n i c a l features and responses to treatment i n p a t i e n t s w i t h AML has been recognized f o r a long time and u n d e r l i e s the n e c e s s i t y f o r new c l a s s i f i c a t i o n systems. One such approach has been the i d e n t i f i c a t i o n of gene products whose expression i s c o r r e l a t e d w i t h e i t h e r a s p e c i f i c l e v e l of d i f f e r e n t i a t i o n or the malignant phenotype i n general. For example, a number of biochemical markers have been i d e n t i f i e d that may be u s e f u l i n d e f i n i n g patterns of hemopoietic c e l l d i f f e r e n t i a t i o n and 38 p o t e n t i a l l y i n the accurate diagnosis of leukemias and lymphomas (259). One notable example i s the enzyme terminal deoxynucleotidyl t r a n s f e r a s e (TdT) which appears to be a u s e f u l marker f o r the i n i t i a l diagnosis and subsequent monitoring of lymphoid neoplasms (259,260). A second approach i s the immunological a n a l y s i s of the c e l l membrane us i n g monoclonal a n t i b o d i e s . Embedded i n the l i p i d b i l a y e r of the c e l l membrane are p r o t e i n s that mediate s p e c i f i c c e l l u l a r f u n c t i o n s . Numerous membrane abno r m a l i t i e s have been i d e n t i f i e d i n n e o p l a s t i c c e l l s (261) and presumably these a l t e r a t i o n s may i n f l u e n c e the n e o p l a s t i c behaviour of a tumour c e l l by modulating c e l l u l a r i n t e r a c t i o n s , i n t e r a c t i o n s w i t h the e x t r a c e l l u l a r matrix, and c e l l u l a r responses to growth f a c t o r s and hormones. The search f o r c e l l surface c o r r e l a t e s of malignancy using immunological approaches has therefore been a major thrust of research i n t o normal and leukemic hemopoietic c e l l d i f f e r e n t i a t i o n . Immunophenotyping of normal and leukemic c e l l populations has r e s u l t e d i n the i d e n t i f i c a t i o n of two major types of c e l l surface antigens: 1) p u t a t i v e tumour antigens and 2) d i f f e r e n t i a t i o n and l i n e a g e - s p e c i f i c antigens. Tumour Antigens: an overview For a tumour to generate s p e c i f i c a l l y s e n s i t i z e d lymphocytes and s t i m u l a t e antibody production, i t must express antigens that are immunogenic i n the host. The demonstration of tumour antigens, and assessment of t h e i r relevance as targets i n t r a n s p l a n t a t i o n r e j e c t i o n has been l a r g e l y r e s t r i c t e d to s t u d i e s of chemically and v i r a l l y - i n d u c e d neoplasms i n animals. These tumour s p e c i f i c t r a n s p l a n t a t i o n antigens (TSTA's) are c l a s s i c a l l y defined by the r e j e c t i o n of tumours that are i n j e c t e d i n t o syngeneic mice p r e v i o u s l y immunized with the same tumour (262). Chemically induced tumours g e n e r a l l y possess an i n d i v i d u a l l y s p e c i f i c r e j e c t i o n antigen that does not c r o s s - r e a c t 39 with rejection antigens expressed on other tumours induced by the same carcinogen. Virus-specific surface antigens characteristic of DNA viruses can also act as TSTA's although in this case immunization confers immunity to a l l tumours induced by the same virus. In some cases the inappropriate expression of embryonic antigens (263) or the modification of histocompatibility antigens (264) on tumour cells may also provide a basis for transplantation rejection. In contrast to v i r a l l y and chemically-induced tumours, spontaneously arising tumours in rodents are less frequently capable of e l i c i t i n g a host rejection response (265) and this must be kept in mind when studying the human situation. A variety of studies in humans have demonstrated that the immune system is capable of mounting a response against autologous tumour cells (266). Although these findings support the contention that some human tumours express c e l l surface components that are immunogenic, almost nothing is known about the molecular nature of these antigens. Despite the extensive efforts to identify human tumour-specific antigens, the d i f f i c u l t i e s associated with demonstrating that a tumour antigen is entirely absent from any normal tissue has led to the term tumour-associated antigen. An antigen detectable only in embryonic cells and tumour cells would thus be operationally defined as a tumour-associated antigen. Leukemia-Associated Antigens Defined by the Patient's Immune Response A number of studies have implied the existence of leukemia-associated antigens (LAA's) by demonstrating that the sera of patients with AML contain antibodies that are directed toward autologous and/or allogeneic leukemic cells (267). Some of these antibodies were shown in vitro to be cytotoxic to host AML cells with the addition of complement or autologous effector cells (268,269). Interestingly the observed cytotoxicity was low at presentation, 40 peaked at the onset of c l i n i c a l remission, and then decreased during continued therapy with the lowest levels being observed at relapse (269). The identification of antibody bound to leukemic cells in vivo and the demonstration that some of the serum-derived antibodies are capable of blocking the ab i l i t y of host lymphocytes to respond to autologous blasts in vitro, lend further support for the existence of leukemia antigens that can e l i c i t an immune response in vivo (270). Cell mediated immunity is generally believed to play a major role in the host resistance against tumour cells. The presence of cellular immunity is demonstrated in vitro in a mixed lymphocyte culture assay (MLC) and in vivo by delayed-type hypersensitivity reactions (DTH). A number of investigators have utilized the MLC assay and reported that remission peripheral blood lymphocytes w i l l respond in vitro to autologous blast cells (271-273), as w i l l the normal lymphocytes from haploidentical siblings (274). Similarly, studies u t i l i z i n g membrane preparations of autologous blast cells demonstrated positive DTH skin reactions in a number of AML patients during both remission and relapse (275). These results are not always confirmed however (276), and there is variation between assays (277). It is possible that these different assays (MLC, cytotoxicity, and skin testing) coupled with the source of antigen (whole blast cells, irradiated blast cells, mitomycin C-treated blast cells, or blast c e l l extracts) are actually measuring different phases of the immune response against different antigens (278). Unfortunately the specificity of a l l these immune reactions is unknown and i t has been suggested that the putative tumour antigens may not be true neo-antigens but rather normal antigens which are usually restricted to infrequent precursor c e l l populations (279). 41 Leukemia-Associated Antigens Defined by Xenoantisera to Leukemic Cells A variety of heteroantisera aimed at detecting leukemia-associated antigens have been reported and have demonstrated varying degrees of specificity for AML cel l s . Unfortunately the preparation of antisera to leukemic cells is subject to a number of technical d i f f i c u l t i e s that limit their c l i n i c a l usefulness. To render these antisera antigen specific, extensive absorptions are required with other hemopoietic c e l l populations and consequently the absorbed reagents are generally of low t i t e r . Other problems reflect the small volumes of antisera available and the lack of reproducible specificity between and within laboratories. Nevertheless, antigens associated with AML have been identified using antisera derived from several sources. Mice rendered tolerant to AML remission cells and then challenged with blast cells from the same patient produced antisera that reacted with leukemic myeloblasts (280,281). This antiserum has been successful in the early diagnosis of relapse in AML before the appearance of morphologically detectable myeloblasts in the bone marrow (282). An antiserum raised in rabbits against membrane components of a Burkitt's lymphoma c e l l line showed specificity for acute leukemia cells (lymphoid and myeloid) and this antiserum did not require prior absorption to prevent reactivity with normal cells (283,284). Other antisera raised in rabbits (285,286), nonhuman primates (287-289), and patients receiving immunotherapy with leukemia c e l l vaccines (290,291) have shown speeifjk y for AML c e l l s . Nonimmunological approaches have also been used to . i t i f y c e l l surface leukemia markers. These techniques involve the radiolabelling of c e l l surfaces followed by either one-dimensional or two-dimensional polyacrylamide gel electrophoresis (PAGE). AML blast cells have been analyzed by the one-dimensional (292) and 2-dimensional techniques (293) and both methods have 42 revealed maturation and leukemia-associated differences in the electrophoretic mobilities of ectoproteins. One of these proteins isolated by the two-dimensional ectolabelling procedure, was preferentially associated with leukemic myeloblasts and had approximately the same localization on 2D gels as one would expect the AML antigen defined by a recently described anti-AML antiserum (286). This antigen has a molecular weight of 68,000 and an is o l e l e c t r i c point of 7.1 (294). Other studies have shown that AML blast cells cultured in vitro continuously shed surface components into supernatant medium (295). Characterization of the glycoproteins shed from AML blasts by gel chromatography, isoelectric focussing, immunoprecipitation and PAGE has defined characteristic profiles that differ quantitatively and qualitatively from compounds shed from normal or other leukemic cells (296). These observations are c l i n i c a l l y relevant since leukemia-associated antigens that are shed into the host circulation may fac i l i t a t e the escape of the malignant cells from immune destruction (297). If antibodies are being formed in vivo against circulating leukemia-associated antigens that are shed from leukemic c e l l surfaces then one would expect antigen-antibody complexes to be formed. A number of studies have detected these immune complexes in the sera of patients with acute leukemia (298-300). In one study of AML the levels of immune complexes correlated significantly with c l i n i c a l course and prognosis (298). Unfortunately the nature of the antigens that are present in these immune complexes is not known. Molecular analysis of these antigens would provide a way to identify leukemia-associated antigens that are immunogenic in the host. 43 Example of a Leukemia-Associated Antigen: CALLA The d e t e c t i o n of the common acute lymphoblastic leukemia antigen (CALLA) on leukemic c e l l populations has proven to be very u s e f u l i n the diagnosis and s u b c l a s s i f i c a t i o n of ALL (301). The antigen was i n i t i a l l y defined by heterologous p o l y c l o n a l a n t i s e r a (302) and l a t e r using a murine monoclonal antibody (303) as a g l y c o p r o t e i n with a molecular weight of 100,000. I n i t i a l s t u d i e s d i d not detect t h i s molecule on normal hemopoietic c e l l s but i t was c l e a r l y h i g h l y expressed on lymphoblasts from p a t i e n t s with ALL. The molecule was therefore designated a leukemia-associated antigen. Subsequent s t u d i e s revealed the expression of the antigen on a small population of mononuclear c e l l s i n normal marrow (304) suggesting that CALLA i s not leukemia s p e c i f i c but rather a normal d i f f e r e n t i a t i o n antigen. CALLA-related surface antigens have now been i d e n t i f i e d on f i b r o b l a s t s and mature granulocytes (weakly) (305), and r e n a l proximal tubule e p i t h e l i u m and breast myoepithelium (306). Within the hemopoietic system the anatomical d i s t r i b u t i o n of CALLA-positive c e l l s i n normal lymphatic t i s s u e and lymphomas (307), and the i d e n t i f i c a t i o n of CALLA-positive mononuclear c e l l s i n normal marrow that co-express cytoplasmic u, the B l d i f f e r e n t i a t i o n antigen, TdT, and l a (308) support the concept that CALLA i s an e a r l y lymphoid d i f f e r e n t i a t i o n antigen. ALL has thus been suggested to represent the c l o n a l expansion of a c e l l that normally expresses t h i s molecule. Data regarding the CALLA antigen i s an example of the accumulating evidence that suggests that i f one looks hard enough, and with o p t i m a l l y s e n s i t i v e reagents, most leukemia-associated antigens w i l l be found to be present on normal c e l l s . D e t a i l e d s c r u t i n y of a l l the human leukemia-associated antigens reported to date has provided no convincing evidence f o r the existence of l e u k e m i a - s p e c i f i c antigens. The a s s o c i a t i o n of 44 these antigens with the leukemic phenotype i s thought to r e f l e c t a) q u a n t i t a t i v e d i f f e r e n c e s between tumour c e l l s and normal c e l l s , b) the i n a p p r o p r i a t e expression of a normal t i s s u e antigen i n another l i n e a g e or t i s s u e , or c) the expression of a normal antigen at an i n a p p r o p r i a t e l e v e l of d i f f e r e n t i a t i o n (309). The p o s s i b i l i t y of a true tumour antigen may e x i s t i n the i d e n t i f i c a t i o n of rearranged oncogene products or products of mutated normal genes. Of course p u t a t i v e tumour antigens that are u l t i m a t e l y shown to be normal antigens under rigorous t e s t i n g may s t i l l be c l i n i c a l l y u s e f u l . The immunoglobulin i d i o t y p e on the surface of a monoclonal B c e l l malignancy provides the best example of an o p e r a t i o n a l l y defined tumour-specific antigen (279). Although immunoglobulin i s a normal gene product of a B c e l l , malignancies of B c e l l o r i g i n express c e l l surface immunoglobulin with a unique V-region c h a r a c t e r i s t i c of the p a r t i c u l a r B c e l l clone from which the tumour was derived. This i d i o t y p i c determinant may thus be defined as a unique tumour s p e c i f i c marker. (B) Normal Myeloid D i f f e r e n t i a t i o n  C e l l Surface Phenotyping of Mature Myeloid C e l l s Membrane g l y c o p r o t e i n s may be common to a v a r i e t y of c e l l types, an example being the t r a n s f e r r i n receptor that i s associated w i t h p r o l i f e r a t i n g c e l l s (310). Other p r o t e i n s are unique to a s p e c i f i c d i f f e r e n t i a t e d c e l l as a r e s u l t of d i s t i n c t patterns of gene expression c h a r a c t e r i s t i c of a p a r t i c u l a r developmental pathway. Molecules with such l i n e a g e - r e s t r i c t e d e xpression may mediate important functions c h a r a c t e r i s t i c of that l i n e a g e . Many of these embedded proteins are exposed on the c e l l surface and are thus amenable to immunological a n a l y s i s . The hybridoma technique developed by Kohler and M i l s t e i n (311) has r e v o l u t i o n i z e d the immunological a n a l y s i s of hemopoiesis by p r o v i d i n g a method f o r the production i n v i r t u a l l y u n l i m i t e d 45 amounts of monoclonal antibody with defined c l a s s , a v i d i t y and s p e c i f i c i t y . These monospecific antibodies do not require extensive absorption procedures, are a v a i l a b l e i n large q u a n t i t i e s e i t h e r as hybridoma supernatant or a s c i t e s f l u i d , and t h e i r s p e c i f i c i t y does not vary between batches. Monoclonal a n t i b o d i e s have now been r a i s e d against every hemopoietic l i n e a g e . Although the greatest amount of work, has centered on the immune system, a n a l y s i s of myeloid d i f f e r e n t i a t i o n using monoclonal a n t i b o d i e s i s now l e a d i n g to the d e f i n i t i o n of myeloid subpopulations on the b a s i s of t h e i r surface phenotypes. A n t i g e n i c expression may be r e s t r i c t e d to one or more myeloid lineages or to morphologically and f u n c t i o n a l l y d i s t i n g u i s h a b l e subsets along the myeloid d i f f e r e n t i a t i o n pathways. An example of a t i s s u e - s p e c i f i c antigen i n the hemopoietic system i s the leukocyte common antigen (T200), an antigen that i s widely expressed on a l l leukocytes (T and B lymphocytes, thymocytes, macrophages, and granulocytes) but not on other t i s s u e s (312). This antigen comprises a f a m i l y of h i g h l y conserved, s t r u c t u r a l l y r e l a t e d molecules, that show molecular weight heterogeneity (180,000-220,000) and a n t i g e n i c d i f f e r e n c e s that are a s s o c i a t e d w i t h d i s t i n c t c e l l types and the s t a t e of maturation (313). The suggestion that T200 mediates important functions at the leukocyte c e l l surface i s supported by the a b i l i t y of anti-T200 antibodies to block NK-mediated c y t o l y s i s (314), B c e l l d i f f e r e n t i a t i o n (315), and CTL d i f f e r e n t i a t i o n and c y t o l y t i c a c t i v i t y (316,317). The recent observation that the r a t T200 molecule spans the l i p i d b i l a y e r and contains a l a r g e cytoplasmic domain of 705 amino acid s makes t h i s antigen a prime candidate f o r membrane-cytoskeleton i n t e r a c t i o n s (318). The i d e n t i f i c a t i o n of maturational and f u n c t i o n a l subsets of the monocytic d i f f e r e n t i a t i o n pathway i s of considerable i n t e r e s t i n view of the 46 major r o l e of these c e l l s i n the immune response. A l a r g e number of monoclonal an t i b o d i e s have now been r a i s e d against c e l l s of t h i s l i n e a g e , some of which are s p e c i f i c f o r monocytes while others may a l s o react w i t h c e l l s of another myeloid lineage (319-321). A number of s t u d i e s have demonstrated f u n c t i o n a l heterogeneity of macrophage populations (322,323) and the c e l l surface phenotyping of these macrophages o f f e r s a unique approach to the c l a s s i f i c a t i o n of these subpopulations (324,325). Monoclonal antibodies have been instrumental i n d e f i n i n g the s t r u c t u r a l and f u n c t i o n a l r e l a t i o n s h i p s of a family of high molecular weight surface g l y c o p r o t e i n s termed LFA-1, mac-1, and pl50,95 that are found on leukocyte c e l l surfaces (326). These molecules are now known to share a common P-subunit and are d i s t i n g u i s h e d by t h e i r noncovalent a s s o c i a t i o n w i t h a d i s t i n c t a-subunit. Of p a r t i c u l a r relevance to the macrophage l i n e a g e i s the mac-1 g l y c o p r o t e i n that was i n i t i a l l y i d e n t i f i e d as a myeloid d i f f e r e n t i a t i o n antigen (327). This molecule i s present on monocytes and granulocytes but i s absent from T and B lymphocytes. Subsequent studies have i d e n t i f i e d mac-1 as the receptor f o r the t h i r d component of complement (CR3), mediating the adherence and phagocytosis of C3b-coated p a r t i c l e s by granulocytes and macrophages. I n t e r e s t i n g l y , a c l i n i c a l syndrome c h a r a c t e r i z e d by rec u r r e n t b a c t e r i a l i n f e c t i o n s i s associated with a c o n g e n i t a l d e f i c i e n c y of t h i s molecule (328). Monoclonal antibodies r a i s e d against myeloid c e l l s have a l s o i d e n t i f i e d c e l l s u rface antigens that are r e s t r i c t e d to mature granulocytes (321,329-332). The mature n e u t r o p h i l plays a c e n t r a l r o l e i n the host defense against b a c t e r i a l i n f e c t i o n s and presumably some of these c e l l s urface g l y c o p r o t e i n s are i n t i m a t e l y involved i n e f f e c t i n g t h i s f u n c t i o n . A number of s t u d i e s have described the a b i l i t y of c e r t a i n monoclonal a n t i b o d i e s 47 to inhibit neutrophil chemotaxis and degranulation (331), oxidative metabolism (333), antibody-dependent cellular cytotoxicity (334), and phagocytosis (335) suggesting that the c e l l surface components defined by these antibodies may play a role in mediating each of these functions. The significance of neutrophil surface glycoproteins is exemplified by the c l i n i c a l disorder characterized by an inherited deficiency of the mac-1, LFA-1 and pl50,95 glycoprotein family (328). The adverse effect of this deficiency on adhesion-dependent functions of myeloid cells (such as attachment and spreading, agglutination, antibody-dependent cellular cytotoxicity, and CR3-mediated adherence and phagocytosis) is thought to account for, at least in part, the recurrent bacterial infections encountered in this disease (328). The observation that many granulocyte antigens are conserved among the major primate families further attests to the functional importance of these structures (336,337). Antibodies that have been found to be specific for the erythroid lineage include those directed at glycophorin (338), the Rh blood group antigen (339), band-3 (340), and two erythroid precursor antigens termed Ep-1 and Ep-2 (341). Another erythroid membrane antigen with a molecular weight of 37,000 was shown to be expressed on a l l hemopoietic cells but arranged in the erythrocyte membrane in a lineage-specific fashion (342). Other monoclonal antibodies that react with erythroid cells appear to be less specific for this lineage (343). Analysis of the normal cellular distribution of myeloid c e l l surface antigens is a powerful technique to study gene products associated with myeloid c e l l differentiation. Although the majority of these antigens have unknown functions, we are now beginning to elucidate the functions of some of these molecules, the LFA-1 family being a prime example. The broad 48 distribution of some of these antigens on many myeloid cells is i t s e l f indicative of an important role (344). Antigens that possess a lineage-restricted pattern of expression, such as band-3 on erythroid cells (340), probably mediate lineage-specific functions, and those that define subpopulations of cells within a lineage may identify functional subgroups of c e l l s . Further development of functional assays for myeloid cells and their precursors should f a c i l i t a t e