Molecular measurement of minimal residual disease in Philadelphia-positive acute lymphoblastic leukaemia

Best Practice & Research Clinical Haematology Vol. 15, No. 1, pp. 91±103, 2002

doi:10.1053/beha.2002.0187, available online at http://www.idealibrary.com on

6 Molecular measurement of minimal residual disease in Philadelphia-positive acute lymphoblastic leukaemia Jerald P. Radich

MD

Associate Member Clinical Research Division, Program in Genetics and Genomics, Fred Hutchinson Cancer Research Center, D4-100; 1100 Fairview Avenue, North Seattle, WA 98109, USA

The Philadelphia chromosome (Ph) is found in approximately 5±25% of acute lymphoblastic leukaemia (ALL) cases and is the harbinger of a poor outcome. Polymerase chain reaction (PCR) assays can detect leukaemia-speci®c genetic lesions down to a sensitivity approaching one leukaemia cell in a background of a million normal cells. In Ph‡ ALL, the unique BCR-ABL translocation is thus a speci®c target for the detection of minimal residual disease (MRD). After chemotherapy or transplantation the detection of residual BCR-ABL transcripts is associated with a high risk of subsequent relapse. With the advent of novel therapeutics that target the structure and function of BCR-ABL, the detection of MRD may allow for targeted therapy that could abort a potential relapse. Key words: minimal residual disease; Philadelphia chromosome; acute lymphoblastic leukaemia.

The Philadelphia chromosome (Ph) is the hallmark genetic lesion found in chronic myeloid leukaemia (CML), and is also found in approximately 25% of adult ALL and 5± 10% of paediatric ALL cases.1,2 The Ph is an example of a speci®c genetic lesion that is associated with a distinctly poor prognosis in ALL. In addition, the Ph serves as a marker for detection of early relapse using sophisticated and sensitive molecular assays, and the speci®c molecular pathogenesis of the Ph may provide the opportunity of truly targeted therapy. THE BIOLOGY OF THE PHILADELPHIA CHROMOSOME (PH) Molecular genetics of Ph‡ ALL The Ph results from the reciprocal translocation of the long arm of chromosome 9 with the long arm of chromosome 22 {t(9;22)(q34;q11)}. This translocation places the 50 portion of the BCR gene from chromosome 22 in juxtaposition with the downstream tyrosine kinase domains of the ABL gene from chromosome 9.3±5 This unique chimeric fusion gene causes the expression of the chimeric BCR-ABL mRNA, which is translated to the functional chimeric BCR-ABL protein. This BCR-ABL fusion protein has an elevated 1521±6926/02/010091‡13 $35.00/00

c 2002 Harcourt Publishers Ltd. *

92 J. P. Radich

tyrosine kinase activity relative to the wild type ABL activity.6,7 In addition, in mouse models, both transgenic and retroviral transplantation experiments have shown the abnormal BCR-ABL activity is sucient to cause leukaemogenesis.8,9 There are two common breakpoints of the BCR and ABL genes (Figure 1).10±13 The heterogeneity in the fusion BCR-ABL genes arises from di€erent breakpoint locations in the BCR gene. The breakpoint in ABL spans a 4 300 kb range and occurs 50 to ABL exon 2 with only rare exceptions. The nomenclature surrounding the types of BCR and ABL breakpoints is confusing. The major breakpoint cluster region (M-BCR) of the BCR was originally identi®ed as the breakpoint area in BCR that fused to ABL. Thus, the initially described breakpoint in BCR occurs in the M-BCR of the BCR gene, producing,

BCR BCR exons 1-11

12 - 16

17 18

19 20 21 22

1 12345 M-bcr

m-bcr

ABL 1b

1a

2

3 4 5 6 7 8

9

10

11

BCR-ABL

p210

1 123 bcr

p190

1

Figure 1. The BCR-ABL translocation. The structures of BCR and ABL are shown in the upper panels. Boxes refer to exons, and lines designate introns. In BCR, breakpoints occur in the major breakpoint cluster area (M-BCR) at the area of the solid descending arrow in the p210 BCR-ABL variant, or in the minor breakpoint cluster region (m-BCR), in the area of the hatched descending arrow forming the p190 BCR-ABL variant. The vast majority of breakpoints in ABL occur between ABL exons 1a and 2. The p210 and p190 BCR-ABL variants are shown in the lower two panels.

