MICHAEL J. SICILIANO PhD Department of Molecular Genetics
The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
The Philadelphia (Ph) chromosome is present in the leukaemic cells of most patients with chronic myelogenous leukaemia. Variant transtocations occur in 10% of patients but breakpoints on chromosomes 9 and 22 remain the same, so prognosis of these patients is unchanged. Clonal evolution is infrequent in chronic phase and its significance depends on the specific chromosome involved, the number of metaphases affected and the timing in the chronic phase. The majority of patients in blastic phase demonstrate clonal evolution; three specific abnormalities (+Ph, +8 and isochromosome 17q) are present in 70% of patients. Loss of the Ph chromosome on therapy is associated with prolonged survival. For monitoring these events conventional G-band cytogenetics (CG) is essential at presenta- tion to characterize the disease cytogenetically, while fluorescence in situ hybridization (FISH) on hypermetaphase preparations (hypermetaphase FISH (HMF)) is important for establishing the specific frequency of Ph ÷ cells. During treatment FISH on interphase cells (I-FISH) can monitor the level of Ph + cells in circulation, while CG may be used to identify any suspected clonal evolution. Where I-FISH is negative, HMF is essential to evaluate minimal residual disease.
Key words: CML; FISH; hypermetaphase FISH; clonal evolution; Philadelphia chromo- some; variant Philadelphia chromosome.
CHROMOSOMAL IDENTIFICATION OF CHRONIC MYELOGENOUS LEUKAEMIA
seen in patients with acute lymphocytic leukaemia, and rarely in acute myeloid leukaemia, its presence in a hypercellular bone marrow without increased blasts clearly defines CML (Kurzrock et al, 1988; Cortes et al, 1996). The Ph chromosome is present in 95% of patients who develop the typical clinical picture of leukocytosis, left shift, basophilia, thrombo- cytosis and splenomegaly. In addition, molecular studies have often revealed the presence of the translocation in the residual 5% of patients where chromatin exchange is too small to be detected by standard G-band- ing (CG) (Cortes et al, 1995).
As discussed in Chapter 1, murine studies have shown that introduction of the aberrant BCR-ABL mRNA into nude mice can recapitulate disease resembling both the chronic and the acute (blastic) phases of CML (Daley et al, 1990; Elefanty et al, 1990; Kelliher et al, 1990). This gives strength to the long-held clinical perception that eradicating cells containing the Ph chromosome is crucial to survival prolongation in CML.
Although haematological remissions may be achieved with hydroxyurea, suppression of bone marrow disease, as evidenced by disappearance of the Ph chromosome, is rare (Kantarjian et al, 1993). Thus the malignant cells persist and undergo as-yet ill-defined molecular events that subsequently lead to progression to blast crisis and death.
The association of the Ph chromosome with CML presents an opportunity for the application of fluorescence in situ hybridization (FISH) in the study and management of the disease. Competitive or suppression in situ hybridization (which blocked repeat sequences in human DNA) made chromosome and/or chromosome region specific FISH possible (Lichter et al, 1988; Pinkel et al, 1988). Initially, chromosome-specific probes were developed from flow-sorted chromosome libraries. Unfortunately, com- petitive hybridization failed to block the very highly repeated centromeric DNA of human chromosome 22 (initially one of the most separable human chromosomes by that method), detracting from the use of such a probe to detect specifically the Ph chromosome (Lichter et al, 1990). Because human chromosomes could also be separated from each other in inter- species somatic cell hybrids (Weiss and Green, 1967), Lichter et al (1990) introduced a polymerase chain reaction (PCR) method, later refined as inter-Alu-PCR by Liu et al (1993), for extracting only the human DNA, minus centromeric sequences, from such hybrids. Because somatic cell hybrids are now available containing only single human chromosomes or specific pieces or regions of human chromosomes, DNA probes may be generated by inter-Alu-PCR to paint those specific regions by competitive FISH. Therefore FISH probes can now be made available to study the various chromosomal aspects of CML. We have had considerable success with such a FISH probe generated from a hybrid (E6B) containing only approximately 5 Mb of human DNA surrounding the ABL locus on human chromosome 9q34 (Henske et al, 1992) as its only human genomic content. Molecular marker analysis had indicated that the human region retained extends from AK1 to ABO, which includes ABL, and that approximately 1/3 of the region is translocated to the Ph chromosome in CML patients. This has provided a sufficiently robust signal so that the Ph chromosome
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can be identified in even the most dismal of metaphases (Seong et al, 1995), This and a number of other probes capable of identifying the Ph trans- location are now available through commercial sources.