functional analysis. Cell Surface Phenotyping of Stem Cell Populations The development of hybridoma technology, in conjunction with clonal assays for hemopoietic progenitors, has allowed the generation of monoclonal antibodies selectively reactive with populations of myeloid cells at specific stages of differentiation. It is now possible to enrich for progenitor populations by taking advantage of differences in c e l l surface antigen expression between progenitor cells and their mature progeny (345,346). Two techniques are commonly used to define the antigenic phenotype of the numerically infrequent progenitor cells found in hemopoietic tissue. The f i r s t technique involves the incubation of bone marrow cells with the monoclonal antibody in question followed by the addition of complement. Stem cells expressing specific antigen are therefore ki l l e d and subsequent colony assays demonstrate a decreased plating efficiency. A major limitation of this assay is that antigens expressed at low density may not render the target c e l l susceptible to complement-mediated k i l l i n g . Nevertheless, this technique has been particularly useful in the HLA-typing of human hemopoietic progenitor populations. The second technique involves the use of the c e l l sorting capabilities of a fluorescence activated c e l l sorter (FACS). Fluorescent-positive and negative cells are separated by this instrument and the respective fractions can be assayed in clonogenic assays. The obvious advantage of this technique is that i t separates viable populations rather than selectively k i l l i n g a population.with antibody and complement. Combinations of these techniques have been used to demonstrate the expression of class II histocompatibility antigens (la-like) on the major progenitor c e l l classes (347). Although the exact proportion of I a + erythroid colony-forming cells has been somewhat controversial, recent studies suggest that i f the conditions of the complement-mediated cytotoxicity are sufficiently optimal one can detect l a antigens on the vast majority of CFU-GM, BFU-E, and CFU-E (348,349). One study reported that DC antigens (DC being a subregion of the HLA-D locus) are not detectable on progenitors but the significance of this observation is not known (349). In contrast to the immune system the function of class II antigens on hemopoietic progenitors is not clear, although there is some evidence to suggest a role in hemopoietic suppression (350-352). The observation that lymphocytes express l a molecules with a unique 'lymphoid' epitope that is not detectable on the l a molecules of monocytes or hemopoietic progenitors, illustrates lineage specific variation in la expression and is a potential basis for selective compartmentalization and regulation of la-associated function (353). While most evidence suggests that committed myeloid and erythroid progenitor cells are Ia +, the more primitive CFU-S in the mouse may not express these antigens (354), and studies with human long term bone marrow culture have suggested that the putative multipotent stem c e l l that is detected by this assay is also Ia~ (63-65). In contrast to this data i t was recently shown in vivo, in a canine autologous transplant model, that I a + bone marrow cells are essential for the successful engraftment of 50 transplanted marrow (355). It should be noted however that i t is not known whether these la-bearing cells are pluripotential stem cells or essential accessory cel l s . Using these techniques i t has been possible to define c e l l surface changes during the course of differentiation from the committed progenitor compartment to the fully differentiated effector c e l l compartment. Dif f i c u l t i e s of analysis have prevented definitive statements as to the antigenic characteristics of the more primitive stem cel l s . Changes in the antigenic pattern on the c e l l surface during differentiation may reflect membrane alterations that mediate cellular responsiveness to extrinsic regulatory signals. These may be quantitative changes in antigen density but this is d i f f i c u l t to assess due to the infrequency of progenitor c e l l s . Several patterns of antigen expression are evident: Some antigens are not expressed on progenitors, appearing only with terminal differentiation (356,357). Of the antigens shown to be expressed on progenitor populations, the vast majority also react with differentiated cells (347,358-360). Others are expressed on progenitors and their differentiating progeny, but absent, or weakly expressed on fully mature cells (360). Central to the immunological analysis of hemopoiesis has been the search for progenitor or stem c e l l specific monoclonal antibodies that would provide a one step purification procedure for these cells. Very few of these monoclonal antibodies have been described. Since many leukemic c e l l lines display immature characteristics, attempts have been made to use these lines as target cells to raise progenitor-specific monoclonal antibodies. Two such monoclonal antibodies possessing narrow specificity were selected from a panel of monoclonal antibodies raised against the K562 erythroleukemia c e l l line (361). These antibodies reacted with <3% of normal bone marrow cell s , 51 and had low or absent binding to mature p e r i p h e r a l blood c e l l s . The a b i l i t y of these a n t i b o d i e s i n the presence of complement to reduce colony formation suggests that they recognize antigens s p e c i f i c to u n d i f f e r e n t i a t e d c e l l s . The HEL c e l l l i n e (362) i s a l s o an erythroleukemia c e l l l i n e that i s considered to be blocked at an e a r l y stage of d i f f e r e n t i a t i o n . The c h a r a c t e r i z a t i o n of a s e r i e s of monoclonal antibodies against t h i s c e l l l i n e i d e n t i f i e d a p r o t e i n with a MW of 24,000 that was present on 4-8% of bone marrow c e l l s i n c l u d i n g the majority of BFU-E, CFU-E, and CFU-E (363). This determinant was a l s o expressed on p l a t e l e t s and megakaryocytes and can not ther e f o r e be considered t r u l y p r o g e n i t o r - s p e c i f i c . Bodger, et a l described a monoclonal antibody, RFB-1, which reacts with hemopoietic progenitor c e l l s (364), i n c l u d i n g CFU-GEMM. RFB-1 i s expressed on approximately 30% of normal bone marrow and weakly on mature p e r i p h e r a l blood T c e l l s . Although the s p e c i f i c i t y of t h i s antibody f o r progenitor populations i s not complete, t h i s antibody has been u s e f u l i n e n r i c h i n g CFU-GEMM up to 150-fold using a combination of l i g h t s c a t t e r and fluorescence i n t e n s i t y of the RFB-1 antigen i n the FACS (365). The best example of a c e l l surface antigen that i s s e l e c t i v e l y expressed on hemopoietic progenitor c e l l s i s C i v i n ' s My-10 (366). The anti-My-10 monoclonal antibody recognizes a c e l l surface antigen with a MW of 115,000 which i s expressed on hemopoietic progenitors and i s undetectable on maturing myeloid and lymphoid c e l l s . Less than 2% of normal bone marrow c e l l s express t h i s antigen, and these My-10-positive c e l l s are b l a s t l i k e i n morphology. The 3C5 monoclonal antibody r e c e n t l y described by Katz et a l (367) may recognize a s i m i l a r s t r u c t u r e to My-10, although a d i r e c t biochemical comparison has not yet been reported. 52 (C) Leukemic Myeloid D i f f e r e n t i a t i o n D i f f e r e n t i a t i o n of malignant c e l l s has been c h a r a c t e r i z e d p r i m a r i l y by morphologic, cytochemical, and to some extent f u n c t i o n a l c r i t e r i a . Attempts to d e f i n e the lineage a f f i l i a t i o n s and maturational l e v e l of leukemic c e l l s has been g r e a t l y improved by the development of monoclonal a n t i b o d i e s that react w i t h normal hemopoietic d i f f e r e n t i a t i o n antigens. This approach i s based on the assumption that the leukemic b l a s t c e l l s , although abnormal i n many resp e c t s , continue to express c e l l surface antigens c h a r a c t e r i s t i c of the l i n e a g e from which they were derived. AML has long been recognized as a heterogeneous disease that shows morphological features of d i f f e r e n t stages of normal myeloid d i f f e r e n t i a t i o n . The FAB c l a s s i f i c a t i o n of AML i s based l a r g e l y on these morphological observations and i t c u r r e n t l y subdivides AML i n t o s i x c a t e g o r i e s (Table I ) . There do not appear to be major prognostic d i f f e r e n c e s among these subtypes although longer remissions i n M3 p a t i e n t s (368), and a somewhat poorer prognosis i n M5 p a t i e n t s (368,369) have been reported. As more and more chemotherapeutic regimens are being developed, i t i s becoming i n c r e a s i n g l y important to define u s e f u l c l a s s i f i c a t i o n schemes that can c o r r e l a t e d i f f e r e n t c l i n i c a l features with s p e c i f i c subtypes. One approach i s the immunological a n a l y s i s of the leukemic c e l l surfaces. Monoclonal a n t i b o d i e s r e a c t i v e w i t h l i n e a g e - r e s t r i c t e d antigens have c l e a r l y been shown to be u s e f u l i n the diagnosis of ALL and such analyses have provided p r o g n o s t i c a l l y u s e f u l i n f o r m a t i o n (370-372). Since the optimal therapy f o r ALL and AML i s accomplished w i t h d i f f e r e n t chemotherapeutic agents (and a l l o g e n e i c bone marrow t r a n s p l a n t a t i o n i s the treatment of choice i n AML) the d i s t i n c t i o n between these diseases i s very important. Such a d i s t i n c t i o n i s not always apparent using morphological and cytochemical c r i t e r i a , but monoclonal 53 antibodies to myeloid and lymphoid antigens have been quite successful in these situations (373). Detailed analysis of lymphoid leukemias with a panel of monoclonal antibodies and other markers has shown that leukemic lymphoid cells express a conservative framework of normal differentiation markers with minimal deviations (230). These consistent features of leukemic phenotypes in relation to normal hemopoietic differentiation have been taken to reflect the imposition of maturation arrest in the leukemic cel l s . The conservation of differentiation-associated phenotypes has also been reported in AML. In one study a series of monoclonal antibodies reactive with normal myeloid cells at different stages of maturation (anti-My-4, My7, My8, Mol, and la) were used to classify 70 patients with AML into four phenotype groups, each corresponding to a normal immature myeloid c e l l (374). Group I corresponded approximately to the normal CFU-C (21%), Group II to the myeloblast (26%), group III to the promyelocyte (8%), and group IV to the promonocyte (45%). Each group contained more than one morphologic type indicating that the level of differentiation on the c e l l surface may not always parallel morphology. Correlations between c e l l surface phenotype and differentiation status have also been reported in studies u t i l i z i n g other panels of monoclonal antibodies (375,376). Not a l l the cells within a given leukemic population express a particular antigen however, and the relative proportion of these antigen-positive and antigen-negative cells varies considerably between patients (377). It is likely that the extent to which AML blasts are reported to adhere to normal differentiation programs is a reflection of which gene products are analyzed. Clearly leukemic cells express a large number of normal c e l l surface antigens but i t is not known whether the observed antigenic heterogeneity represents disorganized gene expression or merely the lack of synchronization in the total blast population. 54 As prev ious ly mentioned a proport ion of the b last populat ion i s capable of forming co lon ies in methy lce l lu lose assays. These colony- forming c e l l s are presumed to be respons ib le , at l eas t in par t , for maintaining the b las t populat ion in v i v o . Immunophenotyping of the clonogenic c e l l s in AML has shown that the c e l l surface p r o f i l e of the t o t a l populat ion does not n e c e s s a r i l y r e f l e c t the phenotype of the clonogenic c e l l s (256). Furthermore, a comparison of the surface phenotype of the c lonogenic c e l l s with normal myeloid progeni tors i d e n t i f i e d three pat terns: (a) a phenotype s i m i l a r to l a t e CFU-GM, (b) a phenotype s i m i l a r to ear ly CFU-GM or CFU-GEMM, and (c) a composite phenotype of ear ly and l a te CFU-GM. The clonogenic c e l l s were a l s o reported to have a r e l a t i v e l y consistent phenotype in contrast to the t o t a l populat ion (256). The leukemic myeloid c e l l s in the chronic phase of CML are capable of d i f f e r e n t i a t i n g in to mature granulocytes and so the c e l l sur face phenotype of CML c e l l s genera l ly r e f l e c t s the dominant populat ion of granulocytes and to a l e s s e r extent the i r d i f f e r e n t i a t i n g precursors (378). When b las t c r i s i s occurs the i d e n t i f i c a t i o n of the lymphoid var iant i s c l i n i c a l l y important s ince th is group of pat ients f requent ly respond to v i n c r i s t i n e and prednisone (129). Several surface antigens have been i d e n t i f i e d which appear to be of value i n th is regard. Lymphoid antigens such as CALLA (379), B - c e l l ant igens such as B l (380) and B4 (381), and T - c e l l antigens such as T101 (378). Two th i rds of b l a s t c r i s e s are of the myeloid v a r i a n t . These are f e l t to represent AML but cytochemical s ta ins are often negative or inconc lus ive (378). A number of myeloid s p e c i f i c monoclonal ant ibodies have been u s e f u l in these equivoca l s i t u a t i o n s , p a r t i c u l a r l y My7 and My9. Panels of these monoclonal ant ibodies have been used to c l a s s i f y CML b las t c r i s i s in to s i x immunologically def ined subgroups: Myeloid, lymphoid, e r y t h r o i d , megakaryocytic, u n d i f f e r e n t i a t e d , and mixed (378). 55 Therapeutic Potential of Monoclonal Antibodies The use of monoclonal antibodies in the treatment of hematologic neoplasia is s t i l l in its infancy. Although monoclonal antibodies that are truly leukemia-specific would be ideal for these purposes, many studies indicate that tumour-specific antigens may not exist or that i t may prove impossible to obtain monoclonal antibodies against them. On the other hand they may not be required i f the antigen is tumour-specific within the context of the hemopoietic system or i f quantitative differences can be taken advantage of. A number of studies have demonstrated that the passive administration of monoclonal antibodies reacting with leukemic cells can be efficient in the treatment of selected animal leukemias (382). The f i r s t monoclonal antibody serotherapy t r i a l in humans utilized an antibody directed against a lymphoma-associated antigen in the treatment of a B-cell lymphoma (383). Subsequent t r i a l s have emphasized the use of monoclonal antibodies against the CALLA antigen in the treatment of ALL (384), against T-cell differentiation antigens in T-cell leukemia or lymphoma (385,386) and against the T101 antigen in B-chronic lymphocytic leukemia (387,388). Unfortunately these c l i n i c a l t r i a l s have reported only limited responses to the infused antibody. Problems encountered generally include the presence of circulating antigen which binds the injected monoclonal antibody, antigenic modulation of the tumour c e l l surface as a direct consequence of antibody binding (particularly true for CALLA) resulting in antigen-negative tumour cell s , and f i n a l l y the immune response against the infused mouse antibodies. Since the process of antigenic modulation depends on the binding of bivalent antibody, Cobbold and Waldmann (389) reported that monoclonal antibodies that are made monovalent are no longer capable of e l i c i t i n g antigenic modulation and may therefore have increased therapeutic potential. 56 The most cited example of successful serotherapy was a report of the use of anti-idiotypic monoclonal antibody to treat a patient with a B-cell lymphoma (390). This patient had entered an accelerated phase of the disease and was no longer responsive to conventional therapy. Following eight i.v. infusions with the anti-idiotypic antibody the patient entered a complete remission and 30 months later remained free of detectable disease (278). One of the d i f f i c u l t i e s associated with autologous bone marrow transplantation is the high risk of contamination of the transplanted remission marrow by residual leukemic cells (278). The goal is to therefore effectively eliminate neoplastic cells ex vivo while sparing the hemopoietic stem cells that are v i t a l for engraftment. Clinical t r i a l s are now using monoclonal antibody and exogenous complement for these purposes (391-393). Monoclonal antibody 'cocktails' containing several antibodies have been particularly effective (394), especially when combined with a chemotherapeutic agent (395). The preparation of immunotoxins may also prove useful in the immunotherapy of leukemia. Monoclonal antibody has been successfully coupled to powerful toxins such as diptheria and r i c i n toxin in the hope of specifically targeting these compounds to leukemic cells (396). Encouraging results have been reported in animal studies (396,397) but the extreme toxicity of these immunoconjugates w i l l necessitate further research before c l i n i c a l t r i a l s can be initiated in humans. 57 6) THESIS OBJECTIVE An i n c r e a s i n g number of l a b o r a t o r i e s have developed monoclonal antibody reagents that recognize antigens on the surfaces of normal myeloid hemopoietic c e l l s and on myeloid leukemic c e l l s at various l e v e l s of maturation. A comparison of the c e l l surface a n t i g e n i c p r o f i l e s of normal d i f f e r e n t i a t i n g myeloid c e l l s w i t h leukemic b l a s t c e l l s has revealed patterns of antigen expression c o n s i s t e n t with models of leukemic hemopoiesis that p o s t u l a t e a block i n normal d i f f e r e n t i a t i o n programs as a mechanism to e x p l a i n the elevated p r o p o r t i o n of these poorly d i f f e r e n t i a t e d c e l l s i n AML. Although the surface antigen p r o f i l e s of the r e l a t i v e l y mature myeloid c e l l s has been e x t e n s i v e l y s t u d i e d , only a l i m i t e d number of antigens which are r e s t r i c t e d i n t h e i r expression to myeloid precursor populations have been c h a r a c t e r i z e d . In view of the f a c t that AML b l a s t c e l l s are thought to represent the leukemic counterpart of myeloid precursors, i t i s p a r t i c u l a r l y i n t e r e s t i n g to i d e n t i f y those antigens that are s p e c i f i c to normal precursor populations s i n c e they may have important r e g u l a t o r y r o l e s i n e a r l y myelopoiesis. As part of a study to i d e n t i f y c e l l surface antigens c h a r a c t e r i s t i c of the more immature stages of myelopoiesis a number of monoclonal a n t i b o d i e s were r a i s e d against an AML c e l l l i n e , HL-60, that i s blocked at an e a r l y stage of myeloid d i f f e r e n t i a t i o n . One of these a n t i b o d i e s , NHL-30.5, was i n i t i a l l y i d e n t i f i e d by i t s i n a b i l i t y to bind HL-60 c e l l s f o l l o w i n g the i n d u c t i o n of g r a n u l o c y t i c d i f f e r e n t i a t i o n i n t h i s c e l l l i n e . Subsequent s t u d i e s showed the antibody to possess considerable s p e c i f i c i t y f o r hemopoietic c e l l s from p a t i e n t s w i t h hematologic d i s o r d e r s c h a r a c t e r i z e d by the presence of immature myeloid c e l l s , i n p a r t i c u l a r AML. These observations i n d i c a t e d that the NHL-30.5 monoclonal antibody might define an e a r l y myeloid d i f f e r e n t i a t i o n antigen that may be of use i n the c e l l surface phenotyping of myeloid 58 leukemias. The i d e n t i f i c a t i o n of the antigen defined by t h i s monoclonal antibody, and the c h a r a c t e r i z a t i o n of i t s c e l l u l a r d i s t r i b u t i o n i s the subject of t h i s t h e s i s . 59 REFERENCES 1. Wu AM, T i l l J E , Siminovitch L, McCulloch EA: A c y t o l o g i c a l study of the capaci ty for d i f f e r e n t i a t i o n of normal hemopoietic colony forming c e l l s . J C e l l Phys io l 69: 177, 1967. 2. 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Greaves MF: Monoclonal ant ibodies as probes for leukemic heterogenei ty and hematopoietic d i f f e r e n t i a t i o n . In: Leukemia Markers (Knapp W, ed ) , Academic Press , Londong, 1981. 380. Stashenko P, Nadler LM, Hardy R: Charac te r i za t ion of a human B lymphocyte s p e c i f i c ant igen. J Immunol 125: 1678, 1980. 381. Nadler LM, Anderson KC, Marti G, BAtes M, Park E , Daley J F , Schlossman SF: B4, a human B lymphocyte associated antigen expressed on normal, mitogen ac t iva ted and malignant B lymphocytes. J Immunol 131: 244, 1983. 382. K i r c h ME: Approaches to cancer therapy using monoclonal a n t i b o d i e s . In: Monoclonal Ant ibodies and Cancer (Wright GL, ed) , Marcel Dekker Inc, NY, 1984. 86 383. Nadler LM, Stashenko P, Hardy R, Kaplan W, Button LN, Kufe DW, Antman KH, Schlossman SF: Serotherapy of a pat ient with a monoclonal antibody against a human lymphoma-associated ant igen. Cancer Res 40: 3147, 1980. 384. 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Nature 297: 594, 1982. 