Molecular measurement of MRD in Philadelphia+ ALL 93

depending on RNA splicing, either a fusion mRNA of exon 2 or 3 (b2 or b3) of the BCR gene with the downstream exon 2 of the ABL sequence (a2). This b3a2 or b2a3 fusion yields the 210 kDa BCR-ABL chimeric protein. This variant is found in the vast majority of CML cases. The M-BCR was characterized before the structure and organization of the entire BCR gene was fully characterized. Therefore the M-BCR exons 1±5 correspond to exons 12±16 of the BCR gene as we now refer to it. Thus, a b3a2 breakpoint may be described as a b14a2 breakpoint in the recent literature. An alternative breakpoint is found in Ph‡ ALL cases that occurs 50 of exon 2 in the minor breakpoint cluster region (m-BCR) of the BCR gene. The juxtaposition of this truncated BCR gene, linked to the downstream ABL exon 2, is referred to as an e1a2 breakpoint. This chimeric mRNA yields a 190 kDa BCR-ABL fusion protein (also referred to as the `p185' breakpoint by some investigators). Modern molecular biology methods using polymerase chain reaction (PCR) based assays can quickly discriminate between the p210 and p190 BCR-ABL breakpoints by using di€erent oligonucleotide primers speci®c to the BCR sequences involved in the di€erent breakpoints.12,14,15 In addition, PCR methods have revealed rare alternative breakpoints utilizing BCR exon 2 (e2) coupled to ABL exon 216, or the use of ABL exon 3 rather than exon 2.17 The biological signi®cance of these rare BCR-ABL variants is unknown. The type of BCR-ABL breakpoint varies with the type of Ph‡ leukaemia. Thus, the p210 variant is almost always associated with CML. In paediatric Ph‡ ALL, nearly 90% of cases are the p190 BCR-ABL, yet in adult ALL approximately 25±50% of cases harbour the p210 BCR-ABL fusion variant and the rest harbour the p190 subtype. Indeed, the ratio of p210/p190 BCR-ABL increases with the decade of the patient's age.2 It is tempting to speculate that some of the p210 Ph‡ ALL cases in adults may well be CML cases presenting in a lymphoid blast crisis. Of interest is that these sensitive PCR reactions have shown that many patients with p210 BCR-ABL can also make variable amounts of p190 BCR-ABL transcripts.18±20 This has been seen in CML and ALL. This suggests that, in those patients with p210 BCRABL, unknown mechanisms result in a transcription preference resulting in a vast predominance of the p210 BCR-ABL transcript. However, because the upstream BCR domains are present and in juxtaposition to ABL, alternative RNA splicing can still produce the p190 transcript. The mechanisms controlling this splicing, and the potential e€ect on disease activity, are unknown. Lineage restriction of the Ph chromosome Because ALL cases can present with the p210 BCR-ABL variant common in CML, the question arises whether many cases of p210 Ph‡ ALL are actually cases of CML presenting in lymphoid blast crisis. In cases of lymphoid blast crisis CML masquerading as ALL, one would expect the Ph to be found in the myeloid lineage as well as the lymphoid blasts. On the contrary, if the disease were true Ph‡ ALL, one would expect to ®nd the Ph only in the lymphoid blasts, and not myeloid lineage cells. Several studies have investigated the level of lineage involvement in Ph‡ ALL, with very disparate results. Some studies have found evidence of `multi-lineage' Ph‡ ALL with both myeloid and lymphoid (blast) cells harbouring the Ph in colony forming assays21, while others have found multilineage Ph‡ ALL as well as cases of pure lymphoid restriction (where the Ph is found solely in the lymphoid population).22±27 Clinical and laboratory data suggest, however, that some of the multilineage patients may have had underlying CML.23 Other experimental methods suggest the involvement of the `stem cell' compartment in Ph‡ ALL. This has been demonstrated by ¯ow cytometry