At presentation cytogenetic analysis usually reveals the Ph chromosome in 100% of cells analysed with standard 20-25 cell analysis. About 2-12% of patients have some evidence of normal diploid cells at diagnosis (Whang-Peng et al, 1968; Sakurai et al, 1976; Ishihara et al, 1983; Swolin et al, 1985). Sakurai et al first reported that these patients might have an improved survival with seven such patients (of 88 Ph ÷ CML patients) all being alive with the longest follow-up at 48 months. In a series of 826 patients with newly diagnosed Ph + CML seen at M.D. Anderson Cancer Center, 52 (6%) had some normal cells present in a median of 10% (range, 5-97%) of metaphases (unpublished data). These patients tended to have more favourable prognostic features such as lower white blood cell (WBC) counts, absence of splenomegaly and less anaemia, and a significantly greater number were asymptomatic at diagnosis (56% versus 36%). Of 35 such patients treated with interferon-based therapy the incidence of complete cytogenetic response was 40% versus 22% of patients with 100% Ph-positive metaphases at diagnosis (unpublished data). Thus, it appears that the level of Ph positivity at diagnosis may be related to long-term prognosis.
The development of a high-resolution quantitative procedure, termed 'hypermetaphase FISH' (HMF) should now make it possible to distinguish different levels of Ph chromosome positivity at presentation. In HMF, FISH was coupled with procedures for increasing the number of bone marrow cells that can be analysed. Hundreds, or even thousands, of metaphase cells were collected onto a slide for Ph chromosome analysis by holding marrow cultures a sufficiently long period of time in the mitotic arrester, colcemid (Figure 1). While such treatment distorted the chromosome spreads beyond the point where CG analysis was possible, the Ph chromosome in such cells could be readily identified by using an appropriate FISH probe (e.g. E6B described above, Figure 2). This was done on a series of 70 samples from CML patients in different clinical states of disease and control samples from putative non-Ph * patients who were undergoing treatment (Seong et al, 1995). The results indicated that scoring 500 or more such cells per sample for presence or absence of the Ph chromosome could be carded out on such preparations in less than 1 hour. HMF clearly identified statistically significant differences between the frequencies of Ph ÷ cells in samples that differed by as little as 4%, recognized significant levels of Ph * cells in 16% of patients characterized as having a complete cytogenetic response (Ph negative) by CG and was informative where insufficient metaphases were obtainable for analysis by CG. With respect to the question at hand, the procedure identified significantly different levels of Ph ÷ cells among patient samples that were all judged 100% Ph ÷ by CG. From these data it is clear that, if applied to new patients, differences in Ph chromosome positivity as low as 5% could be resolved in order to approach further the question of whether different levels of Ph* cells at presentation have clinical signifi- cance with respect to therapy.
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Figure 1. Single microscope field demonstrating the yield of metaphases by the hypermetaphase • procedure. The chromosomes in most such figures were highly over-contracted, and useless for a
karyotype analysis (from Seong et al, 1995).