88 C H A P T E R II MATERIALS AND METHODS 1) PRODUCTION OF THE NHL-30.5 MONOCLONAL ANTIBODY A female BALB/c mouse was immunized twice with the acute promyelocyt ic leukemia c e l l l i n e HL-60 by in t raper i tonea l i n j e c t i o n over a per iod of s e v e r a l weeks. Three days fo l lowing a f i n a l intravenous i n j e c t i o n the immune spleen c e l l s were fused with the NS-1 myeloma c e l l l i n e according to the method of Kohler and M i l s t e i n (1) . The NS-1 c e l l s were cu l tured i n Dulbecco's modified E a g l e ' s minimal e s s e n t i a l medium (DMEM) conta in ing 15% FCS. The immune splenocytes and NS-1 c e l l s were fused in a 50% s o l u t i o n of polyethylene g l y c o l (PEG; Baker Chemical, NJ) in DMEM at a r a t i o of approximately 10^ spleen c e l l s to 10^ NS-1 c e l l s . The fused c e l l s were resuspended in 50 ml of DMEM + 15% FCS and a one ml volume was plated i n each w e l l of a 48-wel l t i ssue cu l ture d ish (Flow l a b o r a t o r i e s , V i r g i n i a ) conta in ing a feeder layer of i r rad ia ted 3T3 f i b r o b l a s t s (~2 x 10^ f i b r o b l a s t s / w e l l ) . The day fo l lowing the f u s i o n , h a l f of the medium was asp i ra ted from the wel ls and 0.5 ml of DMEM conta in ing 15% FCS, 13 ug/ml hypoxanthine, 0.19 Mg/ml aminopterin, and 3.9 ug/ml thymidine (HAT medium) was added to each w e l l . This procedure was repeated three and f i v e days l a t e r . Hybridoma supernatants from 96 wells were screened for r e a c t i v i t y against HL-60 c e l l s in an i n d i r e c t binding assay (see below) us ing 125j_ rabbi t (Fab')2 anti-mouse immunoglobulin as second ant ibody. F i f t y of the 96 wells gave p o s i t i v e r e s u l t s against HL-60 and the c e l l s from some of these 89 cu l tures were subsequently cloned in soft agar. The cloned hybridomas were again screened for r e a c t i v i t y with the HL-60 c e l l l i n e and 34 p o s i t i v e clones were grown up in DMEM + 15% FCS. To enhance the s e l e c t i o n of ant ibodies that might i d e n t i f y antigens associated with ear ly myeloid c e l l d i f f e r e n t i a t i o n the i n i t i a l s e l e c t i o n procedure i d e n t i f i e d monoclonal ant ibodies that reacted with leukemic specimens conta in ing immature myeloid b las t c e l l s . One of the a n t i b o d i e s , NHL-30.5, reacted with HL-60 c e l l s but th is a b i l i t y was l o s t when the c e l l s were induced to d i f f e r e n t i a t e . This hybridoma was subsequently recloned twice and a more de ta i led ana lys is of i t s c e l l u l a r d i s t r i b u t i o n was undertaken. The NHL-30.5 ant ibody-producing hybridoma c e l l s were i n j e c t e d in to pr istane-pr imed BALB/c mice and severa l weeks l a t e r the a s c i t e s f l u i d was harvested from these mice. The pooled a s c i t e s was p r e c i p i t a t e d with ammonium sulphate (50% s a t u r a t i o n ) , d isso lved in 20 mM phosphate buf fer (pH 8.0) and loaded onto a P100 column equ i l ib ra ted with the same b u f f e r . The O D 2 8 O ° f each f r a c t i o n was measured and the f i r s t peak pooled and loaded onto a DEAE-a f f i g e l blue column. Fract ions from the D E A E - a f f i g e l - b l u e column were tested for a c t i v i t y in the b inding assay and for pur i ty by SDS-PAGE, and the peak was again pooled. 2) CELL LABELLING PROCEDURES  Binding Assay Binding of the antibody to a panel of c e l l l i n e s and f resh leukemic t i ssue was tested by an i n d i r e c t binding assay using rad io iod ina ted F ( a b ' ) 2 f r a c t i o n s of rabbi t anti-mouse Ig ant ibodies (RaMIg). One m i l l i o n target c e l l s were incubated with 50 u l of hybridoma supernatant in a 96-wel l m i c r o t i t r e p la te for one hour at 4 °C . Af ter washing twice with 200 u l E a r l e ' s balanced s a l t s o l u t i o n containing 0.5% BSA, 0.1% NaN3, and 10 mM 90 HEPES bu f fe r , the c e l l s were incubated with 50 y l (10 5 cpm) of 1 2 5 I - R a M I g (1-3 x 10? cpm/ug) and fur ther incubated for one hour at 4 ° C . C e l l s were washed again three t imes, t ransfer red to gamma-counter tubes, and counted on a Beckman biogamma counter. Ant igen Est imat ion A modi f ica t ion of the binding assay was used to estimate the number of NHL-30.5 antigens on var ious c e l l types. Twenty y l of chloramine T (0.5 mg/ml) was added to 25-50 yg ( in 25-50 y l ) of p u r i f i e d NHL-30.5 antibody fol lowed by the add i t ion of 1 mCi 125T_ s odium iodide (Amersham, Canada). The r e a c t i o n was stopped a f t e r 15 minutes with the add i t ion of 50 y l of sodium metab isu l f i t e (2 mg/ml) and the iodinated antibody was loaded onto a smal l P10 s i z i n g column. The antibody peak was pooled and th is stock was used in a d i r e c t binding assay. Various concentrat ions of the iodinated antibody (1-3 x cpm/yg) were t i t r a t e d with d i f f e ren t numbers of target c e l l s to determine sa tura t ion l e v e l s , and the s p e c i f i c b inding was c a l c u l a t e d by subt rac t ing the amount of r a d i o a c t i v i t y bound to the c e l l s in the presence of an excess of co ld antibody from the amount bound at sa tu ra t ion in the absence of co ld ant ibody. An estimate of antigen densi ty was then obtained us ing the fo l lowing c a l c u l a t i o n : # m o l e c u l e s / c e l l = s p e c i f i c binding (cpm bound/ce l l ) x 6.02 x 10^3 s p e c i f i c a c t i v i t y of antibody (cpm/mole) FACS Ana lys is C e l l s (1-2 x 10 6 ) were washed with RPMI 1640 conta in ing 10% FCS, 0.1% NaN3, and lOmM HEPES bu f fe r , and incubated for one hour at 4°C with 50 y l of undi luted hybridoma cu l tu re supernatant in a 96-wel l m i c r o t i t r e p l a t e . Media 91 a lone , or an unrelated monoclonal antibody ra ised against mouse lymphocytes was added to the con t ro l c e l l s . The c e l l s were washed twice and resuspended in 50 y l of FITC-conjugated rabbit or goat (Fab'>2 ant ibodies to mouse Ig . A f t e r an a d d i t i o n a l incubat ion for one hour at 4 °C , the c e l l s were incubated with propidium iodide (25 ug/ml) for f i ve minutes before the f i n a l three washes. Dead c e l l s could then be gated out from the a n a l y s i s on the bas is of propidium iodide f luorescence . If the c e l l s were not going to be analyzed the same day they were washed three times in phosphate buf fered s a l i n e (PBS) and f i xed for 30 minutes on ice with IX paraformaldehyde in PBS. In s i t u a t i o n s where the f luorescence p r o f i l e s of the media c o n t r o l and the test sample demonstrated a c lear crossover point when over lapped, the percent p o s i t i v e c e l l s was ca lcu la ted by subt rac t ing the background s t a i n i n g in the media con t ro l (at the crossover point) from those reac t ing with the test sample and d i v i d i n g by the number of c e l l s analyzed (10,000 or 20,000). I f no d i s t i n c t crossover point was evident the top 5% f luorescent c e l l s in the media cont ro l was subtracted from the number of f luorescent c e l l s in the test sample at the same gates. Flow Cytometry The FACS system (Becton Dick inson, CA) was used to measure the s i z e and f luorescence i n t e n s i t y of hemopoietic c e l l s l a b e l l e d with var ious monoclonal a n t i b o d i e s . This i s accomplished by passing a stream of c e l l s , i n s i n g l e f i l e , through the focussed beam of a high power l a s e r ( in th is case a Spectra Physics Argon laser at a power s e t t i n g of 400 mW) that i s coupled with a set of h igh ly s e n s i t i v e detectors (Figure I I ) . These photodetectors t r a n s l a t e o p t i c a l s i g n a l s , emitted by i n d i v i d u a l c e l l s as they pass through the l a s e r beam, in to e l e c t r i c a l impulses that are then stored for d i s p l a y and a n a l y s i s . The i n t e n s i t y of the l i g h t sca t te r s i g n a l depends to a large extent on c e l l 92 s i z e , and th is i s one parameter rout ine ly d isplayed by the instrument; the la rger the c e l l , the greater the l i gh t s c a t t e r . L ight s c a t t e r measurements are a lso use fu l to d iscr imina te between v iab le and nonviable c e l l s . I f the c e l l s have been tagged with a f luorescent probe (such as antibody coupled to a f luorescent dye) , the f luorescence i n t e n s i t y of each c e l l when exc i ted by the l aser can be analyzed separately from l i g h t s c a t t e r measurements. The f luorescence detectors are covered by f i l t e r s that b lock the wavelength of l i g h t emitted by the l aser and th is reduces background s i g n a l s . When the s o r t i n g c a p a b i l i t i e s of the FACS are u t i l i z e d , the stream of c e l l s i s subjected to an u l t r a s o n i c v e r t i c a l v i b r a t i o n of ~40,000 Hz that breaks the stream into small droplets conta in ing , on average, one c e l l fo r every eight droplets (2) . The o p t i c a l measurements made on each c e l l are compared to preset parameter l e v e l s before the c e l l reaches the stream t i p . I f the c r i t e r i a for i n c l u s i o n into a sorted populat ion are met, then the drople t conta in ing the c e l l of in terest i s e l e c t r i c a l l y charged as i t reaches the stream t ip and can therefore be appropr ia te ly de f lec ted as i t passes through an e l e c t r i c f i e l d created by charged d e f l e c t i o n p l a t e s . Since hemopoietic c e l l s are e a s i l y dispersed in to s i n g l e c e l l suspensions, they are i d e a l subjects for FACS a n a l y s i s . A l l c e l l s s ta ined in th is study were analyzed on e i ther a FACS IV (Department of Neurology, U n i v e r s i t y of B .C . ) or a FACS 440 (Terry Fox Laboratory, B . C . Cancer Research Cent re ) . The instruments were rout ine ly standardized using g lu tara ldehyde-f ixed chicken red blood c e l l s and f luorescent microspheres. Debris was gated out on the basis of l i g h t sca t te r measurements. 93 Cell sample Collection tubes FIGURE II Schematic representat ion of the f luorescence a c t i v a t e d c e l l s o r t e r (FACS), From ref (2) . 94 3) IMMUNOPRECIPITATION Target c e l l s (2 x 10?) were washed three times with PBS and resuspended in 0.3 ml of PBS in a g lass tube coated with 100 ug of iodogen (P ierce Chemical , Rockford, IL) (3) . One mCi of 1 2 5 T _ S 0 C i i u m iod ide (Amersham, Canada) was added and the c e l l s were incubated at room temperature for one hour while a g i t a t i n g . The c e l l s were washed twice in EBSS + 0.5% BSA, 0.1% NaN3, and 10 mM HEPES buf fe r , and resuspended in 1.5 ml of PBS conta in ing 50 y l of pheny lmethy lsu l fony l f luor ide . The c e l l s were then lysed with the a d d i t i o n of 0.5 ml of 2% NP40 in PBS and the lysa te was incubated on i c e for f i v e minutes before microfuging (Eppendorf) for f i v e minutes at 4 ° C . The lysa te was incubated with 30 u l of monoclonal antibody for one hour at 4°C and subsequently with 40 y l of a 50% suspension of rabbi t anti-mouse Ig-coupled sepharose beads (approximately 2 mg of anti-mouse Ig/ml of beads) overn ight . The fo l lowing day the beads were washed in EBSS + 0.5% NP40 f i v e times and the bound mater ia l removed by incubat ing the beads with SDS-PAGE sample buf fe r in a b o i l i n g water bath for f i v e minutes. The immunoprecipitate was then analyzed by sodium dodecyl su l fa te -po lyacry lamide ge l e l e c t r o p h o r e s i s (SDS-PAGE) (4) using e i t h e r 10% or 7.5% gels followed by autoradiography with Kodak X-OMAT f i l m and a Dupont Cronex i n t e n s i f y i n g screen. Molecular weight determinations were obtained by comparison to prote in standards (BioRad, Canada). HL-60 c e l l s were metabol ica l ly l a b e l l e d with 35g_met.hionine (800 Ci/mmol) or ^H- leucine (5 Ci/mmol) (Amersham, Canada) to determine i f the NHL-30.5 ant igen i s synthesized by these c e l l s . For 35s_m ethionine l a b e l l i n g , 2 x 10? c e l l s were washed twice in DMEM containing no methionine, and resuspended in 2 ml of methionine- f ree DMEM containing 10% d ia lyzed FCS. For ^H- leucine l a b e l l i n g , medium conta in ing no leucine was used. One hundred yCi of 35g_ 95 methionine or 1 mCi of ^H- leucine was added and the c e l l s were cu l tured for four hours at 37°C. C e l l s were washed three times and resuspended in l y s i s buf fer (1% T r i t o n X-100 in s a l i n e containing 50 mM T r i s - H C l (pH 7 .4) , 1 mM CaCl2> and ImM MnCl2). The lysa te was incubated on ice for 20 minutes, and then microfuged for f i v e minutes at 4°C. The supernatant was then incubated with l e n t i l l e c t i n - c o u p l e d beads (Sigma, St . Lou is , MO), prewashed with l y s i s buf fer conta in ing 0.5% BSA, for one hour. The unbound prote ins were removed i n three washes with l y s i s buf fer and the bound f r a c t i o n in another 3 washes of l y s i s buf fer conta in ing 0.1M a-methyl mannoside. The l e n t i l l ec t in -bound and unbound f r a c t i o n s were then immunoprecipitated with monoclonal ant ibodies as prev ious ly descr ibed for rad io iodinated c e l l s . Phosphorylat ion of the NHL-30.5 antigen was studied by l a b e l l i n g HL-60 with 32p. HL-60 c e l l s (2 x 10?) were washed twice in DMEM conta in ing no phosphate and then incubated with 0.5 mCi of 32p_ o r | - i 1 0 phosphate (50-100 mCi/mg, New England Nuclear , Canada) in phosphate-free DMEM + 15% d i a l y z e d FCS for three hours at 37°C. The c e l l s were then lysed in 0.5% NP40 and immunoprecipitat ion was ca r r i ed out as described for 1 2 5 T _ i a D e i i i n g , 4) HEMOPOIETIC CELL LINES The cu l tu re condi t ions adopted for the c e l l l i n e s u t i l i z e d in th is thes is are summarized in Table II. 5) DIFFERENTIATION OF MYELOID LEUKEMIA CELL LINES  Granu locy t ic D i f f e r e n t i a t i o n Myelogenous leukemia c e l l l i n e s were induced to d i f f e r e n t i a t e us ing a v a r i e t y of inducing agents. Previous studies have ou t l ined the opt imal cu l tu re requirements for the induct ion of g ranu locy t ic d i f f e r e n t i a t i o n i n the HL-60 c e l l l i n e using dimethylsul foxide (DMSO) (13) or r e t i n o i c ac id (13). C e l l s were cu l tured in p l a s t i c t issue cu l ture f l asks (Corning, NY) at a 96 TABLE II Culture Condit ions for Human Hemopoietic C e l l l i n e s CELL LINE ORIGIN CULTURE MEDIUM HL-60 (5) Acute promyelocytic leukemia DMEM + 10% FCS K562 (6) Chronic myeloid leukemia ( P h i - p o s i t i v e ) DMEM + 10% FCS KG-1 (7) Acute myeloblast ic leukemia a + 10% FCS KG- la (8) Acute myeloblast ic leukemia RPMI + 10% FCS HEL (9) Erythroleukemia RPMI + 10% FCS U937 (10) H i s t i o c y t i c lymphoma RPMI + 10% FCS 1,4,6,8 and 10 (11) B - c e l l lymphoma RPMI + 10% FCS Jurkat (12) T - c e l l leukemia RPMI + 10% FCS A l l c e l l l i n e s were cu l tured in an atmosphere of 5% CO2 at 37°C . The above c e l l l i n e s were generous g i f t s from the fo l lowing i n d i v i d u a l s : HL-60: Dr. J . Levy, Dept. of Microbio logy, Un ive rs i t y of B . C . K562: Dr. BB L o z z i o , Dept. of Medical B io logy , U n i v e r s i t y of Tennessee. KG-1: Dr. HP K o e f f l e r , Dept. of Medicine, Un ive rs i t y of C a l i f o r n i a , L . A . HEL/KG-la/U937: Dr. T. Pawson, Dept. of Microb io logy , U n i v e r s i t y of B . C . Jurka t : Dr. DG K i l b u r n , Dept. of Microbio logy, U n i v e r s i t y of B . C . 97 seeding concentrat ion of 2 x 10^ c e l l s / m l in the appropr iate medium (see s e c t i o n on c e l l l i n e s ) contain ing 10% FCS and e i ther 1.25% DMSO ( F i s h e r , Canada) or 1 x 10~6 M r e t i n o i c ac id (Sigma, St . L o u i s , MO). Cont ro l c u l t u r e s were incubated in t i ssue cu l ture medium contain ing no inducing agent. The cu l tu res were incubated for f i v e days and the v i a b i l i t y of the c e l l s on each day was assessed by trypan blue exc lus ion . One, three, and f i v e days fo l lowing the add i t ion of the inducing agent, the c e l l s were harvested for a n a l y s i s of the expression of se lected c e l l surface ant igens . Morphologica l and cytochemical assessment of the extent of d i f f e r e n t i a t i o n was examined independently by a hematopathologist at the Vancouver General H o s p i t a l . Monocytic D i f f e r e n t i a t i o n C e l l s (HL-60, KG-1, and HEL) were cul tured in 100 mm diameter t i s s u e cu l tu re dishes at a seeding concentrat ion of 2 x 10^ c e l l s / m l in appropr ia te medium conta in ing 1.6 x 10~ 8 M (HL-60 or KG-1) , or 10~ 6 to 10" 7 M (HEL) 12-0-tetradecanoylphorbol-13 acetate (TPA). Since the TPA was d i s s o l v e d i n DMSO, equivalent amounts of DMSO were added to cont ro l c u l t u r e s . Induced HL-60 and KG-1 c e l l s were harvested on days one and two and induced HEL c e l l s on days one, three, and f i v e . Nonadherent c e l l s were removed and the adherent c e l l s were treated with s a l i n e contain ing 0.2% EDTA and 0.1% BSA at 4°C for about 30 minutes to f a c i l i t a t e the removal of the adherent c e l l s . Strongly adherent c e l l s were gent ly removed with a rubber policeman. 6) HUMAN CELL PREPARATIONS  Pat ient Specimens Heparinized per iphera l blood and bone marrow asp i ra tes were obtained with informed consent from pat ients with various hematologic mal ignancies . Normal marrow cont ro ls were obtained from i n d i v i d u a l s donating the i r marrow fo r t ransp lanta t ion or from pat ients with malignancies that d id not invo lve the 98 marrow. Bone marrow c e l l s were prepared by one of two techniques. The f i r s t involved spinning at 800g for four minutes fol lowed by the removal of the buffy coat and t rea t ing with ammonium ch lor ide (9 volumes N H 4 C I (8.3 g/1 in water) , 1 volume t r i s base (20.6 g/1 adjusted to pH 7.65 with 1M HCL), and f i n a l l y adjusted to pH 7.2) to remove res idua l red c e l l s . The second technique had a lower y i e l d but enriches for immature populat ions by removing granulocytes by c e n t r i f u g a t i o n on 1.077g/cm3 f i c o l l - h y p a q u e (LSM, L i t t o n B i o n e t i c s , Kensington, MD) or 1.077g/cm^ p e r c o l l (Pharmacia, Uppsala , Sweden). Pe r iphera l blood specimens were genera l ly obtained by f i c o l l - h y p a q u e prepara t ion , although the large number of c e l l s in some pat ient specimens ( p a r t i c u l a r l y CML) made i t eas ier to prepare a buffy coat . A l l specimens for s o r t i n g experiments (Chapter 5) were i so la ted on f i c o l l - h y p a q u e (1.077g/cm3) g rad ien ts . C e l l F rac t iona t ion Granulocytes and erythrocytes were p u r i f i e d from the per iphera l blood of healthy volunteers using a one-step f i co l l -hypaque (1.114 g/cm^) sedimentation procedure (Mono-Poly Resolving Medium, Flow Labora tor ies , Inglewood, CA) . Pur i t y of the enriched populat ions was assessed morpholog ica l ly . For monocyte p u r i f i c a t i o n (15), the l i g h t densi ty f r a c t i o n of f i c o l l - h y p a q u e (1.077g/cm^) separated c e l l s were washed and resuspended at 5 x 10^ c e l l s / m l in RPMI 1640 with 10% FCS and placed in 100 mm diameter t issue cu l tu re d i s h e s . Fol lowing an incubat ion per iod of 90 minutes at 37°C, the adherent c e l l s were washed f i v e times with RPMI 1640 contain ing 10% FCS and treated for one minute with normal s a l i n e conta in ing 0.2% ethylenediaminetetraacet ic ac id (EDTA) and 0.1% bovine serum albumin (BSA). The adherent c e l l s were then gent ly removed with a rubber policeman and shown to be >80% p o s i t i v e for n o n s p e c i f i c es te rase . 99 P l a t e l e t s were obtained from heparin or c i t r a t e ant icoagula ted blood of pooled donors from the Canadian Red Cross (Vancouver, B . C . ) ' P l a t e l e t s from i n d i v i d u a l donors were prepared by spinning 10 ml of pe r iphera l blood at 190 x g for 20 minutes at room temperature and removing the supernatant . Th is p l a t e l e t - r i c h plasma was then spun at 2,500 x g for f i v e minutes at room temperature and the pe l l e ted p l a t e l e t s were resuspended in b inding assay medium adjusted to pH 6.5 with 1M c i t r i c a c i d . Normal bone marrow f i b r o b l a s t s from three separate donors were obtained by p l a c i n g 2-3 x 10^ nucleated marrow c e l l s from each donor in 8 ml of medium (a + 20% FCS) in 60 x 15 mm t issue cul ture dishes (Fa lcon , C a l i f o r n i a ) . The c e l l s were cul tured in an atmosphere of 5% CO2 at 37°C , and f i v e days l a t e r the nonadherent c e l l s were discarded and replaced with f resh medium. When conf luent , the f i b r o b l a s t cu l tures were washed twice in s a l i n e fol lowed by the add i t ion of 5 ml of a 0.25% t ryps in so lu t ion ( in c i t r a t e d s a l i n e ) . Ten minutes l a t e r one ml of FCS was added to stop fur ther t r y p s i n a c t i o n , and the adherent c e l l s were detached by gentle p i p e t t i n g . The c e l l s were washed in a + 20% FCS and cu l tu res were r e i n i t i a t e d with 10^ c e l l s / d i s h . A f t e r one or two such subcul tures the enzymatic detachment of the adherent c e l l s for FACS ana lys is was accomplished using b a c t e r i a l col lagenase type I (200 un i ts /mg, Sigma, MO) (16) instead of t ryps in to prevent d i g e s t i o n of c e l l sur face ant igens. The col lagenase was d isso lved in calcium and magnesium-free Hank's balanced s a l t s o l u t i o n (HBSS-Ca-Mg). Immediately p r i o r to use, FCS was added to g ive a f i n a l concentrat ion of 20% FCS and 0.1% co l lagenase . Confluent cu l tu res were drained of medium, washed twice in HBSS-Ca-Mg, and cu l tured in 10 ml of the col lagenase s o l u t i o n for three hours in an atmosphere of 5% CO2 at 37 C. Adhe rent c e l l s were again removed by gent le p i p e t t i n g . 100 For PHA-st imulat ion of lymphocytes, the mononuclear f r a c t i o n from f i c o l l -hypaque (1.077g/cm3) separated per iphera l blood was cu l tured i n p l a s t i c t i ssue cu l tu re f l a s k s at a concentrat ion of 10^ c e l l s / m l in RPMI 1640 conta in ing 10% FCS and 1% phytohemagglutinin (PHA; Gibco, Calgary , A l b e r t a ) . The c u l t u r e s were incubated in an atmosphere of 5% CO2 and the c e l l s were harvested a f t e r three days. V i a b i l i t y was assessed by trypan blue exc lus ion and s t i m u l a t i o n was confirmed by pu ls ing the c e l l s with ^H-thymidine and counting in a s c i n t i l l a t i o n counter (17). 