Chromosome abnormalities in CML

5 Chromosome abnormalities in CML ANNE

HAGEMEIJER

HISTORY AND CLINICAL SIGNIFICANCE In 1960 Nowell and Hungerford described the presence of a minute chromosome in leukaemic cells of patients with chronic myeloid leukaemia - (CML). This minute chromosome was called Philadelphia chromosome or Ph after the city of discovery. Later, using banding techniques Janet Rowley (1973) discovered that the Ph originated from a translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;ql 1). This translocation, by extension called Philadelphia translocation, generates two derivative chromosomes, the 9q + and 2 2 q - , the latter corresponding to the minute Ph of the prebanding era (Figures IA and 2A). The Ph chromosome is the hallmark of CML, since it is found in more than 90% of cases of CML. The constitutional karyotype of the same patients studied in lymphocyte or skin fibroblast cultures is normal; only the leukaemic cells show the Ph anomaly. The finding ofa Ph in bone marrow cells of a patient with a myeloproliferative disorder has a major diagnostic and prognostic value. CML is a stem cell disease and accordingly Ph is found in all bone marrow cell lineages: m);elocytic, monocytic, megakaryoblastic and erythroblastic precursor cells are Ph positive. The majority of B-lymphocytes also carry the Ph (Martin et al., 1982), in contrast to peripheral blood T lymphocytes which are Ph negative (Bartram et al, 1987). There are a few reports of exceptional cases showing involvement ofT-cell lineage in CM L (Griffin et al, 1983b, Chan et al, 1986). These cases demonstrate that the Ph anomaly most probably occurs in an early stem cell, still capable of proliferation and differentiation along the myeloid and lymphoid pathways. This is also indicated by the various phenotypes of blasts found during blastic transformation, also termed blast crisis (BC) (Griffin et al, 1983a). CML is a biphasic disease. The chronic phase has a median duration of 3.5 years (range 6 months to 20 years) and is followed by a blastic phase characterized by blastic proliferation without maturation that is similar to acute leukaemia. The chronic phase of CML is characterized by a hyperactive myeloproliferation that generates increased numbers ofgranulocytic precursors at all stages ofmaturation. As a consequence cytogenetic analysis of bone Baillibre's Clinical Haematologk~Vol. 1, No. 4, December 1987

963

964

A. HAGEMEIJER

9

A

22 ...

.

4

Figure 1. Partial karyotypes showing (A) the standard Ph translocation (9,22)(q34;q I I) and (B) an example of'simple variant Ph translocation' between chromosomes 4p16 and 22ql 1. The distal part of 22 is visible on 4p and both chromosomes 9 show no apparent changes. Trypsin-Giemsa banding technique. Arrows indicate the rearranged chromosomal regions.

marrow cells shows the Ph chromosome in over 90% (usually 100%) of metaphases. The non-leukaemic stem cells (Ph negative) that are present are suppressed by the proliferation of the Ph positive leukaemic clone. Only specific stem cell assays (Dube et al, 1984) and very aggressive therapy will reveal the presence of these normal Ph negative stem cells (Sharp et al, 1979). Interestingly, recent treatment using recombinant interferon (IFN) may result in partial and probably temporary suppression of the Ph positive cells (Talpaz et al, 1986). Classical approaches to treating the chronic phase of CML using busulphan or hydroxyurea aim to control myeloid proliferation and have no influence at the stem cell level; the bone marrow karyotype remains Ph positive. This contrasts with the situation in acute leukaemia, where complete remission is characterized by the disappearance of the cell clone that carries the leukaemia specific chromosomal abnormality (Hagemeijer et al, 1981a). Based on blast morphology and on immunological marker analysis, BC is divided into two major forms: lymphoid BC in approximately 30% and myeloid BC in 60% of cases; the remaining 10% of cases are of mi~ed phenotype (Griffin et al, 1983a). Lymphoid BC phenotypically resembles common-ALL, while myeloid BC is more heterogeneous, usually myeloblastic, but megakaryocytic and erythroid variants occur (Bain et al, 1977; Rosenthal et al, 1977). In about 80% of the cases, blastic transformation is accompanied and often preceded by additional cytogenetic changes. These are discussed below. VARIANT Ph TRANSLOCATIONS The t(9;22)(q34;ql 1) is found in over 90% of CML cases and is referred to as the standard Ph transloeation (see Figures 1A and 2A). Variant forms of the

965

CHROMOSOME ABNORMALITIES IN CML

9

22

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4

11 40

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Figure 2. Partial karyotypes showing (A) the standard Ph translocation (9;22)(q34;I 1), (B) a complex variant translocation (9;ll;22)(q34;q13;ql 1) and (C) a masked Ph resulting from an apparent t(6;22)(p21;ql I). R-bands with acridine orange. Arrows indicate the chromosomal rearrangements.

