Revisiting gene mutations and prognosis of ex-M6a-acute erythroid leukemia with regard to the new WHO classification

Due to the lack of specific clinical and biological features, M6a-acute erythroid leukemia (M6a-AEL), defined as an erythroid/myeloid type of acute leukemia, is no longer a distinct entity in the last classification of myeloid neoplasms by the World Health Organization (WHO). The diagnosis of M6a-AEL was previously made if a proliferation of erythroid precursors ⩾ 50% with a myeloblast count ⩾ 20% when counted as a percentage of non-erythroid cells, was found in the bone marrow. In 2016, revision of the WHO classification, the denominator used for calculating the blasts percentage was changed from non-erythroid cells to all nucleated cells. Consequently, M6a-AELs are now either myelodysplastic syndromes (MDSs) if the percentage of myeloblasts is ⩾ 20% of non-erythroid cells but o20% of all nucleated cells or acute myeloid leukemia (AML) if the percentage of myeloblasts is ⩾ 20% of all nucleated cells. As for any other AMLs prior therapy, recurring WHO cytogenetic abnormalities, and criteria for AML with myelodysplasia-related changes (AML-MRC) have to be taken into consideration for classification. By using targeted next-generation sequencing (tNGS) and array-comparative genomic hybridization (aCGH), we previously established a molecular classification of 40 M6a-AELs in five classes (C) based on mutations in NPM1 (C1), transcription factors (C2), splicing factors and/or chromatin modifiers (C3), TP53 (C4) or neither (C5). This classification could help in prognosis stratification. We have here re-analyzed our M6a-AEL molecular data according to 2016 WHO classification and compared them to a previously published cohort of MDS. After written consents obtained according to our ethical committee regulations and biobank procedures 11 new M6aAEL patients were added to the cohort and 106 genes were sequenced by tNGS as previously described. The 51 ex-M6a-AELs were reclassified as either MDSs—thereafter named AEL-MDSs (N= 24)—or AMLs (N= 27). To be homogenous in terms of blasts, the comparative cohort of MDS patients—thereafter named typical-MDSs—was made of 19 MDSs with excess of blasts type 2 (MDS-EB-2). Statistical analyzes were done using the survival package (version 2.30) in the R software (version 2.8.0) and the correlations were calculated with the Fisher’s exact test at 5% level of significance. Median age was 61, 59 and 77 years for AMLs, AEL-MDSs and typical-MDSs, respectively. AMLs comprised 9 AMLs with recurrent genetic abnormalities (AML-RGAs), 10 AMLs with myelodysplasiarelated changes (AML-MRCs), 4 AML-NOS, 3 therapy-related AMLs (t-AMLs) and 1 AML unclassified due to lack of data. According to the European LeukemiaNet risk stratification by genetics (ELN 2017), the prognosis was favorable for four patients, intermediate for 10 patients and adverse for the rest of the cohort. All AEL-MDSs were MDS-EB-2, except one case with excess of blasts type 1 (MDS-EB-1). The percentage of myeloblasts was between 10 and 18 (mean= 14%) and dysplasia was observed in all of the cases. Three AEL-MDSs were therapy-related myeloid neoplasms (t-AEL-MDSs). According to the International Prognostic Scoring System Revised (IPSS-R), karyotypes were good/very good for 12 cases, intermediate for six and poor/very poor for six leading to high or very high IPSS-R risk category, except one case that fell in an intermediate category due to a del(5)(q15q35). Typical-MDSs patients had between 10 and 18% myeloblasts (mean = 13%) and the erythroid component varied from 7 to 31% except for two cases (HD-0486 and HD-0982) with 45 and 40%, respectively. According to IPSS-R, karyotype was good for nine cases, intermediate for four and poor/very poor for six leading to high or very high IPSS-R risk category for most of the patients (16/19). The main clinical and biological characteristics of the patients are presented in Supplementary Table 1. Mutations were observed equally in AEL-MDS and AML cases: 87.5% (21/24) and 85% (23/27), respectively (Fisher’s exact test P= 1, Figure 1). The number of mutations was also quite equivalent, 61 and 63 in AEL-MDSs and AMLs, respectively. The median number of mutations in AELMDSs and AMLs was 3 and 2, respectively. All molecular classes C1–C5 were observed in both groups and the number of patients in each class was comparable (Supplementary Figure 1a). No difference in the number of mutations by functional pathways was found between AEL-MDSs and AMLs (Fisher’s exact test, P= 0.08, Supplementary Figure 1b) in spite of a higher frequency of mutations in the cohesin complex genes in AEL-MDSs. The median variant allele frequency (VAF) in NPM1-mutated cases (C1) was similar in AEL-MDSs and AMLs (0.20 and 0.32, respectively) but the most frequent additional mutations were in the cohesin complex genes, especially in SMC3 (3/6) for AEL-MDSs, and in DNMT3A (4/6) in AMLs (Supplementary Figure 2a). Median VAF in TP53-mutated cases (C4) was 0.32 in AEL-MDSs and 0.4 in AMLs. Eighty percent of AEL-MDSs (N= 4/5) and 30% of AMLs (N= 3/10) carried two different TP53 mutations. Homozygous inactivation of TP53 was suspected in three other cases (VAF40.8) due to a del(17p) (N= 3) or by uniparental disomy of chromosome 17 harboring a somatic TP53 mutation (HD-2199) (Supplementary Figure 2b). In spite of a triple alteration of TP53 (double mutations and loss of heterozygosity—LOH), the VAF remained low (o0,5) for four cases suggesting either a dilution of the sample or two different clones harboring a mutation. Double TP53 mutants (sequence variant or LOH) have been described in myeloid diseases but not with the same frequency; the high frequency in our series (N= 10/15) may suggest an implication in the erythroid proliferation, as recently suggested in pure erythroid leukemias. When comparing VAFs in C3, defined as ‘secondary-type’ mutations, medians were not different between AEL-MDSs and AMLs, respectively 0.414 and 0.471 (Supplementary Figure 2c). Finally, we did not find any GATA2-mutations (also verified by Sanger, data not shown) in either AEL-MDSs or AMLs and only one AML patient carried a bi-allelic mutation of CEBPA. These results contrast with a recent report describing a high frequency of mutations in these two genes but are in accordance with other data. These results show that AEL-MDSs and AMLs are similar in terms of molecular profiles and confirm our previous observation: ex-M6a-AELs show some differences with non-erythroid-rich AMLs; they have more TP53 mutations and less DNMT3A and ASXL1 mutations (Supplementary Table 2a). Citation: Blood Cancer Journal (2017) 7, e594; doi:10.1038/bcj.2017.68