V A C A N T TRANSLOCATIONS
Variant translocations involving one to three other chromosomes in addition to 9 and 22 are seen in 5-12% of patients and were first described by Hayat in 1973 (Hayat et al, 1973; Sonta and Sandberg, 1977; Pasquali et al, 1979; Sandberg, 1980; Potter et al, 1981; Oshimura et al, 1982; Ishihara et al, 1983; Bernstein et al, 1984; De Braekeleer, 1987; Ishihara and Minamihisamatsu, 1988; Zaccaria et al, 1989; Hild and Fonatsch, 1990; Kadam et al, 1990; Stopera et al, 1990; Mitelman, 1993). Tradition- ally these variant translocations have been described as simple when they involve chromosome 22 and chromosomes other than chromosome 9, and complex when they involve chromosomes 9, 22 and one or more other chromosomes. Oshimura et al (1982) pointed out that, although simple variant translocations did not clearly involve chromosome 9, the rearrange- ment might be missed because the terminal segments of all human chromosomes (except the Y chromosome) are indistinguishable using common banding techniques. Hagemeijer et al (1984) described three simple variant translocations between chromosome 22 and chromosomes 19, 4 or 12 when G- or Q-banding was used. However, high-resolution R-banding revealed a small deletion of the terminal end of chromosome 9
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in all three cases. In two cases in situ hybridization (ISH) demonstrated c-abl sequences on the Ph chromosome. The authors noted that breakpoints in complex translocations were frequently dispersed throughout the length of the third chromosome but infrequently involved terminal bands whereas the reverse was true for simple variant translocations (Hagemeijer et al, 1984). Similarly to Oshimura et al they believed that translocations of a terminal band would be at the limits of detection with standard cytogenetic techniques. ISH using radioactively labelled probe has suggested that the simple translocations may also involve chromosome 9, although the rearranged c-abl is not detected at the level of chromosome banding Bartram et al (1983) first documented translocation of the c-abl to chromo- some 22 in two cases of complex variant Ph translocation. Morris et al (1988) performed ISH studies on cells from three complex variant Ph translocations and two apparently simple variants, t(19;22) and t(11 ;22). These studies showed transposition of the c-abl gene from chromosome 9q34 to the bcr region of chromosome 22 in all five cases; this was confirmed by rearrangements of the BCR gene. Abe et al (1989) described 10 cases of variant Ph translocations where five were classified as simple exchanges between chromosome 22 and chromosomes 14, 15, 16, or 17 and in one case both chromosome 2 and 13. Chromosome ISH indicated translocation ofc-abl from 9q34 to 22ql 1 in all five cases. In a larger study Dube et al (1989) analysed cells from 33 patients with CML and unusual marrow cytogenetics for rearrangement of the BCR gene. Twenty-three patients had variant Ph translocations and 10 patients had no detectable Ph chromosome. BCR rearrangement was detected in all cases; four of the I0 Ph-negative cases had involvement of 9q34. Seong et al (1993) used probes made by inter-ALU-PCR from somatic cell hybrids to perform FISH in a case of a patient with t(1 ;22) and detected a small translocation involving the distal q arm of chromosome 9.
Most reports in the literature suggest that the prognosis and survival of patients with variant Ph translocations are similar to those of patients with a standard t(9;22) abnormality (Sonta and Sandberg, 1977; Pasquali et al, 1979; Sandberg, 1980; Ishihara and Minamihisamatsu, 1988; Morris et al, 1988). Sandberg et al (1980) reported a median survival of 3--4 years for patients with either a standard or variant translocations. Potter et al (1981) detected 18 (15%) of 119 patients with a variant Ph. They reported that patients with a variant Ph had a shorter benign phase at a median of 23 months versus 43 months in patients with t(9;22). They also found a higher incidence of variant Ph in older patients. No data on survival were provided. Given the heterogeneous definition of disease acceleration still present this conclusion can only be regarded as tenuous.
Berustein et al (1984) noted a difference in the incidence of variant translocations according to race in a series published from South Africa. A variant Ph was detected in 14 (7.8%) of 180 cases. Eleven of the 14 patients were black compared with 83 of the remaining 166 cases. In addition, clonal evolution occurred more frequently in the group with variant trans- locations although the types of clonal changes that occurred were similar for the two groups. De Braekeleer (1987) reviewed 327 variant Ph trans-
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locations reported in the literature. Breakpoints were located in the telomeric bands in 72% of simple translocations versus only 9.6% of complex translocations. No significant difference was found between simple and complex translocations regarding sex, age, ethnic background, geographical distribution, WBC count, haemoglobin level or platelet count. Survival data were available for 152 cases. No significant difference was found in survival between patients with standard, simple or complex Ph translocations.
