Cell culture studies in CML

4 Cell culture studies in C M L C. J. E A V E S A. C. E A V E S

BACKGROUND Clonogenic progenitors -Twenty years ago, a new era of haematology was ushered in with the development of the first in vitro colony assay for a human haemopoietic cell (Senn et al, 1967). Over the next decade this methodology was successfully extended to include all aspects of human myelopoiesis (McCulloch, 1984; Golde and Takaku, 1985). Thus for the first time it became possible to identify, quantitate and characterize an array of primitive human haemopoietic cell types, each able to give rise to a colony containing a specific type (or types) ofmature progeny and each able to express a finite, albeit sometimes extensive, proliferative ability. Haemopoietic progenitors were thus discovered, and are now defined, by the size and composition ofthe colonies they generate. In general, the more primitive the cell, the larger the colony produced, the more numerous the number of lineages represented, and the longer the period of colony growth before maturation is complete (Eaves et al, 1979). This view of primitive haemopoietie cell organization, illustrated in Figure 1, is referred to as the hierarchical model of progenitor differentiation. Extensive studies have revealed consistent differences between the morphological and functional properties of cells that generate different types of colonies, thus bearing out an important prediction of the model (Eaves and Eaves, 1985). In addition, it is becoming evident that the control of mature blood cell production in vivo is probably normally regulated most precisely during terminal differentiation. At this stage, cells either continue to proliferate and/or mature under the control of circulating or local extrinsic regulatory molecules, or they die as non-contributors. In contrast, primitive haemopoietic cells have the option to reversibly enter a quiescent state (Becker et al, 1965; Eaves et al, 1979; Fauser and Messner, 1979). This property allows enormous changes in population turnover, and hence in the potential for subsequent increased mature blood cell output (Cashman et al, 1985). The process that determines the proliferative and differentiative potential of Baillibre's Clinical Haematolog~Vol. 1, No. 4, December 1987

931

C. J. EAVES AND A. C. EAVES

932 ULTIMATE STEM CELL I !

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Figure 1. Diagrammatic representationof the hierarchicalmodel of progenitorcell organization in the haemopoieticsystem inferredfrom the characterization,proliferativepotentialin vitro,and 'relatedness' in vivo of different classes of operationally defined clonogenic cells (Eaves et al, 1979). Reprinted from Eaves and Eaves, 1983 with permission.

primitive haemopoietic cells at the single cell level also appears to include a significant stochastic component (Till et al, 1964; Eaves et al, 1979) even under conditions where extrinsic signals are equal and non-limiting (Humphries et al, 1981; Ogawa et al, 1985). Thus, exceptions to the rigid flow of cellular traffic implied by the hierarchical model of blood cell production should be anticipated. Such exceptions have, in fact, been demonstrated in vitro. The extreme disparity in proliferative and differentiative potential that is sometimes seen when the first two daughter cells of a series of progenitors are analysed individually is one example (Ogawa et al, 1985). The best characterized clonogenic haemopoietic cells, because they are also the most abundant, are those that generate colonies containing only one lineage of mature progeny. Current data suggest that these, like their morphologically recognizable but incompletely differentiated progeny, represent transit populations capable of amplifying the number of mature cells produced in a given cohort but themselves lacking self-sustaining potential. Thus from the point of view of understanding the behaviour of haemopoietic stem cells, either normal or neoplastic, interest has focused on more primitive progenitor types.

I N V I T R O S T U D I E S OF C M L

933

Human cells able to generate primary multi-lineage colonies (CFUGEMM) in vitro are detectable but at relatively low frequencies (Messner, 1984). However, these cells show little or no self-renewal under the conditions used to express their multi-lineage colony-forming ability in vitro. Production of new colony-forming cells, including CFU-GEMM, has recently been described in even rarer human 'blast' colonies (Gordon et al, 1987b; Leary and Ogawa, 1987). These blast colonies can be generated in two different types of semi-solid culture systems. The relationship or extent of overlap between any of these human pluripotent clonogenic populations and cells with long-term self-maintenance capacity in vitro or in vivo is not yet known. Growth factors Extensive analysis of the conditions required to optimize the plating efficiency of different classes of haemopoietic colony-forming progenitors has led to the identification of a number of distinct regulatory molecules. These haemopoietic growth factors are collectively referred to as the colony-stimulating factors or CSFs. Several are also known as interleukins (ILs), because of their production by, and action on, leukocytes. Many of these factors have recently "been purified and their genes cloned and used to produce single factors in large quantities by recombinant DNA techniques. As a result, their actions in vitro and in humans are rapidly being more fully delineated (Clark and Kamen, 1987; Seiler and Schwick, 1988). Table 1 lists 10 of these molecules that have reached this stage of characterization. Initially, many of the CSFs were thought to have highly restricted lineage specificity and, indeed, many were named accordingly. With the availability of large amounts of pure material, actions by most of the haemopoietie growth factors on a much broader range of target cells have now been identified. In some instances, this has depended on the use of factors in combination, since two factors may elicit a response not obtained with either alone. The costimulating effects oflL-I (Stanley et al, 1986), IL-4 (Peschel et al, 1987), and IL-6 (Ikebuchi et al, 1987) in combination with various CSFs are examples of this type ofphenomenon. The synergistic action of two CSFs, e.g. M-CSF and GM-CSF (Pragnell et al, 1988) is another. The use of end points other than stimulation ofproliferation have allowed non-clonogenic haemopoietic target cells to be recognized as also CSF-responsive, including neutrophils, eosinophils and macrophages (Clark and Kamen, 1987). In addition, actions of a variety of haemopoietic growth factors (including IL-I, IL-4, IL-6 and GMCSF) on non-haemopoietic cells have been revealed (e.g. Dedhar et al, 1988; Gauldie et al, 1988; Kaushansky et al, 1988; Lowenthal et al, 1988). These findings complicate even further the possible networks of cell regulation that haemopoietic cells may involve and contribute to, and that may be relevant to disease mechanisms affecting blood cell production.