7) CELL SORTING Normal per iphera l b lood, normal marrow, and CML marrow c e l l s were separated by f i c o l l - h y p a q u e (1.077g/cm3) and sta ined for s o r t i n g as p rev ious ly descr ibed under 'FACS A n a l y s i s ' using s t e r i l e condi t ions and medium conta in ing no NaN3- C e l l s were sorted into p o s i t i v e and negative f r a c t i o n s depending on the i r r e a c t i v i t y with the NHL-30.5 monoclonal antibody according to the fo l lowing c r i t e r i a : In cases where less than 5% of the c e l l s were p o s i t i v e (seen with a l l normal bone marrow and per iphera l blood specimens) the sor t gates were adjusted so that c e l l s with the highest f luorescence i n t e n s i t y , comprising 5% of the t o t a l populat ion, were included in to the p o s i t i v e f r a c t i o n , and c e l l s with the lowest f luorescence i n t e n s i t y (the remaining 95%) were sorted in to the negative f r a c t i o n . In s i t u a t i o n s where greater than 5% of the c e l l s were NHL-30 .5 -posi t ive (seen only with f i c o l l - h y p a q u e separated CML per iphera l blood) then a l l p o s i t i v e c e l l s were sorted in to the p o s i t i v e f r a c t i o n . The sorted f rac t ions were then counted and plated in standard methy lce l lu lose assays to determine the d i s t r i b u t i o n of the var ious myeloid progeni tor c lasses wi th in each sorted populat ion. As a c o n t r o l some specimens were sta ined with media or an isotype-matched monoclonal antibody ( IgGl) s p e c i f i c for phycoerythr in , and the top 5% again sorted and assayed for progeni tors . 101 8) ASSAYS FOR CLONOGENIC MYELOID PROGENITORS C e l l s were plated in standard 0.8% methy lce l lu lose cu l tu re medium conta in ing 30% FCS, 1% deionized BSA, 10"^M 2-mercaptoethanol, 3 un i ts per ml of human ur inary e ry thropo ie t in (100-300 units/mg) (18), and 9% human leukocyte condit ioned medium (19) prepared by a mod i f i ca t ion of the standard agar-serum overlay procedure (20). Normal unsorted bone marrow c e l l s were plated at 1 x 10^ c e l l s per ml and per iphera l blood at A x 10^ c e l l s per ml. Unsorted CML c e l l s and a l l sorted c e l l s were a lso plated at mu l t ip le lower c e l l concentrat ions to ensure obta in ing a condi t ion where colony numbers were s u f f i c i e n t but not excess ive . Cultures were scored using an inver ted microscope and the data obtained consistent with a l i n e a r c e l l dose-colony y i e l d r e l a t i o n s h i p . Adequate burst and granulocyte colony formation by separated c e l l s was achieved by the presence of leukocyte condi t ioned medium. Colonies der ived from CFU-E (1-2 c l u s t e r s of e ry th rob las ts ) and mature BFU-E (3-8 c l u s t e r s of e ry throb las ts ) were counted on day 10, and from p r i m i t i v e BFU-E (>8 c l u s t e r s of e ry throb las ts ) and CFU-C (>20 granulocytes and/or macrophages) on day 18, according to estab l ished c r i t e r i a (21). In order to determine whether binding of the NHL-30.5 monoclonal antibody to progeni tor c e l l sur faces could i n h i b i t or st imulate colony format ion, 1-10 yg/ml of p u r i f i e d NHL-30.5 monoclonal antibody was included in the assay medium. Assays both with and without e ry thropo ie t in and leucocyte condi t ioned medium were used in these experiments. For i n h i b i t i o n s t u d i e s , monoclonal ant ibodies against the t r a n s f e r r i n receptor (NHL-62.14, and NB-2) were used as p o s i t i v e c o n t r o l s , and p u r i f i e d IgG and an an t i -LFA-1 monoclonal antibody were used as negat ive c o n t r o l s . 102 REFERENCES 1. Kohler G, M i l s t e i n C: Continuous cul tures of fused c e l l s s e c r e t i n g antibody of predefined s p e c i f i c i t y . Nature 256: 495, 1975. 2. van den Engh G, V i s s e r J : Flow cytometry in experimental haematology. In: B i b l t h c a Haemat 48: 42, (Karger, B a s e l , 1984). 3. Markwell MAK, Fox CF: Surface s p e c i f i c i o d i n a t i o n of membrane prote ins of v i ruses and eukaryot ic c e l l s using l , 3 , 4 , 6 - t e t r a c h l o r o - 3 -d i p h e n y l g l y c o l u r i l . Biochemistry 17: 4807, 1978. 4. 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In: Hematopoietic Stem C e l l s (Golde DW, Takaku F, eds ) , Marcel Dekker Inc, NY, 1985. 104 C H A P T E R III NHL-30.5: A MONOCLONAL ANTIBODY DEFINING AN ACUTE MYELOGENOUS LEUKEMIA (AML)-ASSOCIATED ANTIGEN 1) INTRODUCTION Heterogeneity i n the c l i n i c a l features and responses of patients with AML has been recognized for a long time and underlies the need for new approaches to disease c l a s s i f i c a t i o n . The most common method involves the use of morphological and cytochemical c r i t e r i a to categorize leukemic blast c e l l populations into subgroups that appear to c o r r e l a t e with d i f f e r e n t stages of normal hemopoietic c e l l d i f f e r e n t i a t i o n . In the l a s t several years, a new method for analyzing hemopoietic c e l l d i f f e r e n t i a t i o n events using monoclonal antibodies (1) has emerged, and a large number of myeloid surface antigens have now been i d e n t i f i e d by this technique (2-5). Some of these are r e s t r i c t e d i n t h e i r expression to c e l l s at various stages of maturation within a p a r t i c u l a r lineage while others may be found on c e l l s of a number of d i f f e r e n t hemopoietic lineages (Chapter I ) . The surface phenotypes of AML blast c e l l s include some of these antigens and, i n general, the pattern of antigen expression i s consistent with the view that the b l a s t s maintain a c e l l surface p r o f i l e c h a r a c t e r i s t i c of a normal myeloid c e l l at an equivalent l e v e l of d i f f e r e n t i a t i o n (3,6), although some exceptions to t h i s have been reported (7-9). To obtain further information about the nature, co n t r o l , and d i s t r i b u t i o n of phenotypes in blast c e l l populations from d i f f e r e n t AML patients, we i n i t i a l l y embarked on a program to develop new monoclonal 1 0 5 antibody reagents with greater s p e c i f i c i t y for c e l l s with immature myeloid c h a r a c t e r i s t i c s . Pre l iminary studies i d e n t i f i e d one such ant ibody, NHL-30.5, which showed an apparent s p e c i f i c i t y for hemopoietic c e l l s from pat ients with AML. The i d e n t i f i c a t i o n of the antigen def ined by th is monoclonal ant ibody, and a n a l y s i s of i t s c e l l u l a r d i s t r i b u t i o n in normal and leukemic hemopoietic t i s s u e i s the subject of th is Chapter. 2) RESULTS R e a c t i v i t y of NHL-30.5 Monoclonal Antibody With Various Human C e l l s The production of monoclonal ant ibodies reac t ive with HL-60 c e l l s i s descr ibed in Chapter II . One of the an t ibod ies , NHL-30.5, showed apparent s p e c i f i c i t y for AML c e l l s and was further charac te r i zed . The r e a c t i v i t y of the NHL-30.5 monoclonal antibody with a v a r i e t y of normal and malignant hemopoietic c e l l s was tested by f luorescence s t a i n i n g . Samples were reacted with the antibody, FITC-conjugated rabbi t (Fab')2 a n t i -mouse Ig , and then analyzed on the FACS. Figure III shows the f luorescence p r o f i l e s of the r e a c t i v i t y of NHL-30.5 with f i c o l l - h y p a q u e separated p e r i p h e r a l blood c e l l s from a representat ive normal, AML, and CML p a t i e n t . Three cont ro ls were used. The f i r s t was a media c o n t r o l in which c e l l s were incubated with the second FITC-conjugated rabbi t (Fab')2 anti-mouse Ig without p r i o r exposure to NHL-30.5. As a p o s i t i v e c o n t r o l we used a monoclonal antibody produced in the same fus ion as NHL-30.5 that reacts with a l l human hemopoietic c e l l s tested to date (except red c e l l s ) , and as a negat ive cont ro l we used an unrelated mouse monoclonal antibody ra ised against mouse lymphocytes. The f luorescence p r o f i l e s of the b inding of NHL-30.5 to the normal and CML samples were the same as the negat ive c o n t r o l s . The p r o f i l e of the AML pat ien t , however, showed s i g n i f i c a n t b inding of the antibody above background. 106 PERIPHERAL BLOOD NORMAL AML CML MEDIA CONTROL POSITIVE CONTROL NEGATIVE CONTROL . \ NHL-30.5 • V o cc 111 CO z UJ O LU > LU FLUORESCENCE INTENSITY FIGURE III FACS a n a l y s i s of the binding of NHL-30.5 monoclonal antibody to f i co l l -hypaque-separa ted per iphera l blood from a normal, AML, and CML pa t ien t . C e l l s were stained with NHL-30.5 and F ITC-rabbi t (Fab')2 anti-mouse Ig. Three cont ro ls were used: The media con t ro l had no test antibody added, the p o s i t i v e c o n t r o l was a monoclonal antibody produced in our laboratory that has reacted with a l l human hemopoietic c e l l s tested to date (except red c e l l s ) , and the negative cont ro l was an unrelated monoclonal antibody ra ised against mouse lymphocytes. 107 MEDIA CONTROL POSITIVE CONTROL NEGATIVE CONTROL NHL-30.5 BONE MARROW NORMAL AML CML FLUORESCENCE INTENSITY CD O DC LU CQ LU O LU > < _l LU CC FIGURE IV FAGS a n a l y s i s of binding of NHL-30.5 to buffy coat samples of bone marrow from a normal, AML, and CML donor. The a n a l y s i s was c a r r i e d out as in Figure III. 108 Figure IV ind ica tes the r e a c t i v i t y of the antibody with marrow c e l l s from the same normal, AML, and CML donors used in F ig I I I . The only c e l l s with detectable b inding above the media and negative cont ro ls are those from the AML pa t ien t . Normal per iphera l blood granulocytes (>90X granulocytes by morphology), monocytes (adherent c e l l s , >80% p o s i t i v e for n o n s p e c i f i c e s t e r a s e ) , lymphocytes (the nonadherent c e l l s from the monocyte p u r i f i c a t i o n procedure) , PHA-stimulated lymphocytes, erythrocytes (ABO), sp lenocytes , and p l a t e l e t s were a lso tested in th is manner. None of these showed any detectable s t a i n i n g (Figure V) . The r e a c t i v i t y of NHL-30.5 was a lso tested in a b inding assay using 1 2 5 i _ r a i j D i t (Fab')2 anti-mouse Ig second an t ibod ies . Table III g ives the binding assay r e s u l t s for the f i r s t 19 AML pat ients tes ted , together with the X p o s i t i v e c e l l s determined by FACS ana lys is and assoc ia ted c l i n i c a l da ta . F i f t e e n of the 19 samples tested were p o s i t i v e by both FACS and b inding assays (pat ients Nos. 1-15). A value of >10% p o s i t i v e was considered a p o s i t i v e r e s u l t in the FACS, and >1,000 cpm was considered p o s i t i v e by b inding assay . Table IV l i s t s a l l of the AML pat ients analyzed to date that have demonstrated r e a c t i v i t y with the NHL-30.5 monoclonal ant ibody. Binding assay data i s not shown s ince these s tudies were not continued a f te r a n a l y s i s of the f i r s t 19 p a t i e n t s . AML pat ients that d id not react ( i . e . showing <10X NHL-30.5-p o s i t i v e c e l l s ) are l i s t e d in Table V. A t o t a l of 48 AML specimens have been s tud ied with th is antibody and 40 of them were c l a s s i f i e d as NHL-30.5-p o s i t i v e . F ive pat ients with AML and re la ted d isorders were studied dur ing the course of the i r d isease and these r e s u l t s are d isp layed in Table VI (see d i s c u s s i o n ) . A summary of a l l the AML pat ients tested with NHL-30.5 i s g iven in Table VI I . A l l of the Ml pat ients studied and most of the M2's and M4's were p o s i t i v e . One of two M3's and only two of s i x M5's were p o s i t i v e . 1 0 9 PHA-STIMULATED GRANULOCYTES MONOCYTES LYMPHOCYTES LYMPHOCYTES ERYTHROCYTES SPLENOCYTES PLATELETS MEDIA CONTROL "\ POSITIVE CONTROL NEGATIVE CONTROL A \ -NHL-30.5 \ < _ J LU CC FLUORESCENCE INTENSITY FIGURE V FACS a n a l y s i s of the binding of NHL-30.5 to populat ions of granulocytes , monocytes, lymphocytes, PHA-stimulated lymphocytes, e ry th rocy tes , splenocytes, and p l a t e l e t s from normal donors. The a n a l y s i s was car r ied out as descr ibed in F igure II I . Mouse a n t i -human serum was used as p o s i t i v e con t ro l for the e r y t h r o c y t e s . PHA-stimulated lymphocytes were cu l tured at a concent ra t ion of 10 6 c e l l s / m l in RPMI 1640 conta in ing 10% FCS and 1% phytohemagglutinin for three days. TABLE III R e a c t i v i t y of the NHL-30.5 monoclonal antibody with the f i r s t 19 AML p a t i e n t s studied FAB Age/sex WBC % B l a s t s FACS a n a l y s i s NHL-30.5 b inding c l a s s i f i c a t i o n x 10 9 /1 % p o s i t i v e (cpm) § PB BM PB BM PB BM 1 Ml 70/F 282 98 97 49 51 2,029 2,033 2 Ml 62/M 9.3 82 94 26 55 1,123 1,734 3 Ml 67/M 17.1 63 71 16 NT 3,617 NT 4 Ml 55/M 0.9 58 81 66 NT NT NT 5 Ml 67/M 3.4 21 35 15 NT 1,327 NT 6 M2 54/F 12.1 51 60 53 46 2,574 3,726 7 M2 55/M 43.8 67 32 21 21 763 1,075 8 M2 88/M 5.3 6 20 53 11 NT 4,316 9 M2 44/F .525 29 71 NT 12 NT NT 10 M2 74/F 4.7 38 41 20 46 1,097 1,588 11 M2 34/F 62 86 89 42H NT NT NT 12 M4 36/F 21.9 42 47 64 26 1,014 NT 13 M4 69/M 10 8 33 26 21 672 695 14 M4 49/F 30.3 57 72 58 NT 1,407 NT 15 M4 43/M 127 94 79 70H NT 4,198 NT 16 M2 15/M 4 9 32 2 NT 843 867 17 M3 26/F 2.3 OCC. 9 <1 NT NT NT 18 M4 42/F 15.5 67 91 <l1 NT 394 NT 19 M5 27/M 112 86 87 <1 NT 11 53 H Leukapheresis samples § B ind ing data represents the r a d i o a c t i v i t i e s of NHL-30 .5 - labe l led c e l l s above background l e v e l s obtained with media c o n t r o l s i n which no f i r s t antibody was used. Media c o n t r o l s were <350 cpm. NT = not tested Pa t ien ts # 5 and 9 were i n re lapse at t e s t i n g . A l l others were tested at p resenta t ion and p r i o r to treatment. I l l TABLE IV A summary of AML specimens contain ing >10% NHL-30 .5 -pos i t ive c e l l s (NHL-30.5-posi t ive) AML FAB °/o BLASTS % POSITIVE BY FACS PATIENT CLASSIFICATION PB BM PB BM 1 U 45 45 58 63 2 Ml 85 90 55 58 3 Ml 94 92 NT 48 4 Ml 11 66 9 51 5 Ml 98 97 49 51 6 Ml 82 94 26 55 7 Ml 63 71 16 NT 8 Ml 58 81 66 NT 9 Ml 21 35 15 NT 10 M2 60 94 54 52 11 M2 51 60 53 46 12 M2 67 32 21 21 13 M2 6 20 53 11 14 M2 29 71 NT 12 15 M2 38 41 20 46 16 M2 86 89 42* NT 17 M3 17 8 36 23 18 M4 4 26 22 NT 19 M4 55 48 NT 42 20 M4 51 79 23 32 21 M4 13 31 NT 47 22 M4 40 95 NT 15 23 M4 2 52 2 63 24 M4 33 66 52 NT 25 M4 78 59 85 71 26 M4 43 78 47 31 27 M4 50 31 48 NT 28 M4 61 74 80 82 29 M4 72 71 36 38 30 M4 42 47 64 26 31 M4 38 49 36 29 32 M4 8 33 26 21 33 M4 22 82 27 17 34 M4 57 72 58 NT 35 M4 88 70 70 NT 36 M4 94 79 70* NT 37 M4 77 78 64 51 38 M5 84 82 58 29 39 M5 99 98 14 NT 40§ u n c l a s s i f i e d 38 28 NT 58 * leukapheresis samples § I n i t i a l l y diagnosed as a monosomy 7 childhood m y e l o p r o l i f e r a t i v e d isease . This pat ient was tested when h is disease had progressed in to AML. NT = not tes ted , U = und i f fe ren t ia ted 112 TABLE V A summary of AML specimens containing <10% NHL-30 .5 -pos i t ive c e l l s (NHL-30.5-negative) AML FAB % BLASTS FACS ANALYSIS PATIENT CLASSIFICATION % POSITIVE PB BM PB BM 1 M2§ 9 32 2 NT 2 M3 Occ. 9 <1 NT 3 M4 67 91 <1* NT 4 M4 88 84 <1 <1 5 M5 86 87 <1 NT 6 M5 6 (85)1 18 (60)H NT 4 7 M5 62 74 <1 1 8 M5 43 50 <1 NT * leukapheresis samples § Relapsed 11 months l a t e r with 46% NHL-30.5 p o s i t i v e c e l l s in the p e r i p h e r a l blood and 18% in the bone marrow. H f igures in parentheses ind ica te % ear ly monocytes. NT = not tested 113 TABLE VI Sequent ia l t es t ing of c e l l s from pat ients with AML and re la ted d isorders PATIENT STATUS DATE % BLASTS FACS ANALYSIS % POSITIVE PB BM PB BM 1 AML-M2 presentat ion Oct 1/1982 11 32 2 NT treated Oct 20/1982 <1 <1 NT <1 remission Nov 10/1982 <1 3 NT 2 re lapse Aug 22/1984 15 65 46 18 2 AML-M1 presentat ion Jan 12/1983 63 71 16 NT remission Apr 20/1983 <1 <1 NT <1 3 AML-M4 presentat ion Oct 12/1983 72 71 36 38 re lapse Dec 13/1984 84 86 43 56 4§ MONOSOMY 7 Sep 13/1984 16 17 33 NT MPD Nov 30/1984 38 28 58 NT 5§ UNSPECIFIED Sep 28/1984 11 NA 20 NT MPD Dec 28/1984 53 NA 66 NT MPD = mye lopro l i f e ra t i ve disease § Pat ients #'s 4 and 5 were being monitored for progression of the i r d isease in to AML. NT = not tested 114 Marrow c e l l s from pat ients with various other malignant hemopoietic d iseases were a lso examined for the i r r e a c t i v i t y with NHL-30.5 by FACS a n a l y s i s and/or b inding assay. The r e s u l t s of these tes ts are summarized together with the AML data in Table VII . F ive non-AML samples reacted with the ant ibody. These were a chronic myelomonocytic leukemia (CMML) (1 /1 ) , a m y e l f i b r o s i s (MF) (1 /2 ) , an acute lymphoblastic leukemia (ALL) (1 /15) , seven chronic myeloid leukemias (CML) (7/26) , and three b las t c r i s e s of chronic m y e l o p r o l i f e r a t i v e d isorders (3 /6, Table VI I I ) . R e a c t i v i t y with c e l l s i n the acute phase of CML was r e s t r i c t e d to one pat ient with myeloid b las t c r i s i s and a biphenotypic var iant conta in ing both a myeloid and a lymphoid b las t populat ion (Table VI I I ) . A l l three lymphoid b last c r i s e s tested were uni formly negat ive. In a d d i t i o n , one pat ient with an u n s p e c i f i e d m y e l o p r o l i f e r a t i v e d isorder entered an unusual b last c r i s i s charac te r i zed by the presence of a CALLA-pos i t i ve , HLA-DR-posi t ive , TdT-negat ive , and NHL-30.5-p o s i t i v e b last popula t ion . D i f f e r e n t i a t i o n of HL-60 Approximately 70-90% of cul tured HL-60 c e l l s showed r e a c t i v i t y with NHL-30.5. When the c e l l s were induced to d i f f e r e n t i a t e by incubat ing i n the presence of DMSO, the f luorescence p r o f i l e s of the induced c e l l s became i d e n t i c a l to those of the media and negative cont ro ls (F igure V I ) , i n d i c a t i n g that a dramatic decrease in antigen expression had occurred . Binding assay data confirmed these r e s u l t s . Control cu l tures bound >1,900 cpm and DMSO-treated c e l l s <100 cpm. Fol lowing exposure of the c e l l s to the DMSO, >80% of the c e l l s showed morphological evidence of d i f f e r e n t i a t i o n beyond the promyelocyte stage. 115 Immunoprecipitation of the antigen The antigen defined by NHL-30.5 monoclonal antibody was immunoprecipitated from the surface of iodinated HL-60 c e l l s as described i n Chapter I I . SDS-PAGE of the p r e c i p i t a t e showed the antigen to have a molecular weight of 180,000 under reducing conditions (Figure VII). Immunoprecipitation of iodinated fresh AML c e l l s from a specimen containing 77% blasts revealed a protein with approximately the same molecular weight on SDS gels (Figure V I I I ) . 3) DISCUSSION In this chapter, the i s o l a t i o n and preliminary c h a r a c t e r i z a t i o n of a murine IgGl monoclonal antibody, NHL-30.5, that reacts with a s i g n i f i c a n t proportion of hemopoietic c e l l s from newly diagnosed or relapsed AML patients i s described. The NHL-30.5 producing hybridoma was derived from a fusion between an NS-1 myeloma c e l l and a spleen c e l l from a BALB/c mouse immunized with HL-60 c e l l s . The antibody i s not cytotoxic for HL-60 c e l l s i n a d i r e c t ^chromium release assay. The apparent molecular weight of the antigen on cultured HL-60 c e l l s i s 180,000, where i t i s expressed on 70-90% of the c e l l s grown under standard culture conditions. The antigen also appears to have the same molecular weight on fresh leukemic c e l l s derived from a patient with M4-AML (Figure VIII). Forty of 48 AML samples tested thus far have been found to contain detectable numbers (>10% above background) of NHL-30.5-positive c e l l s . Greatest consistency was seen between NHL-30.5 p o s i t i v i t y and the Ml c l a s s i f i c a t i o n , although the vast majority of the M2's and M4's were also p o s i t i v e . Notably, one of the two M3 patients (acute promyelocytic leukemia) did not have detectable NHL-30.5-positive c e l l s , even though t h i s antibody was i n i t i a l l y raised against an antigen on leukemic promyelocytic c e l l s (HL-60). 116 TABLE VII Summary of results of testing blood (ficoll-hypaque-separated) and/or marrow cells (buffy coat-separated) from various patient categories with the NHL-30.5 monoclonal antibody NUMBER TESTED NUMBER POSITIVE^ AML - undifferentiated 1 1 AML - Ml 8 8 AML - M2 8 7 AML - M3 2 1 AML - M4 22 20 AML - M5 6 2 AML - unclassified 1 1 Chronic myelomonocytic leukemia 1 Myelofibrosis 2 Chronic myeloid leukemia (chronic phase) 26 (for acute phase see Table VIII) Acute lymphoblastic leukemia 15 AML in remission 5 Acute lymphoblastic leukemia in remission 3 Polycythemia vera 1 Multiple myeloma 1 Myelodysplasia 3 Chronic lymphocytic leukemia 2 Normal bone marrow 10 1 1* 7 1 0 0 0 0 0 0 0 40/48 (83%) * A 66-year-old male who presented with myelofibrosis and a peripheral leucocyte count of 2,700 with 19% blasts. Six months after testing, his peripheral counts rose to 123,000 with 50% blasts, and 2 months later he developed myeloid skin i n f i l t r a t e s and died unresponsive to chemotherapy. § positive: >10% NHL-30.5-positive cells 117 HL-60 HL-60 induced with DMSO MEDIA CONTROL A POSITIVE CONTROL NEGATIVE CONTROL A NHL-30.5 FLUORESCENCE INTENSITY FIGURE VI FACS a n a l y s i s of binding of NHL-30.5 to HL-60 c e l l s before and a f t e r induct ion of d i f f e r e n t i a t i o n with DMSO. C e l l s were incubated in the presence of 1.25% DMSO for 5 days and analyzed as descr ibed in Figure III. 118 TABLE VIII R e a c t i v i t y of NHL-30.5 with hemopoietic c e l l s from pat ien ts in the acute phase of CML PATIENT PHILADELPHIA CHROMOSOME BLAST CRISIS* 3 I BLASTS FACS ANALYSIS X POSITIVE PB BM PB BM 1 CML + myeloid (M4) 40 41 15 34 3§ MPD NA 53 NA 66 NT 4 CML + biphenotypic^ 48 69 10 15 5 CML + lymphoid 19 34 1 NT 6 CML + lymphoid 25 90 2 <1 7 CML + lymphoid 44 84 1 <1 * based on morphological , cytochemical , and c e l l sur face phenotyping c r i t e r i a § A l l pat ients were considered to have t y p i c a l Ph^ -pos i t i ve CML before they entered b last c r i s i s with the exception of pat ient #2 (MPD, u n s p e c i f i e d m y e l o p r o l i f e r a t i v e d i s o r d e r ) . The b last populat ion in th is pat ient was unusual in that i t expressed HLA-DR and CALLA but was TdT-negat ive . NA = informat ion not a v a i l a b l e NT = not tested 11 Biphenotypic in th is context ind icates the presence of both a myeloid and a lymphoid b las t popula t ion . 