Przepiorka and Thomas (1988) examined the cytogenetic data for 126 patients with Ph+ CML in accelerated or blast phase who underwent allo- geneic or syngeneic bone marrow transplantation (BMT). They described additional clonal abnormalities in 84% and a variant Ph in 14% and reported a second Ph, +8, or a variant Ph to be more frequent in patients who relapsed following BMT. The risk of relapse in these patients was 73% at 3 years versus 31% in the other patients. Details of the other chromosomes involved in the variant translocations were not provided and extrapolation to patients in chronic phase is difficult as specific variant translocations could be associated with a poorer prognosis especially given the fact that these were selected patients that already had progressive disease.
Mitelman (1993) noted that over 400 patients with CML and variant translocations characterized by banding techniques had been described. Although all chromosomes except Y had been implicated in these trans- locations, the pattern was not completely random as clustering has been seen involving 3p21, llq13, 12p13, 17q25 and 22q13. Stopera et al (1990) found variant Ph translocations in four (5.5%) of 72 patients with CML involving chromosomes 1, 5, 8, 11, 15, 17, 17 and 21. Three of the eight breakpoints corresponded to known fragile sites and four of the eight corresponded to loci of oncogenes. All of the breakpoints in this study except lq32 corresponded to consistent cancer breakpoints. Recently the first case of a variant translocation involving chromosome Y was described (Gallego et al, 1996). Gallego et al detected a t(Y;22)(p 11 ;ql 1) in a 4 year old boy with leukocytosis and splenomegaly. The breakpoint on chromo- some 22 was in the bcr region but no study was done to detect c-abl involvement.
Thirty-two patients (3.8%) with a variant Ph translocation were identified from a database of 826 newly diagnosed patients with CML at M.D. Anderson Cancer Center (unpublished data). Age and sex distri- butions were similar. Black patients represented 18.8% of variant cases versus 4.5% of standard translocations (P=0.001). There was no signifi- cant difference in presenting features such as WBC count, platelet count, haemoglobin level or splenomegaly. Importantly, responses to interferon (IFN) and survival were identical whether patients had standard or variant translocations.
Given the myriad possibilities for chromosomal involvement in variant translocations it is certainly possible that the overall survival similar to that seen in standard Ph translocation results from a mixture of abnormalities. Breakpoints at sites of oncogenes or suppressor genes might confer a
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poorer prognosis than other abnormalities not involving crucial genes for proliferation or differentiation. Thus, combining heterogeneous groups results in an intermediate prognosis identical to that of patients without a complex translocation. This situation may be akin to that of acute myeloid leukaemia patients with diploid cytogenetics who have an intermediate prognosis. These patients probably have various molecular abnormalities that have not been well defined and so alterations conferring a poor prognosis are combined with aberrations conferring a better prognosis and cancel each other out in the overall schema.
CLONAL EVOLUTION
Additional cytogenetic abnormalities at the time of diagnosis of CML have been associated with shortened survival in two large series (Kantarjian et al, 1985; Sokal et al, 1988). Sokal et al found 58 (8.8%) of 661 patients that had clonal abnormalities in addition to the Ph chromosome. There was no difference in survival in subgroups depending on whether the additional aberration involved reduplication of the Ph chromosome, hyperdiploidy or hypodiploidy. When all patients were combined and compared with control patients the 58 patients with clonal evolution had significantly poorer survival (P < 0.02). Survival curves were identical for the first 2 years but then diverged and the annual death rate among patients with additional cytogenetic abnormalities was 40% higher. At the 25th percentile the difference in survival was more than 2 years (Sokal et al, 1988).
Kantarjian et al (1985) performed a multivariate analysis examining the association between patient characteristics and therapy with survival. Of 303 patients with Ph+ referred within 3 months of diagnosis there were 29 patients (10%) with additional chromosomal abnormalities. Median survival in those 29 patients was 28 months versus 41 months (P = 0.01) in the remaining patients (Kantarjian et al, 1985).
Updating this series to 826 newly diagnosed patients with CML treated at M.D. Anderson Cancer Center, 60 patients (7%) were noted to have clonal evolution in a median of 64% (range 3-100%) of metaphases (unpublished data). Additional clones including +Ph, isochromosome 17q (iso(17q)) and +8 were seen in only 16 patients (27%). Patients with clonal evolution were more frequently male (82%), had a higher incidence of palpable splenomegaly, more peripheral blood basophils and were more often symptomatic at diagnosis. Thirty-eight patients received IFN-based therapy; their cytogenetic response to IFN was inferior and median survival was shorter at 42 months versus 78 months in patients without clonal evolution.