94 J. P. Radich

isolation of CD34‡ , Lin cells from Ph‡ ALL cases, where the Ph has been identi®ed by ¯uorescence in situ hybridization (FISH) in the isolated populations.21 In addition, Nod-SCID mouse models have been generated using CD34‡ cells from Ph‡ ALL patients to reconstitute leukaemia.28 Taken together, Ph‡ ALL appears to be a heterogeneous disease encompassing (1) a lymphoid lineage restricted ALL; (2) a `stem cell' ALL, with evidence of disease in both lymphoid and myeloid lineages; and (3) misclassi®ed CML in lymphoid blast crisis.

TREATMENT OF PH‡ ALL The Ph is a poor prognostic factor for both paediatric and adult patients treated with conventional chemotherapy. Paediatric studies are summarized in Table 1.1,29±34 In paediatric studies, over 75% of Ph‡ ALL patients go into complete remission after induction therapy, but most relapse, resulting in an event-free survival (EFS) of 10± 20%, compared to 470% in patients without the Philadelphia chromosome.1,34 Ph‡ ALL children who have high white blood cells counts, or who respond poorly to an initial course of prednisolone, have an especially poor prognosis.29,33 In adults the outcome after conventional chemotherapy is similarly poor.2,35±44 In general, initial remission rates of 60±80% can be achieved, but the duration of remission in Ph‡ ALL adults is usually very short (512 months), with resultant EFS of approximately 10%. The `hyper-CVAD' therapy38 may have a positive impact on improving CR rates and ®rst CR duration, but the survival after 3 years is not clearly di€erent than with less aggressive regimens. Thus, even with modern chemotherapy, the likelihood of curing Ph‡ ALL in adults appears very limited. Table 1. MRD detection and subsequent relapse in PH‡ ALL.a Author (reference)

Number of BCRABL‡

Number of relapses

Number of BCRABL

Number of relapses

CHEMO Mitterbauer et al (66) Mitterbauer et al (67) Preudhomme et al (68)

6 6 7

6 4 6

1 0 1

1 0 0

ALLO-BMT Kohler et al (69) Miyamura et al (65) Radich et al (20) Synder et al (78) Mitterbauer et al (67) Preudhomme et al (68)

0 5 21 10 3 3

0 5 8 1 2 3

1 4 9 7 0 2

0 1 0 1 0 0

AUTO-BMT Miyamura et al (65) Radich et al (20) Preudhomme et al (68) Mitterbauer et al (67)

2 2 3 2

2 2 2 0

4 4 1 0

0 2 0 0

aNote.

`BCR-ABL‡ ' and `BCR-ABL ' refer to the numbers of patients in each study who were ever BCR-ABLpositive, or always BCR-ABL-negative, respectively, while in a haematological complete remission. If a patient was BCR-ABL-negative, then became positive concurrent with relapse, the result was categorized above as a relapse in a BCR-ABL-negative patient. The numbers of each category of patient are explicitly cited in some studies, but in others the data were extracted from ®gures as best as possible.

