Angela Poerio

Contribution of ABL Kinase Domain Mutations to Imatinib Resistance in Different Subsets of Philadelphia-Positive Patients: By the GIMEMA Working Party on Chronic Myeloid Leukemia

Purpose: ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. Experimental Design: Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. Results: Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated with imatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. Conclusions: We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop or T315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.

Nilotinib for the frontline treatment of Ph(+) chronic myeloid leukemia.

Nilotinib has a higher binding affinity and selectivity for BCR-ABL with respect to imatinib and is an effective treatment of chronic myeloid leukemia (CML) after imatinib failure. In a phase 2 study, 73 early chronic-phase, untreated, Ph(+) CML patients, received nilotinib at a dose of 400 mg twice daily. The primary endpoint was the complete cytogenetic response (CCgR) rate at 1 year. With a median follow-up of 15 months, the CCgR rate at 1 year was 96%, and the major molecular response rate 85%. Responses were rapid, with 78% CCgR and 52% major molecular response at 3 months. During the first year, the treatment was interrupted at least once in 38 patients (52%). The mean daily dose ranged between 600 and 800 mg in 74% of patients, 400 and 599 mg in 18% of patients, and was less than 400 mg in 8% of patients. Dose interruptions were mainly due to nonhematologic and biochemical side effects. Myelosuppression was irrelevant. One patient progressed to blastic crisis after 6 months; one went off-treatment for lipase increase grade 4 (no pancreatitis). Nilotinib is safe and very active in early chronic-phase CML. These data support a role for nilotinib for the frontline treatment of CML. This study was registered at ClinicalTrials.gov as NCT00481052.

Philadelphia-positive patients who already harbor imatinib-resistant Bcr-Abl kinase domain mutations have a higher likelihood of developing additional mutations associated with resistance to second- or third-line tyrosine kinase inhibitors.

Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphia-positive leukemias. To assess how Bcr-Abl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for T315I. Patients already harboring mutations had higher likelihood of relapse associated with development of further mutations compared with patients who did not harbor mutations (23 of 51 vs 8 of 44, respectively, for patients who relapsed on second TKI; 13 of 20 vs 1 of 6, respectively, for patients who relapsed on third TKI).

Achieving a Major Molecular Response at the Time of a Complete Cytogenetic Response (CCgR) Predicts a Better Duration of CCgR in Imatinib-Treated Chronic Myeloid Leukemia Patients

Purpose: Most patients with chronic-phase chronic myeloid leukemia (CML) who receive imatinib achieve a complete cytogenetic remission (CCgR) and low levels of BCR-ABL transcripts. CCgR is durable in the majority of patients but relapse occurs in a subset. Experimental Design: To determine the potential of quantitative reverse transcription-PCR of BCR-ABL to predict cytogenetic relapse, we serially monitored residual disease in 97 CML patients with an imatinib-induced CCgR. Patients with late chronic phase CML after IFN-α failure were treated with imatinib (400 mg daily). Results: During the imatinib median follow-up time of 36 months (range, 12-54 months), disease monitoring occurred by cytogenetics and quantitative PCR. Twenty percent of patients experienced cytogenetic relapse at a median of 18 months after CCgR and a median of 24 months after starting imatinib. None of the possible prognostic factors studied in univariate and multivariate analyses seemed to predict for loss of cytogenetic response but the reduction of BCR-ABL transcript levels at the time of CCgR is an important prognostic factor. Conclusions: In our study, we showed not only that achieving a major molecular remission at 12 months is predictive of a durable cytogenetic remission but also that patients who achieved a major molecular remission (expressed both as the BCR-ABL/β2 microglobulin ratio % <0.0005 and as a 3-log reduction from median baseline value) already at the time of first achieving a CCgR have significantly longer cytogenetic remission durations than those without this magnitude of molecular response (P < 0.05).

Expression of spliced oncogenic Ikaros isoforms in Philadelphia-positive acute lymphoblastic leukemia patients treated with tyrosine kinase inhibitors: implications for a new mechanism of resistance

Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph+ acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non-DNA-binding Ik6 isoform was detected in 49% of Ph+ ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph+ ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph+ ALL patients.

Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis

Background In patients with Philadelphia-positive acute lymphoblastic leukemia, resistance to treatment with tyrosine kinase inhibitors is frequent and most often associated with the development of point mutations in the BCR-ABL kinase domain. We aimed to assess: (i) in how many patients BCR-ABL kinase domain mutations are already detectable at relatively low levels at the time of diagnosis, and (ii) whether mutation detection correlates with subsequent response to therapy. Design and Methods We retrospectively analyzed samples collected at diagnosis from 15 patients with Philadelphia-positive acute lymphoblastic leukemia who subsequently received tyrosine kinase inhibitor therapy (dasatinib) by cloning the BCR-ABL kinase domain in a bacterial vector and sequencing 200 independent clones per sample. Results Mutations at relatively low levels (2–4 clones out of 200) could be detected in all patients – eight who relapsed and seven who achieved persistent remission. Each patient had evidence of two to eight different mutations, the majority of which have never been reported in association with resistance to tyrosine kinase inhibitors. In two patients out of six who relapsed because of a mutation, the mutation (a T315I) was already detectable in a few clones at the time of diagnosis. On the other hand, a patient who was found to harbor an F317L mutation is in persistent remission on dasatinib. Conclusions Our results suggest that the BCR-ABL kinase domain is prone to randomly accumulate point mutations in Philadelphia-positive acute lymphoblastic leukemia, although the presence of these mutations in a relatively small leukemic subclone does not always preclude a primary response to tyrosine kinase inhibitors.