Ph translocation are found in about 5% of cases (Sandberg, 1980). To date using banding techniques more than 300 cases of variant Ph have been documented (Mitelman, 1985). Three major cytogenetic types of variants have been described: 1. Complex translocations involving chromosomes 9 and 22 and at least one (sometimes more) additional chromosome. In these cases, the third chromosome is the recipient of the deleted part of 22q-- while 9 is a recipient of the deleted part of the third chromosome (Figure 2B). 2. Simple translocations of the distal part ofchromosome 22 (22ql l-qter) to another chromosome than 9, without apparent involvement of chromosomes 9 (Figure I B). 3. MaskedPh where the Ph has lost its typical 2 2 q - appearance as a result of chromosome translocation on the q arm, or rarely on the p arm (Figure 2C). Simple or quite complex chromosomal rearrangements may give rise to a 'masked Ph' (Hagemeijer et al, 1985). A fourth type of variant translocation, showing apparently intact chromosomes 22 and a breakpoint at 9q34, has also been reported (Bartram et al,

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1985; Oscier et al, 1985). These cases are considered to be Ph negative (due to the intact 22) and will be discussed in the section dealing with Ph negative CML. A recent survey by de Braekeleer (1987) showed an overall frequency of 4.3% for variant Ph translocations among 4061 Ph positive CML patients collected in 52 series of unselected cases. Approximately 2/3 were complex translocations and 1/3 simple variants. Uneven geographic and ethnic distribution is also suggested in the same study. Distribution of the breakpoints on sites other than 9q34 and 22qi I shows involvement of all chromosomes, excepting Y, in the formation of variant Ph. Chromosomes 18 and X were under-represented while chromosomes 17, 11, 12 and 3 were clearly over-represented (Sandberg, 1980; Heim et al, 1985; de Braekeleer, 1987; Verma and Macera, 1987). A correlation between frequent sites of additional breakpoints and oncogene localization and/or fragile sites has been proposed but is far from established. Clearly additional studies with improved banding techniques allowing better localization of breakpoints and molecular investigations are necessary to confirm or refute these propositions. From a clinical standpoint there is no difference between cases with standard or variant Ph translocations; their chronic phase duration and survival curves overlap (Sandberg, 1980; de Braekeleer, 1987). The mechanisms by which variant translocations arise are unclear and probably heterogeneous. In a few cases there is evidence for secondary occurrence of a variant translocation, e.g. when standard and variant translocations are simultaneously (or sequentially) found (Pedersen, 1984; Ohyashiki et al, 1987), or when a proximal breakpoint on 9q+ is demonstrated by translocation of part of 9q together with the 22q distal (Hagemeijer et al, 1984). In the majority ofcases there is no evidence for sequential events and a rearrangement involving multiple breaks is the most probable explanation. MOLECULAR CHANGES ASSOCIATED WITH Ph TRANSLOCATION Molecular investigations of the standard Ph translocation demonstrated that the A B L oncogene is translocated from its normal location on 9q34 onto chromosome 22q I 1 within a gene called BCR for breakpoint cluster region (de Klein et al, 1982; de Klein and Hagemeijer, 1984). The breakpoints on chromosome 9 are scattered within a DNA segment of about 175 kb, 5' of the A B L exons homologous to v-abl. In contrast, the breakpoints on chromosome 22 are clustered in a 4.8 kb BgllI fragment called BCR (breakpoint cluster region) (Groffen et al, 1984) or M-BCR for major cluster region. The genomic recombination B C R - A B L occurs in frame. A B C R - A B L chimeric gene is formed that is transcribed in a unique 8.5 kb mRNA (Stam et al, 1985), coding for a chimeric BCR-ABL protein of 210 kD with enhanced tyrosine kinase activity, P210 BCR-ABL (Konopka et al, 1984). Molecular investigations of and the significance of BCR, A B L and B C R - A B L fusion genes are described and discussed in detail in the next chapter.

967

CHROMOSOME ABNORMALITIES IN CML

ql I ~

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IGL

U

SIS

5'

" L.._J 5 b c r ABL

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(Ph) 22q-

Figure 3. Scheme of the t(9;22) showing the regional localization of the ABL, SIS, IGL and BCR genes, as well as the probes used for in situ hybridization studies.

