Simultaneous FLT3, NPM1 and DNMT3A mutations in adult patients with acute myeloid leukemia – case study

Abstract

Background: Nowadays, cytogenetics and molecular genetics, but not only, are mandatory in acute myeloid leukemia (AML) management, as a consequence of their impact on AML pathogenesis, classification, risk-stratification, prognosis and treatment. Objective: The aim of our study was to present our algorithm for the analysis of copy number changes, aneuploidies and somatic mutations focusing on a rare AML case positive for four somatic mutations. Methods: Cytogenetic analysis, Multiplex Ligationdependent Probe Amplification (MLPA) analysis, somatic mutation analysis (for FLT3 ITD, FLT3 D835, DNMT3A R882 and NPM1 c.863_864ins) by using several PCR techniques and also next-generation sequencing (NGS) analysis were performed. Results: Cytogenetic analysis did not reveal structural or numerical chromosomal anomalies. The patient’s DNA showed no copy number changes or aberrations (CNAs) following the MLPA analysis. By using several molecular technologies we found four mutations: FLT3-ITD, FLT3 D835 (c.2504A>T, D835V), DNMT3A R882C, and NPM1 c.863_864insTCTG. Challenges, benefits, applications and the limitations of each molecular technique used for the investigation of the mentioned mutation, and not only, are also described. Conclusion: All these techniques can be useful in the diagnosis of AML patients, each of them covering the limits of the other technique. New strategies for a positive, fast, accurate and reliable diagnosis are mandatory in cases with AML.

Cytokine rs361525, rs1800750, rs1800629, rs1800896, rs1800872, rs1800795, rs1800470, and rs2430561 SNPs in relation with prognostic factors in acute myeloid leukemia

“…FLT3 (ITD, D835), DNMT3A (R882), and NPM1 type A (c.863_864insTCTG) mutations were analyzed as previously reported . Fragment analysis of FLT3 ITD and NPM1 type A mutation was performed in all cases and allow us to establish the ratio of mutated to normal alleles.…”

“…20 FLT3 (ITD, D835), DNMT3A (R882), and NPM1 type A (c.863_864insTCTG) mutations were analyzed as previously reported. [21][22][23][24] Fragment analysis of FLT3 ITD and NPM1 type A mutation was performed in all cases and allow us to establish the ratio of mutated to normal alleles. High resolution melting (in-house method) was used to re-genotype 10% of the AML cases for FLT3 and DNMT3A mutations.…”

The Value of FLT3, NPM1 and DNMT3A Gene Mutation Analysis in Acute Myeloid Leukemia Diagnosis

“…Taking into account that there are few data regarding the NPM1, DNMT3A, and FLT3 combined mutations in AML additional mutation studies are necessary, to determine the appropriate prognosis value of concomitant mutations in AML, especially those focused on investigation of all three genes. Recently, a rare AML case with concomitant four somatic mutations [(namely FLT3 ITD, FLT3 D835 (also known as c.2504A>T, D835V), DNMT3A R882C, and NPM1 c.863_864insTCTG)] was reported to be associated with an adverse prognostic and a very short survival (23). In summary, reviewing the published data, it is not recommended to use FLT3 mutation as a marker for minimal residual disease (MRD) monitoring.…”

Presence of copy number aberration and clinical prognostic factors in patients with acute myeloid leukemia: an analysis of effect modification

“…A few MLPA studies have been performed in patients with AML, but they included a small number of samples. 10,11,16 Recurrent CNAs might represent an important prognostic marker in AML and might guide clinical decision making, especially in AML. Their identification by molecular methods could be helpful in cases with inconclusive cytogenetic analysis.…”