Molecular measurement of MRD in Philadelphia+ ALL 95

Because of the dire outcome of Ph‡ ALL following conventional therapy, most experts recommend proceeding to early marrow transplant as the most likely method to obtain a cure. Whereas the outcome for Ph‡ ALL following conventional chemotherapy is poor, the outcomes following transplantation appear surprisingly good.29,45±49 Patients transplanted in ®rst CR appear to have a disease-free survival (DFS) of 40±60%, with patients in second remission having DFS slightly less than 40%.45,47,48 A comparison of post-remission therapy by chemotherapy alone, allogeneic transplantation, and autologous transplantation con®rmed the advantage of allogeneic transplantation on DFS.29 The success of transplantation for Ph‡ ALL suggests that the myeloablative preparative regimen, combined with the immunological e€ect of the allograft, overwhelm the mechanism of resistance in Ph‡ ALL. In addition, the good outcomes following transplantation in Ph‡ ALL may occur from these patients moving expeditiously to transplant compared to other ALL patients. Either way, transplantation is an e€ective therapy in the treatment of Ph‡ ALL, and should be considered in ®rst remission patients with an appropriate allogeneic donor. DETECTION OF MINIMAL RESIDUAL DISEASE The Ph can be detected by conventional cytogenetics, FISH or PCR assays. Several studies have demonstrated that convention cytogenetics underestimates the frequency of Ph detection in diagnostic samples compared to FISH or PCR.36,50,51 This may be because the leukaemic cells grow poorly in culture, or (given the fragile nature of lymphoblasts) are lysed in handling. For diagnostic purposes, FISH and PCR are similarly reliable, with FISH having an advantage secondary to the avoidance of the contamination issue inherent in PCR.52 On the other hand, PCR allows for direct determination of the p190 and p210 variants, which is dicult to do with FISH without speci®c probes. PCR methods are more sensitive than FISH and are probably optimal for minimal residual disease (MRD) studies.53 The exquisite sensitivity of these assays allows the detection of approximately one leukaemic cell in a background of 104±105 normal cells. This allows for the detection of MRD when the patient appears by all other less sensitive assays (morphology, cytogenetics, and ¯ow cytometry) to be in remission. Many studies have demonstrated the importance of MRD detection in ALL using immunoglobulin gene rearrangements as the marker of MRD.54±58 These studies have consistently shown that the detection of MRD, especially above a `threshold' value of one ALL cell in 103 normal cells, is highly associated with subsequent relapse. There is also a large body of literature documenting the importance of MRD testing for BCR-ABL in CML, where both qualitative and quantitative MRD detection is associated with relapse after transplantation.59±64 However, few studies have focused on MRD detection and signi®cance in Ph‡ ALL. A summary of the associations of BCR-ABL detection (or its absence) and subsequent relapse after either chemotherapy, allogeneic, or autologous transplantation, is shown in Table 1. Miyamura et al found that 8/10 Ph‡ ALL patients presenting in `CR' at the time of transplantation were nevertheless BCR-ABL-positive, a ®nding supported by Gehly et al (®ve of eight patients positive for BCR-ABL) and Mitterbauer et al.14,65,66 The appearance of BCR-ABL post-chemotherapy was associated with a very high risk of relapse.66±68 However, most of the data concerning MRD are from the transplant setting.20,65,69 Kohler et al69 ®rst described a single Ph‡ ALL patient who became BCR-ABL-negative following allogeneic transplantation. The patient remained negative and in CR. Miyamura et al65 studied 15 patients post-transplantation,

