A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias.

BACKGROUND: Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor-refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). METHODS: We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months. RESULTS: Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event. CONCLUSIONS: Ponatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440 .).

Mutation detection and typing of polymorphic loci through double-strand conformation analysis.

Variations, such as nucleotide substitutions, deletions and insertions, within genes can affect the function of the gene product and in some cases be deleterious. Screening for known allelic variation is important for determining disease and gene associations. Techniques which target specific mutations such as restriction enzyme polymorphism and oligonucleotide probe or PCR primer reactivity are useful for the detection of specific mutations, but these techniques are not generally effective for the identification of new mutations. Approaches for measuring changes in DNA conformation have been developed, based on the principle that DNA fragments which differ in nucleotide composition exhibit different mobilities after separation by polyacrylamide gel electrophoresis (PAGE). Here we describe a conformation-based mutation detection system, double-strand conformation analysis (DSCA), which provides a simple means to detect genetic variants and to type complex polymorphic loci. We demonstrate the application of DSCA to detect genetic polymorphisms such as a single-nucleotide difference within DNA fragments of up to 979 base pairs in length. We present the application of DSCA in detecting four different mutations in the cystic fibrosis gene (CFTR) and 131 different alleles encoded by HLA class I genes.

Response to donor lymphocyte infusions for chronic myeloid leukemia is dose-dependent: the importance of escalating the cell dose to maximize therapeutic efficacy.

Donor lymphocyte infusions (DLI) are an effective treatment for patients with chronic myeloid leukemia (CML) in relapse after allografting but the optimal cell dose has yet to be identified. To address this question, we investigated the factors affecting the dose required to achieve remission (effective cell dose, (ECD)) in 81 patients treated with an escalating dose regimen. The overall proportion of patients who achieved a molecular remission was 88%. The cumulative proportion of remitters increased significantly at each dose level. With a CD3(+) cell dose < or =10(7)/kg, 56% of patients in molecular/cytogenetic relapse obtained molecular remission, whereas only 20% of those in hematologic relapse did so. At the same cell dose, 58% of patients who received lymphocytes from volunteer unrelated donors achieved remission, as compared to 29% of those who received DLI from sibling donors. We conclude that the response to DLI is dose-dependent and that the ECD is influenced by the quantity and phase of CML at relapse and degree of donor/recipient histocompatibility.

Treatment with interferon-alpha preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors (CFU-GM) from patients with chronic myeloid leukemia but spares normal CFU-GM.

The biological target for interferon (IFN)-alpha in chronic myeloid leukemia (CML) is unknown, but one possibility is that amplification of granulocyte-macrophage colony-forming cells (CFU-GM) is reduced. Replating CFU-GM colonies and observing secondary colony formation provides a measure of CFU-GM amplification. Amplification of CML, but not normal, CFU-GM in vitro was significantly inhibited by IFN-alpha (P = 0.02). In 5 out of 15 CML cases studied by fluorescence in situ hybridization, in vitro treatment with IFN-alpha increased the proportion of CFU-GM, which lacked BCR-ABL. The ability of patients' CFU-GM to amplify, and suppression of this ability by IFN-alpha, predicted responsiveness to IFN-alpha therapy in 86% of cases. Investigation of patients on treatment with IFN-alpha showed a threefold reduction in CFU-GM amplification in responders (P = 0.03) but no significant change in nonresponders (P = 0.8). We conclude that IFN-alpha preferentially suppresses amplification of CML CFU-GM to varying degrees. The differing in vitro sensitivities to IFN-alpha and growth kinetics of individual patients' cells could help differentiate those who will or will not benefit from treatment with IFN-alpha.

The third International Congress on Myeloproliferative and Myelodysplastic Syndromes.

This meeting was convened by Richard T. Silver and co-chaired by Jerry L. Spivak. It was held from 27 to 29 October 2005 in Washington, DC. Thirty-one invited speakers from seven different countries participated in the conference, which was attended by more than 300 individuals from 23 countries. As in previous years, a clinical symposium for patients, held the day before the symposium, was sponsored by the Cancer Research and Treatment Fund, Inc., New York, NY 10021. This meeting report provides a summary of the five sessions prepared and highlighted by one of the session chairs. In addition to the formal presentations on the biology, clinical aspects and management of these diverse marrow stem cell disorders, there was considerable interest generated because of the availability of several new agents that have been recently approved. A special luncheon satellite symposium was devoted to the dramatic changes in the therapeutic options for the myelodysplastic syndromes, sponsored by MGI Pharma, Inc. The keynote address was presented by Dr. George Q. Daley from Harvard Medical School and the Children's Hospital Medical Center. He reviewed the molecular steps in the formation of the Philadelphia chromosome and some of the newly described mutations leading to resistance to chemotherapy (see Section 4).

