Hsa-miR-181a-5p, hsa-miR-182-5p, and hsa-miR-26a-5p as potential biomarkers for BCR-ABL1 among adult chronic myeloid leukemia treated with tyrosine kinase inhibitors at the molecular response.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) as first-line therapy for Chronic Myeloid Leukemia (CML) show a high success rate. However, a low number of patients with long-term treatment-free remission (TFR) were observed. Molecular relapse after imatinib discontinuation occurred at 50% at 24 months, with 80% occurrence within the first 6 months. One of the reasons for relapse is untimely TKIs discontinuation caused by large errors from estimates at very low-level or undetectable disease, thus warranting new biomarkers for CML. METHODS: Next Generation Sequencing (NGS) was used to identify microRNAs (miRNAs) at the molecular response in CML adult patients receiving TKIs treatment. A total of 86 samples were collected, 30 from CML patients responsive and 28 from non-responsive to imatinib therapy, and 28 from blood donors. NGS was conducted whereby 18 miRNAs were selected and validated by real-time RT-qPCR in triplicate. RESULTS: Hsa-miR-181a-5p was expressed significantly (p-value< 0.05) with 2.14 and 2.33-fold down-regulation in both patient groups, respectively meanwhile hsa-miR-182-5p and hsa-miR-26a-5p were significant only in the non-responsive group with 2.08 and 2.39 fold up-regulation. The down-regulation was consistent with decreased amounts of BCR-ABL1 in patients taking TKIs regardless of molecular responses. The up-regulation was consistent with the substantial presence of BCR-ABL1 in CML patients treated with TKIs at the molecular response. CONCLUSIONS: Therefore, these miRNAs have potential as new therapeutic biomarkers for BCR-ABL1 status in adult CML patients treated with TKIs at molecular responses. These could improve current approaches and require further analysis to look for targets of these miRNAs in CML.

Characterization of New Alpha Zero (α0) Thalassaemia Deletion (--GB) among Malays in Malaysian Population.

Malaysia is a multicultural and multiethnic country comprising numerous ethnic groups. From the total population of 32.7 million, Malays form the bulk of the Bumiputera in Malaysia comprise about 69.9%, followed by Chinese 22.8%, Indian 6.6%, and others 0.7%. The heterogeneous population and increasing numbers of non-citizens in this country affects the heterogeneity of genetic diseases, diversity, and heterogeneity of thalassaemia mutations. Alpha (α)-thalassaemia is an inherited haemoglobin disorder characterized by hypochromic microcytic anaemia caused by a quantitative reduction in the α-globin chain. A majority of the α-thalassaemia are caused by deletions in the α-globin gene cluster. Among Malays, the most common deletional alpha thalassaemia is -α3.7 deletion followed by --SEA deletion. We described the molecular characterization of a new --GB deletion in our population, involving both alpha genes in cis. Interestingly, we found that this mutation is unique among Malay ethnicities. It is important to diagnose this deletion because of the 25% risk of Hb Bart's with hydrops fetalis in the offspring when in combination with another α0- thalassaemia allele. MLPA is a suitable method to detect unknown and uncommon deletions and to characterize those cases which remain unresolved after a standard diagnostic approach.

Molecular and hematological studies in a cohort of beta zero South East Asia deletion (ß°-thal SEA) from Malaysian perspective.

