Frequent Mutations and Their Influence on Prognosis in Patients with Acute Myeloid Leukemia

Background. Several genetic alterations such as translocations, gene mutations and deletions play an important role in myeloid leukemogenesis. The cytogenetic information is a very significant tool to classify pts at their initial diagnosis into prognostic categories. For pts with cytogenetically normal AML, prognosis can be specified by mutational status of the genes NPM1, FLT3, CKIT, NRAS and DNMT3A.

The aim of the research was to investigate the frequency and prognostic impact of FLT3, NPM1, CKIT, NRAS and DNMT3A mutations in AML pts and to analyze their interaction with other prognostic markers.

Methods. This study was performed in 200 adult pts (190 pts with de novo and 10 pts with secondary AML), previously untreated, median age 55 years (18-86). According to the results of cytogenetic analyses pts were separated in four groups: with favourable (9,0%), unfavourable (14,0%) prognosis, with normal karyotype (NK) (48,5%) and other aberrations (28,5%). Mutations in FLT3, CKIT and NPM1 were analysed by PCR and in NRAS by sequencing. Mutation analysis of DNMT3A R882 was performed by high-resolution melting curve analysis. Cytogenetic studies were analysed on bone marrow samples using standard GTG-method.

Results. Mutations in FLT3, CKIT, NRAS and NPM1 genes were detected in 105/200 (52,5%) pts. A total of 128 mutations were revealed in this group: 24,0% - FLT3-ITD, 6,5% - FLT3-TKD, 20,5% - in NPM1, 10,0% - in NRAS and 3,0% - in CKIT. 82 pts had single mutations and in 23 pts mutations occurred simultaneously: 17 with FLT3-ITD and in NPM1, 2 with FLT3-ITD and FLT3-TKD, 1 with FLT3-TKD and in NPM1, 3 with NPM1 and NRAS mutations. We found that mutations with the significantly higher incidence (p=0,001) were observed in the group of pts with NK (80/97), whereas there were only 8/28 pts with mutations in the group with complex karyotype. When analyzing the age-related features, it was shown that the majority of mutations were detected in the group of pts at the age from 60 to 69 years.

Mutations FLT3-ITD and FLT3-TKD were associated with higher WBC count comparing with pts without mutations (p=0,001 and p=0,014, respectively). The median follow-up for overall (OS) and relapse-free (RFS) survival for pts with FLT3-ITD against ptswith FLT3-ITD- was: 5,4 vs 12,8 months and 4,9 vs 10,0 months (p=0,001 and p=0,001), respectively. Mutation FLT3-TKD was also found to be prognostically unfavorable, but only comparing with pts with FLT3-ITD- genotype.

As the result of OS and RFS analyses in pts with and without NPM1 mutations we revealed the significant favorable influence of NPM1 mut on the prognosis (p=0,002 and p=0,020, respectively). However pts with genotype FLT3-ITD+/NPM1+ were found to get to the group with an intermediate risk.

We detected the significant adverse influence of CKIT mut on RFS (p=0,041). Mutations in NRAS didn't impact on prognosis; we only showed the tendency towards worsening of OS and RFS in group of pts with favorable cytogenetics (p=0,214 and p=0,160, respectively).

Mutations DNMT3A R882 were investigated in group of 143 AML pts and were detected in 23 (16,1%) pts. Pts with DNMT3A R882 had higher WBC (p=0,001) and platelets (p=0,020) count at diagnosis and more frequently belonged to FAB groups M5 (p=0,003), as compared with DNMT3A wt. Of 23 pts who had AML with DNMT3A mutations, 17 had tumors with NK profiles (24,3% of a total of 70 cytogenetically normal samples) (p=0,009). Pts with isolated DNMT3A mutations were seen in 4 cases, whereas in the rest of pts they were detected simultaneously with mutations in genes FLT3, NPM1, NRAS and CKIT. DNTM3A mutations were significantly more prevalent in NPM1 mut (p=0,005) and FLT3-ITD (p=0,005) positive cases than wild type. DNMT3A mutations were associated with negative influence on pts OS and risk of relapse, compared with DNMT3A wt (р = 0,031 and р = 0,045, respectively).

Summary. Mutations in FLT3 and NPM1 had a significantly higher incidence in the group of pts with a normal karyotype. FLT3 mutations showed the adverse prognostic value. Insertions in NPM1 were shownto be the favorable factor, correlating with prolonged RFS in all pts excepting pts with FLT3-ITD+/ NPM1+ genotype. CKIT mut was associated with higher relapse incidence in AML pts, while NRAS mut showed lack of prognostic significance. AML with DNMT3A mut represent the group, homogeneous on a number of clinical and laboratory parameters, associated with adverse prognosis and a high risk of the relapse.