The mechanism by which the production of a BCR-ABL fusion protein relates to leukaemogenesis in CML is not yet fully understood but it appears to be highly specific for the disease and thus clinically relevant. Furthermore, it provides us with new tools to investigate CML in addition to cytogenetics. For example, the demonstration of the 8.5 kb BCR-ABL mRNA or of the P210 BcR-AuLprotein are alternative ways to show the presence of the Ph rearrangement, at least at the molecular level (Kurzrock et al, 1986; 1987a,b,c). Southern blot analyses of leukaemic DNA, using BCR specific probes, allow the demonstration of a breakpoint in the 4.8 kb BgllI BCR region ofchromosome 22ql I (Groffen et al, 1984), while in situ hybridization of specific DNA probes onto metaphase chromosomes allows the mapping of the genes on particular chromosome bands and therefore makes possible a detailed analysis of complex or unclear cytogenetic rearrangements. We studied by Southern blot analysis more than 65 CML Ph positive cases with standard and variant translocations. All but one showed a breakpoint in the BCR region (de Klein and Hagemeijer, 1984 and unpublished data). These results were confirmed by other investigators; all together DNA from about 600 Ph positive CML patients has been analysed using BCR specific probes (Collins, 1986; Kurzock et al, 1986; Andrews et al, 1987; Blick et al, 1987; Chan et al, 1987b; Bartram et al, 1987 and personal communication; Groffen, personal communication). In at least 98% of the cases a breakpoint in the 4.8 kb BgllI BCR fragment was demonstrated; such CML cases are P h + , "BCR+. The few cases without BCR breakpoint ( P h + , BCR--) are very intriguing and warrant further investigation (Selleri et al, 1987; Saglio et al, 1987).

In situ hybridization studies of cytogenetic variants Single copy DNA probes specific for the oncogenes ABL and SIS were labelled with 3H-deoxynucleotides and hybridized to metaphase chromo-

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A. HAGEMEIJER

Table I. Molecular investigations of CML cases with standard and variant Ph translocations.

Type of Ph

BCR Localization of specificprobes* by in situ hybridization Number breakstudied point A BL SIS 5' BCR 3" BCR

Standard t(9;22) 65t Complex variant t(?;9;22) 8 Simple variant t(?;22) 8 MaskedPh 6

+

9, 22q--

22, 9q+

+

9, 22q-

22, t(22q)** 22, 22q-

+

9, 22q-22, t(22q)**) 22, 22q-22, t(22q)** 9,maskedPh 22, t(22q)** 22, maskedPh 22, t(22)**

+

22, 22q-

22, 9q+ 22,t(22q)**

* ABL was always studied; the three other probes were not investigated in all cases. t only 5 cases studied with in situ hybridization technique. ** t(22q) stands for distal part of 22 (ql 1--*qter)wherever it is translocated.

somes o f patients with standard and variant Ph translocations (Figure 3). When needed, probes for the proximal (5' B C R ) and distal (3' B C R ) part of B C R region were also used (Bartram et al, 1983, 1984; Hagemeijer et al, 1984, 1985) (Figure 3). Using the in situ hybridization technique we studied many variant translocations from our own series o f patients and cases referred to us from other European centres, particularly from France, Italy, Belgium and the Netherlands (Table 1). All cases showed essentially the same results: A B L was translocated onto the 2 2 q - in complex as well as in simple variant translocations where involvement o f chromosome 9 was not cytogenetieally evident, or to a masked Ph chromosome, and S I S was situated on the distal part of chromosome 22, wherever it was translocated. When investigated, 5' B C R mapped on the Ph at the same location as A B L , while 3' B C R was found on the 9 q + or at other sites corresponding to the distal part o f chromosome 22. These findings demonstrated that in all variant Ph cases chromosome 9 is involved, a necessary step to explain translocation of A B L to the Ph. Transloeation o f A B L on the Ph appears also as an essential feature o f the Ph. On the other hand, translocation of the S I S oncogene occurs to various sites, together with the distal part o f 22, and therefore is not consistent between cases. In the light o f these investigations simple variant translocations appear to be complex translocations involving 9, 22 and at least one other chromosome, but the breakpoint on the 3rd chromosome receiving the distal part 22 (22ql 1-qter) is subterminal or terminal so that translocation on 9q34 is not visible unless a higher resolution banding technique is successfully used. A masked Ph chromosome sometimes results from very complex breaks and exchanges but there is always co-location of 5' B C R and A B L (Hagemeijer et al, 1985). These findings are perfectly in line with the molecular analyses found in standard Ph (see next chapter) and emphasize the fact that the 'phenomenon of Ph is in fact a genomic rearrangement of 5' B C R - A B L . It usually takes place on the Ph chromosome as a result o f a t(9;22) but it can sometimes result from much more complicated cytogenetic events.