96 J. P. Radich

13 of these studied within the ®rst year following transplant. Of the seven patients who were PCR-positive for MRD, all seven subsequently relapsed. Of the eight patients who were PCR-negative, only one relapsed. In addition, Radich et al20 studied 36 Ph‡ ALL patients following marrow transplantation. Thirteen of these 36 patients were persistently BCR-ABL-negative post-transplant, and 8/13 were disease-free survivors. Two of these BCR-ABL-negative patients subsequently relapsed, both after a prolonged period without MRD testing. Of the 23 patients with at least one BCR-ABL-positive assay post-transplantation, 10 relapsed, and 11 remained in remission. The relative risk (RR) of relapse associated with BCR-ABL positivity compared to BCR-ABL-negative patients was 5.9. The median time from ®rst BCR-ABL-positive assay to relapse was 94 days, with a range of 28 to 416 days. The increased RR among BCR-ABL-positive patients was found predominantly in patients with the p190 BCR-ABL variant, with a RR of relapse of 11.2 in this group compared to PCR-negative patients. In a multivariable model, BCR-ABL positivity remained statistically signi®cant after adjusting for other co-variables known to be associated with a high relapse risk (i.e. remission status, type of donor, graftversus-host disease). The evidence that p210 BCR-ABL is associated with a lesser risk of relapse was indirectly supported by Sierra et al; these authors reported on the success of unrelated donor transplantation in Ph‡ ALL.47 Of 18 patients transplanted, nine were diseasefree survivors. Of these nine, four were BCR-ABL-positive at least once post-transplant (all within the ®rst 3 months). All had the p210 BCR-ABL variant, and all subsequently became BCR-ABL-negative and remained disease-free. Snyder et al48 published impressive results concerning transplantation for Ph‡ ALL. Of 17 patients studied using molecular testing, 10 were BCR-ABL-positive at some time after transplantation. Of these 10, ®ve had the p210 variant, four the p190, and one had both the p190 and p210 BCR-ABL variants. Only one relapse occurred, in a patient bearing the p190 BCRABL. The timing of PCR assays was not given. ISSUES IN MRD MONITORING While comprehensive studies of MRD detection in Ph‡ ALL have not been performed, there are some insights into the `nuts and bolts' of monitoring that are relevant to discuss. Blood versus bone marrow The qualitative BCR-ABL assay appears to be concordant between blood and bone marrow approximately 70% of the time.20 Discordance in qualitative assay (yes or no for BCR-ABL) can occur in either direction, but is most common with a negative result in the peripheral blood associated with a positive result in the bone marrow. In general, BCR-ABL levels derived from semi-quantitative PCR assays are similar between peripheral blood and bone marrow.67,70 When discordance occurred, levels in bone marrow were usually greater than in peripheral blood. Frequency of monitoring While maximum sensitivity is obviously important in the detection of MRD, an overlooked crucial variable is frequency of monitoring. Unfortunately, the published data o€er little guidance. In our study of transplant patients, the median time from

Molecular measurement of MRD in Philadelphia+ ALL 97

®rst detection of BCR-ABL to relapse was 90 days.20 Similar results on few cases suggest that most relapses occur 2±6 months after an increase in BCR-ABL burden has been detected (either negative to positive, or a quantitative increase).67,68 However, all of these measurements of BCR-ABL MRD were a re¯ection more of the arbitrary schedule of sampling than the biology of disease. Given that (1) physicians must balance the accuracy of frequent bone marrow testing with the patient discomfort associated with frequent aspirations, and (2) there is a reasonable correlation between peripheral blood and bone marrow testing, one possible solution is to perform bone marrows at clinically relevant points (after induction, consolidation, etc.) with frequent peripheral blood sampling. Any major change in peripheral blood BCR-ABL status would be an indication for an additional bone marrow assay. Quanti®cation It is clear that the quanti®cation of MRD burden is informative in CML, where detection is focused on the p210 BCR-ABL transcript. Similarly, quanti®cation of Ph‡ ALL would seem useful, especially for determining the disease burden for patients who appear in remission.67 The advent of `real-time' PCR using ¯uorescent probes has made PCR quanti®cation faster and more reliable in CML.62±64,71 We are currently employing this real-time PCR to quantify p190 BCR-ABL in Ph‡ ALL, and our preliminary data suggest that the method is as reliable as the p210 assay.