Regulation by stromal cells While colony assays have been a powerful aid for delineating pathways of haemopoietic cell development and have provided exquisitely sensitive

C. J. EAVES A N D A. C. EAVES

934

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IN VITRO STUDIES OF CML

935

methods for measuring haemopoietic growth factors (at concentrations of < 1 ng/ml), colony assays have been of limited value for investigating mechanisms of primitive haemopoietic cell regulation in vivo. Considerable evidence indicates that these regulatory mechanisms are primarily of a localized nature and may involve direct or close range interactions between primitive haemopoietic cells and certain poorly characterized, fixed elements of the marrow (reviewed in Eaves et al, 1987b). These cells, which are of nonhaemopoietic origin, are generically referred to as the 'stroma'. They include a number of mesenchymally-derived cell types, usually classified histochemically and/or immunologically as endothelial cells, fat cells (adipocytes), and adventitial reticular cells (fibroblasts) (Lichtman, 1981). Assays for their precursors, which are present in marrow but not blood and can be detected in vitro as fibroblast colony-forming cells (CFU-F) or reticular-fibroblast colony-forming ceils (CFU-RF), have also been reported (Friedenstein et al, 1978; Castro-Malaspina et al, 1980; Lim et al, 1986; Mclntyre and Bjornson, 1986). Recent studies suggest that many of these stromal-type cells when perturbed in vitro (e.g. by the products of activated macrophages, such as IL-I or tumour-necrosis factor (TNF)) may respond by increasing their production .ofat least two haemopoietic growth factors (Broudy et al, 1986; Munker et al, 1986; Rennick et al, 1987; Kaushansky et al, 1988; Zsebo et al, 1988). In addition, it is now known that some growth factors may be localized on the surface ofstromal cells (Roberts et al, 1987), either as integral components of the plasma membrane (Rettenmier et al, 1987), or as external moieties bound to extracellular matrix components (Gordon et al, 1987c) which are also secreted by stromal cell types. Thus, at least two mechanisms for geographically concentrating haemopoietic growth factors within the marrow have been identified. The cell biology of the stroma of haemopoietic tissue is, however, still in its infancy. The possibility that some stromal cells produce additional growth factors, either constitutively or following activation, has barely been explored, although several new leads have been reported recently (Hunt et al, 1987; Quesenberry et al, 1987; Landreth and Dorschkind, 1988; Lemoine et al, 1988). The nature and origin of possible negative regulators is similarly just beginning (Zipori et al, 1986; Eaves et al, 1987a). Key to progress in delineating mechanisms of stromal-mediated regulation of haemopoiesis has been the development of a marrow culture system that supports primitive haemopoietic cell proliferation and differentiation for extensive periods of time (months) in the absence of exogenously added haemopoietic growth factors. This so-called 'long-term marrow culture system', developed originally by Dexter and colleagues for mouse marrow (Dexter et al, 1977), was successfully adapted for human marrow three years later (Gartner and Kaplan, 1980; Greenberg et al, 1981). Initiation of the cultures with a high concentration of marrow cells fosters the formation of a confluent adherent layer that contains many cells with the phenotypic characteristics of a variety of stromal elements (Keating et al, 1982; Eaves et al, 1987b). These cells appear to be crucial for the continued maintenance of a clonogenic haemopoietic cell population for periods in excess of a month (Dexter et al, 1980; Andrews et al, 1986; Eaves et al, 1986). This is probably

936

C. J. EAVES AND A. C. EAVES

related to the fact that the most primitive haemopoietic cells become localized in the adherent layer (Mauch et al, 1980; Coulombel et al, 1983a). Over time, these cells proliferate (Dexter et al, 1980; Cashman et al, 1985) and differentiate to give rise to cells with a lower proliferative capacity. The latter are then released into the overlying medium (Greenberger, 1979; Slovick et al, 1984). There they continue to mature, or die because the factors required to support their terminal maturation are not present. Under standard conditions of long-term human marrow culture maintenance, neutrophils and macrophages are produced in large numbers, whereas, for example, erythropoiesis is arrested at an early clonogenic progenitor cell stage (BFU-E) (Gregory and Eaves, 1978; Dexter et al, 1981; Eaves et al, 1983). With each medium change, the most primitive (high proliferative potential) colony-forming cells in the adherent layer of normal marrow cultures are transiently activated into S-phase and presumably complete at least one mitotic cycle. In contrast, cells of lower proliferative potential remain continuously in cycle, suggesting loss of responsiveness to whatever mechanism causes more primitive cells to re-enter Go. All progenitors including occasional primitive cells in the non-adherent fraction also remain continuously in cycle, providing evidence that the stromal elements of the adherent layer play a significant role in regulating the kinetic behaviour of primitive haemopoietic cells and utilize short-range interactions to do so in this system (Cashman et al, 1985; Eaves et al, 1986).

Use and limitations of in vitro methodologies Clonogenic assays allow the number of haemopoietic progenitors in a given suspension to be measured, if conditions to support colony formation are non-limiting and independent of the number of progenitors present. The validity of these assumptions is checked in most laboratories by establishing a linear relationship between colony yield and the number ofcells plated, and by the use ofstimulatory conditions that are supraoptimal for colony growth, i.e. on the plateau of the dose response curve for each component of the culture medium. These requirements frequently differ for different types of colonies and hence for the detection of different types of progenitors (Eaves and Eaves, 1985). Fortunately conditions optimized for the growth of one type of colony are often not incompatible with the growth of another. For example, the concentration of erythropoietin that is optimal for the formation of large (BFU-E-derived) erythroid colonies is higher than that required for small (CFU-E-derived) erythroid colonies, whereas the development of granulocyte-macrophage colonies is not influenced by the addition of erythropoietin to the medium. On the other hand, addition of high concentrations of adequately purified erythropoietin can provide optimal stimulation of all erythroid colonies without affecting otherwise optimally stimulated granulocyte-macrophage colonies (Krystal et al, 1984). Thus, the detection of many types of myeloid progenitors can be optimized in a single assay.

IN V I T R O STUDIES O F C M L

937

In our work, we have routinely used a fetal calf serum based methycellulose medium (Eaves and Eaves, 1985) that has allowed standardized measurements of different categories ofclonogenic erythropoietic and granulopoietic progenitors (CFU-E, BFU-E and CFU-GM) to be accumulated on large numbers ofnormal and patient samples over more than a decade. Pluripotent progenitors (CFU-GEMM) are also detected under these conditions (Messner, 1984), but their much lower frequency makes obtaining as precise quantitative information on individual patients more difficult and hence has less commonly been undertaken. The test cells have to be plated at an appropriately low concentration to minimize colony overlap and this means that more plates have to be set up to allow minority colony types to be detected. Optimal growth of megakaryocyte colonies (CFU-M) requires the inclusion of additional factors in the medium. This can be achieved without altering the growth of other colony types by substituting a suitable human plasma for the fetal calf serum supplement (Messner et al, 1982)9 Even when all technical parameters are optimized, interpretation of colony assay data is bedevilled by a number of other limitations of the methodology. First, most scoring criteria allow the inclusion of a rather broad spectrum of progenitor types. This makes it difficult to use colony data to distinguish "between altered input (or exit) of cells into (or out of) a given progenitor compartment, and altered amplification within the compartment. Similarly, apparent alterations in other progenitor parameters, e.g. surface antigen expression, may reflect changes in the distribution of cells within a given compartment rather than an overall shift in the behaviour of the population as a whole9 To some extent, this can be minimized by the adoption of more refined colony sizing criteria during scoring (Eaves et al, 1979; Cashman et al, 1985). Second, it must be remembered that colony assay data yield values only forprogenitor concentrations, i.e. the number of progenitors relative to the other nucleated cells in the suspension. To extrapolate from such information to estimates of absolute compartment size requires some method of quantitating the original sample. This is readily achieved with peripheral blood where defined aliquots can be removed, processed and assayed and progenitor measurements then expressed on a per ml basis9 The situation is more difficult with marrow, since the degree of contamination of marrow aspirates with peripheral blood is highly variable and a quantitative estimate of the absolute cellularity of the marrow itself is not readily obtained. Finally, there is the problem that colony growth can be affected by other cells present in the suspension, a variable that is not readily controlled. 9 Interpretation of long-term marrow culture data is further complicated by the fact that more than one cell type is required to establish a functional culture. In addition, there is as yet no information about the 'plating efficiency' ofprimitive haemopoietic progenitors in this system, even when the non-haemopoietic constituents are made non-limiting, as can be achieved by the use ofpre-established irradiated marrow adherent layers as feeders (Eaves et al, 1986). In spite of these limitations, long-term marrow culture studies have provided many new insights about how primitive haemopoietic cells may be regulated (Dexter et al, 1980; Eaves et al, 1986; Eaves and Eaves, 1988), and