CHROMOSOME ABNORMALITIES IN CML

969

Ph NEGATIVE C M L Many series of C M L patients reported before 1980 show as many as 10% to 15% of cases without a Ph chromosome in leukaemic metaphases (Ezdinli et al, 1970 and reviewed by Kantarjian et al, 1986). As a group, these patients present with slightly atypical features such as thrombocytopenia and monocytosis, absence of basophilia, and most importantly, poor response to standard C M L therapy with significantly shorter median survival than the Ph positive cases (Ezdinli et al, 1970; Kantarjian et al, 1986). More recently, morphological and clinical criteria for myelodysplastic syndromes and myeloproliferative syndromes have been codified (Galton, 1982; Bennett et al, 1982; see also Chapter 2. This has led to a clear characterization of juvenile C M L as a subacute myelomonocytic leukaemia of childhood distinct from C M L (Brodeur et al, 1979) and to the reassessment o f m a n y Ph negative C M L cases as myelodysplastic syndromes, mostly chronic myelomonocytic leukaemia (CMML) (Pugh et al, 1985; Travis et al, 1986). Nevertheless, some of the ;Ph negative C M L patients show all the classic features of Ph positive CML. Furthermore we may not always be as critical regarding aberrant clinical features in Ph positive cases as in Ph negative ones. But the distinction between Ph positive and Ph negative C M L cases is not only semantic; indeed it carries important prognostic implications and probably in future significant differences in therapeutic approach.

Cytogenetic and molecular findings in Ph negative C M L We studied 14 cases of presumed Ph negative C M L and reviewed 63 additional cases (Bartram et al, 1985; Bartram and Carbonell, 1986 and personal communication; Kurzrock et al, 1986; Dreazen et al, 1987; Wiedemann et al, 1988) that were studied cytogenetically and molecularly. Sixty cases (about 75%) presented with a normal karyotype; 20 of these cases showed a breakpoint in the BCR region when studied by Southern blot analyses. In six of the latter cases in situ hybridization studies revealed the presence of a transloeation of the A B L oncogene to an unusual location i.e. chromosome 22ql 1 in 4 cases (Morris et al, 1986; Dreazen et al, 1987) and chromosome Ip36 in 2 cases (personal unpublished data). Seventeen cases presented with cytogenetic abnormalities and in six cases a remarkable translocation involving 9q34 and a chromosome other than 22 was observed. A breakpoint in the BCR region was found in these latter cases as well as in five other patients with cytogenetic abnormalities. Extensive in 9situ hybridization studies o f a C M L patient with t(9;12)(q34;q21) revealed colocation of 5" B C R and A B L probes on the 12q-- derivative chromosome, indicating a Ph type of genomic rearrangement, although at an aberrant chromosomal site (Bartram et al, 1985). In three other patients similar studies revealed the presence of complex rearrangements with a masked Ph mimicking a normal 22. In summary recent studies indicate that when stricter clinical and morphological criteria are applied less than 5% of C M L cases are Ph negative. At least one third and probably half of these cases show molecular evidence

