Molecular Genetic Classification of Brazilian Acute Myeloid Leukemia and Myelodysplastic Syndromes Patients

Introduction:

Until the last decade, AML diagnoses relied on cell morphology and immunophenotyping by flow cytometry. Patients were stratified into good risk, intermediate or unfavorable according to karyotype. Treatment consisted of induction, consolidation and intensification or hematopoietic stem cell transplantation. Despite, rigorous attempt to follow standard therapeutic protocols and overcome adversities, results among Brazilian patients were dismal. In the last ten years, with the introduction of molecular tests for FLT3, NPM1, CEBPA and C-Kit, some of the good and intermediate risk patients moved to unfavorable, justifying the adverse response to therapy, especially in the favorable group. However, more recently, the advent of next generation sequencing (NGS) can provide a still better stratification in order to the optimization of therapy. MDS patients follow a somewhat similar pathway in respect to diagnosis, classification, prognosis and therapy decisions.

Objective:

The objective of this study was to reclassify AML/MDS Brazilian patients according to molecular genetic aberrations.

Methods:

14 AML (excluding promyelocytic leukemia) and 16 MDS patients were diagnosed according to WHO, 2016, after morphological analysis, immunophenotyping (with a wide panel of MoAb) and G-banding karyotype (ISCN, 2016). AML patients mean age was of 49,8 years and M:F ratio was 0.75 while MDS mean age was 62.5 yrs and M:F ratio = 1.3. One AML patient was classified as favorable risk, 11 as intermediary and two unfavorable. According to IPSS-R, among MDS, 14 patients were favorable and two intermediary risk. NGS and Sanger sequencing were performed in peripheral blood or bone marrow samples collected in EDTA tubes, for the following mutations: in RNA-splicing machinery (SF3B1, SRSF2, ZRSR2, U2AF1, U2AF2); DNA methylation (TET2, DNMT3A, IDH1/2); chromatin modification (ASXL1, EZH2); transcription factor (TP53, RUNX1); signal transduction/kinases ( FLT3, JAK2); RAS pathway (KRAS, NRAS, CBL, NF1, PTPN11); cohesin complex (STAG2, CTCF, SMC1A, RAD21); and DNA repair (ATM, BRCC3, DLRE1C, FANCL)

Results: All patients presented at least one or more molecular mutations with a mean of 2.43 mutations per AML case and 1.87 per MDS. Nine out of 11 AML patients classified as intermediary risk group (karyotype with +8 or normal) were moved into unfavorable prognosis after the molecular analyses, since they presented SRSF2, RUNX1, FLT3, TP53 and U2AF1 mutations. Besides that, one initially good prognosis patient (presence of inv16) showed FLT3 -ITD mutation, and was then considered as unfavorable risk. Only one patient classified as intermediary risk (normal karyotype) was reclassified into favorable (presence of NPM1 mutation). The genetics classification ended in only one favorable, one intermediary and 12 unfavorable patients. Referring to MDS, four patients with good prognosis (normal karyotypes) were moved to unfavorable due to the presence of ASXL1 mutations. Still, one MDS patient with 7% of ring sideroblasts and normal karyotype presented SF3B1 mutation and remained in the favorable risk.

Conclusion and discussion:

Genetic classification allowed a better allocation of AML patients and showed that the majority of them (85%) were stratified to unfavorable risk. This scenario explains the dismal treatment results obtained when patients were classified by karyotyping, FLT3, NPM1, CEBPa and CKIT only. Furthermore, approximately 40% to 50% of patients with AML have a normal karyotype at diagnosis, and this represents the largest subset of AML (Mitelman et al, 2010). All such cases are categorized in the intermediate-risk group; yet, this group is quite heterogeneous, and not all patients present the same response to treatment. This is likely a result of the large variability in gene mutations and gene expression in this population. Similarly, 50% of patients with MDS have normal karyotype, a heterogeneous group as well, and molecular alterations allowed classifying them differently. With the genetic classification approach precision therapy is being introduced aiming for better results.