938

C. J. EAVES A N D A. C. EAVES

recently have provided evidence that the cells responsible for initiating longterm haemopoiesis in human marrow cultures are different from the majority of cells detected by current clonogenic assays (Sutherland et al, 1988). APPLICATION OF CLONOGENIC ASSAYS

Expansion of neoplastic progenitor numbers in chronic phase Standard colony assay procedures to detect progenitors defined for normal individuals have been widely applied to marrow and blood specimens from CML patients. Results from plating marrow cells from newly diagnosed or treated chronic phase patients have revealed no significant abnormality in colony growth, i.e. typically the colonies formed are indistinguishable from those derived from normal individuals and the concentration ofall progenitor types (relative to more terminally differentiated cells) in the marrow does not appear to be markedly altered (Eaves et al, 1980). This is not surprising as the blood cell differentiation process appears to be proceeding relatively normally in these patients, although some aspect of haemopoietic cell kinetics must be altered since the number of circulating granulocytes is abnormally increased and all the other mature myeloid cells are also derived from the neoplastic clone (Adamson, 1984; Fialkow, 1985). In most instances where CML marrow colony data have been collected, confirmation that the progenitors measured were neoplastic has not been undertaken. However, this can be generally assumed to be the case since several studies have demonstrated the clonal origin of virtually all progenitors detectable either by glucose-6phosphate-dehydrogenase (G6PD) isoenzyme analysis (Singer et al, 1979; Adamson, 1984) or by revealing the Philadelphia chromosome in their progeny (Chervenick et al, 1971; Aye et al, 1973; Moore and Metcalf, 1973; Dub6 et al, 1984b). Occasional exceptions to this and their explanation are discussed later (see below). Although there is no evidence that the number of neoplastic clonogenic cells is increased relative to other cell types in the marrow, the absolute progenitor content of the marrow may well be considerably higher than normal. Increased cellularity of the marrow is a recognized feature of marrow biopsies from CML patients at diagnosis or with poorly controlled disease. Application of colony assays to peripheral blood samples provides additional evidence for progenitor compartment expansion in CML. Interestingly, not only are granulopoietic progenitor numbers in the blood increased (Paran et al, 1970; Moore et al, 1973; Goldman et al, 1974; Moberg et al, 1974), so also are all other types of circulating clonogenic myelopoietic cells, including erythropoietic (Eaves and Eaves, 1979; Goldman et al, 1980), megakaryopoietic (Vainchenker et al, 1982), and pluripotent (Messner, 1984) progenitors. Examples for different patients taken from our experience are illustrated in Figures 2 and 3. It can be seen that considerable variation occurs amongst individual patients. Nevertheless, both circulating progenitor types shown are highly increased when the peripheral WBC count is elevated. In fact, the peripheral blood content of each type of progenitor appears on average to be

939

IN VITRO STUDIES OF CML 106

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W B C per ml of B l o o d Figure 2. BFU-E concentration in the peripheral blood as a function of the WBC count 9 Each solid point represents a measurement on a different C M L patient in chronic phase, including treated as well as newly diagnosed cases. Open points correspond to measurements obtained during the same period and under the same conditions for a series of normal individuals9 The dotted lineindicatesthe slopeexpectedfor a linear relationshipbetweenBFU-E/mland the W B C count 9

exponentially (x z) related to the WBC count, i.e. with each doubling of the .WBC count the progenitor content of the blood quadruples, with each tenfold increase in the WBC count the number of circulating progenitors per ml increases approximately 100 times, etc. Note also that normal values fit the same relationship. One might speculate on two different mechanisms that could contribute to this marked increase in circulating progenitors in CML: a generalized amplification of all progenitor compartments, and a decreased progenitor retention in (or increased mobilization from) the extravascular spaces of the marrow, spleen and other sites. A generalized amplification of progenitors in

940

C. J. EAVES AND A. C. EAVES 106

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C M L seems likely. Consistent with this is the fact that all types o f n e o p l a s t i c p r o g e n i t o r s in the circulation, like those in the m a r r o w , a r e actively cycling, even p r i o r to initiation o f a n y t r e a t m e n t ( T a b l e 2). This c o n t r a s t s m a r k e d l y with the s i t u a t i o n in n o r m a l i n d i v i d u a l s w h e r e the m a j o r i t y o f the m o s t p r i m i t i v e (high proliferative p o t e n t i a l ) p r o g e n i t o r s in the m a r r o w are quiescent, as are all p r o g e n i t o r s f o u n d in the circulation. O n the o t h e r h a n d , the r e l a t i o n s h i p between the W B C c o u n t a n d the n u m b e r o f c i r c u l a t i n g C F U G M suggests that the p r o d u c t i o n in vivo o f g r a n u l o c y t e s a n d m a c r o p h a g e s on

941

IN VITRO STUDIESOF CML Table 2. Alteration of the proliferative state of neoplastic haemopoietic colony-forming cells in CM L*. % of the population in S-phaset Tissue

Progenitor

Marrow CFU-E BFU-E (total) CFU-GM (total) BFU-E (> 16 clusters):~ CFU-GM (> 500 cells)~ CFU-GEMM Blood

CFU-E BFU-E (total) CFU-GM (total) CFU-GEMM

Normal

CML

43+2(20) 27_+2 (20) 37_+3 (20) 4-+2 (20) 4+2(20) Not done

33+2(13) 33_.+4 (13) 31_+4 (13) 40-+4 (13) 44_+7 (5) Not done

1+3 0__+1 0__+2 0+20

(9) (9) (9) (2)

33+3(16) 34+3 (16) 30__+4(16) 48___5 (5)