970

A. HAGEMEIJER

for a genomic rearrangement analogous to the Ph translocation, but this is not evident cytogeneticaily. Biologically, these patients belong to the category of Ph positive C M L and should be treated as such. Prospective studies of Ph negative C M L are now needed to ascertain the clinical and prognostic significance of these findings. Yet another group of patients should be mentioned, i.e. the rare cases with late appearance ofa Ph (Lisker et al, 1982) and cases with early disappearance of Ph (Hagemeijer et al, 1979). Both types of events do not apparently affect the course of the disease. These cases are extremely rare. They have been well documented clinically and cytogenetically but unfortunately not studied molecularly. The usual interpretation of these findings is based on the 'multistep' pathogenesis of carcinogenesis and implies a primary mutation of the stem cell before (and leading to) the occurrence of the Ph (Fialkow et al, 1981) (see Chapter 1). At this stage the absence of molecular data makes it impossible to comment on the general or exceptional biological significance of these observations. CYTOGENETIC EVOLUTION IN CHRONIC AND BLASTIC PHASE OF C M L Over 90% of the patients show metamorphosis of C M L from a relatively benign chronic phase to an acute and usually rapidly fatal stage. In 80% of the cases blast crisis (BC) is heralded and accompanied by non-random clonai chromosomal aberrations superimposed on the Ph chromosome. In the remaining 20% no additional chromosomal changes are seen (Lawler, 1977; Hagemeijer et al, 1980). The four most frequent and clearly non-random abnormalities observed are duplication of the Ph, trisomy 8, trisomy 19 and i(17q) replacing a normal 17. These four abnormalities are found in various combinations in different patients. Sequential studies have also shown clonal progression with successive appearance of the non-random abnormalities (Hagemeijer et al, 1980; Watmore et al, 1985). These aberrations have been extensively discussed in a recent review by Bernstein (1988). The onset of BC may be abrupt, may follow a gradual acceleration, or may be primarily an extramedullary transformation. The extramedullary sites mostly involved are spleen and lymph nodes, rarely skin or central nervous system (Swolin et al, 1983). Morphological and immunological characterization of the blasts reveal that all the differentiative patterns ofthe pluripotential stem cell can be found: about 30% are of lymphoid origin, and 70% ofmyeloid origin with 10 to 20% mixed forms (Janossy et al, 1978; Griffin et al, 1983a). In general, cytogenetic clonal evolution may be considered as a reliable predictive or diagnostic parameter of BC. However, it is not sufficient in itself, as clonal chromosome aberrations in addition to Ph can also be observed in 10 to 20% of cases during the chronic phase. Abnormal clones have been shown to arise but not evolve or expand immediately. Sequential studies showing expansion and increase in complexity of the karyotype due to additional trisomy or aneuploidy are of fundamental value as an indication of acute transformation (Hagemeijer et al, 1980; Watmore et al, 1985). An i(l 7q) is also

CHROMOSOME ABNORMALITIES IN CML

971

highly characteristic of impending transformation and never found as a stable abnormality in the chronic phase (Figure 4). Other types of cytogenetic modifications are observed in relapse occurring after bone marrow transplantation. These are invariably Ph positive and accompanied by various clonal changes resulting in bizarre and unusual translocations and structural abnormalities, probably induced at the time of conditioning treatment before transplantation. Experience with these cases is still limited (Zaccaria et al, 1987; personal unpublished data).

Clonal chromosomal aberrations during chronic phase of CML The presence of additional karyotype abnormalities during the chronic phase of CML has been considered to be an unfavourable feature independent of all other disease characteristics (Sokal et al, 1984). In our experience and in other surveys, 10 to 20% of chronic phase patients may show mosaicism with chromosomal changes in addition to the Ph. These secondary abnormalities are most often duplication of Ph or i(Ph), loss of Y chromosome and sometimes trisomy 8, and are found as a single abnormality in a certain 9 percentage of cells. They appear to be compatible with a normal chronic phase; they are characterized by their stability, absence of progression and may even be transient. They may disappear spontaneously or with chronic phase therapy. Therefore, these stable subclones do not necessarily predict impending transformation. Study of a large number of metaphases (more than 300) during chronic phase may also demonstrate the presence of additional changes in a very small percentage of cells (Sonta and Sandberg, 1977). When blast crisis develops it may or may not show clonal progression of these minimal secondary clones. Loss of Y has been proposed as a favourable feature during chronic phase o f C M L (Sakurai and Sandberg, 1976). It is usually confined to chronic phase and in BC cional progression seems to develop in the Y-positive cell line.

Cional progression in myeloid BC 70% of CML cases evolved into BC of myeloid origin, mostly myeloblastic. Usually a hyperdiploid karyotype is present with acquired non-random abnormalities already discussed, i.e., + 8, + 19, i(17q) and +22q- in various combinations. Other changes are occasionally seen, such as structural .abnormalities of chromosome 1, rearrangements of chromosome 17 with translocation of 17q onto another chromosomal segment such as a marker (5p:: 17q) (Hagemeijer et al, 1981b); trisomies of chromosome 10, chromosome I 1 or chromosome 21 are also relatively frequent. Certain subtypes of myeloid transformation are occasionally seen that show cytogenetic changes analogous to the abnormalities found in acute leukaemia of the same FAB subtype, e.g. erythroblastic BC showing multiple aberrations similar to the findings in erythroleukaemia (Rosenthal et al, 1977; O'Malley et al, 1983). Cases of promyelocytic BC have been described with acquired t(15;17) in