The presence of additional cytogenetic changes, while uncommon in the chronic phase of CML, is frequent during the accelerated and blastic phases (Canellos et al, 1976; Mandelli et al, 1977; Rosenthal et al, 1977; Rowley, 1978; Sandberg, 1978; Sonta and Sandberg, 1978; Stoll and Oberling, 1979; Bemstein et al, 1980; Alimena et al, 1982; Ishihara et al, 1983; Sadamori et al, 1983; Haas et al, 1984; Godde-Salz et al, 1985; O'Malley
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and Garson, 1985; Sadamori et al, 1985; Swolin et al, 1985; Cervantes et al, 1986; Parreira et al, 1986; Singh et al, 1986; Alimena et al, 1987; Kantarjian et al, 1987; Krulik et al, 1987; Sessarego et al, 1987; Bernstein, 1988; Cervantes et al, I990; Hild and Fonatsch, 1990; Nanjangud et al, 1994; Ruff et al, 1995). In a series of 68 patients from South Africa, Bernstein et al (1980) noted additional chromosomal abnormalities in 9% of patients in the chronic phase and 86% of 14 patients with acute-phase disease. The three most frequently observed patterns of clonal evolution were trisomy 8, duplication of the Ph chromosome and iso(17q), any one of which occurred in 75% of patients with clonal evolution. After trans- formation there was no correlation between the pattern of clonal evolution, blast morphology or survival with the median survival being 4 months.
Alimena et al (1982) reported additional chromosomal abnormalities in 43 (62%) of 69 patients with blastic-phase CML. Thirty-three (76%) patients had one of the three common abnormalities as defined above, either alone or with additional changes. Basophilia was a characteristic feature for patients with an iso(17q). Nine patients (13%) had a lymphoid blast crisis but there was no correlation between karyotypic pattern and blast morphology. The presence of additional chromosomal abnormalities had no effect on survival although there was a trend within the lymphoid blast crisis group for a shorter survival (4 months versus 16 months) when clonal evolution was present (Alimena et al, 1982).
In a large multicentre series, Ishihara et al (1983) noted additional clonal changes in 48 of 434 (11%) patients in chronic phase whereas 106 of 158 (67%) patients evidenced such changes in the blastic phase. Additional Ph, trisomy 8 or iso(17q) was noted in 73% of blast phase patients.
Swolin et al (1985) conducted a prospective study in 32 patients with Ph- positive CML. Eight patients developed additional clonal abnormalities in chronic phase. Seven of the eight patients developed clinical transformation of disease within 2-8 months of evidencing clonal evolution. In total, 25 of 32 patients (78%) developed abnormalities in addition to the Ph, with 80% of those abnormalities involving double Ph, trisomy 8 or iso(17q). Interestingly, the number of additional abnormalities per case with abnormalities tended to be higher among busulphan-treated patients (3.3) than in those receiving hydroxyurea or no therapy (2.0). There was no difference in cytogenetic abnormalities between patients with a lymphoid (N= 6) or myeloid blast crisis, although iso(17q) was not seen in patients with lymphoid phenotype (Swolin et al, 1985).
Alimena et al (1987) reported a higher incidence of the three common changes (+Ph, +8, iso(17q)) in patients receiving busulphan therapy (82%) versus hydroxyurea (29%). Similarly, Nanjangud et al (1994) noted a higher incidence of clonal evolution at the time of blast crisis in patients previously treated with busulphan (70%) versus hydroxyurea (44%). They also detected a difference in survival depending on whether patients had only the Ph chromosome (median survival 7.7 months), additional abnormalities in a fraction of metaphases (median survival 4.1 months) or clonal evolution in all metaphases (median survival 2.1 months) (Nanjangud et al, 1994).
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Sadamori et al (1983) analysed 10 parameters for an association with response and survival in 64 patients with blast phase CML. Only chromo- some findings were important in predicting outcome. Complete or partial remission was achieved in 79% of patients without clonal evolution versus 53% of patients with clonal evolution in < 100% of metaphases and 30% of patients with additional clones in all cells analyzed. The median survival in the respective groups was 5.7 months, 4.9 months and 2.5 months. No information on blast phenotype was provided.
