Mixed Phenotype Acute Leukemia, T/Myeloid, NOS (MPAL-TM) Has a High DNMT3A Mutation Frequency and Carries Further Genetic Features of Both AML and T-ALL: Results of a Comprehensive Next-Generation Sequencing Study Analyzing 32 Genes

According to WHO classification Mixed Phenotype Acute Leukemia, T/myeloid, NOS (MPAL-TM) is defined by the immunophenotype with expression of both myeloid and T-lymphatic antigens. Only limited data is available on genetic aberrations.

Molecular and cytogenetic characterization of MPAL-TM in comparison to AML and T-ALL.

We studied 18 patients with MPAL-TM (7 female, 11 male; median age 61.6 yrs, range 19.6–89.3). Five patients had complex karyotypes (≥3 aberrations), 4 had other chromosomal abnormalities and 8 had a normal karyotype (n=1 no data available). Survival data was available in 16 cases (median survival 19.0 months). Two cases were selected for whole-exome sequencing (NimbleGen SeqCap EZ Human Exome, Roche NimbleGen, Madison, WI; HiSeq 2000, Illumina, San Diego, CA), mutation data was subsequently validated in the 16 remainder cases. The detailed genetic characterization included also a comprehensive targeted mutation screening of 32 genes with relevance in myeloid malignancies. Using microdroplet-based library construction (RainDance, Lexington, MA) next-generation amplicon sequencing data was generated for ASXL1, BCOR, BCORL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, NOTCH1, NPM1, NRAS, PHF6, PRPF40B, PTPN11, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, ZRSR2 (MiSeq instrument; Illumina, San Diego, CA). MLL-PTD was analyzed by quantitative real time PCR.

In both cases selected for whole-exome sequencing mutations in DNMT3A were detected. When extending DNMT3A analysis to the complete cohort, mutations were detected in 10/18 cases (55.6%). This represents the largest mutation frequency reported thus far both for DNMT3A itself and for any gene mutation in MPAL-TM. DNMT3A mutations were associated with a more mature T-lymphatic immunophenotype with CD2 expressed more often (8/10 cases) as compared to cases without DNMT3A mutations (2/8 cases, p=0.054). The majority of DNMT3A mutations were missense mutations (n=9) with 3 mutations affecting the hotspot codon Arg882 in the methyltransferase domain, followed by 2 splicesite mutations, 1 nonsense, and 1 frame-shift mutation. Notably, a biallelic mutation status occurred at high frequency in these cases (7/10). In detail, except for three cases with a mutation in Arg882, all other mutated cases showed either two distinct mutations (n=3) or one homozygous mutation (n=4). This contrasts AML which is known to virtually lack biallelic DNMT3A mutations (3/49 cases (6.1%), Grossmann et al., ASH 2011) and suggests a closer association of MPAL-TM to T-ALL in which half of cases show a biallelic DNMT3A mutation status (8/16 cases (50.0%), Grossmann et al., ASH 2011). Contrasting data for both AML and T-ALL where DNMT3A mutations are related to an adverse prognosis, DNMT3A mutations in MPAL-TM were associated with a longer overall survival (12.0 vs. 3.1 months), although this difference was not significant.

Moreover, the broad targeted mutation screening resulted in the detection of mutations in the following genes: TP53 (n=3), IDH1/2 (n=3), BCOR (n=3), NOTCH1 (n=3), NRAS (n=3), BRAF (n=2), PHF6 (n=2), ETV6 (n=2), RUNX1 (n=1), FLT3 (n=1), TET2 (n=1), ASXL1 (n=1), ZRSR2 (n=1), and SF1 (n=1). The median number of mutations per patient was 2 (range 0–7), only one patient had no mutation. TP53 mutations were observed only in cases with complex karyotypes. Interestingly, the genetic mutations encountered were predominantly limited either to genes known to play a role in epigenetic processes (DNMT3A, TET2, IDH1/2, ASXL1; n=11) or to transcription factors (TP53, RUNX1, ETV6; n=6). Furthermore, these two groups of mutations occurred virtually mutually exclusive, except for one case harboring both a mutation in DNMT3A and ETV6. There were only two remaining cases not carrying aberrations in these two gene categories.

1. A remarkably high mutation frequency for DNMT3A (55.6%) was observed in MPAL-TM. 2. In MPAL-TM DNMT3A mutations were most frequently biallelic and lacked an adverse prognostic impact. 3. Other molecular mutations in MPAL-TM were limited to genes with epigenetic function or to transcription factors. 4. Cytogenetic abnormalities in MPAL-TM were neither typical for AML nor for T-ALL. 5. Analysis of DNMT3A mutations should be considered in the diagnostic work-up of patients with MPAL-TM in the future.

Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schoeck:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.