Integrative genomic analysis reveals cancer-associated mutations at diagnosis of CML in patients with high-risk disease.

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P = .007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1 The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P = .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach.

Dynamic modeling of imatinib-treated chronic myeloid leukemia: functional insights and clinical implications.

Treatment of chronic myeloid leukemia (CML) with the tyrosine kinase inhibitor imatinib represents a successful application of molecularly targeted cancer therapy. A rapid hematologic and cytogenetic response can be induced in the majority of people, even in advanced disease. However, complete eradication of malignant cells, which are characterized by the expression of the BCR-ABL1 fusion protein, is rare. Reasons for the persistence of the malignant clone are currently not known and provide a substantial challenge for clinicians and biologists. Based on a mathematical modeling approach that quantitatively explains a broad range of phenomena, we show for two independent datasets that clinically observed BCR-ABL1 transcript dynamics during imatinib treatment of CML can consistently be explained by a selective functional effect of imatinib on proliferative leukemia stem cells. Our results suggest the general potential of imatinib to induce a complete elimination of the malignant clone. Moreover, we predict that the therapeutic benefit of imatinib can, under certain circumstances, be accelerated by combination with proliferation-stimulating treatment strategies.

Comparison of bone marrow high mitotic index metaphase fluorescence in situ hybridization to peripheral blood and bone marrow real time quantitative polymerase chain reaction on the International Scale for detecting residual disease in chronic myeloid leukemia.

BACKGROUND: Recently, an International Scale was proposed for standardizing BCR-ABL transcript measurements and reporting in the assessment of minimal residual disease by real-time quantitative polymerase chain reaction (RQ-PCR). Here we present the setting up of the International Scale conversion factors for a national laboratory by performing both a cross-analysis of a set of standard samples from a reference laboratory and an analysis of bone marrow and peripheral blood samples at diagnosis (from 32 and 27 patients, respectively). DESIGN AND METHODS: A total of 222 bone marrow and 173 peripheral blood mononuclear cell samples from 96 patients with chronic myeloid leukemia were analyzed with RQ-PCR according to Europe Against Cancer protocols. Additionally, 291 bone marrow samples were analyzed with high mitotic index metaphase fluorescence in situ hybridization (metaphase FISH). RESULTS: Major molecular response according to the International Scale in BCR-ABL/GUS transcript levels corresponded to a ratio of 0.035% in peripheral blood and 0.034% in bone marrow, yielding the same conversion factor of 2.86 for both types of sample. Based on metaphase FISH, values of 10%/-1.0 log, 1%/-2.0 log and 0.1%/-3.0 log on the International Scale, corresponded to 13%, 2%, and 0.3% of Philadelphia chromosome positive cells in bone marrow, respectively. CONCLUSIONS: In conclusion, conversion factors can be determined either by cross-analyzing a number of samples with a laboratory that has already established the International Scale or utilizing sufficient numbers of reference samples from chronic myeloid leukemia patients at diagnosis, or using the upcoming international standards.

Phospho-CRKL monitoring for the assessment of BCR-ABL activity in imatinib-resistant chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia patients treated with nilotinib.

Actual BCR-ABL kinase inhibition in vivo as determined by phospho-CRKL (pCRKL) monitoring has been recognized as a prognostic parameter in patients with chronic myelogenous leukemia treated with imatinib. We report a biomarker sub-study of the international phase I clinical trial of nilotinib (AMN107) using the established pCRKL assay in imatinib-resistant chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia. A minimum dose (200 mg) required for effective BCR-ABL inhibition in imatinib resistant/intolerant leukemia was determined. The pre-clinical activity profile of nilotinib against mutant BCR-ABL was largely confirmed. Substantial differences between peripheral blood baseline pCRKL/CRKL ratios were observed when comparing chronic myeloid leukemia with Ph+ acute lymphoblastic leukemia. Finally, rapid BCR-ABL-reactivation shortly after starting nilotinib treatment was seen in acute lymphoblastic leukemia patients with progressive disease carrying the P-loop mutations Y253H, E255K, or mutation T315I. Monitoring the actual BCR-ABL inhibition in nilotinib treated patients using pCRKL as a surrogate is a means to establish effective dosing and to characterize resistance mechanisms against nilotinib.