Kantarjian et al (1987) analysed 242 patients with Ph+ blast crisis. Clonal evolution was present in 60% and +Ph, +8 and/or iso(17q) in 73% of those patients (Kantarjian et al, 1987). Median survival was 4.5 months and several factors predicting for remission and survival were found. On multivariate analysis the presence of iso(17q) was independently associated with a decreased response rate even after adjusting for its association with myeloid phenotype; double Ph was independently associated with shorter survival (Kantarjian et al, 1987).
A multivariate analysis was also conducted by Cervantes et al (1990) in 80 patients with Ph-positive blast crisis. Forty-seven patients (59%) displayed clonal evolution. Trisomy 8 was an independent predictor for shorter survival; as in most series the overall survival was short at 4.8 months.
The significance of clonal evolution occurring in chronic phase is unclear, especially on interferon therapy. In a multivariate analysis, Kantarjian et al (1988) described cytogenetic clonal evolution as an independent characteristic indicative of accelerated-phase CML. However, cytogenetic analysis was not done routinely throughout therapy but was often obtained at a time when other features of disease evolution were present. Additional clones are heterogenous, may occur in a small fraction or up to 100% of examined metaphases and may be transient. Few data exist analysing the relevance of these specific factors to the prognosis of clonal evolution.
Recently Majlis et al (1996) examined the significance of cytogenetic clonal evolution in CML excluding patients in blast phase. Between 1967 and 1993, 264 patients were found from a denominator of 1371 patients in the CML database. Fifty patients had features of accelerated disease as defined previously. The median time from diagnosis of CML to clonal evolution was 24 months and the median survival of patients from the time of clonal evolution was 19 months. Shorter survival time was associated with several factors including chromosome 17 abnormalities, a higher percentage of abnormal metaphases, features of accelerated disease and no previous IFN therapy. Longer time from diagnosis to clonal evolution was also associated with poorer survival except for newly diagnosed patients who fared worse than those developing clonal evolution in the first two years.
A proportional-hazards regression model also determined the above characteristics to be significant and when patients were grouped according to the number of unfavourable factors (excluding IFN) they segregated into groups with different median survivals ranging from 51 months in patients
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without any unfavourable factors to only 7 months in patients with three or four poor prognostic features. Of significance was the fact that median survival from the time of clonal evolution had increased from 8 months in patients diagnosed before 1985 to 34 months in patients seen since 1988. However, the authors noted that, prior to IFN therapy and its associated requirement for periodic cytogenetic analysis to investigate response, cytogenetic analyses were often performed only when there was a sign of disease progression. This confounds any potential analysis investigating the effect of IFN on survival with clonal evolution. To try to address this question patients in the three risk groups defined by the analysis were sub- divided according to whether they had received IFN. In the largest group consisting of intermediate-prognosis patients the median survival was 30 months in patients treated with IFN versus 17 months in those not receiving IFN. There was little difference in survival associated with IFN treatment in either the best or the worst prognostic groups.
Interestingly, although the previously defined common abnormalities (double Ph, isochromosome 17 and trisomy 8) also accounted for 59% of all cases seen, the authors noted a decrease in these abnormalities over the time period of the study such that they accounted for 74% of abnormalities seen in patients before 1985 and only 50% since 1988. This suggested that improvement in prognosis with clonal evolution may not be solely attributed to changes in therapy.
Slovak et al (1995) analysed the influence of clonal evolution on outcome of allogeneic BMT. Eleven patients in second chronic or accelerated phase had no evidence of clonal evolution whereas 10 patients had additional abnormalities at the time of transplant. Pseudodiploidy and hyperdiploidy were common in the latter group although complex abnormalities (more than three plus Ph) were not seen. In the first group one patient relapsed, five died without relapse and five remained in remission. One patient with clonal evolution relapsed, five died without relapse and four remained in remission. Median follow-up for both groups was 52 months. A similar analysis could not be done for patients transplanted in blastic phase because only two of 10 had no additional cytogenetic abnormalities. Interestingly, only one of those 10 patients was alive at 57 months and that patient did not have clonal evolution at the time of transplant.