* All patients assessed prior to initiation of any treatment. I As determined by standard thymidine suicide assays where cells are exposed to a pulse (20 minutes) of high specific activity tritiated thymidine just before plating. Data from Cashman et al (1985) and Eaves et al (1986). Values shown are the means _SEM of (n) normals or CML patients, assessed individually. :~Subdivision of BFU-E and CFU-GM in the marrow into low and high proliferative potential sub-populations allows a minor category of lineage-restricted progenitors that are quiescent in normal marrow to be revealed. a per progenitor basis decreases as the Ph positive clone expands. Alternatively, there m a y be an increasing tendency o f all progenitors to enter the circulation (or both possibilities m a y apply). Very little is k n o w n a b o u t how haemopoietic cell migration in and out o f the circulation is determined. ' H o m i n g ' molecules have recently been identified on the surface o f some T lymphocytes (Gallatin et al, 1983) and a n u m b e r o f structures responsible for specific cell-cell recognition and binding in the haemopoietic system have been characterized (Hynes, 1987; Simmons et al, 1988). The fact that circulating haemopoietic progenitors in n o r m a l individuals do not represent a r a n d o m sampling o f their counterparts in the m a r r o w suggests that the traffic o f these populations m a y also have a specific molecular basis and this might well be perturbed in C M L . Evidence for altered surface properties in neutrophils from C M L patients with high W B C counts has been reported (Baker et al, 1985) and altered adhesiveness o f their primitive progenitors has also been described ( G o r d o n et al, 1987a).

Abnormalities of neoplastic progenitor regulation in chronic phase The very large variation in circulating progenitor n u m b e r s observed in patients with different W B C counts (Figures 2 and 3), or even in the same patients before and after treatment (unpublished observations), allows a

942

C. J. EAVES AND A. C. EAVES 106

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comparison of the increase in the number of one type of progenitor relative to another in individual patients, i.e. in different Ph-positive clones. Figure 4 shows such a plot for the same data shown in Figures 2 and 3. Again considerable variation between individual patients is observed but the ratio between primitive erythropoietic progenitors and granulopoietic progenitors in the circulation remains, on average, constant. Note also that the data for progenitors in CML patients overlap with those obtained for normal individuals. Thus, whatever mechanism determines the number of circulating progenitors in a given patient at a given time, appears to influence both erythroid and granulopoietic progenitors more or less equally. A comparison of the ratio of the same two classes of progenitors in the marrow also shows this to be within the normal range regardless of the WBC count (Eaves et al, 1980).

IN V I T R O STUDIES O F C M L

943

These findings reinforce tile concept of a consistent, lineage non-specific amplification of neoplastic progenitors in CML originating from the inappropriate expansion of a single, primitive pluripotent cell. In addition, they suggest perturbation of a control mechanism normally operative not only at the level of stem cells, but also effective in regulating the cycling and/or viability of some early lineage-restricted progenitor populations (Table 2). On the other har~d, the lack of lineage selectivity of progenitor amplification in CML also suggests that the process of lineage restriction itself is not affected by the molecular changes that characterize the neoplastic (Ph positive) pluripotent cell. The same reasoning is consistent with current concepts that unique regulatory mechanisms exist to control the number of mature blood cells produced from lineage-restricted clonogenic progenitors and that these are differentially affected in CM L. Thus, the potential for an enormous increase in granulocyte and monocyte production, which is the hallmark ofthis disease, is probably due to defective restraint of the terminal differentiation of a greatly expanded compartment of granulopoietic progenitors. In contrast, red cell levels may be subnormal, in spite of equivalent increases in the population of CFU-E, a cell type believed to be the immediate precursor of the proerythroblast (Eaves et al, 1984). The fact that members of the neoplastic CFU-E population frequently exhibit the capacity to undergo terminal maturation in vitro in the absence of added erythropoietin (Eaves and Eaves, 1979) suggests that the mechanism restricting red cell output in the CML patient does not involve deficiencies in the availability or response of erythroid cells to this regulator. Indeed, it is possible that the mechanism is one intrinsic to the terminally differentiating neoplastic erythroid cell uniquely manifested in this context. Alternatively, it may simply result from an altered balance between the number of developing erythroid cells and the number of competent accessory macrophages available (Lichtman, 1981). Obviously, it is more intellectually appealing to focus on abnormal features of neoplastic progenitors common to all CML patients, since these may point to molecular targets of the B C R - A B L gene product. However, investigation of the sometimes striking heterogeneity between different CML patients may be equally informative, by providing insights into normal variations in individual stem cell properties preserved during transformation, and/or by suggesting targets of additional genetic changes. Two cases in our experience illustrate this point (see Table 3). Both are female patients who presented with a low WBC count ( < 40 x 109/litre) and normal levels or marginally elevated numbers ofcirculating progenitor numbers. On examination of direct marrow metaphases both showed > 90% Ph positive dividing cells. The first patient has remained in this state without treatment for over 4 years. Expansion ofher neoplastic clone thus appears to have plateaued early on at a level barely above normal limits. The second patient exemplifies the opposite extreme. Two months after diagnosis she returned with a significant increase in all of her circulating myeloid progenitors concomitant with an equally unanticipated lymphoid blast crisis and an associated peripheral WBC count of 190 x 109/litre. Thus, in this case, a very rapid expansion of the neoplastic

944

c . J . EAVES AND A. C. EAVES

Table 3. Variation in the behaviour of the neoplastic clone exemplified in two different CML patients. Disease parameters

Case number i

A t diagnosis: 9 Age (years)/sex

52 F

41 F

95% 270f 90:~

100% 1300t 890:~

Trealmenl:

None

None

Later: Disease status WBC ( x 109/I) BM cytogenetics (% Ph+ve)* No. of circulating BFU-E (per ml of blood) No. ofcirculating CFU-GM (per ml of blood) Increase in circulating progenitors

+ 4 years Chronic phase 10.0 94% 100t 200~ None

+ 7 weeks Lymphoid blast crisis 190.0 100%w 3600t 9900~ Sixfold

WBC ( x 109/1) BM cytogenetics (% Ph +ve)* No. of BFU-E (per ml of blood) No. of CFU-GM (per ml of blood)

12.0

Case number 2

37.0

* Remainder: normal. t Normal range defined by the mean +__2s.d. for BFU-E is 300-500/ml (Eaves and Eaves, 1985). :~ Normal range defined by the mean __.2 s.d. for CFU-GM is 4-150/ml (Eaves and Eaves, 1985). wAll metaphases also showed various additional abnormalities.