972

A. HAGEMEIJER

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CHROMOSOME ABNORMALITIES IN CML

973

addition to the Ph (Berger et al, 1983; Hogge et al, 1984) and megakaryoblastic BC shows abnormalities of3q21-26 (Carbonell et al, 1982; Bernstein et al, 1986). Clonal evolution in lymphoid BC About 30% 9f CML cases evolve into lymphoid BC. The phenotype usually corresponds to that of a precursor-B cell with rearrangement of immunoglobulin heavy chain gene and positivity for terminal deoxynucleotidyl transferase (TdT) and HLA-DR, CDI9 (B4) and CD10 (common ALL) cell markers (Bakhsi et al, 1983). Whether specific cytogenetic markers correspond to the lymphoid phenotype is not yet clear. The i(17q) seems restricted to myeloid transformation; duplication of Ph and trisomy 8 are found in both myeloid and lymphoid transformations (Alimena et al, 1987), while at least 40% of the cases with lymphoid BC show only the Ph without additional changes (Yao, 1985). Monosomy 7 and trisomy 21 are also frequent in lymphoid BC (Parreira et al, 1986). Too few cases of Ph positive CML with lymphoid BC exhibiting T-cell characteristics have been described to establish whether this type of transformation is associated with specific cytogenetic secondary changes (Herrmann et al, 1984). Ten rare cases of near-haploid karyotypes have been reported (Cibull et al, 1987). They showed persistence of 9q+ and 2 2 q - , disomy or trisomy 8 and 21. Near haploid karyotypes are associated with lymphoid BC and with acute lymphoblastic leukaemia, but in at least one case the phenotype of the blast was myeloid (Andersson et al, 1987). Molecular investigations of blast crisis Only a small number of cases have been investigated. The presence of the abnormal P210 BcR-AnLprotein has been demonstrated in at least 15 patients as well as in cell lines derived from BC cases, i.e. K562, KCL22, etc. (Konopka et al, 1985; Clark et al, 1987; Kurzrock et al, 1987c; Maxwell et al, 1987). The specificity of B C R rearrangements has been studied by Southern blot analyses of chronic phase and blastic phase DNAs of the same patients (Bartram et al, 1986; Andrews et al, 1987; personal unpublished data). All but two cases showed identical restriction fragments in both phases. The two exceptional cases have been interpreted as secondary BC rearrangements, unique to these given patients. These data suggest that the Ph translocation is pathogenetic for CML in chronic phase, but that in BC new oncogenetic factors dominate the picture. Evidence for R A S mutation in some cases of CML in BC has been reported by Liu et al (1988). Ph POSITIVE ACUTE LEUKAEMIAS The Ph translocation is not restricted to CML but is also observed in about 20% ofadult acute lymphoblastic leukaemia (ALL), 2-3% of childhood ALL and 1-2% of AML. Because of the clinical similarities between acute leukaemias and BC of CML, it has been proposed that Ph positive acute

974

n. tlAGEMEIJER Table 2. Cytogeneticstudies of three Ph positive ALL patients in complete remission.

Sex/age (years) Ph translocation % Ph+ cells at diagnosis Remission duration (months) Number ofcytogeneticstudies during complete remission Total no. of metaphases analysed No. ofPh+ metaphases (%)

Patient 1

Patient2

Patient3

F, 50 t(9;14;22) 66 26

M, 43 M, 52 t(X;5;9;22) t(9;22) 75 80 10 12

5 6 5 128 144 174 3 (1.4%)* 5 (3.5%) 0 (5/5 CDl0+)t

* t(9;22)(q34;q I 1) instead of variant t(9; 14;22)(q33;q32;q I 1); "["after immunolabellingof the metaphases, all CDI0+ metaphaseswere Ph positive, but the overallmajority of cellswere CDI0-.

leukaemias were in fact BC o f C M L with a clinically silent or undiagnosed chronic phase (Catovsky, 1979). M a n y features argue against this interpretation, among which are (a) the rapid onset of acute leukaemia, (b) the usual persistence of a substantial percentage of Ph negative metaphases, and (c) the fact that complete haematological remission is usually attained in response to treatment with disappearance of the Ph positive metaphases from bone m a r r o w (Sandberg et al, 1980). In contrast, in BC of C M L more than 95% of the bone marrow metaphases are Ph positive, and successful treatment of BC is followed by a return o f the chronic phase with persistence o f Ph positive cells. Furthermore, very recently differences in molecular rearrangements have been found in 50% of the cases of Ph positive A L L (de Klein et al, 1986; Hermans et al, 1987).