ARE P190 AND P210 BCR-ABL DIFFERENT? There are a few reasons to suspect that p190 and p210 BCR-ABL might be biologically di€erent diseases. First, the in vitro tyrosine kinase activity of p190 BCR-ABL is greater than that of p210 BCR-ABL.72,73 Second, in animal models, p190 BCR-ABL appears to induce leukaemia that is more virulent than p210 leukaemia. Few clinical studies in humans have addressed the biology of these variant breakpoints. Kantarjian et al74 and Secker-Walker et al2 could ®nd no di€erence in outcome between p190 and p210 Ph‡ ALL cases. Data from a more contemporary chemotherapy trial suggested that p190 and p210 Ph‡ ALL behaved somewhat di€erently.51 In the p190 cases, seven of eight patients went into remission, and six of these seven relapsed. This is the usual expectation for Ph‡ ALL, where remission can often be achieved, but relapse generally follows. On the other hand, only 5/12 patients with p210 disease went into remission, yet four of ®ve remained disease-free. In addition, as noted above, the signi®cance of p190 BCR-ABL post-transplantation has also been reported to be worse than that of p210 disease. Of the eight patients with p190 BCR-ABL detected post-transplant, seven relapsed, whereas only one of eight patients with p210 detection went on to relapse.20 Larger studies of patients treated uniformly will need to be performed to address this interesting clinical and biological question.

THE FUTURE Ph‡ ALL is an ideal disease on which to launch studies of novel therapeutic approaches because it has a poor prognosis and has a well-characterized genetic lesion. The de®ned

98 J. P. Radich

role of BCR-ABL in leukaemogenesis has prompted innovative ways to target this chimeric gene for focused attacks. One such approach is by targeting BCR-ABL mRNA, either by antisense oligonucleotides or ribozymes.75±79 The mechanism of the antiproliferative e€ects of BCR-ABL antisense oligonucleotides is unclear, and may stem from direct inhibitory e€ect on target mRNA, or from non-speci®c toxicity for the degradation products of the therapeutic oligonucleotides.79 By themselves, antisense molecules to BCR-ABL can inhibit BCR-ABL protein synthesis and decrease leukaemic colony formation, but they do not extinguish leukaemia. The combination approach of chemotherapy and antisense appears more e€ective in eliminating leukaemic cells than either approach alone, both in colony assays and in SCID mouse models.77 Ribozymes are synthetic mRNA molecules, which, after annealing to a target mRNA catalytically cleave the target. Hairpin ribozymes designed to target p190 BCR-ABL mRNA have been designed.78 When mixed with a liposomal vector, the ribozyme is e€ectively taken up by p190 BCR-ABL-bearing cell lines with a resultant decrease in BCR-ABL protein expression and an inhibition of cell growth in culture. These approaches hold promise in combination with conventional therapy, especially during remission, or as a way to purge stem cell collections in vitro prior to an autologous transplant. Another interesting approach is to target BCR-ABL activity speci®cally with the tyrosine kinase inhibitor STI571 (commercial name: Glivec), which inhibits the ABL tyrosine kinase domain by binding the ATP binding site of ABL. STI571 is an inhibitor of other tyrosine kinases, including normal ABL, BCR-ABL, c-Kit and PDGF, but not other protein kinases such as Src, Fms, or Her-2/neu.80 STI571 was initially studied in p210 BCR-ABL cellular systems, and was found to inhibit cellular proliferation, inhibit colony formation of both cell lines and patient samples, and promote apoptosis.80,81 Similar e€ects were found in p190 BCR-ABL systems. STI571 was found to decrease auto-phosphorylation of the p190 protein as well as to inhibit growth of both Ph‡ ALL cell lines and patient cells.82 Protein levels were not decreased, indicating a direct e€ect of the drug on the protein enzymatic activity. Phase I and II trials in CML have shown that this agent is remarkably free of toxicity.83 In addition, in CML the drug has been shown to a€ect haematological remissions (return of the WBC to normal) in 490% of patients and to cause complete cytogenetic remissions (disappearance of the Ph in all metaphases studied by cytogenetics) in 15% of patients.83 In addition, drug discontinuation for adverse reactions was quite rare. Experience in Ph‡ ALL has shown a response rate of 50%, with relatively short duration of remission.84 While the mechanism of resistance in ALL is not known, a recent study has demonstrated the acquisition of a new point mutation in ABL in relapsed samples of blast crisis CML.85 In the future, these novel agents may be used in combination with chemotherapy, or, especially in the context of treatment of MRD, in Ph‡ leukaemia. SUMMARY The detection of residual BCR-ABL transcripts in Ph‡ ALL patients otherwise depicted in CR is associated with a high risk of subsequent relapse. The advent of newer quanti®cation strategies may further re®ne the detection and predictive value of measuring MRD and signal patients who would bene®t from novel therapy. The ongoing development of novel agents that target the RNA structure of BCR-ABL, or the rogue enzymatic activity of the BCR-ABL chimeric protein, appear especially promising. These approaches may eventually move Ph‡ ALL from the bad to the favourable prognostic category.