clone occurred. The progenitor findings suggest that this expansion originated in the Ph positive pluripotent stem cell compartment although there was also during this period a dramatic increase in a pre-B cell subcione(s) characterized by additional chromosomal changes. What allows one clone (Case 1) to remain almost normal for 4 years whereas another may increase many fold in a few weeks (Case 2)? Are differences in genetic changes other than B C R - A B L the total explanation? A discussion of abnormalities in neoplastic progenitor regulation in CML would not be complete without mention of other (than erythropoietin) growth factor requirements. Analysis of these, as an explanation for the deregulated amplification of the cionogenic compartments has, however, been relatively unrewarding. Autonomous or autocrine growth of Ph positive cells has riot been found, nor has evidence of altered growth factor responsiveness of Ph positive granulopoietic progenitors been demonstrable (Metcalf, 1977). Some resistance to prostaglandin El-mediated suppression of granulopoiesis has been documented (Aglietta et al, 1980) and high concentrations ofinterferonceor 7 can lead to a selective suppression of the neoplastic clone in vivo (Talpaz et al, 1986; Kurzrock et al, 1987). How, or if, these relate to the mechanism of clonal overgrowth in vivo, or the manifestation ofany of the other phenotypic changes that accompany this process is unknown. Residual normal progenitors In early studies failure to detect normal metaphases (Sandberg, 1980) or progenitors (Aye et al, 1973; Moore and Metcalf, 1973) fostered the beliefthat

IN VITRO STUDIES OF CML

945

the growth of the Ph positive clone led to the irreversible suppression, and even possible extinction, of normal haemopoietic cells. However, even the first cytogenetic studies of haemopoietie colonies from CM L patients (Chervenick et al, 1971) suggested the persistence of normal, functionally intact haemopoietic stem cells in some patients. This finding, although highly controversial, nevertheless spurred the development of intensive chemotherapeutic regimens with curative intent. These proved largely unsuccessful clinically but served the important purpose of establishing the presence of normal haemopoietic stem cells in a significant fraction of CML patients (Singer et al, 1980; Goto et al, 1982). At about the same time, our group discovered that a persisting population of primitive normal haemopoietic cells could be similarly revealed when marrow aspirates from many CML patients were used to initiate longterm cultures (see below). Why then are normal progenitors not detectable in marrow or blood samples from most CML patients? The answer is suggested from a reexamination of Figures 2 and 3. At diagnosis, the WBC count is usually sufficiently elevated that even normal levels of circulating normal clonogenic progenitors (see Figures 2 and 3) would be expected to constitute only a very minor proportion of the total circulating progenitor population. Following 9sustained treatment with conventional chemotherapy, the relative number of these might decline even further. Thus, labour-intensive analytical procedures that in practice restrict the number ofindividual colonies that can be examined and thereby limit the sensitivity of detecting minority progenitor populations (Hook, 1977) might well fail to detect normal progenitors. If, as a result of the presence of Ph positive cells, the number of normal clonogenic cells were reduced, they would constitute an even smaller proportion of the total clonogenic population and hence be even more difficult to detect. On the other hand, if amplification of Ph positive progenitors were accompanied by a significant increase in analogous normal progenitor compartments, then detection of the latter might be expected even in patients presenting with WBC counts > 40 x 109/litre. To test the relative importance of progenitor amplification on the detectability of normal clonogenic cells, we have analysed cytogenetically the colonies generated by haemopoietic cells obtained from a large series of untreated CML patients with peripheral leukocyte counts ranging from 12490 x 109/litre. The findings (Dub6 et al, 1984b; Kalousek et al, 1984) indicate that when the total number of progenitors in the circulation was low enough for normal levels of Ph negative progenitors to be detectable (given the number of colonies that could be analysed), such cells were consistently demonstrable but when the total number of progenitors in the circulation exceeded that limit, such progenitors were not revealed. Thus in patients with limited amplification of their Ph positive clone, as suggested by a WBC count of less than 40 x 109/litre, and whose haemopoietic cells had not yet been influenced by exposure to cytotoxic agents, no evidence of a significant increase or decrease in Ph negative progenitors has been obtained. The majority of the dividing cells in the marrow of CML patients, as in normal individuals, can be assumed to be terminally differentiating elements due to the exponential expansion these cells undergo at this final stage of their

946

Tm ie "I-I

i ~~ =

f

im

Stem ce,,s

I progenitors

1

Comm,.e

r-

~

C. J. EAVES A N D A. C. EAVES

~

~Endcells

Figure 5. Diagrammatic representation of the two-dimensional expansion of the neoplastic clone (hatched areas) in CML: in the stem cell compartment (horizontally) over time, and with differentiation (vertically) into mature progeny. Also illustrated is the suppression of primitive normal haemopoietic progenitors from further differentiation.

development. When direct marrow preparations from those patients where normal progenitors could be demonstrated were analysed, the proportion of Ph negative cells was usually considerably lower or below the limit of detection. Thus there appears to be a selective amplification in favour of the neoplastic clone not only over time but also with increasing differentiation (Figure 5). A similar phenomenon has been described in patients with polycythaemia vera (Adamson, 1984). Whether this simply reflects a superior proliferative and/or survival capacity of differentiating neoplastic cells, or the operation ofsuppressive mechanisms exerted by neoplastic cells selectively on normal cells, or both, is not yet known. The fact that normal progenitors form colonies ofmature progeny that are indistinguishable in size and morphology from analogous colonies of Ph positive cells suggests that whatever mechanisms confer an advantage on the latter in vivo are relaxed under conditions used to support colony formation in vitro. Blast crisis The blast crisis phase of CML is characterized by the rapid accumulation of cells that retain a primitive 'blast' morphology and do not undergo further morphological differentiation. It is generally assumed that this results from the acquisition of additional genetic changes. Cytogenetic studies (Sandberg,

IN V I T R O STUDIES O F C M L

947

1980) and, more recently, gene transfer approaches (Liu et al, 1988) ofpatients in blast crisis provide strong support for this view. Thus, in many respects, CML in blast crisis resembles acute myeloid leukaemia. Progenitor data bear out this analogy. Marked patient-to-patient heterogeneity is seen in both disease categories. In some, only morphologically normal colonies are obtained; in others, only morphologically abnormal colonies or a mixture is seen. Cytogenetic studies of these in the blast crisis phase of CML (Moore and Metcalf, 1973; Kalousek et al, 1984) suggest the following. The formation of normal-appearing colonies is due to the persistence of chronic phase progenitors. However, like normal progenitors in chronic phase patients, these may be diluted by the blast population to levels below detectability. Lack of abnormal colonies presumably indicates culture conditions that are inadequate to support the proliferation of the cytogenetically more evolved blast crisis progenitors characteristic of some patients, although in others these too can be detected in vitro. However, abnormal colonies may, even for patients with cytogenetic clonal evolution, be derived from cells that are karyotypically indistinguishable from those typical of chronic phase. Thus alterations in differentiation potential may not always correlate with karyotypically detectable changes. Ph positive ALL Approximately 20% of adults presenting with a diagnosis of acute lymphoblastic leukaemia (ALL) determined by the B, T or pre-B phenotypic characteristics of their blasts are found upon cytogenetic analysis to have Ph positive disease (Greaves, 1982). Some of these cases undoubtedly represent patients who have had a silent course of CML prior to the secondary development of blast crisis in a B-lineage (usually) or T-lineage (rarely) derivative of the primary clone. Lack of symptoms in CML patients who are diagnosed 'by accident' is not unusual and the likelihood of escaping diagnosis in chronic phase would be increased in patients who, by chance, progressed into blast crisis relatively early during expansion ofthe Ph positive clone (Greaves, 1982). Historically, Ph positive ALL patients have been reclassified retrospectively as CML patients presenting in lymphoid blast crisis in those cases where induction of 'remission' reveals an underlying chronic phase picture. This is clearest when the dividing cells in the marrow remain Ph positive, as is typical of most CML patients in chronic phase either preceding or following therapeutic control of a blast crisis (Sandberg, 1980). Much more controversial has been the interpretation applied to patients presenting as Ph positive ALL whose Ph positive metaphases decrease to very low levels or disappear following remission induction. In the case of CMLs undergoing a rapid transition to blast crisis, it might be expected that effective treatment of the blast population could also lead to a Ph negative or mosaic (Ph negative/Ph positive) remission because the parent chronic phase clone had not yet become sufficiently large to remain dominant after therapy. On the other hand, the possibility that some patients presenting with Ph positive ALL have a different disease entity arising in a lymphoid-restricted precursor, has also been widely entertained (Figure 6).