Abstract: We report the haematological parameters and molecular characterization of beta zero (ß°) South East Asia (SEA) deletion in the HBB gene cluster with unusually high levels of Hb F compared to a classical heterozygous beta zero (ß°)-thalassaemia. Methods: Retrospective study on 17 cases of (ß°) South East Asia (SEA) deletion from 2016 to 2019 referred to Institute for Medical Research were conducted. The clinical information and haematological profiles were evaluated. The mutation was analyzed, and the results were compared with other ß°-thalassaemia groups. For HBB gene genotyping, all the cases were subjected for multiplex gap-PCR, 5 cases were subjected for HBB gene sequencing for exclusion of compound heterozygous with other beta variants. Co-inheritance of α-thalassaemia were determined using multiplex gap-PCR and multiplex ARMS-PCR. Results: Seventeen cases were positive for ß°-thal SEA deletion. Fifteen cases were heterozygous and two were compound heterozygous for ß°-thal SEA deletion. The results were compared with 182 cases of various heterozygous ß° deletions and mutations. The mean Hb for heterozygous ß°-thal SEA deletion (13.44 ± 1.45 g/dl) was normal and significantly higher than heterozygous IVS 1-1 and Codon 41/42 (post hoc test, p < 0.05). The medians for the MCV and MCH of ß°-thal SEA deletion were significantly higher than for all heterozygote ß°-thalassaemia traits (Mann Whitney test, p < 0.05). Patients with ß°-thal SEA deletion had elevated levels of Hb A2 consistent with ß-thalassaemia traits, with Hb F levels consistent with HPFH or δß-thalassaemia carriers. The median for Hb A2 was 4.00 + 1.00%, similar to that observed in other ß°-thalassaemia groups except for IVS 1-1 mutation (median 5.30 + 0.45%) and ß°-Filipino (∼45 kb deletion) deletion (median 6.00 + 0.58). Interestingly, we found that Hb F levels for ß°-thal SEA deletion were statistically higher than other ß°-thalassaemia mutations (median 19.00 + 5.50%, p < 0.05), except for the ß°-thal 3.5 kb deletion group. Conclusion: We conclude that ß°-thal SEA deletion has a unique haematological parameters of beta zero thalassaemia trait. We affirm to classifying this deletion as SEA-HPFH based on previous studies considering the phenotype features rather than the molecular defect of ß°-thal SEA deletion, as this will make it easier to offer genetic counselling to affected individuals.

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.

Construction of a microRNA-mRNA Regulatory Network in De Novo Cytogenetically Normal Acute Myeloid Leukemia Patients.

Background: The association between dysregulated microRNAs (miRNAs) and acute myeloid leukemia (AML) is well known. However, our understanding of the regulatory role of miRNAs in the cytogenetically normal AML (CN-AML) subtype pathway is still poor. The current study integrated miRNA and mRNA profiles to explore novel miRNA-mRNA interactions that affect the regulatory patterns of de novo CN-AML. Methods: We utilized a multiplexed nanoString nCounter platform to profile both miRNAs and mRNAs using similar sets of patient samples (n = 24). Correlations were assessed, and an miRNA-mRNA network was constructed. The underlying biological functions of the mRNAs were predicted by gene enrichment. Finally, the interacting pairs were assessed using TargetScan and microT-CDS. We identified 637 significant negative correlations (false discovery rate <0.05). Results: Network analysis revealed a cluster of 12 miRNAs representing the majority of mRNA targets. Within the cluster, five miRNAs (miR-495-3p, miR-185-5p, let-7i-5p, miR-409-3p, and miR-127-3p) were posited to play a pivotal role in the regulation of CN-AML, as they are associated with the negative regulation of myeloid leukocyte differentiation, negative regulation of myeloid cell differentiation, and positive regulation of hematopoiesis. Conclusion: Three novel interactions in CN-AML were predicted as let-7i-5p:HOXA9, miR-495-3p:PIK3R1, and miR-495-3p:CDK6 may be responsible for regulating myeloid cell differentiation in CN-AML.

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.

Whole-Exome Sequencing of ETV6/RUNX1 in Four Childhood Acute Lymphoblastic Leukaemia Cases

Background: ETV6/RUNX1 gene fusion is the most frequently seen chromosomal abnormality in childhood acute lymphobastic leukamia (ALL). However, additional genetic changes are known to be required for the development of this type of leukaemia. Therefore, we here aimed to assess the somatic mutational profile of four ALL cases carrying the ETV6/RUNX1 fusion gene using whole-exome sequencing. Methods: DNA was isolated from bone marrow samples using a QIAmp DNA Blood Mini kit and subsequently sequenced using the Illumina MiSeq system. Results: We identified 12,960 to17,601 mutations in each sample, with a total of 16,466 somatic mutations in total. Some 15,533 variants were single nucleotide polymorphisms (SNPs), 129 were substitutions, 415 were insertions and 389 were deletions. When taking into account the coding region and protein impact, 1,875 variants were synonymous and 1,956 were non-synonymous SNPs. Among non-synonymous SNPs, 1,862 were missense, 13 nonsense, 35 frameshifts, 11 nonstop, 3 misstart, 15 splices disrupt and 17 in-frame indels. A total of 86 variants were located in leukaemia-related genes of which 32 variants were located in the coding regions of GLI2, SP140, GATA2, SMAD5, KMT2C, CDH17, CDX2, FLT3, PML and MOV10L1. Conclusions: Detection and identification of secondary genetic alterations are important in identifying new therapeutic targets and developing rationally designed treatment regimens with less toxicity in ALL patients.