Long-Term Outcome of Complete Cytogenetic Responders After Imatinib 400 mg in Late Chronic Phase, Philadelphia-Positive Chronic Myeloid Leukemia: The GIMEMA Working Party on CML

PURPOSE Imatinib mesylate (IM) has rapidly become the front-line treatment of Philadelphia-positive (Ph-pos) chronic myeloid leukemia, but the number of patients who were treated and are being treated with IM second-line is still substantial. PATIENTS AND METHODS We have monitored and analyzed the cytogenetic and molecular response to IM 400 mg/d in a cohort of 277 late chronic phase (LCP) patients who were resistant or intolerant to interferon-alpha and were observed for 48 to 79 months (median, 72 months). RESULTS One hundred fifty-three patients (55%) achieved a complete cytogenetic response (CCgR). Seventy-seven percent of them were still in CCgR after 5 years. The rate of response loss did not increase over time. The 6-year progression-free survival and overall survival of these 153 complete cytogenetic responders were 90% and 91%, respectively. Molecular response was less than major in 21%, major in 78%, and complete in one patient only. CONCLUSION These data confirm that, in LCP the CCgR rate to IM is 50% to 60%, and show that CCgR is stable and is associated with a prolonged survival, even if leukemia continues to be molecularly detectable.

Front-line treatment of Philadelphia positive chronic myeloid leukemia with imatinib and interferon-α: 5-year outcome

This study confirms the excellent response to imatinib front-line therapy in patients with chronic myeloid leukemia. By contrast, most patients discontinued pegylated interferon-α due to its side effects. In 2004, we reported the short-term results of a multicentric, phase 2 study of imatinib 400 mg daily and pegylated interferon-α in the treatment of 76 early chronic phase Philadelphia-positive chronic myeloid leukemia patients. In this report, we update the results with an observation time of five years. After two years of treatment, all but 10 patients (13%) had discontinued pegylated interferon-α. The complete cytogenetic response rate at five years was 87%, and 94% of complete cytogenetic responders maintained the complete cytogenetic response after five years. All but one complete cytogenetic response also achieved a major molecular response. These data confirm the excellent response to imatinib front-line and the stability of the complete cytogenetic response. Any possible additional benefit of the combination with interferon-α remains uncertain, due to low patient compliance.

Comparison Between Patients With Philadelphia-Positive Chronic Phase Chronic Myeloid Leukemia Who Obtained a Complete Cytogenetic Response Within 1 Year of Imatinib Therapy and Those Who Achieved Such a Response After 12 Months of Treatment

PURPOSE Imatinib mesylate is a potent inhibitor of BCR-ABL, the constitutively active tyrosine kinase protein critical for the pathogenesis of chronic myeloid leukemia. PATIENTS AND METHODS We reviewed 284 patients with late chronic-phase Philadelphia chromosome (Ph) -positive chronic myeloid leukemia treated with imatinib 400 mg daily after interferon-alpha failure. In a retrospective study, we evaluated the pattern and rapidity of the response to imatinib, comparing the cytogenetic and molecular responses, progression-free and overall survival rates in patients who obtained a complete cytogenetic response within 1 year of treatment (early responders), and in patients where a complete cytogenetic response was detected after 12 months (late responders). RESULTS After 3 or 4 years of treatment, the molecular response of the late cytogenetic responders was similar to that of the early cytogenetic responders. At 36 months of treatment the amount of residual disease measured by standardized quantitative reverse-transcriptase polymerase chain reaction was 0.00047 in late responders versus 0.00022 in early responders, and at 48 months it was 0.00019 versus 0.00026 (median values, P value = nonsignificant). The estimated 4-year progression-free survival rate was 88% for early responders and 100% for late responders, while the estimated 4-year overall survival rates were 92% and 100% for early and late responders, respectively. CONCLUSION The sensitivity and the response (cytogenic and molecular) to imatinib may require 1 year or more. Long-term follow-up results continue to improve in terms of rates and durability of the complete cytogenetic response, major or complete molecular response, and progression-free and overall survival.

The response to imatinib and interferon-α is more rapid than the response to imatinib alone: a retrospective analysis of 495 Philadelphia-positive Chronic Myeloid Leukemia patients in early chronic phase

Before the introduction of imatinib, interferon α-based regimens were the gold standard for treatment of early chronic phase chronic myeloid leukemia patients. The combination of IFN-α with imatinib is currently being investigated in at least two large clinical trials, the German CML Study IV and the French SPIRIT trial. We reviewed the cytogenetic and molecular responses of 76 early chronic phase chronic myeloid leukemia patients who were treated with imatinib and interferon-α and of 419 early chronic phase chronic myeloid leukemia patients treated with imatinib alone front-line. The complete cytogenetic response rate was higher in the IM+IFN-α group than in the imatinib group at six months (60% vs. 42%; P=0.003), but not at 48 months (88% vs. 88%). The durability of the complete cytogenetic response was similar in the two groups with 94% and 91% of complete cytogenetic responders in continuous complete cytogenetic response at 48 months (P=0.56). The major molecular response rate was higher in the IM+IFN-α group at six months (58% vs. 34%; P=0.0001) and 12 months (67% vs. 47%; P=0.001) but not later on (65% vs. 57% at 48 months; P=0.25). Overall and progression free survival were comparable in the two groups; a significant trend to a better event free survival was observed in patients treated with PegIFNα (91% vs. 78%; P=0.02). These data suggest that the response to the combination treatment is more rapid. It is not yet known how much a rapid reduction will influence the longer-term overall and progression free survival, and the cure rate.