MINIMAL RESIDUAL DISEASE
Up to now minimal residual disease studies have been focused on the detection of the Ph translocation associated with CML rather than any of the additional cytogenetic events discussed above. The small number of cells available for Ph chromosome analysis by CG has made cytogenetic quantification of MRD in CML by that method an impractical enterprise. This is due to the statistical fact that a small number of cells cannot provide a reliable estimate of the true percentage of Ph+ cells in a patient with MRD. Using HMF, where readings can be obtained on 5 0 cells, one can reliably estimate parameters which characterize MRD. I :~r instance, if one
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detected five Ph+ cells out of 500 cells (1.0%) by HMF, the 99% confidence interval on the true percentage of Ph+ cells in the patient ranges from 0.3% to 2.8%. To put these numbers in perspective, detecting 0 Ph+ cells out of 20 cells studied by CG (the usual number analysed by CG in patients under treatment) yields a 99% confidence interval ranging from 0.0% to 20.6%. No false positives or false negatives were observed in studying 5000 cells by HMF from the marrows of normal controls in the studies of Seong et al (1995). Therefore because 0/5000 yields an exact Clopper-Pearson 99% upper confidence bound of 0.000926, one may use 0.001 (0.1%) as a conservative upper bound on the false-positive probability for further evaluating HME It can be concluded that HMF can give investigators considerable power in identifying MRD in CML.
The power of HMF in detecting clinically significant MRD has been evaluated in BMT recipients. Application to BMT was considered particularly propitious because much of the benefit of allogeneic BMT is mediated by an immune graft-versus-leukaemia effect (Snyder and McGlave, 1990) which can be enhanced by donor lymphocyte infusion (DLI). The importance of detecting early relapse is underscored by the observation that the results of DLI have been best when given early in the course of relapse (Kolb et al, 1995). Studying 51 BMT recipients by HMF and CG at multiple time points post-BMT, Seong et al (submitted, a) detected low levels of Ph+ cells (0.5-5.2%) by HMF in 10 recipients who had not been found to have Ph+ cells by CG analysis performed simul- taneously. In two such patients residual Ph+ cells were detected within a few months post-BMT by HMF, while subsequent HMF tests (after 3 months post-BMT) determined those patients to be in complete HMF remission (0% Ph+ cells out of 500 cells studied), probably because of a graft-versus leukaemia effect (Radich et al, 1995). However, when such low levels were detected beyond three months post-BMT, relapse ensued. Similar results were also achieved in a smaller patient set by EI-Rifai et al, (1996). In the one case (Seong et al, submitted, a) when DLI was employed based on the HMF information (1.8% Ph+ cells) the patient remained in long-term remission. Thus HMF can detect MRD in CML at far lower levels than Ph+ cells can be reliably detected by CG and at levels that are useful in planning clinical interventions. It might also be expected that, as additional chromosomal elements are identified by CG as being associated with progressive disease, probes for identifying such elements should be readily attainable through the genome project and could also be incorpo- rated in a multidimensional approach to detecting MRD by HMF.
It is often noted that strictly molecular techniques such as reverse- transcriptase PCR (RTPCR) are exquisitely sensitive for the detection of chimeric message (BCR-ABL) and may be even more useful than HMF in detecting MRD. This raises several issues that might have clinical interest. Because it has been shown that some patients remain in durable complete remission despite persistently positive RTPCR results (Miyamura et al, 1993 Radich et al, 1995), RTPCR might be too sensitive to be useful clinically. That is, making clinical judgements on data that are below the threshold of clinical significance might not be efficacious. It should also be
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recognized that even where quantitative RTPCR is developed (Hochhaus et al, 1996), it is quantitative only for what it is measuring, the level of chimeric message, and is not quantitative for the frequency of cells with the Ph chromosome (because of possible variable expression of the chimeric message in the cell population being assayed). On the other hand, the case can be made that HMF is not strictly quantitative for the frequency of cells with the Ph chromosome, because it can evaluate only cycling cells. HMF cannot count Ph+ cells that do not enter division. That can only be done by quantitative genomic PCR for the chimeric gene or by interphase FISH (I-FISH). Both approaches are independent of cycling cells or chimeric gene expression. Unfortunately, a general assay for the former has yet to be developed. I-FISH will be discussed below.