119 FIGURE VII Ana lys is of the NHL-30.5 antigen by immunoprecipi tat ion. HL-60 c e l l s were surface labeled with a c e l l l y s a t e was prepared, and the antigen was immunoprecipitated with NHL-30.5 monoclonal ant ibody. Ana lys is was car r ied out on SDS-PAGE (10%) under reducing cond i t ions . Two contro ls were used: a p o s i t i v e c o n t r o l (descr ibed in Figure I I I ) , and a negative c o n t r o l (no monoclonal antibody added). 120 a b e d 4 5 - * » 3 1 -FIGURE VIII Immunoprecipitation of the NHL-30.5 antigen from the p e r i p h e r a l blood c e l l s of a pat ient with AML (pat ient #37, Table IV) . The sample contained 77% b last c e l l s (M4 c l a s s i f i c a t i o n ) . The l y s a t e from the iodinated c e l l s was immunoprecipitated with a c o n t r o l antibody descr ibed in Figure III ( lane a ) , with a n t i - t r a n s f e r r i n r e c e p t o r - s p e c i f i c monoclonal ant ibodies ( lanes b and c ) , and with NHL-30.5 ( lane d, arrow). 121 Since th is per iphera l blood sample contained only the occas iona l b las t c e l l and bone marrow c e l l s were not tes ted , the negative r e s u l t may have been due to the s e n s i t i v i t y l i m i t of the FACS and binding assays. Th is might a l s o exp la in the negative r e s u l t obtained for one of the M2 p a t i e n t s . Six other AML samples with s i g n i f i c a n t b last counts however, d id not show detectable numbers of NHL-30.5 p o s i t i v e c e l l s . Four of these were monocytic leukemias (M5) and two were myelomonocytic (MA). Since NHL-30.5 does not react with d i f f e r e n t i a t e d monocytes, i t i s poss ib le that AML b l a s t s expressing more d i f f e r e n t i a t e d c h a r a c t e r i s t i c s , such as M5-AML, no longer express NHL-30.5. It should be noted that even amongst p o s i t i v e pat ients there was no c o r r e l a t i o n between the b last count in the sample and the X p o s i t i v e c e l l s determined by FACS a n a l y s i s . F ive pat ients with AML and re la ted d isorders were tested at l eas t twice dur ing the course of the i r disease (Table VI ) . Pat ient #1 (AML) d id not react upon presentat ion (poss ib ly due to the low b last count i n the specimen ana lyzed) , nor d id he react when tested fo l lowing treatment, and l a t e r when he was i n remiss ion . Upon relapse the patient was s t rong ly p o s i t i v e . Pat ient #2 (AML) was p o s i t i v e at presenta t ion , and negative in remiss ion , and pat ient #3 (AML) was p o s i t i v e both at presentat ion and relapse ( remission not t es ted ) . Pat ient #4 was i n i t i a l l y diagnosed as a childhood m y e l o p r o l i f e r a t i v e d isease with c e l l s monosomic for chromosome #7. Monosomy 7 i s the d i a g n o s t i c c r i t e r i o n for one of the more common mye lopro l i f e ra t i ve s ta tes i n chi ldhood and i t i s known to carry a high r i s k of progression to AML (10,11) . Th is pat ient u l t imate ly developed AML and on the two occasions that he was tested (2.5 months apar t ) , r e a c t i v i t y with the NHL-30.5 monoclonal antibody increased with the increased b las t count. The f i f t h pa t ien t , i n i t i a l l y diagnosed as an unspec i f i ed m y e l o p r o l i f e r a t i v e d isease , entered an acute phase charac ter i zed 122 by the presence of HLA-DR and CALLA-positive blast c e l l s that were TdT-negative ( t y p i c a l CALLA-positive ALL i s also TdT-positive). This might be interpreted as the anomalous expression of both myeloid and lymphoid markers since the blast c e l l s can not be c l e a r l y designated as eith e r myeloid or lymphoid. Nevertheless, the r e a c t i v i t y of these c e l l s with NHL-30.5 was shown to increase when the blast count increased. Although only f i v e patients were analyzed sequentially, the results obtained with these patients suggest that expression of NHL-30.5 correlates with the active phase of th e i r disease. NHL-30.5, i n addition to reacting with c e l l s from patients with a diagnosis of AML, bound to c e l l s from a number of other patients not considered to have AML at the time of study. The f i r s t of these was a patient with MF who had 19% blasts in his peripheral blood and whose blood and marrow c e l l s produced numerous abnormal colonies i n methylcellulose assays. Eight months l a t e r , this patient developed overt AML. The second 'non-AML' but NHL-30.5-positive patient was diagnosed as a CMML with 28% bla s t s i n his bone marrow. This patient also produced abnormal colonies i n methylcellulose cultures, but remained i n stable condition eight months a f t e r t e s t i n g . Since CMML i s considered to be a preleukemic disorder, the demonstration of some phenotypic s i m i l a r i t i e s with AML may not be su r p r i s i n g . The t h i r d patient was an adult ALL with 92% bl a s t s . At present we have no explanation for t h i s f i n d i n g since 14 other ALL patients with s i g n i f i c a n t blast populations were a l l c l e a r l y negative. The expression of the antigen on the p o s i t i v e ALL sample might be interpreted as a form of lineage i n f i d e l i t y (8). A l t e r n a t i v e l y , the NHL-30.5 antigen may be a d i f f e r e n t i a t i o n antigen expressed on c e r t a i n primitive lymphoid as well as myeloid c e l l s . A fourth patient appeared to be an ALL on i n i t i a l examination, but cytochemical studies and c e l l surface phenotyping demonstrated a myeloid, as 123 w e l l as a lymphoid, b las t populat ion. Subsequently the pat ient was shown to be P h i - p o s i t i v e suggesting ear ly b last c r i s i s in a pat ient whose primary d isorder was CML. Another pat ient with an unspec i f i ed m y e l o p r o l i f e r a t i v e d isease a lso entered a biphenotypic b last c r i s i s and c e l l s from th is i n d i v i d u a l s t rongly reacted with NHL-30.5. Four other pat ients with CML i n b l a s t c r i s i s were tested and one of them was shown to conta in NHL-30.5-p o s i t i v e c e l l s . This p a r t i c u l a r patient was in myeloid b l a s t c r i s i s . The three pat ients with NHL-30.5-negative b last c r i s i s had the lymphoid v a r i a n t . R e a c t i v i t y with f ico l l -hypaque-separa ted hemopoietic c e l l s from pa t ien ts i n the chronic phase of CML appeared to be l ess frequent. Of 26 CML's s tud ied only seven of them possessed greater than 10% (but no more than 20%) NHL-30.5-p o s i t i v e c e l l s . I f buffy coat preparations of CML c e l l s were used, very few NHL-30 .5 -pos i t ive c e l l s could be detected (<2%). Since f i c o l l - h y p a q u e treatment of CML suspensions (which removes mature granulocytes) enr iches for NHL-30 .5 -pos i t ive c e l l s , the negative resu l t observed with buffy coat preparat ions might r e f l e c t a d i l u t i o n e f fec t mediated by the excess of mature granulocytes predominating in th is d isorder . The r e s u l t s presented in th is Chapter have led to the ten ta t ive des ignat ion of the NHL-30.5 antigen as a leukemia-associated marker. Such a des ignat ion i s based pr imar i l y on the ana lys is of i t s c e l l u l a r d i s t r i b u t i o n on hemopoietic c e l l s from normal and leukemic i n d i v i d u a l s . The molecule i s expressed predominantly on leukemic populat ions from pat ients with AML and to a l e s s e r extent other hematologic d isorders character i zed by the presence of immature myeloid b las t c e l l s . Normal mature hemopoietic c e l l s are c l e a r l y negat ive , as are the vast majori ty of the d i f f e r e n t i a t i n g c e l l s found i n normal bone marrow. Since the NHL-30.5 antigen i s a lso found on und i f f e ren t i a ted HL-60 c e l l s but not fo l lowing DMSO-induced HL-60 124 d i f f e r e n t i a t i o n , i t i s poss ib le that th is antigen i s a normal d i f f e r e n t i a t i o n an t igen , present on a minor populat ion of p r im i t i ve myeloid c e l l s that i s subsequently l o s t dur ing the d i f f e r e n t i a t i o n process. I f th is hypothesis i s c o r r e c t , then one would not expect a normal bone marrow to show r e a c t i v i t y with the antibody (Table VII) because of inherent l i m i t a t i o n s i n the s e n s i t i v i t y of the assays used. Expression of the ant igen on myeloid leukemic c e l l s could then be in terpreted as i n d i c a t i v e of a type of d i f f e r e n t i a t i o n block lead ing to the s e l e c t i v e a m p l i f i c a t i o n of a clone of c e l l s expressing the NHL-30.5 marker. A second p o s s i b i l i t y i s that , at l eas t w i th in the context of the hemopoietic system the NHL-30.5 antigen i s a novel molecule assoc ia ted with aberrant gene expression in the leukemic c e l l s . Whichever mechanism i s postu la ted , the r e a c t i v i t y of the NHL-30.5 monoclonal antibody with c e l l s from pat ients with AML, and i t s lack of r e a c t i v i t y with the major i ty of normal hemopoietic c e l l s , suggests a p o t e n t i a l use for th is reagent in the immunophenotyping of myeloid leukemias. Approaches designed to determine whether NHL-30.5 i s l eukemia -spec i f i c or d i f f e r e n t i a t i o n - s p e c i f i c are presented in Chapters IV and V. 125 REFERENCES 1. Kohler G, M i l s t e i n C: Continuous cultures of fused c e l l s s e c r e t i n g antibody of predefined s p e c i f i c i t y . Nature 256: 495, 1975. 2. B a l l ED, Fanger MW: The expression of myeloid-specific antigens on myeloid leukemia c e l l s - c o r r e l a t i o n s with leukemia subclasses and implications for normal myeloid d i f f e r e n t i a t i o n . Blood 61: 456, 1983. 3. Foon KA, Schroff RW, Gale RP: Surface markers on leukemia and lymphoma c e l l s : Recent advances. Blood 60: 1, 1982. 4. Skubitz KM, Zhen Y, August JT: A human granulocyte s p e c i f i c antigen characterized by use of monoclonal antibodies. Blood 61: 19, 1983. 5. Todd RF, Schlossman SF: Analysis of antigenic determinants on human monocytes and macrophages. Blood 59: 775, 1982. 6. G r i f f i n JD, Mayer RJ, Weinstein HJ, Rosenthal DS, Coral FS, Beveridge RP, Schlossman SF: Surface marker analysis of acute myeloblastic leukemia: I d e n t i f i c a t i o n of d i f f e r e n t i a t i o n associated phenotypes. Blood 62: 557. 7. Bettelheim E, Paietta E, Majdic 0, Gadner H, Schwarzmeier J , Knapp W: Expression of a myeloid marker on TdT-positive acute lymphocytic leukemia c e l l s : Evidence by double-fluorescence s t a i n i n g , blood 60: 1392. 8. Smith LJ, Curtis JE, Messner HA, Senn JS, Furthmayr H, McCulloch EA: Lineage i n f i d e l i t y i n acute leukemia. Blood 61: 1138. 9. Paiett a E, Dutcher JP, Wiernik PH: Terminal transferase p o s i t i v e acute promyelocytic leukemia: In v i t r o d i f f e r e n t i a t i o n of a lymphocytic/promyelocytic hybrid phenotype. Blood 65: 107, 1985. 10. S i e f f CA, Chessells JM, Harvey BAM, P i c k t h a l l VJ, Lawler SD: Monosomy 7 in childhood: A myeloproliferative disorder. Br J Haematol 49: 235, 1981. 11. Pasquali F, Bernasconi P, Casalone R, Fraccaro M, Bernasconi C, Lazzarino M, Morra E, Alessandrino EP, Marchi MA, Sanger R: Pathogenetic s i g n i f i c a n c e of 'pure' monosomy 7 in myeloproliferative diseases. Analysis of 14 cases. Hum Genet 62: 40, 1982. 126 C H A P T E R IV DIFFERENTIATION-LINKED EXPRESSION OF AN AML-ASSOCIATED ANTIGEN ON MYELOID LEUKEMIA CELL LINES 1) INTRODUCTION The concept that AML c e l l s are unable to d i f f e r e n t i a t e or mature normally i n vivo i s an old one. The imposition of maturation arrest upon the malignant c e l l s appears to abrogate normal d i f f e r e n t i a t i o n yet at the same time allows the maintenance of the p r o l i f e r a t i v e a b i l i t y that characterizes normal immature c e l l s at a corresponding l e v e l of d i f f e r e n t i a t i o n . Current evidence suggests that leukemogenesis results i n an uncoupling of p r o l i f e r a t i o n from d i f f e r e n t i a t i o n rather than the loss of genes that regulate the control of normal growth and d i f f e r e n t i a t i o n (1). The morphological, cytochemical, and c e l l surface phenotype of leukemic c e l l s i s thus generally believed to r e f l e c t that of a corresponding normal c e l l lineage and stage of maturation (2). It i s therefore of considerable importance to determine to what extent AML c e l l s remain subject to the regulatory mechanisms that control the growth and development of normal myeloid precursors. In one study the introduction of myeloid leukemia c e l l s into the mouse embryo at an appropriate stage of development produced healthy mice whose granulocytes contained a marker derived from the leukemic clone (3). The implication of this result i s that the leukemic c e l l s were able to p a r t i c i p a t e i n normal myeloid d i f f e r e n t i a t i o n programs, presumably due to the influence of an appropriate combination of signals derived from the developing fetus. This phenotypic reversion of malignancy has been confirmed 127 i n v i t r o by inducing the normal sequence of myeloid d i f f e r e n t i a t i o n i n clones of leukemic c e l l s using a physiological inducer of d i f f e r e n t i a t i o n termed 'macrophage and granulocyte inducer' (MGI) (1). The analysis of the control of gene expression and fun c t i o n a l a c t i v i t y during human myeloid d i f f e r e n t i a t i o n has been greatly f a c i l i t a t e d with the establishment of c e l l l i n e s derived from patients with myeloid leukemias. Although these l i n e s appear to be blocked at s p e c i f i c stages of d i f f e r e n t i a t i o n , a number of studies have shown that i n some cases the c e l l s may be released from maturation arrest and induced to terminally d i f f e r e n t i a t e i n the presence of a variety of inducing agents (4). Table IX summarizes the d i f f e r e n t i a t i o n potential of these c e l l l i n e s as defined by such inducing agents. Induction of Granulocytic D i f f e r e n t i a t i o n The acute promyelocytic leukemia c e l l l i n e HL-60 i s unique among the myeloid c e l l l i n e s i n that i t i s capable of undergoing d i f f e r e n t i a t i o n along e i t h e r the granulocyte or macrophage lineages (5-7). Other myeloid l i n e s demonstrate at best only p a r t i a l granulocytic d i f f e r e n t i a t i o n p o t e n t i a l (4). Numerous ph y s i o l o g i c a l and nonphysiological agents are capable of inducing t h i s pathway of d i f f e r e n t i a t i o n i n HL-60 c e l l s , including: dimethylsulfoxide (DMSO), dimethylformamide, hexamethylene bisacetamide, butyric acid, hypoxanthine, actinomycin D, methotrexate, 5-azacytidine, vitamins A and D and th e i r metabolites, c y c l i c nucleotides, p r o t e o l y t i c enzymes, and a factor derived from various conditioned media believed to be a member of the CSF family (4,8,9). Unfortunately the wide variety of compounds that t r i g g e r d i f f e r e n t i a t i o n i n these c e l l s makes i t d i f f i c u l t to i d e n t i f y common features that may play a ro l e i n the i n i t i a t i o n of the granulocytic d i f f e r e n t i a t i o n program. 128 TABLE IX 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 established human myeloid leukemia c e l l l i n e s . Adapted from ref (4). CELL LINE LEVEL OF DIFFERENTIATION BLOCK DIFFERENTIATION POTENTIAL INDUCING AGENTS KG-la KG-1 HL-60 ML-1&3 U937 K562 HEL Early myeloblast Myeloblast Promyelocyte Myelomonoblast monocytoid Early b l a s t / erythroblast Erythroblast Macrophages Granulocytes or macrophages Macrophages Macrophages Erythroblasts Erythroblasts or macrophages Phorbol esters Numerous Phorbol esters Phorbol esters Hemin/butyrate Hemin Phorbol esters 129 Commitment of HL-60 to granulocytic d i f f e r e n t i a t i o n occurs very r a p i d l y , within 8-18 hours (10), and the lineage into which the c e l l s w i l l d i f f e r e n t i a t e can be switched by passaging the c e l l s at pH 7.2 to become ne u t r o p h i l i c granulocytes, and pH 7.6 to become e o s i n o p h i l i c granulocytes (11). The induced HL-60 c e l l s display phenotypic and fun c t i o n a l c h a r a c t e r i s t i c s of mature granulocytes and these are summarized i n Table X. It appears that the capacity for terminal d i f f e r e n t i a t i o n i s p a r t i a l l y defective however since at least two well known markers of mature granulocytes, namely l a c t o f e r r i n and leukocyte a l k a l i n e phosphatase, are not expressed i n d i f f e r e n t i a t e d HL-60 c e l l s and there also appears to be incomplete expression of some c e l l surface antigens (12). Induction of Monocytic D i f f e r e n t i a t i o n The tumour-promoting phorbol diesters, the prototype of which i s 12-0-tetradecanoylphorbol 13-acetate (TPA), can induce the KG-1, ML-3, HL-60, U937, and HEL myeloid c e l l l i n e s to d i f f e r e n t i a t e into c e l l s possessing markers of monocytic d i f f e r e n t i a t i o n (Table XI) (4,13). The erythroleukemia c e l l l i n e HEL i s p a r t i c u l a r l y i n t e r e s t i n g i n that i t can be induced to express e i t h e r e r y t h r o i d - s p e c i f i c markers when cultured i n the presence of hemin, or monocyte-specific markers in the presence of TPA (14). Current evidence suggests that the phorbol esters may trigger d i f f e r e n t i a t i o n through an i n t e r a c t i o n with protein kinase C, and that this i n t e r a c t i o n leads to a cascade of c e l l u l a r events that are involved i n the monocyte d i f f e r e n t i a t i o n program (15). TPA has also been shown to induce the d i f f e r e n t i a t i o n of the majority of fresh AML blasts into macrophage-like c e l l s (16,17). Most AML c e l l s do not appear to r e t a i n granulocytic potential however, since the culture conditions known to induce HL-60 c e l l s into granulocytes trigger no or minimal granulocytic d i f f e r e n t i a t i o n i n fresh AML bla s t s . Nevertheless, the 130 TABLE X Ch a r a c t e r i s t i c s of HL-60 leukemic promyelocytes before and a f t e r induction of d i f f e r e n t i a t i o n along the granulocyte lineage. Adapted from refs (4) and (8). CHARACTERISTIC UNINDUCED INDUCED Morphology promyelocytes d i f f e r e n t i a t i n g granulocytes DNA synthesis + P r o l i f e r a t i o n + Colony formation (soft agar) + Tumorigenicity + Chemotaxis - + Phagocytosis (bacteria) - + NBT reduction* - + B a c t e r i c i d a l a c t i v i t y - + Fc receptors + + Complement receptors - + Alk a l i n e phosphatase L a c t o f e r r i n Expression of d i f f e r e n t i a t i o n antigens consistent with granulocytic d i f f e r e n t i a t i o n . A l t e r a t i o n s i n oncogene expression with d i f f e r e n t i a t i o n . * Nitro-blue tetrazolium (NBT) i s a histochemical s t a i n that i d e n t i f i e s a functional phagocytic system i n mature granulocytes. 131 TABLE XI Ch a r a c t e r i s t i c s of HL-60 leukemic promyelocytes before and a f t e r induction of d i f f e r e n t i a t i o n along the monocytic lineage. Adapted from refs (4) and (8). CHARACTERISTIC UNINDUCED INDUCED Morphology promyelocytes adherent monocytes DNA synthesis + Colony formation + Phagocytosis (bacteria) Phagocytosis (latex beads) - + Non-specific esterase - + NADase - + Cyt o t o x i c i t y for tumour c e l l s - + Expression of d i f f e r e n t i a t i o n antigens consistent with monocytic d i f f e r e n t i a t i o n . A l t e r a t i o n s i n oncogene expression with d i f f e r e n t i a t i o n . 132 therapeutic implication of these b i o l o g i c a l modifiers of myeloid d i f f e r e n t i a t i o n i s stimulating a systematic search for agents that could possibly induce terminal d i f f e r e n t i a t i o n of myeloid leukemia c e l l s i n vivo (18-21). Oncogenes and D i f f e r e n t i a t i o n The i d e n t i f i c a t i o n of s p e c i f i c genes that play a ro l e i n normal myeloid d i f f e r e n t i a t i o n i s c r u c i a l to an understanding of how the gross chromosomal abnormalities observed i n leukemic c e l l s can influence the pathogenesis of myeloid leukemia. Several authors have suggested that a number of c e l l u l a r oncogenes might encode products that control c e l l growth and d i f f e r e n t i a t i o n . In support of this view are reports of a l t e r a t i o n s i n the t r a n s c r i p t i o n a l expression of a number of these genes i n myeloid leukemia c e l l l i n e s following induction of d i f f e r e n t i a t i o n (Table XII). Although induction of d i f f e r e n t i a t i o n r e s u l t s i n the cessation of p r o l i f e r a t i o n , the decreased expression of at least the c-myc gene appears to be d i r e c t l y related to the d i f f e r e n t i a t i o n process rather than to a c e l l c y c l e - r e l a t e d phenomenon (22). C e l l Surface Antigens and D i f f e r e n t i a t i o n A number of studies have shown that the morphological and fu n c t i o n a l maturation of myeloid leukemia c e l l l i n e s , as induced by various agents, involves a coordinated seri e s of a l t e r a t i o n s i n c e l l surface antigen expression that r e f l e c t s the normal myeloid d i f f e r e n t i a t i o n scheme. The HL-60 c e l l l i n e for example i s known to express a promyelocyte antigen termed Pro-Iml, but does not express the monocyte antigen 0KM1. Induction of monocytic d i f f e r e n t i a t i o n i n these c e l l s results i n a loss of the Pro-Iml antigen, and the subsequent expression of the 0KM1 molecule (23). Induction of granulocyte d i f f e r e n t i a t i o n on the other hand r e s u l t s i n the induction of C3 receptors, the increased expression of a number of granulocytic markers, TABLE XII Alte r a t i o n s i n the t r a n s c r i p t i o n a l expression of a number of c e l l u l a r oncogenes i n HL-60 c e l l s following induction of d i f f e r e n t i a t i o n . Adapted from refs (4) and (29). STAGE OF DIFFERENTIATION Oncogene Promyelocytes Granulocytes macrophages myc + 1 myb + J- 4 fes + + 4 abl + + + ras^ + + + fos T + = t = t r a n s c r i p t i o n a l l y active marked decrease i n t r a n s c r i p t i o n marked increase i n t r a n s c r i p t i o n 134 and the diminished expression of HLA-ABC and 6 2-microglobulin determinants, as well as a v a r i e t y of other myeloid d i f f e r e n t i a t i o n antigens (6,12,24-28). Although the pattern of expression of these c e l l surface antigens i s generally consistent with that of a normal myeloid d i f f e r e n t i a t i o n pathway, p a r t i a l defects i n antigen expression have been reported, and the d i f f e r e n t i a t i o n -linked changes observed i n a p a r t i c u l a r c e l l l i n e i s not n e c e s s a r i l y reproduced i n another (12). It has been suggested that heterogeneity i n antigen expression observed between the d i f f e r e n t myeloid l i n e s when induced to d i f f e r e n t i a t e might r e f l e c t heterogeneity i n the d i f f e r e n t i a t i o n block of each c e l l l i n e , and that i n d i v i d u a l phenotypic c h a r a c t e r i s t i c s are responsible for the extent of d i f f e r e n t i a t i o n obtained with a given inducer (12). Nevertheless, these molecular changes on the c e l l surface may have important roles i n the sequential stages of the myelopoietic d i f f e r e n t i a t i o n program, and they o f f e r a powerful approach to the study of myeloid d i f f e r e n t i a t i o n at the molecular l e v e l . In the preceding chapter the c e l l u l a r d i s t r i b u t i o n of the NHL-30.5 antigen was shown to be r e s t r i c t e d to leukemic specimens containing immature myeloid blast c e l l s . In this section the expression of the NHL-30.5 antigen was analyzed i n d e t a i l on a series of myeloid leukemia c e l l l i n e s induced to d i f f e r e n t i a t e i n v i t r o . 