Introduction: Mixed phenotype acute leukemia (MPAL) is a relatively rare and difficult to treat leukemia with features of at least two cell lineages (typically myeloid combined with B-ALL or T-ALL). Early abnormalities in hematopoiesis appear to result in specific types of bone marrow disorders and leukemias. Epigenetic changes, such as aberrant DNA methylation facilitated by mutated DNA methyltransferases, have been implicated in the pathogenesis of many hematopoietic disorders. A recent study of adult patients with T-ALL/myeloid mixed phenotype acute leukemia demonstrated a 50% mutation frequency in the DNMT3A gene (Kern et al., ASH 2012). Interestingly, a Dnmt3a-null mouse model in our lab develops both myeloid and lymphoid malignancies. We hypothesize that epigenetic genes are an important regulator of early stem cell differentiation and that mutations in these genes may cause abnormal stem cell differentiation resulting in the ambiguous phenomenon of mixed phenotype acute leukemia.

Methods: We analyzed DNA from a total of 19 MPAL samples (12 T/myeloid, 6 B/myeloid, and 1 B-ALL/T-ALL), with an age range of 20-74 years old. Pathology and flow cytometry records were reviewed by a hematopathologist to ensure that all samples met WHO 2008 criteria for MPAL prior to analysis. Five of the samples were cytogenetically normal, 7 had complex karyotypes or other high risk features, 3 had intermediate risk cytogenetic abnormalities, 1 was Philadelphia chromosome positive, 2 had MLL rearrangement and 2 samples did not have cytogenetic data available. Exome capture array was performed with Nimblegen and sequencing runs were performed in paired-end mode using the Illumina HiSeq 2500 platform for next generation sequencing. Samples achieved >96% of the targeted exome bases covered to a depth of 20x or greater. Results were mapped to Human Reference Genome, common SNPs were eliminated and mutations were identified with variant allele frequency >0.05 (most were >0.2). Over 500 previously reported genes with mutations in leukemias and other cancers were assessed.

Results: Twelve of the 19 samples (63%) had mutations in epigenetic regulatory genes. The most commonly mutated gene was DNMT3A with 6 patients (32%); 3 of these patients had T/myeloid subtype, 2 of these patients had B/myeloid subtype and 1 patient and B-ALL/T-ALL subtype. Other mutations in epigenetic regulatory genes were found in EZH2 (3 patients, 16%), IDH1 (2 patients, 11%), IDH2 (3 patients, 16%, all T/myeloid), TET1 (3 patients, 16%, all T/myeloid), and TET3 (3 patients, 16%). Interestingly, DNMT3A was co-mutated with TP53 in 5 patients, IDH2 in 3 patients, NRAS in 2 patients, TET3 in 1 patient, and NOTCH1 in 1 patient. Mutations were also frequently found in PRPF40B (6 patients, 32%), TP53 (5 patients, 26%), BRAF (4 patients, 21%), NOTCH1 (4 patients, 21%). Other genes found to be mutated at a lower frequency were RUNX1, GATA2, JAK2, KRAS, NRAS, SIRPA, LRP1B, and NF1.

Conclusions: Identifying abnormalities in epigenetic regulators may provide additional understanding of the pathogenesis of these rare and difficult to treat disorders. Adults with AML and DNMT3A mutations have demonstrated worse clinical outcomes (Ley et al., 2010), suggesting that standard chemotherapy may not be sufficient to treat malignancies which arise from hematopoietic stem cells. This understanding may guide future decisions regarding the necessity of personalized genetic testing at the time of diagnosis, the appropriateness of stem cell transplantation as well as providing prognostic information and future therapeutic targets for these patients.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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