Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes

Abstract

Introduction: Myelodysplastic syndromes encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress toward acute myeloid leukemia. The accumulation of genetic alterations is closely associated with the progression of myelodysplastic syndromes toward acute myeloid leukemia.

Objective: To investigate the presence of mutations in the points most frequent for mutations (hotspot mutations) in phosphatidylinositol-3-kinase (PI3K), Janus kinase 2 (JAK2), FMS-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1), which are involved in leukemia and other cancers, in a population of Brazilian MDS patients.

Methods: Fifty-one myelodysplastic syndromes patients were included in the study. According to French-American-British classification, the patients were distributed as follows: 31 with refractory anemia, 8 with refractory anemia with ringed sideroblasts, 7 with refractory anemia with excess blasts, 3 with refractory anemia with excess blasts in transformation and 2 with chronic myelomonocytic leukemia. Bone marrow samples were obtained and screened for the presence of hotspot mutations using analysis based on amplification with the polymerase chain reaction, sequencing, fragment size polymorphisms or restriction enzyme digestion. All patients were screened for mutations at the time of diagnosis, and 5 patients were also screened at the time of disease progression.

Results: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression.

Conclusions: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression.

Figure 1

PCR and Sequencing of exons 9 and 20 of PI3K. The fragment size of the exon 9 (A) and exon 20 (B) of PI3K are indicated in the figure. In both figures A and B, lane 1: Ladder 100bp fragments; lane 2: negative control; lanes 3 and 4: amplicons obtained from genomic DNA of patient MDS patients (RA). Representative PI3K sequencing from MDS patients, determined by automated sequence analysis of exon 9 (C) and 20 (D). The localization of the most frequent hotspot mutations are highlighted in the figure.

Figure 2

Fragment analysis of FLT3-ITD and NPM1 mutations. Representative fragment size analysis of a MDS patient with wild-type alleles for FLT3 (A), an AML patient with the FLT3-ITD mutation (B), an MDS patient with wild-type NPM1 (C) and an AML patient with a mutation in exon 12 of NPM1 (D). The arrows indicate the presence of the mutant allele.

Figure 3

JAK2 V617F genotyping. (A) PCR amplification of JAK2: lane 1: 100 bp ladder; lane 2: negative control; lanes 3 to 6 – 460-bp amplicons obtained from the genomic DNA of a patient with PV (3), a CMML patient after disease progression (4) and two MDS patients (with RA) (5 and 6). (B) BsaXI digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in a PV patient (3) and in the CMML patient positive for the JAK2 V617F allele after disease progression (4); lanes 5 and 6: digestion pattern observed in two MDS patients (with RA) with wild-type JAK2 alleles.

Figure 4

FLT3-D835 genotyping. (A) PCR amplification of FLT3: lane 1: 100 bp ladder; lane 2: negative control; lanes 4 to 5: 114-bp amplicons obtained from the genomic DNA of patients with MDS (3-4) and AML (5). (B) Eco321 digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in two MDS patients negative for the FLT3-D835 allele, lane 5: digestion pattern observed in an AML patient with the FLT3-D835 mutation.