Molecular measurement of MRD in Philadelphia+ ALL 99

Practice points ‡

. Ph is a high-risk leukaemia that is rarely curable by conventional chemotherapy . stem cell transplantation is often curative in Ph‡ ALL and should be considered for patients when they achieve ®rst remission . the detection of BCR-ABL in patients otherwise believed to be in remission is associated with an elevated risk of subsequent relapse

Research agenda . the treatment of minimal residual disease post-transplantation should be performed under a clinical trial . careful documentation of p190 versus p210 BCR-ABL is encouraged in order to study the biological and clinical di€erences between these two subtypes of Ph‡ ALL . novel therapeutic approaches speci®cally attacking BCR-ABL should be considered in patients not eligible for transplantation

Acknowledgement I would like to thank Dr Derek Stirewalt for his insightful and helpful comments. The work was supported by National Institute of Health grant CA18029. REFERENCES 1. Uckun FM, Nachman JB, Sather HN et al. Clinical signi®cance of Philadelphia chromosome positive pediatric acute lymphoblastic leukemia in the context of contemporary intensive therapies: a report from the Children's Cancer Group. Cancer 1998; 83: 2030±2039. 2. Secker-Walker LM, Craig JM, Hawkins JM & Ho€brand AV. Philadelphia positive acute lymphoblastic leukemia in adults: age distribution, BCR breakpoint and prognostic signi®cance. Leukemia 1991; 5: 196±199. 3. Heisterkamp N, Jenkins R, Thibodeau S et al. The bcr gene in Philadelphia chromosome positive acute lymphoblastic leukemia. Blood 1989; 73: 1307±1311. 4. De Klein A, Hagemeijer A, Bartram CR et al. bcr rearrangement and translocation of the c-abl oncogene in Philadelphia positive acute lymphoblastic leukemia. Blood 1986; 68: 1369±1375. 5. Chan LC, Karhi KK, Rayter SI et al. A novel abl protein expressed in Philadelphia chromosome positive acute lymphoblastic leukaemia. Nature 1987; 325: 635±637. * 6. Clark SS, McLaughlin J, Crist WM et al. Unique forms of the abl tyrosine kinase distinguish Ph1-positive CML from Ph1-positive ALL. Science 1987; 235: 85±88. 7. Konopka JB, Watanabe SM & Witte ON. An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell 1984; 37: 1035±1042. 8. Voncken JW, Morris C, Pattengale P et al. Clonal development and karyotype evolution during leukemogenesis of BCR/ABL transgenic mice. Blood 1992; 79: 1029±1036. * 9. Voncken JW, Kaartinen V, Pattengale PK et al. BCR/ABL P210 and P190 cause distinct leukemia in transgenic mice. Blood 1995; 86: 4603±4611. 10. Rubin CM, Carrino JJ, Dickler MN et al. Heterogeneity of genomic fusion of BCR and ABL in Philadelphia chromosome-positive acute lymphoblastic leukemia. Proceedings of the National Academy of Sciences of the USA 1988; 85: 2795±2799. 11. Kurzrock R, Shtalrid M, Gutterman JU et al. Molecular analysis of chromosome 22 breakpoints in adult Philadelphia-positive acute lymphoblastic leukaemia. British Journal of Haematology 1987; 67: 55±59.

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