2) RESULTS R e a c t i v i t y of NHL-30.5 with Leukemic C e l l Lines A number of hemopoietic c e l l l i n e s were tested for r e a c t i v i t y with the NHL-30.5 monoclonal antibody. None of the lymphoid c e l l l i n e s tested (SU-DHL 1, 4, 6, 8, and 10, and Jurkat) showed any fluorescence above background. A number of the myeloid l i n e s did react however, and these are l i s t e d i n Table XIII. Since the HL-60 c e l l l i n e has been the most extensively characterized, 135 these c e l l s were i n i t i a l l y chosen to study the e f f e c t of d i f f e r e n t i a t i o n on the expression of the NHL-30.5 antigen. Induction of Granulocytic D i f f e r e n t i a t i o n Approximately 70-90% of HL-60 c e l l s showed r e a c t i v i t y with NHL-30.5 under normal conditions and the binding of 1 2 5 j . _ i a D e ; Q e Q p u r i f i e d NHL-30.5 antibody gave an estimation of 4,000 molecules/cell. When the l i n e was induced to d i f f e r e n t i a t e along the granulocyte lineage by incubating i n the presence of DMSO (Figure IX) or r e t i n o i c acid (Figure X), the number of c e l l s expressing the antigen decreased. This decrease began on the f i r s t day following the addition of the inducing agent and continued to decline s t e a d i l y u n t i l the fluorescence p r o f i l e s of the induced c e l l s became i d e n t i c a l to those of the negative c o n t r o l . By the f i f t h day only 3% of the DMSO-induced c e l l s and <1% of the r e t i n o i c acid-induced c e l l s were p o s i t i v e by FACS an a l y s i s . Furthermore, only 250 NHL-30.5 molecules/cell could be detected on the induced c e l l s using iodinated antibody. An addit i o n a l peak, was observed i n the fluorescence p r o f i l e of NHL-30.5-labelled c e l l s from the r e t i n o i c acid-treated cultures on day f i v e (Figure X). This was also present i n the negative control however, and may r e f l e c t the dec l i n i n g health of the cultures associated with overgrowth (control c e l l s ) or d i f f e r e n t i a t i o n ( r e t i n o i c a c i d -treated c e l l s ) . Following exposure to the d i f f e r e n t i a t i n g agents >80% of the c e l l s showed morphological evidence of d i f f e r e n t i a t i o n beyond the promyelocyte stage. NHL-62.14, a monoclonal antibody with apparent s p e c i f i c i t y for the t r a n s f e r r i n receptor (produced i n the same fusion as NHL-30.5) was used as a p o s i t i v e c o n t r o l . Greater than 95% of cultured HL-60 c e l l s were p o s i t i v e for NHL-62.14, but i f induced to d i f f e r e n t i a t e only 60% of the c e l l s were p o s i t i v e and fluorescence i n t e n s i t y was much weaker. 136 TABLE XIII Re a c t i v i t y of NHL-30.5 monoclonal antibody with various hemopoietic c e l l l i n e s CELL LINE CHARACTERISTICS FACS ANALYSIS % NHL-30.5-P0SITIVE HEL erythroleukemia (AML) >90% HL-60 promyelocytic leukemia (AML) 70-90% KG-1 myeloblastic leukemia (AML) 30-50% KG-la immature variant of KG-1 (AML) <10% K562 early blast/erythroleukemia (CML) <10% U937 early monocytoid ( h i s t i o c y t i c lymphoma) <1% 137 UNINDUCED DAY 1 DAY 3 DAY 5 CONTROL NHL-30.5 NHL-62.14 A A A A 69% A 36% A 9% A 3% A 99% 73% A. 62% 55% 7A FIGURE IX FACS analysis of the reactivity of NHL-30.5 monoclonal antibody with HL-60 cells induced to differentiate with DMSO. Cells were cultured in the presence of 1.25% DMSO and analyzed for reactivity on days one, three, and five following the addition of DMSO. The control antibody is an unrelated monoclonal antibody raised against mouse lymphocytes and NHL-62.14 is an anti-transferrin receptor antibody. Percentages refer to the X positive c e l l s . The horizontal axis represents fluorescence intensity (log) and the vertical axis represents c e l l number. 138 UNINDUCED DAY 1 DAY 3 DAY 5 CONTROL NHL-30.5 NHL-62.14 A A A 69% A 38% A 23% A <1% 99% 81% 57% 61% A FIGURE X FACS analysis of the reactivity of NHL-30.5 monoclonal antibody with HL-60 cells induced to differentiate with retinoic acid. Cells were treated with 1 x 10~^ M retinoic acid and analyzed for reactivity on days one, three, and five following induction as in Figure IX 139 The NHL-30,5 and NHL-62.14 antigens were immunoprecipitated from the surface of iodinated HL-60 c e l l s before and af t e r induction with DMSO. Figure XI shows that NHL-30.5 could only be precipitated from the uninduced c e l l s . The NHL-62.14 antigen was detectable before and a f t e r induction, although there was less p r e c i p i t a t e d from the induced c e l l s . Immunoprecipitation of c e l l s l a b e l l e d with 35s_Methionine or ^ H-leucine was unsuccessful. Metabolic l a b e l l i n g with 32p f a i i e d to demonstrate phosphorylation of the NHL-30.5 antigen under conditions where the tr a n s f e r r i n receptor was c l e a r l y l a b e l l e d (Figure XII). Induction of Monocytic D i f f e r e n t i a t i o n The HL-60 c e l l l i n e has previously been shown to acquire monocytoid c h a r a c t e r i s t i c s following incubation i n the presence of TPA (13). Figure XIII shows the fluorescence p r o f i l e s of TPA-induced HL-60 c e l l s stained with the NHL-30.5 and NHL-62.14 antibodies. Within one day following induction of d i f f e r e n t i a t i o n , 85% of the c e l l s were adherent and had to be removed with a rubber policeman. One and two days following addition of the TPA <1% of the c e l l s (both adherent and nonadherent) expressed the NHL-30.5 antigen. E s s e n t i a l l y a l l of the uninduced c e l l s expressed high l e v e l s of the tr a n s f e r r i n receptor but two days following induction only h a l f of the c e l l s were p o s i t i v e and fluorescence i n t e n s i t y was much weaker. The KG-1 c e l l l i n e was i n i t i a l l y derived from a patient with myeloblastic leukemia and these c e l l s r e t a i n the morphology of leukemic myeloblasts. Under normal culture conditions 30-50% of the KG-1 c e l l s expressed the NHL-30.5 antigen and estimations of the number of molecules per c e l l varied between 500 and 2,300. Incubation of the KG-1 c e l l s i n the presence of DMSO had no e f f e c t on the expression of NHL-30.5. This confirms previous observations that KG-1 c e l l s are re s i s t a n t to agents known to induce granulocytic d i f f e r e n t i a t i o n i n 140 FIGURE XI Immunoprecipitation of NHL-30.5 and NHL-62.14 antigens on HL-60 c e l l s before and af t e r induction with DMSO (day 5). Target c e l l s were l a b e l l e d with 125T and the antigens were immunoprecipitated from the c e l l lysates with the NHL-30.5 and NHL-62.14 monoclonal antibodies. The analysis was carried out on SDS-PAGE (5%) under both reducing (A) and nonreducing (B) conditions. 141 FIGURE XII Immune-precipitation of NHL-30.5 and NHL-62.14 ( t r a n s f e r r i n receptor) antigens from HL-60 c e l l s l a b e l l e d with 32p t A n a l y s i s was c a r r i e d out on SDS-PAGE (7.5%) under reducing c o n d i t i o n s . 142 UNINDUCED DAY 1 DAY 2 FIGURE XIII FACS analysis of the reactivity of NHL-30.5 monoclonal antibody with HL-60 c e l l s induced to differentiate with TPA. Cel ls were harvested on days one and two following addition of the TPA and analysis was carried out as in Figure IX. 143 HL-60 c e l l s (12). Agents that induce monocytic d i f f e r e n t i a t i o n can induce KG-1 c e l l s however. Within one day of treatment with TPA, 60% of the induced KG-1 c e l l s became adherent although they were e a s i l y resuspended following a f i v e minute incubation i n saline containing 0.2% EDTA and 0.1% BSA. This i s in contrast to the HL-60 c e l l l i n e which became very strongly adherent i n the presence of TPA and most c e l l s had to be removed with a rubber policeman. Nevertheless, both the adherent and nonadherent c e l l s from TPA-treated KG-1 cultures lose the NHL-30.5 antigen (Figure XIV). The KG-la c e l l l i n e i s a spontaneous variant of the KG-1 l i n e and i s considered to be more immature than i t s parental l i n e . The c e l l s are morphologically undifferentiated blast c e l l s and are negative for a l l histochemical stains (12,30). No inducer has been described that i s capable of stimulating a d i f f e r e n t i a t i o n program i n these c e l l s . The NHL-30.5 antigen i s weakly expressed on a small subpopulation of KG-la c e l l s (5%). This population appears to represent a d i s t i n c t subset of NHL-30.5-positive c e l l s (rather than background staining) since this proportion was increased to 15% by s o r t i n g the NHL-30.5-positive c e l l s i n the FACS followed by expansion of the c e l l s i n tissue culture. The K562 c e l l l i n e was established from a CML patient i n blast c r i s i s (31) and was i n i t i a l l y f e l t to be blocked at a very early myeloid blast stage. The subsequent demonstration of the synthesis of the erythroid marker glycophorin (32) and the i n d u c i b i l i t y of globin gene expression i n these c e l l s suggested at least p a r t i a l erythroid d i f f e r e n t i a t i o n p o t e n t i a l . Only 10% of the K562 c e l l s express NHL-30.5. The HEL c e l l l i n e was i n i t i a l l y derived from a patient with erythroleukemia and these c e l l s strongly express the NHL-30.5 marker. Under normal culture conditions >90% are NHL-30.5-positive with an estimated 1.5 x 144 UNINDUCED DAY 1 DAY 2 CONTROL NHL-30.5 NHL-62.14 FIGURE XIV FACS analysis of the reactivity of NHL-30.5 monoclonal antibody with KG-1 cells induced to differentiate with TPA. Cells were harvested on days one and two following addition of the TPA and analysis was carried out as in Figure IX. 145 10 4 molecules/cell. The c e l l l i n e generally grows i n suspension although i t i s s l i g h t l y adherent i n tissue culture dishes. In the presence of TPA t h i s adherence i s greatly increased and the c e l l s express markers of monocytic d i f f e r e n t i a t i o n (33). Figure XV i l l u s t r a t e s the morphology of HEL cultures p r i o r to, and f i v e days following, the addition of TPA into the medium. The fluorescence i n t e n s i t y of induced HEL c e l l s stained with NHL-30.5 began to decrease within one day following induction and continued to decline u n t i l approximately 3,000 NHL-30.5 molecules/cell were remaining on day f i v e . Likewise, the number of fluorescing c e l l s progressively decreased to 40% and this p a r a l l e l e d the decreased expression of the t r a n s f e r r i n receptor (Figure XVI). The nonadherent c e l l s i n the tissue culture dishes behaved i n a s i m i l a r manner (Figure XVII). When the nonadherent c e l l s were removed and cultured i n tissue culture dishes containing fresh medium they r a p i d l y became adherent (within several hours) suggesting that a) their lack of adherence i n the i n i t i a l culture dishes was due to overcrowding on the dish and b) they were committed to become adherent since TPA was no longer required i n the medium. 3) DISCUSSION Expression of the NHL-30.5 antigen was examined on c e l l l i n e s derived from patients with myeloid leukemias. Two of these l i n e s , K562 and the undifferentiated variant of the KG-1 l i n e (KG-la) expressed the antigen weakly and on <10% of the c e l l s . Further characterization of the NHL-30.5-positive and NHL-30.5-negative populations i n these c e l l l i n e s may reveal some important functional differences. Three AML-derived c e l l l i n e s (HEL, HL-60, and KG-1) expressed the antigen on a s i g n i f i c a n t l y l a rger proportion of c e l l s ; HEL being the strongest, followed by HL-60 and then KG-1. These l i n e s are phenotypically immature although they re t a i n the capacity to acquire some of the d i f f e r e n t i a t e d c h a r a c t e r i s t i c s of the myeloid lineage under the influence of an appropriate stimulus. 146 FIGURE XV Cultures of HEL c e l l s before (A) and following (B) induction of d i f f e r e n t i a t i o n with TPA. Cultures were stained with May-Grunwald-Giemsa. 147 UNINDUCED DAY 1 DAY 3 DAY 5 CONTROL NHL-30.5 NHL-62.14 A I 1 ! L a lA, A 96% | j \ ] 84% LA 62% AY 43% A 94% ! A U A 84% 73% J — ' 49% A FIGURE XVI FACS analysis of the reactivity of the NHL-30.5 monoclonal antibody with HEL cells induced to differentiate with TPA. Adherent cells were harvested one, three, and five days following the addition of TPA and analysis carried out as in Figure IX. 148 UNINDUCED DAY 1 DAY 3 DAY 5 CONTROL j i i I : V V A , A \J A K 96% I | 88% 72% i 1 53% i NHL-30.5 I ^ • a \-J V K \J \ 94% 88% 76% 60% NHL-62.14 i -A i A K u V FIGURE XVII FACS analysis of the reactivity of the NHL-30.5 monoclonal antibody with the nonadherent cells from a TPA-induced culture of HEL ce l l s . One, three, and five days following the addition of TPA the nonadherent cells were harvested and analyzed as in Figure IX. 149 3x1031-Amount of Labelled Antibody Added (cpm/well) FIGURE XVIII Saturation curve for the binding of 1 2 5 I - l a b e l l e d NHL-30.5 monoclonal antibody to HL-60 c e l l s . An estimate of antigen density was obtained by substracting the amount of r a d i o a c t i v i t y bound to the c e l l s i n the presence of an excess of cold antibody (B) from the amount bound at saturation i n the absence of cold antibody (A). 150 I f the HL-60 c e l l l i n e i s induced to d i f f e r e n t i a t e using DMSO or r e t i n o i c a c i d , the c e l l s acquire properties of mature granulocytic c e l l s . This process takes f i v e days and, as demonstrated by fluorescent s t a i n i n g (Figures IX and X) and immunoprecipitation (Figure XI), involves the loss of the NHL-30.5 molecule beginning on the f i r s t day. The HL-60 c e l l s can also be induced to d i f f e r e n t i a t e into c e l l s with markers of mature monocytic c e l l s by incubating i n the presence of TPA. C e l l s treated i n this manner are also negative for NHL-30.5 antigen expression. The KG-1 c e l l l i n e on the other hand i s incapable of granulocytic d i f f e r e n t i a t i o n but can be induced with TPA to become adherent and express monocytoid properties. The NHL-30.5 antigen i s undetectable on both the adherent and nonadherent c e l l s from TPA treated KG-1 cultures. In contrast to the HL-60 and KG-1 c e l l l i n e s , induction of monocytic d i f f e r e n t i a t i o n i n the HEL c e l l s does not reduce NHL-30.5 expression to undetectable l e v e l s . Approximately 1.5 x 10^ molecules/cell are present on the uninduced c e l l s and this i s reduced f i v e f o l d following induction of d i f f e r e n t i a t i o n . The functional s i g n i f i c a n c e of the d i f f e r e n t i a t i o n - a s s o c i a t e d decrease i n NHL-30.5 antigen expression i s not known, although i t lends support to the idea that NHL-30.5 i s an early myeloid d i f f e r e n t i a t i o n antigen that i s progressively l o s t during the maturation process. This hypothesis would provide an explanation for the expansion of NHL-30.5-positive c e l l s seen i n AML and other hematologic disorders that are characterized by the accumulation of immature myeloid precursors. These observations lead into the following Chapter which w i l l analyze the expression of this putative d i f f e r e n t i a t i o n antigen on normal and leukemic myelopoietic progenitor populations. 151 REFERENCES 1. Sachs L: Control of growth and normal d i f f e r e n t i a t i o n i n leukemic c e l l s -Regulation of the developmental program and re s t o r a t i o n of the normal phenotype i n myeloid leukemia. J C e l l Physiol (suppl 1): 151, 1982. 2. Greaves MF: Leukaemogenesis and d i f f e r e n t i a t i o n : A commentary on recent progress and ideas. Cancer Surveys 2: 189, 1982. 3. Gootwine E, Webb CG, Sachs L: P a r t i c i p a t i o n of myeloid leukaemic c e l l s i n j e c t e d into embryos in haemopoietic d i f f e r e n t i a t i o n i n adult mice. 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Gallo RC, Breitman TR, Ruscetti FW: P r o l i f e r a t i o n and d i f f e r e n t i a t i o n of human myeloid leukemia c e l l l i n e s i n v i t r o . In: Maturation Factors and Cancer (Moore MAS, ed), Raven Press, NY, 1982. 9. Fibach E, Treves A, Kidron M, Mayer M: Induction of d i f f e r e n t i a t i o n i n human myeloid leukemic c e l l s by pr o t e o l y t i c enzymes. J C e l l Physiol 123: 228, 1985. 10. T s i f t s o g l o u AS, Wong W, Hyman R, Minden M, Robinson SH: Analysis of commitment of human leukemia HL-60 c e l l s to terminal granulocytic maturation. Cancer Research 45: 2334, 1985. 11. Fischkoff SA, Condon ME: Switch i n d i f f e r e n t i a t i v e response to maturation inducers of human promyelocytic leukemia c e l l s by p r i o r exposure to a l k a l i n e conditions. Cancer Research 45: 2065, 1985. 12. Ferrero D, Pessano S, Pa g l i a r d i GL, Rovera G: Induction of d i f f e r e n t i a t i o n of human myeloid leukemias: Surface changes probed with monoclonal antibodies. Blood 61: 171, 1983. 13. Perussia B, Lebman D, Pegoraro G, Lange B, Damsky C, Aden D, Var t i k a r J, T r i n c h i e r i G, Rovera G: Induction of d i f f e r e n t i a t i o n of human myeloid leukemia c e l l s by phorbol di e s t e r s . In: Maturation Factors and Cancer (Moore MAS, ed), Raven Press, NY, 1982. 152 14. Papayannopoulou Th, Nakamoto B, Yokochi T, Chait A, Kannagi R: Human erythroleukemia c e l l l i n e (HEL) undergoes a d r a s t i c macrophage-like s h i f t with TPA. Blood 62: 832, 1983. 15. Nishizuka Y: The r o l e of protein kinase C in c e l l surface s i g n a l transduction and tumour promotion. Nature 308: 693, 1984. 16. K o e f f l e r HP, B a r - E l i M, T e r r i t o M: Phorbol ester-induced macrophage d i f f e r e n t i a t i o n of leukemic blasts from patients with myelogenous leukemia. J C l i n Invest 66: 1101, 1980. 17. Pegoraro L, Abrahm J, Cooper R, Levis A, Lange B, Meo P, Rovera G: D i f f e r e n t i a t i o n of human leukemias in response to 12-0-tetradecanoylphorbol 13-acetate in v i t r o . Blood 55: 859, 1980. 18. K o e f f l e r HP, Yelton L, Prokocimer M, H i r j i K: Study of d i f f e r e n t i a t i o n of fresh myelogenous leukemia c e l l s by compounds that induce a human promyelocytic leukemia c e l l l i n e (HL-60) to d i f f e r e n t i a t e . Leuk Res 9: 73, 1985. 19. P o l l i N, O'Brien M, Tavares de Castro J, Rodriquez B, McCarthy D, Catovsky D: Monocytic d i f f e r e n t i a t i o n induced by 1,25 dihydroxyvitamin D 3 in myeloid c e l l s . An u l t r a s t r u c t u r a l immunocytochemical study. Leuk Res: 259, 1985. 20. Lotem J, Berrebi A, Sachs L: Screening for induction of d i f f e r e n t i a t i o n and t o x i c i t y to blast c e l l s by chemotherapeutic compounds i n human myeloid leukemia. Leuk Res 9: 249, 1985. 21. Shkolnik T, Schlossman SF, G r i f f i n JD: Acute u n d i f f e r e n t i a t e d leukemia: Induction of p a r t i a l d i f f e r e n t i a t i o n by phorbol ester. Leuk Res 9: 11, 1985. 22. Filmus J, Buick RN: Relationship of c-myc expression to 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 HL-60 c e l l s . Cancer Research 45: 822, 1985. 23. Chiao JW, Wang CY: D i f f e r e n t i a t i o n antigens of HL-60 promyelocytes during induced maturation. Cancer Res 44: 1031, 1984. 24. Boss MA, Delia D, Robinson JB, Greaves MF: D i f f e r e n t i a t i o n - l i n k e d expression of c e l l surface markers on HL-60 leukemic c e l l s . Blood 56: 910, 1980. 25. Perussia B, Lebman D, Ip SH, Rovera G, T r i n c h i e r i G: Terminal d i f f e r e n t i a t i o n surface antigens of myelomonocytic c e l l s are expressed i n human promyelocytic leukemia c e l l s (HL-60) treated with chemical inducers. Blood 58: 836, 1981. 26. Murao S, Epstein AL, Clevenger CV, Huberman E: Expression of maturation-s p e c i f i c nuclear antigens in d i f f e r e n t i a t i n g human myeloid leukemia c e l l s . Leuk Res 45: 791, 1985. 27. Stockbauer P, Gahmberg CG, Andersson LC: Changes i n c e l l surface glycoproteins and antigens during d i f f e r e n t i a t i o n of the human myeloid leukemia c e l l l i n e s ML-1, ML-2 and HL-60. Cancer Res 45: 2821, 1985. 153 28. Sugimoto T, Tatsurai E, Takeda K, Minato K, Sagawa K, Minowada J : Modulation of c e l l surface antigens induced by 12-0-tetradecanoyl-phorbol 13-acetate i n two myeloblastic c e l l l i n e s , a promyelocytic c e l l l i n e , and a monoblastic c e l l l i n e : Detection with f i v e monoclonal antibodies. J Natl Cancer Inst 72: 923, 1984. 29. M i t c h e l l RL, Zokas L, Schreiber RD, Verma I: Rapid induction of the expression of proto-oncogene fos during human monocytic d i f f e r e n t i a t i o n . C e l l 40: 209, 1985. 