The presence of a BCR-ABL mutant allele in CML does not always explain clinical resistance to imatinib.

The expansion of a leukemia clone bearing a Bcr-Abl kinase domain mutation is associated with acquired resistance to imatinib and may also predict disease progression in patients with Philadelphia-positive chronic myeloid leukemia (CML). Here we report results of pyrosequencing to quantitate the non-mutated and mutant alleles in 12 CML patients monitored over periods ranging from 11 to 58 months, and describe three contrasting kinetic patterns: Group 1 - in four patients total BCR-ABL transcript numbers remained high with the mutant allele predominating; Group 2 - in four patients the total number of BCR-ABL transcripts fell to low levels but the mutant allele predominated; and Group 3 - in four other patients the total level of transcripts remained high (n = 2) or fell (n = 2) but the mutant clone persisted at relatively low level. In Group 2 the mutant leukemia clone was presumably still relatively sensitive to imatinib but in Group 1 the leukemia could be classified as resistant. In Group 3 patients the imatinib sensitivity of the leukemia was variable. We conclude that a mutant clone does not necessarily have a proliferative advantage and its presence does not always account for resistance to imatinib. Other mechanisms underlie resistance in at least some patients.

Effects of bromodichloromethane on ex vivo and in vitro luteal function and bromodichloromethane tissue dosimetry in the pregnant F344 rat.

Bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss, i.e. full-litter resorption, in F344 rats when treated during the luteinizing hormone (LH)-dependent period. This effect is associated with reduced maternal serum progesterone (P) and LH levels, suggesting that BDCM disrupts secretion of LH. To test the hypothesis that BDCM also affects luteal responsiveness to LH, we used ex vivo and in vitro approaches. For the ex vivo study (i.e., in vivo exposure followed by in vitro assessment), dams were dosed by gavage on gestation days (GD) 6-9 (plug day=GD 0) at 0 or 100 mg/kg/d. One hour after the GD-9 dose, rats were killed, blood was collected, and tissue concentrations of BDCM were assessed. Corpora lutea (CL) were incubated with or without hCG, an LH agonist, to stimulate P secretion. For the in vitro study, CL were pooled from untreated F344 rats on GD 9 and cultured with BDCM at 0, 0.01, 0.10 or 3.0 mM. BDCM was found at highest concentrations in adrenal, ovarian, adipose, and hypothalamic tissues. BDCM treatment decreased serum P and LH levels in vivo. Ex vivo, however, BDCM-exposed CL showed >2-fold increases in P secretion relative to controls. Both control and BDCM-exposed CL displayed a 2.4-fold increase in P secretion in response to hCG challenge. In contrast, in vitro exposures reduced CL responsiveness in a dose-related fashion while baseline levels were unaffected. It is unclear if the ex vivo 'rebound' reflects the removal of the CL from a possible direct inhibitory influence of BDCM, or a response to diminished LH stimulation in vivo. Thus, these data suggest that BDCM disrupts pregnancy in F344 rats via two modes: disruption of LH secretion, and disruption of the CL's ability to respond to LH.

Topical issues in unrelated donor haematopoietic stem cell transplants: a report from a workshop convened by the Anthony Nolan Trust in London - 2005.

Over more than three decades, The Anthony Nolan Trust (ANT) has provided an unrelated donor (UD) for over 4000 children and adults lacking a suitable family member donor, and has remained at the forefront of developments in haematopoietic stem cell transplantation (HSCT) and bone marrow register management. These three decades have seen major changes in clinical practice of UD-HSCT, including new indications, increased use of alternative haematopoietic cell sources, significant improvement of the outcome as a result of better support care, less-toxic conditioning regimens, and better donor selection, and expansion to older patients with higher comorbidities. In order to foster our goal of improving UD-HSCT availability and outcome in a progressively more complex clinical scenario, a new initiative from ANT was launched in 2005 to convene an experts workshop to address the topical issues in this field. Four consecutive panels addressed factors influencing donor selection and transplant outcome, the use of cord blood, regulatory and accreditation issues, and future developments in this field. This report summarizes the discussions held in this workshop, which will likely develop into a periodic event where transplant clinicians, scientists and registry members will meet to share their experience and vision in the field of UD-HSCT.