Because the ability to screen large numbers of cells has been shown to be the key for quantitating MRD, detecting the Ph translocation in inter- phase cells by FISH (I-FISH) is an approach that has been attempted over the last few years. Two differently coloured cosmid-size (30-60kb) genomic probes from the two different chromosomal sites coming together as a result of the translocation have been applied. Ph+ cells were scored by observing a spot of intermediate colour (Tkachuk et al, 1990; Nacheva et al, 1993; Bentz et al, 1994). However, the small signals produced by the cosmid-sized probes against the background of the interphase cells made the quantitation of that approach difficult. Using larger probes (containing 0.5-5.0 Mb of human DNA) from yeast artificial chromosomes (Lengauer et al, 1992) or somatic cell hybrids (Seong et al, 1994) that overlaid one of the Ph translocation breakpoints, investigators looked for one of the two FISH signals present in normal cells being split as an indication of the Ph translocation. While the probes were more robust, the results lack the confirmation that one of the split signals brings ABL together with BCR. Using a second, differently coloured cosmid probe that would be brought together with the initial probe has proven useful in validating such trans- locations and reducing the false-positive reading (Seong et al, 1994).
In order to exploit the positive aspects of each of these I-FISH approaches, Seong et al (submitted, b) used two differently coloured cosmid contigs (> 200 kb) that would be brought together by the Ph trans- location producing intermediately coloured bright spots in interphase Ph+ cells. This was used to determine the frequency of Ph+ polymorphonuclear leukocytes (PMNs) in the circulation of CML patients. Because HMF for the detection of Ph+ cells in the bone marrow had been validated as a sensitive measure of tumour burden in CML patients undergoing therapy (Seong et al, 1995), the percentage of cells detected with BCR-ABL fusions in PMN was compared with HMF results from the marrow of the same 26 patients in order to determine the usefulness of I-FISH on peripheral blood for evaluating the cytogenetic status of CML patients. The two data sets correlated well (r=0.983, P<0.0001). However, the low false-positive frequency of HMF which allowed the statistically significant detection of minimal residual disease (< 1% Ph+ cells), and changes in frequency of < 4%, was not possible by I-FISH which had a significantly higher false positive rate. It was concluded that I-FISH on PMNs is a sensitive and
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reliable method for monitoring the frequency of Ph+ cells in CML patients undergoing therapy and reduces the need for repeated marrow aspirations. However, for the detection of MRD or for monitoring small changes in the frequency of Ph-positive cells, I-F/SH on PMN could not be substituted for HMF on short-term marrow cultures.
It will be of interest to study the relationship between RTPCR, HMF and I-FISH with respect to clinical relevance on the same patient set to evaluate these different approaches to measuring clinically significant MRD. In addition to assay development, such studies should help us to a better understanding of roles of the different biological phenomena associated with the cytogenetics of CML.
Finally, the results of the studies reviewed in this chapter lead us to suggest the following algorithm for cytogenetic approaches to monitoring disease in CML patients. Initial diagnosis needs to be done by CG to characterize the different cytogenetic elements associated with the disease and by HMF with appropriate probe (e.g. E6B) in order to determine the specific frequency of Ph+ cells cycling in the patient's marrow. During treatment, CG needs to be done only to identify any suspected clonal evolution or variant translocations. I-FISH on PMNs should be adequate to estimate the frequency of Ph+ cells in circulation during this phase. When statistically significant levels of Ph+ cells are not detected by I-FISH, HMF on bone marrow cells needs to be conducted in order to determine and quantitate the presence of MRD.
Acknowledgements We are indebted to former post-docs, students and fellows who have helped the laboratory to implement and exploit the molecular cytogenetic technologies described in this review, especially Drs Gaudenz Dolf, Pu Liu, David Seong and Paula Marlton. This work was supported in part by NIH Grants CA34936, CA49639, CA16672 and CA65164 and a gift from Mr Kenneth D. Muller.
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