Ph positive ALL The Ph translocation is found in about 20% of adult A L L (Bloomfield et al, 1978) and less than 5% ofchildhood A L L (Ribeiro et al, 1987). Morphologically, these leukaemias are classified as FAB LI or L2. The large majority of cases are immunologically early precursor-B cells that show rearrangements of immunoglobulin heavy chain gene, express T d T and the membrane markers C D I 9 (B4) and C D I 0 (common A L L antigen). Only exceptional cases exhibit a T-cell phenotype (Roozendaal et al, 1981; Louwagie et al, 1985). Ph positivity constitutes a p o o r prognostic factor. Remission can be obtained in most cases but is o f short duration and the median survival is still only 10 to 12 months in recent series (Bloomfield et al, 1986; Ribeiro et al, 1987). Cytogenetically, the Ph translocation in A L L is indistinguishable from the t(9;22) found in CML. In 50% of the cases the Ph is the only cytogenetic aberration. Thc pcrcentage of variant translocations is higher in acute leukaemia (about 15%) compared to 5% in C M L (Ribeiro et al, 1987;

CHROMOSOME ABNORMALITIES IN CML

975

personal unpublished observations). In particular, complex variants involving 14q32 have been reported a few times (e.g. de Klein et al, 1986). In about 50% of the cases, additional aneuploidy is present together with the Ph. The most frequent associated changes are monosomy or deletion of chromosome 7, and secondary structural rearrangements of one or both chromosomes 9. When complete remission is obtained, the Ph disappears from the bone marrow metabhases, but even in long-lasting remissions of more than a year analysis of a large number of cells discloses the presence of residual Ph in 1-3% of the mitoses (Table 2). These data strongly suggest that remission bone marrow of Ph positive ALL is probably inadequate for use in autologous bone marrow transplantation. Usually, Ph positive A L L will sooner or later relapse as an acute leukaemia of the same or slightly less differentiated phenotype as the primary leukaemia. Rare cases of Ph positive ALL relapsing as A M L or C M L have been reported (Tanzer et al, 1975). These exceptions support the contention that Ph positive leukaemia is a stem cell leukaemia with various phenotypic expressions, probably determined by yet unknown other factors. Sequential molecular studies of such cases may disclose the mechanism of these various phenotypic expressions. Ph positive A L L cases have been studied molecularly using the tools and the knowledge acquired in the study of C M L (for details see Chapter 6). About 50% of the cases revealed a breakpoint in the B C R region analogous to the findings in CML, so-called P h + , B C R + cases (de Klein et al, 1986). In these cases a 8.5 kb aberrant m R N A was also expressed coding for the P210 BcRABL hybrid protein. Analysis of P h + , B C R - - cases revealed that the breakpoint on 22ql I was more proximal in the B C R gene (Hermans et al, 1987). This new breakpoint also results in genomic recombination with transcription of a 7.4 kb m R N A and abnormal PI90 BcR'ABL protein with enhanced tyrosine kinase activity (Chan et al, 1987; Kurzrock et al, 1987a). The normal human ABL protein has a MW of 145 kD. Ph positive A M L About 1% of A M L are Ph positive. Two thirds of cases are FAB-MI with immature features including peroxidase negative granules; the remaining cases are mostly M2, rarely M4, exceptionally M5 and never M3. Standard and variant translocations have been reported. As in ALL, the Ph is a poor prognostic factor (Bloomfield et al, 1978; Maddox et al, 1981). The number of cases studied molecularly is small (Kurzrock, 1987c; Bartram, 1988; Chen et al, 1988). As in Ph positive ALL, half of the cases revealed the presence of a 9 breakpoint in the C M L BCR region with production ofa P210 BcRABL protein. To the best of my knowledge, Ph positive, BCR-negative A M L cases have not yet been further characterized molecularly. DISCUSSION The Ph chromosome was discovered 28 years ago and was relatively easy to demonstrate by cytogenetic methods even before the use of banding