948

C. J. EAVESAND A. C. EAVES CML IN LYMPHOID

B-LINEAGE RESTRICTED

BLAST CRISIS

Ph + ve A L L

Figure 6. Alternative models of Ph positive ALL based on proposed differences in the cell initially transformed. Black circles represent populations containing neoplastic cells; open circles: exclusively normal populations.

Most cases of ALL appear to originate in a committed B or T lineage cell and hence do not show involvement of myeloid cells in the neoplastic clone (Fialkow, 1985; Kalousek et al, 1988). Moreover, the cytogenetic changes they exhibit are distinct and different from those generally associated with the myeloid leukaemias and in some instances can be seen to be specifically related to genetic events characteristic of early lymphoid cell development (Third International Workshop on Chromosomes in Leukemia, 1981). However, simple analysis of direct marrow metaphases does not allow ready discrimination of the particular type of cell in which a chromosomal abnormality may be found. Thus resolution of the lineages involved in cases of Ph positive ALL not readily reclassified as CML has had to await the application of newer approaches. Two of these have recently emerged. The first relates to the discovery that the Ph translocation, especially in patients who first present as ALL, may represent one of two major classses of B C R - A B L gene rearrangement (described in detail in Chapter 6 of this volume). These are distinguished hy different regions of the B C R gene in which the breakpoints are clustered, and the resultant generation of two types of abnormal B C R - A B L message (Shtivelman et al, 1985; Hermans et al, 1987) and protein product (BenNeriah et al, 1986; Walker et al, 1987; Hermans et al, 1987). The vast majority ofpatients with 'classical' CML are found to be ofthe same type (P210 BcR'ABL) when examined from this point of view (De Klein et al, 1986). Thus, one possibility was that the other type of molecular change (P 185Bc~ABL)might be associated with de novo lymphoid transformation. If this were the case, then identification of a subset of Ph positive ALL patients with lymphoid-restricted clones would be possible by appropriate molecular investigations. The second and, in the end, more informative approach has been the use of cytogenetic and molecular methods to associate clonality (and/or Ph positivity or B C R - A B L rearrangement) with specific haemopoietic cell

IN VITRO STUDIES OF CML

949

lineages. Cytogenetic analysis of cells in individual granulopoietic and erythroid colonies obtained from patients presenting with Ph positive ALL has now established unequivocally the pluripotent stem cell origin of the Ph positive clone in a majority of the cases examined to date, including examples with both types ofmolecular alteration (Tachibana et al, 1987; Kalousek et al, 1988). However, it has also been shown that Ph positive disease may occasionally'arise in a pre-B cell. We have documented one case of Ph positive ALL where throughout the 8 month course between diagnosis and terminal relapse, Ph positive myeloid progenitors could never be found (Kalousek et al, 1988). Purified granulocytes were also consistently polyclonal and showed no evidence of B C R - A B L rearrangement (Turhan et al, 1988). Interestingly, the type of B C R - A B L rearrangement observed in this case was the same as that seen in most patients with multi-lineage disease (CML) involving the major breakpoint cluster region (Turhan et al, 1988). It thus appears that neither type of molecular change will be predictive of the origin of the clone in individual patients. LONG-TERM CULTURE FINDINGS General When marrow cells are obtained from newly diagnosed CML patients and used to initiate long-term cultures, a typical albeit sometimes incomplete adherent layer is usually produced. However the number of non-adherent granulocytes and macrophages present after 4-6 weeks and the number of clonogenic progenitors detectable are highly variable. When marrow cells are obtained from treated, chronic phase patients, even greater heterogeneity in all of these parameters is encountered and 'failures' (poor adherent layer and/ or little or no long-term haemopoiesis) are not uncommon (Coulombel et al, 1983b; Eaves et al, 1983; Dub6 et al, 1984c). A number of possible reasons for this can be considered: I. Variable dilution of critical mesenchymal precursors by the neoplastic clone (since cultures are initiated with a fixed number of nucleated cells in the aspirate rather than a fixed sampling of the marrow stroma). 2. Abnormalities in stromal cell function. 3. Differential behaviour of normal and neoplastic progenitors. 4. Variable effects of treatment on normal and neoplastic haemopoietic and stromal progenitors populations.

Differences in the maintenance of normal and neoplastic progenitor populations Cytogenetic analysis of individual colonies generated in assays of cells from long-term CML marrow cultures has allowed a more consistent picture to be revealed (Coulombel et al, 1983b; Eaves et al, 1985). In cultures established from most patients, the Ph positive progenitors rapidly disappear to reach undetectable levels within 4-6 weeks. Why this occurs is not yet clear. Indeed,