30. K o e f f l e r HP, b i l l i n g R, Sparkes RS, Golde DW: An u n d i f f e r e n t i a t e d variant derived from the human acute myelogenous leukemia c e l l l i n e KG-1. blood 56: 265, 1980. 31. Lozzio CB, Lozzio BB: Human chronic myelogenous leukemia c e l l l i n e with p o s i t i v e Philadelphia chromosome. Blood 45: 321, 1975. 32. Mikko J, Gahmber C, Andersson L: Biosynthesis of the major human red c e l l s i a l o g l y c o p r o t e i n , glycophorin A, i n a continuous c e l l l i n e . Nature 279: 604, 1979. 33. Papayannopoulou Th, Brice M, Yokochi T, Rabinovitch PS, Lindsley D, Stamatoyannopoulos G: Anti-HEL c e l l monoclonal antibodies recognize determinants that are also present on hemopoietic progenitors. Blood 63: 326, 1984. 154 C H A P T E R V RESTRICTED EXPRESSION OF AN ACUTE MYELOGENOUS LEUKEMIA-ASSOCIATED ANTIGEN (NHL-30.5) ON NORMAL HEMOPOIETIC PROGENITOR CELLS 1) INTRODUCTION A v a r i e t y of c i r c u l a t i n g blood c e l l types are now recognized by t h e i r unique morphologic, antigenic, and functional properties. Current evidence indicates that the production of these c e l l s begins with the a c t i v a t i o n i n primitive pluripotent progenitors of a unique program of gene expression that may take many c e l l generations to complete (1). Execution of such programs leads f i n a l l y to the a c q u i s i t i o n of s p e c i a l i z e d properties: although preliminary changes, including changes i n responsiveness to various e x t r i n s i c growth factors, are also thought to occur. Some changes have been correlated with the loss of d i f f e r e n t i a t i v e and p r o l i f e r a t i v e p o t e n t i a l that d i s t i n g u i s h d i f f e r e n t populations of committed and pluripotent hemopoietic progenitors i n clonogenic assays (2). Nevertheless, r e l a t i v e l y l i t t l e i s known about the changes that d i s t i n g u i s h these c e l l s from more p r i m i t i v e hemopoietic progenitors or from their progeny i n which evidence of terminal maturation can f i r s t be detected. A widely adopted approach to i d e n t i f y gene products associated with early stages of hemopoietic c e l l d i f f e r e n t i a t i o n has been to prepare monoclonal antibodies against antigens found on leukemic c e l l populations (3-9). Such an approach i s based on the assumption that a morphologically recognized leukemic blast phenotype may be associated with the continued 155 expression of surface antigens normally r e s t r i c t e d to p r i m i t i v e hemopoietic c e l l types. In the preceding Chapters the i d e n t i f i c a t i o n and p a r t i a l c h aracterization of a c e l l surface antigen expressed on hemopoietic c e l l s from patients with AML was presented (9,10) (Chapters I I I and IV). The monoclonal antibody defining this molecule was shown to react with a subs t a n t i a l proportion of blood and marrow c e l l s from over 80% of patients with newly diagnosed or relapsing AML. In contrast, normal mature blood c e l l s i ncluding granulocytes, lymphocytes, monocytes and p l a t e l e t s were a l l found to be negative. S i m i l a r l y , by FACS analysis, a detectable (>2%) p o s i t i v e population could not i n i t i a l l y be demonstrated i n buffy coat preparations of marrow aspirates from either normal or CML patients. Subsequent studies with l i g h t density peripheral blood preparations from patients with CML revealed a small but c l e a r l y detectable NHL-30.5 p o s i t i v e population to be present i n some cases (7/26) (Table VII, Chapter I I I ) . A preliminary study with more highly p u r i f i e d populations of CML clonogenic granulopoietic progenitor c e l l s also indicated that these reacted with NHL-30.5 (11). In Chapter IV the expression of NHL-30.5 was analyzed on myeloid leukemia c e l l l i n e s induced to d i f f e r e n t i a t e , and was shown to have a maturation-linked pattern of expression. Together with the c l i n i c a l data presented i n Chapter I I I , these observations suggest that the NHL-30.5 marker i s an early myeloid d i f f e r e n t i a t i o n antigen, and that i t s increased expression on AML marrow or blood r e f l e c t s the expanded population of immature myeloid c e l l s c h a r a c t e r i s t i c of AML. If this hypothesis i s correct then one might predict that the small (<5%) NHL-30.5-positive population i n normal marrow would include myeloid progenitor c e l l s . In th i s Chapter, the expression of NHL-30.5 on erythropoietic (CFU-E/BFU-E) and granulopoietic 156 (CFU-C) c e l l s from both normal and leukemic i n d i v i d u a l s was evaluated using FACS so r t i n g procedures and i n v i t r o assays for clonogenic myeloid progenitors. 2) RESULTS FACS A n a l y s i s / C e l l Sorting Since the peripheral blood of patients with CML t y p i c a l l y contains elevated numbers of a l l progenitor classes (12-14), CML blood was f i r s t used to investigate the expression of the NHL-30.5 antigen on these pr i m i t i v e c e l l types. C e l l s from four patients were studied. R e a c t i v i t y of NHL-30.5 with CML peripheral blood was variable depending on the patient (Table XIV). In cases where less than 5% of the c e l l s were p o s i t i v e the sort gates were adjusted so that c e l l s with the highest fluorescence i n t e n s i t y , comprising 5% of the t o t a l population, were sorted into the po s i t i v e f r a c t i o n and c e l l s with the lowest fluorescence (the remaining 95%) were sorted into the negative f r a c t i o n . The sorted f r a c t i o n s were then plated i n standard methylcellulose assays to determine the d i s t r i b u t i o n of the various myeloid progenitors within each sorted population. Table XIV i l l u s t r a t e s the number of CFU-C, BFU-E, and CFU-E colonies per 10 5 c e l l s from the four CML patients sorted i n this manner. In the p o s i t i v e f r a c t i o n s granulocyte/macrophage progenitors were enriched an average of four f o l d while BFU-E and CFU-E were enriched s i x and seven f o l d r e s p e c t i v e l y . Both the enrichment of progenitors i n the po s i t i v e f r a c t i o n , and t h e i r corresponding depletion from the negative f r a c t i o n was s i g n i f i c a n t (p< 0.05, Table XIV). Sorted c e l l s from two addi t i o n a l CML patients were plated i n the absence of erythropoietin and a ten f o l d enrichment of CFU-C was observed i n the NHL-30.5-positive f r a c t i o n (data not shown). TABLE XIV The number of progenitors per 10^ c e l l s in sorted fractions from CML peripheral blood PATIENT NO. 1 2 3 4 Mean of (4.4Z)§ (1.2%) (6.8%) (10.9%) 4 experiments* Control 207 121 184 1535 290 (164-512) CFUC (+) Fraction 1092 414 1120 3750 1174 (748-1843) (-) Fraction 17 14 34 205 36 (20-67) Control 262 443 46 1055 275 (142-530) CFU-E (+) Fraction 2530 3200 36 5610 2012 (1107-3654) (-) Fraction 83 132 19 173 78 (48-127) Control 80 167 39 1780 175 (76-401) BFU-E (+) Fraction 1050 979 290 3120 982 (605-1596) (-) Fraction 30 79 20 263 60 (34-106) Control 1.7 5.3 0 0 CFU-GEMM (+) Fraction 164.4 6.5 2.5 0.5 (-) Fraction 0 1.2 1 1 . u 0 0 m 0 § Figures in parentheses indicate X NHL-30.5-positive * Geometric mean (Range defined by ± 1 S.E.M.) Enrichment of a l l progenitors in the positive fraction and corresponding depletion from the negative fraction was significant when compared to the control in a one-tailed paired-sample t test (p<0.05). 158 Ficoll-hypaque separated normal bone marrow and peripheral blood c e l l s were also stained, sorted, and assayed i n a s i m i l a r fashion. Although a d i s t i n c t NHL-30.5-positive population was not observed i n normal marrow, the sort gates were adjusted i n each case so that c e l l s with the highest fluorescence i n t e n s i t y , again comprising 5% of the t o t a l population, were sorted into the p o s i t i v e f r a c t i o n . Table XV shows the frequency of myeloid progenitors i n sorted f r a c t i o n s from six d i f f e r e n t normal marrow specimens. CFU-C were consistently, and s i g n i f i c a n t l y (p< 0.05), enriched i n the NHL-30.5-positive f r a c t i o n (on average, approximately 17 f o l d ) . S i g n i f i c a n t (p< 0.05) enrichment of BFU-E and CFU-E was also demonstrated, with corresponding depletion of these progenitors from the negative f r a c t i o n s . Although the anomalous behaviour of erythroid progenitors from bone marrow #3 (Table XV) suggests the existence of NHL-30.5-negative erythroid progenitors i n this patient, the p o s s i b i l i t y that this r e s u l t was an arte f a c t can not be ruled out. Table XVI shows the res u l t s of the same so r t i n g experiments on f i c o l l -hypaque separated normal peripheral blood. The p o s i t i v e f r a c t i o n again showed s i g n i f i c a n t (p< 0.05) enrichment of a l l progenitor types (ten f o l d for CFU-C, f i v e f o l d for CFU-E, and f i v e f o l d for BFU-E). C e l l s plated from the negative f r a c t i o n were also usually reduced i n t h e i r content of these progenitors. Small numbers of CFU-GEMM (progenitors of mixed granulocyte-erythroid-macrophage colonies) were observed i n some of the above experiments where the general trend suggested a s i m i l a r s t a i n i n g behaviour to that of other clonogenic progenitors. A summary of the d i s t r i b u t i o n of the various progenitor classes i n sorted f r a c t i o n s from CML peripheral blood, normal peripheral blood, and normal bone marrow i s gr a p h i c a l l y i l l u s t r a t e d i n Figures XIX-XXI. TABLE XV The number of progenitors per 10 5 c e l l s in sorted fractions from normal bone marrow. PATIENT NO. 1 2 3 4 5 6 Mean of (3.4%)§ (3.8%) (4.9%) (2.6%) (3.8%) 6 experiments* Control 103 93 102 247 127 197 135 (114-159) CFU-C (+) Fraction 2267 2054 1630 2640 3308 2350 2320 (2105-2556) (-) Fraction 20 15 72 15 69 19 28 (20-37) Control 221 182 225 278 190 67 178 (145-219) CFU-E (+) Fraction 2856 3243 340 3460 3487 1220 1896 (1296-2772) (-) Fraction 144 21 229 28 268 17 67 (40-112) Control 55 65 93 171 27 197 82 (61-111) BFU-E (+) Fraction 250 946 75 1530 450 2350 554 (330-930) (-) Fraction 34 13 105 42 64 19 37 (27-50) Control 1 1.5 0.5 0.4 0 0 CFU-GEMM (+) Fraction 0 13.5 0 0 0 0 (-) Fraction 0 0.4 0 0 0 0 § Figures in parentheses indicate X NHL-30.5-positive * Geometric mean (Range defined by ± 1 S.E.M.) There was significant enrichment of a l l progenitors in the positive fraction when compared to the control fraciton in a one-tailed paired-sample t test (p<0.05). Depletion from the negative fraction was significant for both CFU-C and CFU-E but was not significant for BFU-E (experiments 3 and 5 were not depleted of BFU-E in the negative fraction). TABLE XVI The number of progenitors per 10^ cells in sorted fractions from normal peripheral blood. DONOR NO. 1 2 3 4 Mean of (<1%)§ « U ) «1Z) (1.5Z) 4 experiments* Control 6 11 4 11 7 (6-9) CFU-C (+)Fraction 75 114 33 71 67 (52-87) (-) Fraction 3 3 1 1 2 (1-3) Control 4 23 6 7 8 (5-12) CFU-E (+) Fraction 54 105 12 48 43 (27-67) (-) Fraction 2 7 <1 1 2 (1-3) Control 8 40 16 30 20 (14-28) BFU-E (+) Fraction 94 176 66 127 109 (88-134) (-) Fraction 14 18 3 6 9 (6-13) Control 0.3 0.9 0.8 1.3 CFU-GEMM (+) Fraction 4.5 0.8 22.0 1.2 (-) Fraction 0.8 0.1 0.4 0.1 § Figures in parentheses indicate X NHL-30.5-positive * Geometric mean (Range defined by ± 1 S.E.M.) There was significant enrichment of a l l progenitors in the positive fraction when compared to the control fraction in a one-tailed paired-sample t test (p<0.05). Depletion from the negative fraction was significant for both CFU-C and CFU-E but was not sigificant for BFU-E (patient #1 showed no depletion of BFU-E from the negative fraction). 161 CFU-C CFU-E BFU-E FIGURE XVIII Graphical representation of the frequency of myeloid progenitors i n sorted fractions from CML peripheral blood. (C) control (unsorted) fraction, (+) NHL-30.5-positive fraction, (-) NHL-30.5-negative fraction. 162 V) "55 o in o a « o c o O) o o z CFU-C CFU-E BFU-E FIGURE XIX Graphical representation of the frequency of myeloid progenitors in sorted fractions from normal bone marrow. (C) control (unsorted) fraction, (+) NHL-30.5-positive fraction, (-) NHL-30. 5-negative fraction. 163 m o in O 0) a (0 o 'c CD O) O O b CFU-C CFU-E BFU-E FIGURE XX Graphical representation of the frequency of myeloid progenitors in sorted fractions from normal peripheral blood. (C) control (unsorted) fraction, (+) NHL-30.5-positive fraction, (-) NHL-30.5-negative fraction. 164 In control experiments, some of these specimens were stained with media or an unrelated monoclonal antibody s p e c i f i c for phycoerythrin (IgGl) and the top 5% again sorted and assayed for progenitors. In such experiments, no enrichment of any progenitor type was obtained, providing d i r e c t evidence that the progenitor enrichment observed with NHL-30.5 s t a i n i n g was due to t h e i r s e l e c t i v e r e a c t i v i t y with this antibody. To test whether marrow f i b r o b l a s t s and other minor adherent c e l l components of normal marrow aspirates express NHL-30.5, marrow adherent layers were established in 20% FCS in alpha medium by seeding 1-2 x 10 7 c e l l s into 60 mm tissue culture dishes and maintaining them at 37°C with weekly feeding. Af t e r one or two subcultures the c e l l s were allowed to reach confluence and then collagenased (15) prior to stain i n g for FACS an a l y s i s . Such c e l l suspensions contained no detectable (<1%) NHL-30.5-positive c e l l s (three separate marrows tested) whereas concurrently analyzed, and collagenase-treated, HL-60 c e l l s were 40% p o s i t i v e . In order to determine whether AML blast c e l l progenitors express NHL-30.5, the same sorting experiments were performed on peripheral blood and bone marrow c e l l s from a patient with AML. Figure XXII i l l u s t r a t e s the fluorescence p r o f i l e s of the r e a c t i v i t y of the NHL-30.5 monoclonal antibody with c e l l s from this patient. Three d i s t i n c t populations were observed: a negative population (comprising ~40% of the t o t a l population), a p o s i t i v e population (~50%), and a strongly p o s i t i v e population (~10%). C e l l s were sorted at gate 'a' (top 60% p o s i t i v e ) or gate 'b' (top 10%) and plated i n standard methylcellulose assays. The patient produced numerous abnormal b l a s t - l i k e colonies i n culture and the d i s t r i b u t i o n of these colonies i n the various sorted frac t i o n s i s i l l u s t r a t e d i n Table XVII. The vast majority of the colony-forming c e l l s sorted into the NHL-30.5-positive population, comprising the top 60% fluorescent c e l l s , 165 Peripheral Blood Bone Marrow FIGURE XXII Fluorescence profiles of the reactivity of the NHL-30.5 monoclonal antibody with peripheral blood and bone marrow c e l l s from a patient with AML. Cells were sorted at gate 'a' (60% NHL-30.5-positive cells) or gate 'b' (10% NHL-30.5-positive ce l l s ) and plated in methylcellulose assays. Colony data i s presented in Table XVII. 1 6 6 TABLE XVII The number of blast colony progenitors per 10^ c e l l s i n sorted f r a c t i o n s from the blood and marrow of a patient with AML. Ce l l s were sorted at gate 'a' (top 60% NHL-30.5-positive), or gate 'b' (top 10% NHL-30.5-positive). See Figure XXI. Fraction peripheral blood bone marrow Unsorted 141 79 Gate 'a' p o s i t i v e (60%) 130 183 negative (40%) 0 5 Gate 'b' p o s i t i v e (10%) 53 75 negative (90%) 98 122 167 although a few were present i n the highly fluorescent population (top 10%). Functional Studies of NHL-30.5 Additional experiments were undertaken to investigate whether the NHL-30.5 antigen might be involved i n progenitor p r o l i f e r a t i o n or d i f f e r e n t i a t i o n responses. To test these p o s s i b i l i t i e s p u r i f i e d NHL-30.5 monoclonal antibody was incorporated into the assay medium (up to 10 ug/ml). This, however, had no detectable i n h i b i t o r y or stimulatory e f f e c t on colony formation by normal bone marrow (Table XVIII) or peripheral blood (Table XIX) progenitors i n comparison to control cultures plated both with and without stimulators (erythropoietin and leukocyte conditioned medium). Addition of an equivalent concentration of mouse Ig (Sigma) was also without e f f e c t . Immunoprecipitation Since the pattern of r e a c t i v i t y of NHL-30.5 i s most reminiscent of that observed with the My-10 monoclonal antibody (7), both antigens were compared d i r e c t l y by immunoprecipitation of surface components of KG-1 c e l l s . The KG-1 l i n e was selected for immunoprecipitation purposes since the My-10 antigen i s known to be absent from HL-60 c e l l s . The proteins immunoprecipitated with these two antibodies were c l e a r l y d i f f e r e n t i n the i r mobility i n SDS-PAGE (Figure XXIII). The NHL-30.5 antigen was shown to have a MW of 180,000 under both reducing and nonreducing conditions as previously demonstrated f o r HL-60 c e l l s (9), whereas the My-10 antigen had an approximate MW of 115,000. The NHL-30.5 antigen was further compared with My-10 by the r e a c t i v i t y of the respective antibodies with two AML c e l l l i n e s i n an i n d i r e c t binding assay (Table XX). The NHL-30.5 monoclonal antibody reacted with both HL-60 and KG-1 c e l l s , while My-10 reacted with KG-1 c e l l s only. The My-10 monoclonal antibody was also unable to i n h i b i t the binding of 12^1-labelled NHL-30.5 antibody to HL-60 c e l l s i n a blocking assay (Table XXI). 168 TABLE XVIII The number of progenitors per 10^ bone marrow c e l l s plated i n the presence of p u r i f i e d NHL-30.5 monoclonal antibody. PROGENITORS / 10 5 ANTIBODY CFU-E BFU-E CFU-C MIXED EXPERIMENT #1 control ND ND ND ND 1 ug/ml anti-LFA-1 41 40 141 0 10 ug/ml NHL-30.5 38.5 19 197 0 1 yg/ml NHL-30.5 38.5 29 116 0 NHL-62.14.4* 13.5 5.5 126 0 NB-2* 0 9 101 0 EXPERIMENT #2 control 200 100.5 59 1 20 yg/ml IgG 169 64.5 62.5 0.5 10 yg/ml IgG 183 59 61 2.5 1 yg/ml IgG 25 59 61 2.5 10 yg/ml NHL-30.5 161 68.5 48 2 1 yg/ml NHL-30.5 110 61.5 46.5 0.5 EXPERIMENT #3 control 87 128 39 3 20 yg/ml IgG 90 118 36.5 1 10 yg/ml IgG 89.5 109 38 0 1 yg/ml IgG 70.5 128 51 0.5 10 yg/ml NHL-30.5 50.5 93 55 0 1 yg/ml NHL-30.5 45 89.5 35 0 * NHL-62.14 and NB-2 are two monoclonal antibodies produced i n th i s laboratory with s p e c i f i c i t y for the t r a n s f e r r i n receptor (unpublished). 169 TABLE XIX The number of progenitors per 4 x 10 5 peripheral blood c e l l s plated i n the presence of p u r i f i e d NHL-30.5 monoclonal antibody. PROGENITORS / 4 X 10 5 ANTIBODY CFU-E BFU-E CFU-C MIXED EXPERIMENT #1 control 25.5 40.3 42 1 20 yg/ml IgG 42 59.5 39.5 3 10 yg/ml NHL-30.5 41 56 37.5 3.5 EXPERIMENT #2 control 11.5 28.5 5 1 20 yg/ml IgG 10 34 6.5 0 10 yg/ml IgG 12 35.5 5.5 0.5 1 yg/ml IgG 7.5 32.5 6 1.5 10 yg/ml NHL-30.5 6.5 22.5 6 0.5 1 yg/ml NHL-30.5 13 25.5 10 1 EXPERIMENT #3 control 4 25.5 7.5 1 20 yg/ml IgG 4.5 21.5 6.5 1 10 yg/ml IgG 5 23 9 1 1 yg/ml IgG 3.5 21 5.5 0 10 yg/ml NHL-30.5 4 13.5 3.5 1 1 yg/ml NHL-30.5 5 18.8 6 0 170 a b c FIGURE XXIII A comparison of the NHL-30.5 and My-10 antigens by immunoprecipitation. KG-1 c e l l surfaces were l a b e l l e d with 1 2 5 I and the antigens immunoprecipitated from the c e l l l y sates using the NHL-30.5 and My-10 hybridoma supernatants. The a n a l y s i s was ca r r i e d out on SDS-PAGE (7.5%) under reducing conditions. Lane a: NHL-30.5, Lane b: My-10, and Lane c: co n t r o l antibody r a i s e d against mouse lymphocytes (does not react with KG-1 c e l l s ) . 171 TABLE XX A comparison of the r e a c t i v i t y of the NHL-30.5 monoclonal antibody and My-10 monoclonal antibody on two AML c e l l l i n e s using an in d i r e c t binding assay. Antibody HL-60 binding (cpm) KG-1 binding (cpm) media a n t i - t r a n s f e r r i n receptor (NHL-62.14) negative c o n t r o l * My-10 NHL-30.5 213 13,760 482 320 2837 261 13,488 291 9,616 855 * an unrelated antibody raised against mouse lymphocytes 172 T A B L E X X I A b i l i t y of the My-10 monoclonal antibody to block the binding of l 25l-NHL-30.5 monoclonal antibody to HL-60 c e l l s i n a dire c t binding assay. F i r s t incubation Second incubation Binding (cpm) media 1 2 5 I - N H L - 3 0 . 5 2 8 0 5 N H L - 3 0 . 5 1 2 5 T _ N H L - 3 0 . 5 2 1 0 My -10 1 2 5 T _ N H L - 3 0 . 5 2 5 4 3 negative control 1 2 5 I - N H L - 3 0 . 