DNMT3A, FLT3, KIT, KRAS, NRAS, NPM1, TP53 and WT1 Genes Mutations Detection in Acute Myeloid Leukemia with Normal Karyotype
A.V. Vinogradov, A.V. Rezaykin, D.R. Salakhov, D.V. Izotov, S.E. Ioschenko, A.G. Sergeev
pp. 89-101
Sverdlovsk Regional Ministry of Health, Yekaterinburg, Russian Federation;
Ural State Medical University, Yekaterinburg, Russian Federation;
Sverdlovsk Regional Clinical Hospital No. 1, Yekaterinburg, Russian Federation;
Institute of Medical Cell Technology, Yekaterinburg, Russian Federation
DOI: 10.22138/2500-0918-2016-14-2-89-101
Abstract. Aim: to estimate the frequency of mutations in DNMT3A, FLT3, KIT, KRAS, NRAS, NPM1, TP53 and WT1 genes in acute myeloid leukemia (AML) patients (pts) with normal karyotype.
Materials and methods. Bone marrow and peripheral blood samples were obtained from 45 AML pts aged 17 to 81, treated in Sverdlovsk Regional Hematological Centre during the period 2008 – 2015. Distribution of the pts according to FAB-classification was as follows: AML M0 — 1, M1 — 3, M2 — 17, M4 — 21, M5 — 1, M6 — 1, M7 — 1. Detection of mutations in in DNMT3A, FLT3, KIT, KRAS, NRAS, NPM1, TP53 and WT1 genes was performed by direct sequencing. Sequencing was realized using an automatic genetic analyzer ABI Prism 310. Additional analysis of normal (in nuclear) and abnormal (in cytoplasm) Npm1 protein localization in leukemic cells was performed in 10 bone marrow samples using immunohistochemical technique.
Results. The average frequency of functionally significant mutations in all investigated genes among the treated AML pts was 42.2%, and correlate with cumulative frequency of point mutation in AML with abnormal karyotype. Average frequency of mutations in NPM1 gene exons 9–12 was 50.0%, FLT3 gene exons 12–15 and 19–21 — 22.0%, KRAS gene exons 1–4 — 22.2%, KIT gene exons 7–12 and 16-19 — 16.7%, NRAS gene exons 1–4 — 13.0%, DNMT3A exons 18–26 – 8.3%, WT1 gene exons 6–9 — 4.5%, TP53 gene exons 4–11 — 3.1%. Multiple point mutations in investigated genes were detected in 24.4% cases AML pts with normal karyotype (usually NPM1 gene exon 12 insertion and FLT3 gene internal tandem duplication, ITD). These patients characterized by more dismal prognosis and poor response to standard chemotherapeutic regimens. In 1 case, AML M4, 4 point mutations were co-existed in investigated sample: NPM1 gene exon 12 tetranucleotide insertion, KRAS gene non-synonymous substitution c.88G>C and c.311A>C, DNMT3A gene transversion c.2141C>G. In 2 cases (AML M1 and AML M6) 3 mutations were co-existed in samles: NPM1 gene exon 12 tetranucleotide insertion, FLT3 ITD, KIT gene transversion c.1621A>C. In 4 samples (26.7%) 2 novel variants of NPM1 gene exon 12 tetranucleotide insertion (named types Ural-1and Ural-2) were detected. Interestingly, in Ural-1 type of NPM1 gene exon 12 tetranucleotide insertion, in 3 cases, typical NES-motif associated with partially damage of NoLS-motif and characterized by abnormal cytoplasm localization of Npm1 protein using validated immunohistochemichal technique.
Keywords: acute myeloid leukemia, normal karyotype, DNMT3A gene, FLT3 gene, KIT gene, KRAS gene, NRAS gene, TP53 gene, WT1 gene, sequencing, immunohisochemistry
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Authors:
Vinogradov Alexander V.
Sverdlovsk Regional Ministry of Health,
Department of Specialized and Hi-Tech Medical Care
MD, chief therapist, scientific advisor
Russian Federation, 620014, Yekaterinburg, Wainer str., 34b
vinogradov-av@russia.ru
Rezaykin Alexey V.
Ural State Medical University,
Department of Microbiology, Virology and Immunology, Laboratory of Molecular Diagnostic
MD, assistant
Russian Federation 620028, Yekaterinburg, Repin str., 3
alexrez@usma.ru
Salakhov Denis R.
Sverdlovsk Regional Clinical Hospital No.1,
Department of Hematology And Chemotherapy
hematologist
Russian Federation, 620102, Yekaterinburg, Volgogradskaya str., 185
noo_namee@mail.ru
Izotov Denis V.
Ural State Medical University
intern-hematologist
Russian Federation 620028, Yekaterinburg, Repin str., 3
phoenus@yandex.ru
Ioschenko Sergey E.
Institute of Medical Сell Technologyes
MD, scientific advisor
Russian Federation 620026, Yekaterinburg, Karl Marx str., 22a
ioshhenko@inbox.ru
Sergeev Alexander G.
Ural State Medical University,
Department of Microbiology, Virology and Immunology, Laboratory of Molecular Diagnostic
MD, professor, chief of the department, scientific chief of the laboratory
Russian Federation 620028, Yekaterinburg, Repin str., 3
sergeev@usma.ru
Received – 20.05.2016
For citation:
Vinogradov A.V., Rezaykin A.V., Salakhov D.R., Izotov D.V., Ioschenko S.E., Sergeev A.G. DNMT3A, FLT3, KIT, KRAS, NRAS, NPM1, TP53 AND WT1 Genes Mutations Detection in Acute Myeloid Leukemia with Normal Karyotype [Detektsiya mutatsij genov DNMT3A, FLT3, KIT, KRAS, NRAS, NPM1, TP53 i WT1 pri ostrykh mieloidnykh lejkozakh s normal’nym kariotipom blastnykh kletok] Vestnik uralskoi meditsinskoi akad-emicheskoi nauki – Vestn. Ural. Med. Akad. Nauki. 2016, no. 2, pp. 89-101. DOI: 10.22138/2500-0918-2016-14-2-89-101 [In Russ.]