950

C. J. EAVES A N D A. C. EAVES

the answer may be multi-factorial and hence difficult to ascertain. To date no simple explanation has emerged. Badly needed are more quantitative assays for 'long-term culture initiating cells' in which the variation of adherent layer formation is suppressed. These may then allow enumeration and characterization of these primitive haemopoietic cells in normal individuals and provide a basis for their assessment in CML. 9Since the anomalous behaviour of the neoplastic clone in long-term CML marrow cultures contrasted so markedly with the situation in vivo, it seemed likely to us that some manipulation of the culture conditions and/or the use of a different source of haemopoietic progenitors (possibly more enriched in Ph positive stem cells), might allow long-term maintenance of Ph positive haemopoiesis to be achieved. We were also encouraged along this line of thought by the early identification of one patient who exhibited no particular distinguishing features but whose Ph positive progenitor population appeared to be relatively well maintained in long-term marrow culture (Dub6 et al, 1984c; Eaves et al, 1986). To eliminate any potential disease-related variation in adherent layer formation (Singer and Keating, 1983), we used culture dishes containing a pre-established feeder of normal marrow adherent layer cells subjected when at, or near, confluence to a dose of irradiation sufficient to inactivate the clonogenic capacity of any residual haemopoietic progenitors. As an alternative source of Ph positive haemopoietic progenitors, we tried peripheral blood. In sharp contrast to the results obtained in standard CML marrow cultures, even for some of the same patients, Ph positive progenitor numbers were well maintained in these reconstructed long-term cultures, but only when a pre-established feeder was used (Eaves et al, 1986). This system thus provided a basis for future exploration of differences between primitive Ph negative and Ph positive progenitors, and their interactions with stromal elements as well as each other. In spite of the rapid decline of Ph positive progenitors in long-term CML marrow cultures, co-existing Ph negative progenitors when present at detectable levels are usually maintained as in normal long-term marrow cultures (Coulombel et al, 1983b). Thus, even when Ph negative progenitors are below the level of detection in the primary aspirate, due to their dilution with Ph positive progenitors, the ratio between these two genotypes is rapidly altered in culture and, by 4-6 weeks, Ph negative progenitors may become predominant. The absolute number of Ph negative progenitors may, however, not attain the level seen in normal cultures. To date we have analysed progenitors from long-term marrow cultures from over 30 CML patients (including some in chronic phase after an initial diagnosis of Ph positive ALL). The results remain the same as those published for fewer patients (Kalousek et al, 1984; Eaves et al, 1985). In the majority of untreated patients, Ph negative haemopoiesis takes over. After treatment, this is still demonstrable in a significant, albeit somewhat smaller proportion ofpatients, and, in general, the number of Ph negative progenitors per culture is lower. However, patient heterogeneity, particularly in the treated group, makes it difficult to assign much biological significance to these differences and clear-cut correla-

IN VITRO STUDIES OF CML

951

tions with duration of diagnosed disease or extent of treatment have not yet been found. Two patients have afforded us the opportunity to investigate the clonality of the Ph negative progenitors detected in long-term CML marrow cultures. In both, the data suggested that these were all polyclonal (Dub6 et al, 1984a; Hogge et al, 1987). While unequivocal evidence of a Ph negative but monoclonal .precursor population in CML has not yet been obtained, secondary acquisition of the Ph chromosome in a case of acute myeloid leukaemia has been reported (Kahn et al, 1975; Miller et al, 1984) and the possibility that a Ph negative preneoplastic clonal population exists in some CML patients therefore remains open. Moreover, monoclonal haemopoiesis in acute leukaemia in remission has now been documented in several patients (Fearon et al, 1986; Fialkow et al, 1987), and in one case was shown to be associated with reversion to cytogenetic normalcy (Jacobson et al, 1984). Given the interest in using long-term culture as a method for clinical purging of marrow harvested for autologous transplantation (see below), the clonal status of the emergent Ph negative progenitors in long-term CML cultures was, and remains, an important question to address. In the past, such studies have been limited by the infrequency of CML patients with suitable genetic markers to allow clonality analysis independent of the Ph chromosome (or B C R - A B L rearrangement). However, with the availability of probes for methylation-sensitive regions of X-linked genes bearing restriction fragment length polymorphisms (RFLPs), approximately half of all female CML patients can now be investigated (Vogelstein et al, 1987). Thus, a more definitive answer on a larger number of patients is now practical.

Differences in the regulation of normal and neoplastic progenitor proliferation The demonstration that all classes of Ph positive progenitors are proliferating continuously in vivo provides a tantalizing, albeit unverified, clue as to how the neoplastic clone might gain a selective advantage over the most primitive co-existing normal cells, since in normal individuals these populations show a much slower rate of turnover (see Table 2). In order to pursue this possibility, more information is needed about the mechanisms that normally maintain primitive haemopoietic cells in a quiescent state and how these are overcome or bypassed by Ph positive cells. The ability to maintain primitive haemopoietic cells in standard long-term marrow cultures has allowed some progress along these lines, since the cycling status of the same types of primitive haemopoietic progenitors whose proliferative state in vivo varies following appropriate perturbation can be monitored and manipulated. Investigation of the mechanisms operative in vitro has suggested that the establishment of a quiescent state is mediated by close range interactions with 'unperturbed' mesenchymal cells since this does not occur when such cells are not present (Eaves et al, 1986). On the other hand, for primitive haemopoietic cells integrated into an adherent layer, reentry into a proliferative cycle requires some stimulation of the mesenchymal cells. This is normally mediated by a constituent of horse serum when the

952

C . J. EAVES A N D A. C . EAVES

cultures are fed with fresh medium, but is also achievable with a variety of other mesenchymal cell activators (Cashman et al, 1985; Eaves and Eaves, 1988). The availability of a culture procedure that reproducibly allows primitive Ph positive progenitor maintenance for periods of more than 4-6 weeks has made possible a first look at their cycling behaviour in this system. The cycling status of CML progenitor classes has been examined as a function both of the presence or absence of normal marrow mesenchymal cells and as to whether they are consequently integrated or not into an adherent layer of normal marrow origin. The ability of a normal feeder to support the long-term maintenance o f a Ph positive population was found to not be associated with an ability to regulate (negatively) the proliferative state of primitive Ph positive progenitors, even those present in the adherent fraction (Eaves et al, 1986). Thus this system should be ofconsiderable value to analyse the cellular and molecular basis of this defective control. Stromal cells in CML

Genetic, phenotypic and functional analysis of the marrow stroma in CML has had to rely primarily on comparisons ofcells produced in vitro with those obtained from normal individuals. In addition to the caveats listed above, it will also be obvious that analysis ofpopulations generated rapidly in vitro will not necessarily reflect the features, or even distribution, of phenotypes that comprise the fixed elements of the marrow in vivo. Cells identified as 'fibroblasts' obtained when marrow is cultured in minimal medium supplemented with fetal calf serum are clearly not members of the Ph positive clone (Fialkow et al, 1978). Such cells can also be obtained by subculturing the adherent layer of long-term marrow cultures (Turhan et al, 1988). However, direct examination of cells in normal adherent layers reveals a complex mixture, including many that exhibit varying degrees of endothelial cell and adipocyte differentiation and other mesenchymal cell markers, in addition to primitive haemopoietic ceils, macrophages and T cells (Keating et al, 1982; Eaves et al, 1987b; Coulombel et al, unpublished findings). How these cells may influence the development ofeach other during adherent layer formation is not known, although clearly the composition of the original inoculum, cell density, and the composition of the medium used are significant factors. Thus, simple phenotypic comparisons of total adherent layers from CML and normal long-term cultures are likely to be plagued with problems of interpretation (Singer and Keating, 1983; Simmons et al, 1987). Such comparisons may also not prove to be as useful as originally anticipated. There is now a growing body of evidence that the capacity to support the proliferation and differentiation of primitive haemopoietic cells of either mouse or human origin may be a generalized property of fibroblast-like cells derived even from non-haemopoietic tissues (Bleiberg et al, 1987; Brockbank and deJong, 1987; Roberts et al, 1987; Sutherland et al, unpublished findings). More tightly regulated functions must therefore be sought to explain the specific localization patterns of normal haemopoiesis and the erosion of this specificity in CML. It is also possible that the marrow

953

IN V I T R O STUDIES O F C M L

/

/

100 flasks (2x10 '~

I ~176176

.,..~ ~

10 Days ~'J/~

Ph p o s i t i v e Stem Cells

/ Treatment

/

T

Ph ne a t i v e Stem Cells

Figure 7. Protocol for using long-term culture to purge autologous marrow for the transplantation of patients with malignancies. In this case CML is chosen as the example and details are those currently being tried in our centre for treating this disease.

stroma plays a relatively insignificant role with regard to influencing clonal expansion in which case greater focus on the changes exhibited by Ph positive cells and their effects on stromai interactions m a y be a more informative line o f investigation.