5 2 6 5 0 173 3) DISCUSSION The purpose of the experiments described i n this Chapter was to investigate the possible d i s t r i b u t i o n of NHL-30.5 on rare but normal marrow elements whose presence would have escaped detection i n standard binding assays or FACS analyses (Chapter I I I ) . Previous studies (Chapter III) had shown that p u r i f i e d populations of lymphocytes, monocytes, granulocytes, erythrocytes, and p l a t e l e t s from normal in d i v i d u a l s did not react with t h i s antibody and that normal bone marrow buffy coat preparations contained l e s s than 2% NHL-30.5-positive c e l l s . In the studies described i n this Chapter, the l i g h t density f r a c t i o n of a l l specimens was obtained by c e n t r i f u g a t i o n of ficoll-hypaque (density = 1.077g/cc). This appeared to increase the number of NHL-30.5-positive c e l l s by a few percent. As a r e s u l t a small but d i s t i n c t (>5X) p o s i t i v e population could sometimes, although not always, be demonstrated i n the peripheral blood of CML patients i n chronic phase. However, this was not the case for any normal marrow or peripheral blood sample, where NHL-30.5-positive c e l l s f a i l e d to reach the 5% l e v e l . Nevertheless, when c e l l s with the highest fluorescence i n t e n s i t y (comprising 5% of the t o t a l population) were sorted and assayed, a s i g n i f i c a n t and s e l e c t i v e enrichment of clonogenic progenitors including CFU-E and BFU-E as well as CFU-C, was obtained. These findings suggest that expression of NHL-30.5 i s part of an ea r l y stage of hemopoietic c e l l d i f f e r e n t i a t i o n that p e r s i s t s even a f t e r lineage r e s t r i c t i o n . Consistent with these r e s u l t s i s the observation that, at least i n the one patient studied, the majority of the blast colony-forming progenitors i n AML (that presumably help to maintain the bla s t population i n vivo) are also NHL-30.5-positive (Figure XXI and Table XVII). In contrast to the p o s i t i v e results obtained with clonogenic progenitors, assessment of adherent marrow c e l l populations that are capable 174 of regulating progenitor turnover i n v i t r o (16) showed these to be NHL-30.5-negative. Few monoclonal antibody reagents are currently a v a i l a b l e that i d e n t i f y antigens r e s t r i c t e d i n their expression to early stages of myeloid c e l l d i f f e r e n t i a t i o n (3,7-9). The My-10 monoclonal antibody (7) has a pattern of r e a c t i v i t y that shows some s i m i l a r i t y to that of NHL-30.5 i n that both react with immature myeloid populations and some leukemic b l a s t s , but not with d i f f e r e n t i a t e d myeloid c e l l s . The present study establishes that the antigen detected by My-10 i s e l e c t r o p h o r e t i c a l l y d i s t i n c t from that detected by NHL-30.5. The former has an approximate molecular weight of 115,000 i n contrast to 180,000, the molecular weight of the NHL-30.5 antigen. The i n a b i l i t y of the My-10 monoclonal antibody to block the binding of iodinated NHL-30.5 antibody to KG-1 c e l l s supports the idea that these antigens are d i s t i n c t molecules. Also consistent with this difference i s the f i n d i n g that NHL-30.5 reacts with c e l l s from most (80%) patients with AML (Chapter I I I ) , whereas My-10 appears to react with c e l l s only from a minority (28%) of AML patients (7). The stage of normal hemopoietic c e l l d i f f e r e n t i a t i o n at which NHL-30.5 f i r s t appears remains to be determined. Since the KG-1 c e l l l i n e (myeloblastic) expresses both the NHL-30.5 and My-10 antigens, while the HL-60 c e l l l i n e (promyelocytic) expresses only NHL-30.5 (which i t loses i f induced to d i f f e r e n t i a t e ) , i t i s possible that myeloid progenitor c e l l s (NHL-30.5-p o s i t i v e and My-10-positive) become My-10-negative somewhere a f t e r the myeloblast stage but remain NHL-30.5-positive u n t i l sometime following the promyelocyte stage. Our lim i t e d observations to date suggest that clonogenic pluripotent progenitors (CFU-GEMM) l i k e their various l i n e a g e - r e s t r i c t e d but clonogenic progeny are NHL-30.5-positive. However, even CFU-GEMM are not believed to represent stem c e l l s with long-term repopulating p o t e n t i a l . In 175 standard short-term assays most CFU-GEMM display l i m i t e d self-renewal capacity (17-19) and thus d i f f e r from high self-renewal pluripotent c e l l s that have recently been shown to be i n i t i a l l y refractory to growth s t i m u l i i n v i t r o (20,21). Recently i t was reported that the l i n e a g e - r e s t r i c t e d progenitors observed i n four to s i x week old long-term marrow cultures may be derived from a population of c e l l s that d i f f e r i n HLA-DR expression from CFU-GEMM as well as CFU-C and BFU-E (22). Experiments are currently underway to determine whether such differences also extend to NHL-30.5 expression. The normal functional role of NHL-30.5 i s also unknown. Since most clonogenic progenitors i n normal peripheral blood are not a c t i v e l y c y c l i n g , i n contrast to those i n CML blood or a l l but the most p r i m i t i v e classes present i n normal bone marrow (23-27), i t seems u n l i k e l y that regulation of NHL-30.5 expression i s d i r e c t l y related to c e l l cycle status. Tests to evaluate whether NHL-30.5 binding could i n h i b i t or stimulate normal progenitor 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 also showed no e f f e c t , although t h i s might r e f l e c t the p a r t i c u l a r determinant recognized by the NHL-30.5 monoclonal antibody rather than the physiologic role of the antigen of which i t i s a part. The data presented in this Chapter, together with the r e s u l t s outlined i n Chapters III and IV, provide a strong basis for the designation of the NHL-30.5 marker as an early myeloid d i f f e r e n t i a t i o n antigen. AML appears to represent the expansion of a neoplastic clone that continues to express NHL-30.5 because the c e l l s are unable to d i f f e r e n t i a t e to those stages characterized by the loss of the NHL-30.5 antigen. P r o g e n i t o r - r e s t r i c t e d antigens such as My-10 and NHL-30.5 may have important roles i n modulating progenitor c e l l behaviour. 176 REFERENCES 1. Eaves AC, Eaves CJ: Erythropoiesis. In: Hematopoietic Stem C e l l s , Marcel Dekker Inc, New York, 1985. 2. Eaves AC, Eaves CJ: Erythropoiesis in culture, i n : C l i n i c s i n Haematology (EA McCulloch, Ed), Vol 13, WB Saunders, Eastborne, England, 1984. 3. Young NS, Hwang-Chen S: Anti-K562 c e l l monoclonal antibodies recognize hematopoietic progenitors. Proc Natl Acad Sci USA 78: 7073, 1981. 4. G r i f f i n JD, R i t z J, Nadler LM, Schlossman SF: Expression of myeloid d i f f e r e n t i a t i o n antigens on normal and malignant myeloid c e l l s . J C l i n Invest 68: 932, 1981. 5. Bernstein ID, Andrews RG, Cohen SF, McMaster BE: Normal and malignant human myelocytic and monocytic c e l l s i d e n t i f i e d by monoclonal antibodies. J Immunol 128: 876, 1982. 6. Ferrero D, Broxmeyer HE, Pag l i a r d i GL, Venuta S, Lange B, Pessano S, Rovera G: A n t i g e n i c a l l y d i s t i n c t subpopulations of myeloid progenitor c e l l s (CFU-GM) i n human peripheral blood and marrow. Proc Natl Acad Sci USA 80: 4114, 1983. 7. C i v i n CI, Strauss LC, B r o v a l l C, Fockler JO, Schwartz JF, Shaper JH: Antigenic analysis of hematopoiesis I I I . A hematopoietic progenitor c e l l surface antigen defined by a monoclonal antibody raised against KG-la c e l l s . J Immunol 133: 157, 1984. 8. Papayannopoulou T, Brice M, Yokochi T, Rabinovich PS, Lindsley D, Stamatoyannopoulos G: Anti-HEL c e l l monoclonal antibodies recognize determinants that are also present i n hemopoietic progenitors. Blood 63: 326, 1984. 9. Askew DS, Eaves AC, Takei F: NHL-30.5: A monoclonal antibody re a c t i v e with an acute myeloid leukemia (AML)-associated antigen. Leuk Res 9: 135, 1985. 10. Askew DS, Eaves AC, Takei F: Expression of an acute myelogenous leukemia-associated antigen (NHL-30.5) on immature myeloid c e l l s . In: Leukocyte Typing I I , Proceedings of the Second International Workshop on Human Leukocyte D i f f e r e n t i a t i o n Antigens (Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds), Springer-Verlag, NY, ( i n press). 11. Dr. P.M. Lansdorp, personal communication. 12. Eaves CJ, Eaves AC: Erythroid progenitor c e l l numbers i n human marrow -Implication for regulation. Exp Hematol 7(suppl 5): 54, 1979. 13. Goldman JM, Th'ng KH, Lowenthal RM: In v i t r o colony-forming c e l l s and colony-stimulating factor i n chronic granulocytic leukemia. Br J Cancer 30: 1, 1974. 177 1A. Vainchenker W, Guichard J, Deschamps JF, Bouguet J , Titeux M, Chapman J, McMichael AJ, Breton-Gorius J: Megakaryocyte cultures i n the chronic phase and in the blast c r i s i s of chronic myeloid leukemia: Studies on the d i f f e r e n t i a t i o n of the megakaryocyte progenitors and on the maturation of megakaryocytes i n v i t r o . Br J Haematol 51: 131, 1982. 15. Coulombel L, Eaves AC, Eaves CJ: Enzymatic treatment of long-term 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, 1983. 16. Dr. Connie J . Eaves, personal communication. 17. Humphries RK, Eaves AC, Eaves CJ: Self-renewal of hemopoietic stem c e l l s during mixed colony formation i n v i t r o . Proc Natl Acad Sci USA 78: 3629, 1981. 18. Messner HA, Fauser AA: Culture studies of human pluripotent hemopoietic progenitors. Blut A l : 327, 1980. 19. Ash RC, Detrick RA, Zanjani ED: Studies of human pluripotent hemopoietic stem c e l l s in v i t r o . Blood 58: 309, 1981. 20. Nakahata T, Ogawa M: Hemopoietic colony-forming c e l l s i n um b i l i c a l cord blood with extensive c a p a b i l i t y to generate mono- and mu l t i p o t e n t i a l hemopoietic progenitors. J C l i n Invest 70: 132A, 1982. 21. Kerk DK, Henry EA, Eaves AC, Eaves CJ: Two classes of p r i m i t i v e pluripotent hemopoietic progenitor c e l l s . Separation by adherence. J C e l l Physiol ( i n press). 22. Keating A, Powell J, Takahashi M, Singer JW: The generation of human long-term marrow cultures from marrow depleted of l a (HLA-DR) p o s i t i v e c e l l s . Blood 6A: 1159, 198A. 23. Tebbi K, Rubin S, Cowan DH, McCulloch EA: A comparison of granulopoiesis in culture from blood and marrow c e l l s on non-leukemic i n d i v i d u a l s and patients with acute leukemia. Blood A8: 235, 1976. 2A. Ogawa M, Grush 0C, O'Dell RF, Hara H, MacEachern MD: C i r c u l a t i n g erythropoietic precursors assessed i n culture: Characterization i n normal men and patients with hemoglobinopathies. Blood 50: 1081, 1977. 25. Lepine J, Messner HA: Pluripotent hemopoietic progenitors (CFU-GEMM) i n chronic myelogenous leukemia. Int J C e l l Cloning 1: 230, 1983. 26. Cashman J, Eavaes AC, Eaves CJ: Regulated p r o l i f e r a t i o n of p r i m i t i v e hemopoietic progenitor c e l l s i n long-term human marrow cultures. Blood ( i n press). 27. 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 erythroid precursor c e l l s and the erythropoietic d i f f e r e n t i a t i o n process, i n : C e l l u l a r and Molecular Regulation of Hemoglobin Switching (G Stamatoyannopoulos and AW Nienhuis, eds), Grune and Stratton, New York, 1979. 178 C H A P T E R VI SUMMARY AND CONCLUSIONS Analysis of c e l l u l a r d i f f e r e n t i a t i o n r e l i e s upon the i d e n t i f i c a t i o n of phenotypic c h a r a c t e r i s t i c s that are associated with s p e c i f i c l e v e l s of maturation. Since the vast majority of normal marrow c e l l s are i n the terminal stages of d i f f e r e n t i a t i o n and are therefore recognizable as precursors of the granulocyte, monocytic, megakaryocytic, or erythroid lineages, the developmental sequence of these myeloid c e l l s has been e a s i l y studied using morphological and cytochemical c r i t e r i a . A more powerful approach i s the immunological analysis of d i f f e r e n t i a t i o n - a s s o c i a t e d c e l l membrane proteins using monoclonal antibodies. Such antibodies have proven useful i n the study of the terminal myeloid d i f f e r e n t i a t i v e events (Chapter I, 5(B)) but also o f f e r a unique approach to study gene products expressed on the numerically infrequent stem c e l l population (1). Regulation of p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i n these stem c e l l s i s thought to reside i n c e l l surface receptors that are responsive to e x t r i n s i c growth factors or c e l l u l a r interactions. Very l i t t l e i s known about the c e l l surface antigenic changes that d i s t i n g u i s h stem c e l l s from t h e i r d i f f e r e n t i a t i n g progeny and consequently c e l l s within the stem c e l l or progenitor c e l l compartment have remained rather e l u s i v e . The i d e n t i f i c a t i o n of stem c e l l - r e s t r i c t e d surface antigens would provide an easy route to the p u r i f i c a t i o n of these c e l l s and, more importantly, might i d e n t i f y c e l l surface molecules that mediate s p e c i f i c c e l l u l a r functions. 179 In AML the neoplastic c e l l s are unable to follow normal d i f f e r e n t i a t i o n pathways, although the continued expression of some normal features of myeloid d i f f e r e n t i a t i o n are usually evident. Current evidence indicates that the c e l l surface phenotype of the blast population can generally be viewed within the framework of normal myelopoietic d i f f e r e n t i a t i o n , and t h i s provides a useful basis for current immunological c l a s s i f i c a t i o n schemes (2). As an approach to i d e n t i f y unique gene products associated with the early stages of myeloid c e l l d i f f e r e n t i a t i o n , a number of monoclonal antibodies were raised against an AML-derived c e l l l i n e (HL-60) considered to be blocked at an immature stage of myeloid maturation. One of these antibodies, NHL-30.5, i d e n t i f i e d a c e l l surface antigen with a c e l l u l a r d i s t r i b u t i o n r e f l e c t i n g that of a myeloid d i f f e r e n t i a t i o n antigen. The i d e n t i f i c a t i o n of t h i s antigen and analysis of i t s c e l l u l a r d i s t r i b u t i o n i s discussed i n this thesis. C l i n i c a l Association of the NHL-30.5 Antigen with AML The NHL-30.5 monoclonal antibody detected a c e l l surface antigen with a molecular weight of 180,000 on the HL-60 c e l l l i n e and on fresh leukemic c e l l s from a patient with the M4 c l a s s i f i c a t i o n of AML. The antibody reacted with hemopoietic c e l l s from 40/48 patients with AML and two patients with preleukemic disorders characterized by the presence of myeloid blast c e l l s (a chronic myelomonocytic leukemia (1/1) and a myelofibrosis (1/2) ). In contrast i t did not react with normal mature hemopoietic c e l l s , i n c l u d i n g lymphocytes, monocytes, granulocytes, erythrocytes, p l a t e l e t s , and splenocytes. Lymphoid r e a c t i v i t y appeared to be a rare event since only one of 15 acute lymphoid leukemias demonstrated r e a c t i v i t y and a l l lymphoid c e l l l i n e s were uniformly negative. Reactivity with c e l l s i n the chronic phase of CML was also rare (7/26) and, when pos i t i v e , the number of NHL-30.5-positive 180 c e l l s was low (1-20%). C e l l s from the acute phase of CML reacted i n a manner analogous to AML i f the blast c r i s i s was of the myeloid variant (1/1), but were c l e a r l y negative i n lymphoid blast c r i s i s (3/3). An unusual case characterized by a CALLA+, TdT~, HLA-DR+ blast population was also p o s i t i v e , as was a biphenotypic blast c r i s i s containing both myeloid and lymphoid b l a s t s . Notably the vast majority of normal d i f f e r e n t i a t i n g bone marrow c e l l s were negative (<5% p o s i t i v e ) , including bone marrow f i b r o b l a s t s . D i f f e r e n t i a t i o n - L i n k e d Expression of the NHL-30.5 Marker Three AML-derived c e l l l i n e s , considered to be blocked at an early stage of maturation were shown to express the NHL-30.5 antigen. The HEL c e l l l i n e (erythroleukemia) contained over 90% NHL-30.5-positive c e l l s with an estimated 1.5 x 10^ molecules/cell. The HL-60 (promyelocytic) c e l l l i n e was 70-90% NHL-30.5-positive with ~4,000 molecules/cell and the KG-1 l i n e (myeloblastic) was 30-50% po s i t i v e and antigen estimations varied between 500 and 2,300 antigens per c e l l . C e l l l i n e s derived from lymphoid leukemias were c l e a r l y negative, as was the U937 (early monocytoid) l i n e . K562 (early b last/erythroid) and KG-la (variant of KG-1) were weakly p o s i t i v e (1-10%). The three NHL-30.5-positive AML c e l l l i n e s are known to possess myeloid d i f f e r e n t i a t i o n p o t e n t i a l . HL-60 c e l l s are unique i n that they respond to stimulation with DMSO or r e t i n o i c acid by i n i t i a t i n g a granulocytic d i f f e r e n t i a t i o n program, or to stimulation with TPA by entering a monocytic d i f f e r e n t i a t i o n program. Induction of either pathway of d i f f e r e n t i a t i o n was shown to result i n the loss of the NHL-30.5 antigen, as detected by flow cytometry and immunoprecipitation. The KG-1 and HEL c e l l l i n e s are incapable of granulocytic d i f f e r e n t i a t i o n but are inducible along the monocyte pathway with TPA. D i f f e r e n t i a t e d KG-1 cultures were also shown to lack the NHL-30.5 molecule. 181 In contrast to HL-60 and KG-1, induction of monocytic d i f f e r e n t i a t i o n i n the HEL c e l l l i n e did not reduce antigen expression to undetectable l e v e l s , although a f i v e f o l d decrease i n expression was observed. Expression of the NHL-30.5 Marker on Normal Myeloid progenitors In order to determine whether the NHL-30.5 antigen i s a normal myeloid d i f f e r e n t i a t i o n marker, i t s expression was studied on clonogenic e r y t h r o p o i e t i c and granulopoietic c e l l s from both normal and leukemic i n d i v i d u a l s . Analysis of normal d i f f e r e n t i a t i n g bone marrow c e l l s and mature peripheral blood mononuclear c e l l s stained i n d i r e c t l y with the NHL-30.5 monoclonal antibody and FITC-second antibodies did not reveal a d i s t i n c t l y p o s i t i v e population. However, the c e l l s with the highest fluorescence i n t e n s i t i e s (comprising 5% of the t o t a l population) sorted on a fluorescence activated c e l l sorter were highly enriched in both e r y t h r o p o i e t i c (CFU-E/BFU-E) and granulopoietic (CFU-C) progenitors. Similar patterns of enrichment were observed i n suspensions of c e l l s from CML peripheral blood suggesting that the NHL-30.5 antigen i s expressed on both normal and leukemic progenitors detectable as CFU-E, BFU-E, and CFU-C. Since the NHL-30.5 antigen has a c e l l u l a r d i s t r i b u t i o n s i m i l a r to the My-10 antigen, both molecules were compared by immunoprecipitation and shown to have c l e a r l y d i f f e r e n t molecular weights. Conclusion The data presented in this thesis suggest that the NHL-30.5 antigen i s a novel myeloid d i f f e r e n t i a t i o n antigen r e s t r i c t e d i n i t s expression to the e a r l y stages of myelopoiesis, and that the loss of this molecule from a myeloid c e l l can be viewed as a stage i n the normal myeloid d i f f e r e n t i a t i o n program. The precise stage of maturation at which the NHL-30.5 antigen i s l o s t i s not clear, but the vast majority of terminally d i f f e r e n t i a t i n g marrow 182 c e l l s are c l e a r l y negative, as are the f u l l y mature c e l l s i n the peripheral blood. The data support the concept that NHL-30.5-positive c e l l s accumulate i n AML patients due to a block i n their capacity to d i f f e r e n t i a t e into the stages characterized by loss of the NHL-30.5 molecule. A number of questions regarding this antigen remain unanswered. With respect to i t s c e l l u l a r d i s t r i b u t i o n , i t i s not yet known whether a l l of the myeloid progenitors express NHL-30.5. I d e n t i f i c a t i o n of a c l e a r l y NHL-30.5-negative population of progenitors might i d e n t i f y a unique functional subset of progenitors analogous to the immunologically defined T - c e l l subsets i n the peripheral blood (3). S i m i l a r l y , and of relevance to p o t e n t i a l use of th i s antibody i n the treatment of AML, i s whether or not the precursors of these myeloid progenitors are NHL-30.5-positive. Preliminary data suggests that CFU-GEMM are NHL-30.5-positive, although i t remains to be established whether stem c e l l s with long-term repopulating po t e n t i a l are also p o s i t i v e . I f the antigen i s expressed on these high self-renewal pluripotent stem c e l l s , then the NHL-30.5 mononclonal antibody can not be used as part of an 'antibody c o c k t a i l ' to purge leukemic marrow for autologous bone marrow transplantation. Nevertheless, the c l i n i c a l data presented i n Chapter I II suggests that the NHL-30.5 antibody, i n conjunction with other monoclonal antibodies, i s p o t e n t i a l l y a useful reagent to monitor patients with preleukemic disorders for possible evolution into AML, and to d i s t i n g u i s h myeloid leukemias from lymphoid leukemias ( p a r t i c u l a r l y in the acute phase of CML). Since the NHL-30.5 antigen i s expressed at r e l a t i v e l y low l e v e l s , techniques designed to increase the s e n s i t i v i t y of i t s detection, such as antibody bridging procedures (5), may be b e n e f i c i a l i n this regard. The antibody i s cur r e n t l y being used i n this laboratory for the c e l l surface phenotyping of leukemias 183 to further evaluate i t s use in the diagnosis and c l a s s i f i c a t i o n of hematologic malignancy. F i n a l l y , and perhaps the most i n t e r e s t i n g question, i s the function of thi s molecule. The r e s t r i c t e d expression of the antigen suggests that i t may play an important role i n modulating progenitor c e l l behaviour but th i s has yet to be proven. In Chapter V i t was shown that the NHL-30.5 monoclonal antibody was unable to i n h i b i t or stimulate colony formation i n v i t r o . However, not a l l monoclonal antibodies raised against a c e l l surface receptor are capable of i n h i b i t i n g i t s function (5), and so i t w i l l be important to r a i s e monoclonal antibodies against d i f f e r e n t determinants on the p u r i f i e d antigen. Further characterization of this molecule, such as amino acid sequencing, determination of i t s orientation i n the plasma membrane, analysis of associated tyrosine kinase a c t i v i t y , and ultimately the cloning of i t s gene would help determine i f NHL-30.5 i s a c e l l surface receptor for an as yet unspecified ligand. 184 REFERENCES 1. C i v i n CI, Strauss LC, Brova l l C, Fockler JO, Schwartz JF, Shaper JH: Antigenic analysis of hematopoiesis I I I . A hematopoietic progenitor c e l l surface antigen defined by a monoclonal antibody raised against KG-l a c e l l s . J Immunol 133: 157, 1984. 2. Foon KA, Bottino GC: Immunology of Acute Leukemia. In: Neoplastic Diseases of the Blood v ol 1 (Wiernik PH, Canellos GP, Kyle RA, S c h i f f e r CA, eds), C h u r c h i l l Livingstone, 1985. 3. Thomas Y, Rogozinski L, Chess L: Relationship between human T c e l l f unctional heterogeneity and human T c e l l surface molecules. Immunol Rev 74: 113, 1983. 4. Lansdorp PM, van der Kwast, TH, de Boer M, Zeijlemaker WP: Stepwise amplified immunoperoxidase (PAP) st a i n i n g . I. C e l l u l a r morphology i n r e l a t i o n to membrane markers. J Hisochem Cytochem 32: 172, 1984. 5. Trowbridge IS, Newman RA, Domingo DL, Sauvage C: T r a n s f e r r i n receptors: Structure and function. Biochem Pharmacol 33: 925, 1984. 

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