Resistance to targeted therapy in chronic myelogenous leukemia.

The advent of the Bcr-Abl selective tyrosine kinase inhibitor imatinib mesylate (Glivec, Gleevec, Novartis, East Hanover, NJ) has substantially changed the treatment landscape for chronic myelogenous leukemia (CML). However, some patients, primarily those with advanced disease, are either initially refractory to imatinib or eventually develop imatinib resistance. Imatinib resistance or intolerance frequently depends on the re-emergence of Bcr-Abl kinase activity, but can also indicate Bcr-Abl-independent disease progression. Results from phase II/III trials suggest rates of resistance and relapse correlate with stage of disease and with the monitoring parameters: hematologic, cytogenetic, and molecular responses. To date, more than 40 different point mutations that code for distinct single amino acid substitutions in the Bcr-Abl kinase domain have been isolated from imatinib-resistant patients. These mutations affect amino acids involved in imatinib binding or in regulatory regions of the Bcr-Abl kinase domain, resulting in decreased sensitivity to imatinib while retaining aberrant kinase activity. Early mutation detection may aid in risk stratification and molecular-based treatment decisions. To overcome imatinib-resistant disease, novel tyrosine kinase inhibitors with activity against imatinib-resistant mutations and/or with inhibition of alternative pathways, such as Src activation, have recently been developed. Additional strategies include imatinib dose escalation, combination therapy, and treatment interruption to stop clonal selection of resistant cells.

Enhanced ABL-inhibitor-induced MAPK-activation in T315I-BCR-ABL-expressing cells: a potential mechanism of altered leukemogenicity.

BACKGROUND: Targeted treatment of chronic myelogenous leukemia using imatinib has dramatically improved patient outcome. However, residual disease can be detected in the majority of patients treated with imatinib. Compensatory activation of MAP kinases (MAPK1/2) in response to BCR-ABL-inhibitors has been reported as a potential cytokine-dependent resistance mechanism leading to the rescue of leukemic progenitor cells. METHODS: Differential MAPK-modulating activity of clinically approved tyrosine kinase inhibitors was assessed in vitro using BCR-ABL-transformed cells. CD34+-enriched progenitors of newly diagnosed chronic myelogenous leukemia patients were exposed to tyrosine kinase inhibitors. MAPK-signaling was studied by Western blot technique. Proliferation assays were used to analyze response to antileukemic treatment. RESULTS: The ABL-inhibitors imatinib and nilotinib activate MAPKs in CD34+ chronic myelogenous leukemia progenitor cells, whereas treatment with the SRC/ABL-inhibitor dasatinib does not affect MAPK-activation at clinically relevant concentrations. Similar results are seen in BCR-ABL-transformed cells in the presence of interleukin-3 (IL-3). Experiments using BCR-ABL-mutant T315I, a resistance mutation not amenable to tyrosine kinase inhibitor binding, demonstrate that ABL-inhibitor-induced MAPK-activation does not depend on BCR-ABL-inhibition and cannot be prevented by selective SRC-inhibition. However, BCR-ABL-T315I enhances MAPK-activation, suggesting a T315I-dependent positive feedback of MAPK-activation. An autocrine IL-3-loop as trigger for aberrant T315I-dependent MAPK-activation was excluded. CONCLUSIONS: Aberrant MAPK-activation triggered by ABL-inhibitors and positively regulated by BCR-ABL kinase mutation T315I might be an experimental explanation for the clinical observation that patients carrying high-resistance mutations show a highly aggressive course of their disease when tyrosine kinase inhibitor treatment is not discontinued in time.