Clinicalapplications The possibility that long-term marrow culture of h u m a n leukaemic cells might have therapeutic potential in the setting of autologous marrow transplantation (Figure 7) was obvious from the very first data demonstrating the dramatic 'switch' in favour of normal haemopoiesis that frequently occurs in cultures established from both A M L and C M L patients (Eaves et al, 1983; Coulombel et al, 1983b; Coulombel ct al, 1985). Transplantation of cells from long-term m a r r o w cultures o f murine origin had established that cells with

954

C. J. EAVES AND A. C. EAVES

long-term in vivo repopulating potential could be obtained after several weeks (Spooncer and Dexter, 1983), although detailed time course studies have indicated that those cells with the highest self-renewal capacity decline rapidly (Mauch et al, 1980; Harrison et al, 1987). Successful long-term cultures initiated with human marrow have historically been more difficult to establish and do not appear to match the extent or duration of haemopoiesis typical of rrturine cultures. This is particularly evident if a comparison of the maintenance of multi-lineage progenitors is made between murine and human long-term marrow cultures. Thus, the risk of ending up with too few repopulating cells to obtain sufficient elimination of leukaemic stem cells appeared to represent a major practical impediment to further exploration of this approach. However, in 1986 the Manchester group reported that they had applied this method to a patient with AML in relapse. About I0 I~ marrow cells were cultured under conventional but scaled-up conditions while the patient received marrow ablative treatment. At the end ofthe I0 days ofculture, both adherent and non-adherent fractions were collected and about 7 x 10 9 recovered cells re-infused. The patient recovered with normal haemopoiesis, although this was delayed (Chang et al, 1986). Nine months later, the leukaemia recurred and the patient died. Since that time, results for five additional AML patients treated with a similar protocol have been reported by this group (Coutinho et al, 1988). In all cases, regeneration of normal haemopoiesis has been obtainable, although rapid recovery which would provide more convincing evidence of engraftment by the cultured cells has not been observed. Our group has focused on patients with CML. Minor but potentially significant modifications to the protocol for harvesting cells from the adherent layer have been introduced. To date two patients in chronic phase, preselected by assessment of experimental cultures of their marrows, have been treated. In the first, regeneration occurred very rapidly (Barnett et al, 1988) and the patient continues off therapy with a normal WBC count 5 months later, although a small number of Ph positive cells were detected at 3 months posttransplant. The second patient, treated more recently, showed trilineage recovery in the marrow by 1 month post-transplant and attained a normal WBC I week later. These encouraging preliminary results have established the practical potential of this procedure in CML, although longer follow-up on a larger patient population will be essential to evaluate its clinical usefulness. These preliminary results also emphasize the need to investigate the biological basis of the apparent selection of cell types observed. Methods for improving the procedure also warrant exploration. In addition, this approach may offer possibilities for autologous marrow transplantation in patients with other types of malignancies. SUMMARY AND FUTURE DIRECTIONS The last I0 years have yielded significant new information about the process of clonal expansion in Ph positive CML. Lineage-indiscriminate expansion

IN VITROSTUDIESOF CML

955

occurs, both in time and with increasing differentiation, although marked heterogeneity between individual patients is seen. Deregulation o f the mechanism(s) that normally maintain the majority of primitive haemopoietic cells in a quiescent state is another aspect of this disease which can now be reproduced in vitro. Altered requirements for responsiveness to haemopoietic growth factors have not been demonstrated (with the exception of variable expression oferythropoietin-independent erythropoiesis). However, failure o f primitive Ph positive clonogenic cells to respond to reversible inhibitors, or abnormal production of mesenchymal cell activators leading to continuous stimulation o f primitive neoplastic cells, represent other mechanisms that might contribute to clonal amplification in vivo. These possibilities are now accessible to examination. Evidence for altered interactions with stromal cells has also been obtained. Analysis of the functional significance of this defect warrants further investigation. Thus a variety o f key changes characterizing primitive Ph positive cells have been revealed. These should facilitate future investigation of the role of the B C R - A B L tyrosine kinase in gene transfer experiments. In addition, they may facilitate the identification o f o t h e r genetic changes that m a y contribute to the evolution o f the C M L clone. The failure o f Ph positive cells to be maintained in long-term cultures initiated with C M L m a r r o w remains an interesting enigma. Does this mean that the marrow content of Ph positive stem cells is highly reduced? Alternatively, do Ph positive stem cells have a decreased potential for selfrenewal but gain ascendancy by an increased rate of turnover and/or viability? H o w can these possibilities be exploited clinically to develop less toxic procedures with curative potential? A start has been made with autologous transplantation of C M L m a r r o w placed in long-term culture. We look with optimism to significant progress in these areas over the next decade.

Acknowledgements The expert secretarial assistanceof M. Coulombe is gratefully acknowledged.The authors" work referred to in this reviewincluding previouslyunpublished data was obtained with the support of grants from the National Cancer Institute of Canada (NCIC) and core support from the Cancer Control Agency of British Columbia and the British Columbia Cancer Foundation. C.J.E. is a Terry Fox Cancer Research Scientist of NCIC.

REFERENCES Adamson JW (1984) Analysis of haemopoiesis: The use of cell markers and in vitro culture techniques in studies ofclonal haemopathiesin man. Clinics in Haematology 13(2),489-502. Aglietta M, Piacibello W & Gavosto F (1980) Insensitivityof chronic myeloid leukemiacells to inhibition of growth by prostaglandin El. Cancer Research 40:2507-2511. Andrews RG, Singer JW & Bernstein ID (1986) Monoclonal antibody 12-8 recognizesa ! 15-kd moleculepresent on both unipotent and multipotent hematopoieticcolony-formingcellsand their precursors. Blood 67" 842-845. Aye MT, Till JE & McCulloch EA (! 973) Cytologicalstudies of granulopoieticcoloniesfrom two patients with chronic myelogenousleukemia. Experflnental Hematology i: 115-118.

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