Monitoring patients in complete cytogenetic remission after treatment of CML in chronic phase with imatinib: patterns of residual leukaemia and prognostic factors for cytogenetic relapse.

We monitored BCR-ABL transcript levels by quantitative real-time PCR in 103 patients treated with imatinib for chronic myeloid leukaemia in chronic phase for a median of 30.3 months (range 5.5-49.9) after they achieved complete cytogenetic remission (CCyR). The patients could be divided into three groups: (1) in 32 patients transcript levels continued to decline during the period of observation (nadir BCR-ABL/ABL ratio 0.015%); in five of these patients BCR-ABL transcripts became undetectable on repeated testing, (2) in 42 patients the transcript levels reached a plateau and (3) in 26 patients transcript numbers increased and the initial CCyR was lost. Three patients were not evaluable. Patients who remained in CCyR for at least 24 months appeared to have a low risk of subsequent cytogenetic relapse. We conclude that the pattern of 'residual' disease after achieving CCyR on imatinib is variable: some patients in CCyR show a progressive reduction in the level of residual disease, some reach a plateau where transcript numbers are relatively stable and others relapse with Ph-positive metaphases.

The involvement of "tumor suppressor" p53 in normal and chronic myelogenous leukemia hemopoiesis.

We investigated the expression of p53 in paraformaldehyde-lysine-periodate fixed normal and chronic myelogenous leukemia (CML) hemopoietic cells with flow cytometry and two monoclonal antibodies, PAb1801 and the mutant-conformation-associated PAb240. With both antibodies p53 proteins were detected in more than 50% of CD34+ cells and in more than 95% neutrophils but were undetectable in the CD34- myeloid precursors. The expression of a p53 protein reactive with PAb240 was closely associated with CD34+/HLA-DR+ cells and with cells in active cell cycle, while the p53 protein recognized by PAb1801 was mainly found in CD34+/HLA-DR- cells and in cells in the G0/G1 phases of the cell cycle. Treatment of chronic-phase CML cells with p53 antisense oligonucleotides resulted in significantly increased numbers of granulocyte-macrophage colony-forming unit colonies in 12 of 17 cases studied. Slightly reduced granulocyte-macrophage colony-forming unit colony numbers were observed in one case and no change in the four others. In eight samples of normal bone marrow cells, treatment with antisense oligonucleotides showed no consistent changes in granulocyte-macrophage colony-forming unit numbers. Our data suggest that the expression of the tumor suppressor p53 is involved in the regulation of both normal and CML hemopoiesis and that the inhibition of p53 expression could modulate the proliferation of CML hemopoietic cells and possibly of normal cells.

Selective induction of leukemia-associated fusion genes by high-dose ionizing radiation.

There is strong clinical and epidemiological evidence that ionizing radiation can cause leukemia by inducing DNA damage. This crucial initiation event is believed to be the result of random DNA breakage and misrepair, whereas the subsequent steps, promotion and progression, must rely on mechanisms of selective pressure to provide the expanding leukemic population with its proliferative/renewal advantage. To investigate the susceptibility of human cells to external agents at the genetic recombination stage of leukemogenesis, we subjected two hematopoietic cell lines, KG1 and HL60, to high doses of gamma-irradiation. The irradiation induced the formation of fusion genes characteristic of leukemia in both cell lines, but at a much higher frequency in KG1 than in HL60. In KG1 cells, the AML1-ETO hybrid gene [associated with the t(8;21) translocation of acute myeloid leukemia] occurred significantly more often than the BCR-ABL [associated with t(9;22) chronic myeloid leukemia] or the DEK-CAN [associated with t(6;9) acute myeloid leukemia] fusion genes. These findings support the notion that ionizing radiation can directly generate leukemia-specific fusion genes but emphasize the differing susceptibility of different cell populations and the differing frequency with which the various fusion genes are formed. The selectivity observed at the primary level of gene fusion formation may explain at least in part the differential risk for development of some but not other forms of leukemia after high-dose radiation exposure.

BCR-ABL tyrosine kinase activity regulates the expression of multiple genes implicated in the pathogenesis of chronic myeloid leukemia.

