Comprehensive analysis of mutations and clonal evolution patterns in a cohort of patients with cytogenetically normal acute myeloid leukemia.

BACKGROUND: Relapsed acute myeloid leukemia (AML) is associated with the acquisition of additional somatic mutations which are thought to drive phenotypic adaptability, clonal selection and evolution of leukemic clones during treatment. We performed high throughput exome sequencing of matched presentation and relapsed samples from 6 cytogenetically normal AML (CN-AML) patients treated with standard remission induction chemotherapy in order to contribute with the investigation of the mutational landscape of CN-AML and clonal evolution during AML treatment. RESULT: A total of 24 and 32 somatic variants were identified in presentation and relapse samples respectively with an average of 4.0 variants per patient at presentation and 5.3 variants per patient at relapse, with SNVs being more frequent than indels at both disease stages. All patients have somatic variants in at least one gene that is frequently mutated in AML at both disease presentation and relapse, with most of these variants are classic AML and recurrent hotspot mutations including NPM1 p.W288fs, FLT3-ITD, NRAS p.G12D and IDH2 p.R140Q. In addition, we found two distinct clonal evolution patterns of relapse: (1) a leukemic clone at disease presentation acquires additional mutations and evolves into the relapse clone after the chemotherapy; (2) a leukemic clone at disease presentation persists at relapse without the addition of novel somatic mutations. CONCLUSIONS: The findings of this study suggest that the relapse-initiating clones may pre-exist prior to therapy, which harbor or acquire mutations that confer selective advantage during chemotherapy, resulting in clonal expansion and eventually leading to relapse.

Identification of FLT3 and NPM1 Mutations in Patients with Acute Myeloid Leukaemia.

Objective: The most frequent acquired molecular abnormalities and important prognostic indicators in patients with Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1) mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method for detection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patients with cytogenetically normal (CN) AML in our population. Methods: A total of 199 samples from AML patients (95 women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction (PCR) and gene sequencing. Result: Sixty-eight patients were positive for the mutations. FLT3-ITD mutations were detected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations of NPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencing and showed 100% concordance with in house method. Conclusion: The optimized in-house PCR assays for the detection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and cost effective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification of these mutations are important for prognostication and optimization of patient care.

Prevalence of BCR-ABL T315I Mutation in Malaysian Patients with Imatinib-Resistant Chronic Myeloid Leukemia

Objective: Chronic Myeloid Leukemia (CML) is caused by a reciprocal translocation between chromosomes 9 and 22, t(9;22) (q34;q11) which encodes for the BCR-ABL fusion protein. Discovery of Imatinib Mesylate (IM) as first line therapy has brought tremendous improvement in the management of CML. However, emergence of point mutations within the BCR-ABL gene particularly T315I mutation, affects a common BCR-ABL kinase contact residue which impairs drug binding thus contribute to treatment resistance. This study aims to investigate the BCR-ABL T315I mutation in Malaysian patients with CML. Methods: A total of 285 patients diagnosed with CML were included in this study. Mutation detection was performed using qualitative real-time PCR (qPCR). Results: Fifteen out of 285 samples (5.26%) were positive for T315I mutations after amplification with real-time PCR assay. From the total number of positive samples, six patients were in accelerated phase (AP), four in chronic phase (CP) and five in blast crisis (BC). Conclusion: Mutation testing is recommended for choosing various tyrosine kinase inhibitors (TKIs) to optimize outcomes for both cases of treatment failure or suboptimal response to imatinib. Therefore, detection of T315I mutation in CML patients are clinically useful in the selection of appropriate treatment strategies to prevent disease progression.