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techniques. As a consequence, Ph is the most frequently reported malignancyassociated chromosomal aberration: it occurs in CML, in acute leukaemia and incidentally in other myeloproliferative disorders. In CML, the presence of the Ph chromosome has played a unifying role for diagnosis, but at the same time it has blurred other nosological differences between patients. Recent molecular studies have demonstrated the existence 6f a breakpoint cluster region on chromosome 22 (GrolTen et al, 1984) and also revealed subtle difference in breakpoints. The P210 BcR'ABLhybrid protein is formed as a consequence. (See Chapter 6). Despite the past abundance of reported cases, there is still a need for large (multicentre) controlled prospective studies using clinico-morphological criteria, cytogenetic data and the new molecular tools now available. In particular, in view of the new treatments becoming available using recombinant molecules (interferon, growth factors, etc.) and bone marrow transplantation, we need to know which features at presentation or within 3 to 6 months from diagnosis are prognostically important e.g. the persistence o f Ph negative stem cells, the early appearance o f secondary clones, the type o f secondary cytogenetic abnormalities, etc. The long-term benefit of Ph negative conversion, the extent of chimerism after bone marrow transplantation, the factors predisposing to late relapse, and other factors are still unknown but hopefully within reach of stPdy. The pathogenesis o f Ph positive C M L is still unknown. The Ph is the first visible event in CML; it is remarkably consistent cytogenetically and molecularly and it is a stem cell abnormality. How the Ph aberration induces the C M L disease is still not understood. We must await further study on the biological activity of the P210 BcR'ABLprotein. The mechanism that causes the Ph translocation is also unknown. A primary mutation preceding the Ph has been suggested as explaining the rare cases of late appearance and o f early disappearance of Ph in CML. If so, this primary mutational event must occur in stem cells and be a strong inducer o f the Ph translocation in order to explain the remarkable consistency o f Ph in CML. Regarding the evolution to blast crisis, no progress has yet been made. It seems obvious that the Ph translocation must play a role in determining its inevitability but that at the time o f BC other oncogenic factors must be involved, possibly controlled by the additional non-random cytogenelic changes. Remarkably, molecular investigation has shown mutation of one of the R A S oncogenes in 50% o f the cases of acute myeloid leukaemia as well as in some ofthe 16 cases of C M L in BC investigated thus far (Janssen et al, 1987; Liu et al, 1988). SUMMARY The Ph chromosome is the hallmark o f CML, where it is found in more than 90% o f the cases. Cytogenetically, it usually results from a t(9;22)(q34;ql 1). The Ph arises in a stem cell and in chronic phase is found in all haematopoietic cell lineages, although it causes only increased granulopoiesis, and sometimes increased thrombopoiesis; furthermore blast crisis may occur in all differentiative patterns o f the pluripotent stem cell.

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Recently, molecular investigations o f Ph positive C M L cases have revealed a consistent genomic recombination between two genes, B C R on c h r o m o s o m e 22 and the A B L oncogene. The latter is translocated f r o m 9q34, its normal site, to the 2 2 q - or Ph c h r o m o s o m e . This molecular rearrangement expresses a unique 8.5 kb B C R - A B L hybrid m R N A transcript, that encodes an altered B C R - A B L protein o f approximately 210 k D with enhanced in vitro tyrosine kinase activity. The breakpoints on c h r o m o s o m e 2 2 q - - are clustered in a 5 kb D N A fragment, allowing their study using Southern blot analysis. Cytogenetic variant forms o f the Ph translocation involving three or more c h r o m o somes are f o u n d in a b o u t 5% o f the cases. Southern blot and in situ hybridization studies have demonstrated that these variants are cytogenetically m o r e complex than the standard t(9;22) but molecularly they show the same essential genomic recombination. This is also true for a small n u m b e r o f cases o f Ph negative C M L . Clonal progression, indicated by the presence o f clonal, n o n - r a n d o m c h r o m o s o m e abnormalities, in addition to the Ph is rare during chronic phase but is f o u n d in 80% o f blast crisis. These additional aberrations may precede BC by weeks or m o n t h s and have therefore a clear prognostic value. Ph is not restricted to C M L , since it is also f o u n d in A L L (20% o f adult .cases) and rarely in A M L . Ph in acute leukaemia is cytogenetically indistinguishable f r o m Ph in C M L , but molecular studies have shown that in 50% o f the cases the breakpoint on c h r o m o s o m e 22 is different from the very consistent and characteristic breakpoint in C M L . Nevertheless genomic recombination takes place that results in a novel A B L protein at least in some o f the cases. Despite extensive cytogenetic and molecular investigations, the mechanisms underlying the formation o f the Ph as well as the pathogenesis o f Ph positive C M L are still u n k n o w n but are n o w the object o f intensive research.

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