The BCR-ABL chimeric protein is thought to play a central role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias, notably chronic myeloid leukemia (CML). There is compelling evidence that malignant transformation by BCR-ABL is critically dependent on its protein tyrosine kinase (PTK) activity. As a result, multiple signaling pathways are activated in a kinase-dependent manner, and thus the activation of such pathways may affect the expression of genes that confer the malignant phenotype. In this study, we used differential display to investigate the alterations of gene expression in BV173, a CML cell line derived from lymphoid blast crisis, after exposure to ST1571, which selectively inhibits ABL PTK activity. We show that the expression of a set of 12 genes is correlated with the kinase activity and that the profile of these genes reflects mechanisms implicated in the pathogenesis of CML. Several of the genes show a consistent pattern of altered regulation in all Ph-positive lymphoid cell lines, whereas others appear to be unique to BV173 cells. We conclude that BCR-ABL PTK activity drives the expression of specific target genes that contribute to the malignant transformation of Ph-positive cells. The identification of downstream molecules with a consistent regulation pattern may provide suitable targets for therapeutic intervention in the future.

Frequent deletion of hSNF5/INI1, a component of the SWI/SNF complex, in chronic myeloid leukemia.

During routine two-fusion fluorescence in situ hybridization analysis of patients with blast crisis of chronic myeloid leukemia (CML), we observed that yeast artificial chromosome 29GD7, which is distal to BCR at 22q11, failed to hybridize to the 9q+ derivative chromosome in 3 of 11 (27%) cases. This deleted region is close to hSNF5/INI1 (SMARCB1), a gene that encodes a widely expressed component of the SWI/SNF chromatin remodeling complex and that suffers biallelic mutations in malignant rhabdoid tumors. To determine whether hSNF5/INI1 was also deleted in patients with CML, we performed fluorescence in situ hybridization analysis with a specific cosmid probe. Deletion of hSNF5/INI1 on the 9q+ chromosome was found in 9 of 25 (36%) cases in blast crisis (lymphoid, n = 3; myeloid, n = 6). For the three of these nine patients for whom material was available prior to transformation, deletions were also seen in chronic phase, indicating that they are early events. Analysis of an additional 21 patients in chronic phase revealed heterozygous loss of hSNF5/INI1 in 5 (24%) cases. Of the 14 patients who had hSNF5/INI1 deletions, 7 showed a mosaic pattern of hybridization in which only a proportion of CML cells that harbored both the t(9;22) derivative chromosomes had a deletion, indicating that loss of hSNF5/INI1 was acquired during the course of the disease. Single-strand conformation polymorphism analysis of all nine hSNF5/INI1 exons and splice junctions failed to reveal any mutations for 31 patients in transformation, including 8 who had deletions, although two polymorphisms were identified. We conclude that deletions of hSNF5/INI1 are frequent in patients with CML. Such deletions may be associated with reduced levels of hSNF5/INI1 expression, which could contribute to leukemogenesis by altering chromatin-mediated transcriptional control. Alternatively, the deletions could target another unidentified gene at 22q11 that plays a role in the pathogenesis of CML.

Direct relation between BCR-ABL tyrosine kinase activity and cyclin D2 expression in lymphoblasts.

Leukemia cells bearing the Philadelphia (Ph) chromosome express a Bcr-Abl fusion protein with deregulated protein tyrosine kinase (PTK) activity, which plays a central role in the malignant transformation. Many different signal transduction pathways are activated by Bcr-Abl, but little is known about their downstream targets in specific cell lineages. We show here that Ph-positive cell lines as well as primary cells derived from chronic myeloid leukemia (CML) in lymphoid blast crisis or from acute lymphoblastic leukemia (ALL) consistently express high levels of cyclin D2, whereas expression of this protein is low or absent in comparable Ph-negative lines and Ph-positive myeloid lines. Inhibition of Bcr-Abl with STI571 resulted in down-regulation of cyclin D2 and reduction of the number of cells in S phase, although complete G1 arrest was not induced. The expression of cyclin D2 in Ph-positive lymphoblasts was mediated via the phosphatidyl-inositol-3 kinase pathway. Analogous results were seen in murine BaF/3 cells transfected with a BCR-ABL expression vector. In contrast to the human cell lines, murine Baf/BCR-ABL cells exposed to STI571 inhibitor were all arrested in G1. This arrest could be abrogated by exogenous expression of cyclin D2 from a transfected cDNA construct. We conclude that a direct connection exists between Bcr-Abl PTK activity and cell cycle progression in which cyclin D2 plays a critical role. However, cell cycle progression in human Ph-positive lymphoid cells is not entirely dependent on Bcr-Abl PTK, and additional genetic lesions must be present.

