ALBERT

Description: Homoharringtonine is a plant alkaloid that inhibits the synthesis of proteins and results in apoptosis. In vitro homoharringtonine shows synergistic or additive effects with imatinib in CML cell lines and in blast cells derived from patients with advanced disease. We undertook a phase I/II study in patients who had achieved a sub-optimal response to imatinib alone to investigate whether the addition of semi-synthetic homoharringtonine (sHHT) (Myelostat®) would reduce the level of residual disease. Patients with CML who had achieved at least 35% Ph-negativity on imatinib were included. All patients were treated with imatinib at ≥ 400mg/day for at least 2 years; and achieved a plateau in BCR-ABL transcripts defined by measuring BCR-ABL transcripts on at least 4 occasions over a minimum period of 1 year with the latest value not lower than the previous minimum value. When sHHT was introduced, imatinib was continued at the previous dosage. sHHT was given subcutaneously at a dose of 1.25 mg/m2 twice daily for one day. Courses were repeated every 28 days. The dose of sHHT was escalated by adding one day of treatment every two courses (eg dose level 2: 1.25mg/m2 BD x 2 days) if the ANC was ≥2.5x109/l and the platelet count ≥200x109/l by day +28 of the previous course, or adding half a day if the ANC was ≥1.5x109/l and the platelet count ≥ 100x109/l. Patients were considered to have reached the maximal tolerable dose (MTD) when the sHHT could not be escalated further. Response was assessed by serial monitoring of peripheral blood levels of BCR-ABL transcripts assayed by RT-PCR at the start of each course. Results 10 patients have been enrolled so far. Patients received a median of six courses of sHHT (range 4–8). The MTD has been achieved in all cases (9 cases in account of myelosuppression and in one case in account of grade III asthenia). The median MDT was 2 days (range 1.5–3 days). The transcript level decline in all patients (table 1). In 7 patients the transcript levels declined by 〉0.5 log and in 5 patients by 〉1 log with respect the baseline value. In 2 patients transcripts became undetectable. The combination was relatively well tolerated with the principal toxicities being grade II asthenia (n=9), grade III astenia (n=1), grade III neutropenia (n=3) and grade III thrombocytopenia (n=1). We believe these preliminary clinical observations justify further study of the use of sHHT in patients on imatinib who fail to obtain low levels of minimal residual disease. Table 1 n Time from onset of imatinib to sHHT therapy (months) Median R-PCR over the year preceding sHHT therapy Minimal R-PCR during the year preceding sHHT therapy R-PCR at screening for sHHT R-PCR at last follow up Transcript levels are expressed as a BCR-ABL/ABL percentage 1 27.2 7.3 5.4 16.9 0.6 2 35.9 2.2 0.04 4.5 1.18 3 34.1 0.012 0.01 0.028

Description: BCR-ABL kinase domain mutation in a CML patient with clinical resistance to imatinib is generally accepted as ‘causal’. Here we report the kinetics of the mutant BCR-ABL transcripts in 6 CML patients with the M244V mutation that are at variance with the accepted view. Prior to starting imatinib, one patient in chronic phase (CP) was previously untreated, 4 patients in CP had previously received alfa-interferon, and the sixth had received an autograft but had subsequently progressed to accelerated phase (AP). Patients were started on 400 mg/day dosage which was escalated to 600, 800 or 1000 mg/day following little or no response. Philadelphia chromosome was the only cytogenetic abnormality detected at the time of diagnosis and at start of imatinib treatment. The median time from diagnosis to starting imatinib was 19 months (range: 3 – 120 months). The M224V mutations were detected initially as a result of routine screening by Sanger’s direct sequencing of cDNA from blood of patients on imatinib, at which time the mutant clone in each patient already constituted 〉80% of BCR-ABL transcripts, measured by QR-PCR. We then retrospectively measured levels of mutated BCR-ABL transcripts in all the stored samples, median 14.5 samples (5 – 28), by pyrosequencing. Of the 4 patients for whom there were pre-imatinib samples the BCR-ABLM244V allele was identified in one case (previously treated with IFN-alfa) at 3%, but was not detected in the other three. In the 2 patients for whom there were no pre-imatinib samples, the BCR-ABLM244V allele was present at 3.0% and 93% respectively in the earliest available sample. In the 5 patients whose earliest samples showed low or absent mutant transcripts the non-mutated BCR-ABL population was progressively replaced by BCR-ABLM244V-positive cells, eventually becoming the dominant clone with a median time from first sample to maximal level of 35 months (11 – 54 months). In three patients total BCR-ABL transcripts decreased and they achieved complete cytogenetic remission (CCyR). In the other three patients (including the newly diagnosed patient) there was little or no reduction in BCR-ABL transcript numbers and all failed to achieve CCyR. One patient in this latter group acquired trisomy 8, reported 40 months prior to blastic phase; she died following allogeneic SCT. The response to increased imatinib dose in three patients is consistent with the reported IC50 for the M244V mutation, i.e. moderate resistance, but the response in the other three patients is not. In summary, the response to imatinib in 6 patients with the same BCR-ABL mutation is variable. Furthermore, these observations imply the mutation is unlikely to be the sole cause of the resistance in at least in 3 of the patients. The observed heterogeneous phenotype of these 6 CML patients is likely to be due to a myriad of undefined contributory factors.