Fusion of H4/D10S170 to the platelet-derived growth factor receptor beta in BCR-ABL-negative myeloproliferative disorders with a t(5;10)(q33;q21).

We have studied a patient who presented with clinical features suggestive of chronic myeloid leukemia in accelerated phase. BCR-ABL transcripts were undetectable by reverse transcription-PCR, but a novel reciprocal translocation, t(5;10)(q33;q21.2), was seen by standard cytogenetic analysis. Chromosome band 5q33 contains the gene encoding the platelet-derived growth factor beta receptor (PDGFbetaR), the receptor tyrosine kinase that is disrupted by the t(5;7), t(5;12), and t(5;14) in myeloid disorders, resulting in the fusion of PDGFbetaR to HIP1, TEL/ETV6, and CEV14, respectively. Southern analysis with PDGFbetaR cDNA revealed novel bands in patient but not control DNA after digestion with several restriction enzymes, indicating that this gene is also targeted by the t(5;10). Fluorescence in situ hybridization analysis of chromosome 5 indicated that a small inversion at 5q33 had taken place in addition to the interchromosomal translocation. The site of the chromosome 10 breakpoint fell within YAC 940e4. Because all PDGFbetaR fusions described thus far result in splicing to a common exon of this gene, we performed 5'-rapid amplification of cDNA ends PCR on patient RNA. Several clones were isolated in which PDGFbetaR fused in frame to H4/D10S170, a previously described ubiquitously expressed gene that is fused to the ret protein tyrosine kinase to form the PTC-1 oncogene in approximately 20% of papillary thyroid carcinomas. The presence of H4-PDGFbetaR chimeric mRNA in the patient was confirmed by reverse transcription-PCR; reciprocal PDGFbeta1R-H4 transcripts were not detected. We conclude that t(5;10)(q33;q21.2) is a novel translocation in BCR-ABL-negative chronic myeloid leukemia and that this abnormality results in an H4-PDGFbetaR fusion gene. This finding further strengthens the association between myeloproliferative disorders and deregulated tyrosine kinases.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE: To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS: A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS: Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION: Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

Bone marrow transplantation for chronic myeloid leukaemia.

Only a minority of all patients with CML can today be treated by allogeneic bone marrow transplantation (BMT) but the probability of cure for such patients is high. The complications of BMT are similar to those that occur following transplant for other diseases, notably GVHD, pneumonitis and infections. Of special interest is the demonstration that a graft-versus-leukaemia effect plays a role in the cure of CML. Studies using the polymerase chain reaction to detect minimal residual disease (BCR/ABL transcripts) may prove useful in predicting relapse and optimizing conditioning schedules. It is now important to test whether BMT can be equally successful in older patients (over 50 years) and in those lacking HLA-identical sibling donors. For other patients autografting may offer the possibility of achieving complete cytogenetic remission and perhaps prolonging life.

Specific point mutations that activate v-abl are not found in Philadelphia-negative chronic myeloid leukaemia, Philadelphia-negative acute lymphoblastic leukaemia or blast transformation of chronic myeloid leukaemia.

The involvement of the BCRlABL fusion gene in patients with Philadelphia (Ph) chromosome positive chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL) is well characterised, but the molecular events underlying the cases of Ph-negative CML and ALL that lack BCR gene involvement and those that cause transformation of Ph-positive CML are unknown. The murine ABL gene can be activated by genetic events that do not involve the BCR gene, including the introduction of two specific point mutations in exons VII and XI respectively, as found in the homologous sequence of the v-abl oncogene. We therefore sought evidence for analogous point mutations in the ABL gene in patients with Ph-negative, BCR-negative CML (n = 25), Ph-negative ALL (n = 18) and in Ph-positive CML in transformation (n = 28). We used restriction fragment length polymorphism and single strand conformational polymorphism techniques to analyse DNA amplified fragments of selected ABL coding regions from leukaemia cells. We identified only normal wild-type DNA sequences. The absence of these transforming point mutations does not exclude the possibility that the ABL gene in such patients could be activated by other means.