PHF6 Mutations Defines a Subgroup of Mixed Phenotype of Acute Leukemia with Aberrant T-Cell Differentiation

Mixed phenotype acute leukemia (MPAL) is a rare form of acute leukemia, and comprises 2% of all acute leukemia diagnoses. With the exception of a small subset harboring BCR/ABL1 or MLL translocations (around 15% in combination), the genetic aberrations of MPAL are largely unknown. PHF6 gene is located at Xq26-27 and encodes plant homeodomain finger 6, a zinc finger containing protein. Recurrent somatic PHF6 mutations are frequent in T-ALL (up to 30%) and rarely in AML (~3%). Here we report recurrent PHF6 mutations in approximately 20% of MPAL patients, making PHF6 the most common genetic aberrations identified in MPAL.

From 2013 to 2016, a total of 1329 patients with myeloid neoplasms were sequenced at the MSKCC with a targeted next generation sequencing panel containing 30 genes that are frequently mutated in acute leukemia. We identified 38 PHF6 mutations with predicted pathologic significance in 33 patients (2.5%) spanning the whole coding sequence: 10 frameshift, 11 nonsense, 1 splicing site, and 16 missense (Figure 1). The most common co-mutant gene observed in cases with PHF6 mutations was RUNX1 (30%, 11/37). No TP53 mutations are detected within the PHF6-mutant cohort(Figure 2). The 33 patients with PHF6 mutations included patients with a diagnosis of MPAL (n=5), AML (n=19), MDS (n=5), and MPN (n=4).

The disproportional distribution of PHF6 mutations in MPAL prompted us to further analyze these patients. During the same period of time, 24 MPAL patients were identified and confirmed based on WHO 2016 classification (excluding AML-MRC and t-AML). Within this cohort, 3 (12%) had BCR-ABL1 (p190) fusion, and 1 (4%) had MLL translocation, two distinct genetically defined subgroups. Importantly, 5 (21%) harbored PHF6 mutations, these mutations were mutually exclusive with BCR-ABL1 or MLL. Four of the 5 patients showed T-cell lineage differentiation, in contrast to MPAL with BCR-ABL1 or MLL translocations which were characterized by B/myeloid differentiation. The 2-year overall survival for the PHF6 mutation positive MPAL was markedly reduced compared to non-mutated cases (40% vs. 100% in PHF6 unmutated cases (p=0.005)).

In the larger subset of AML cases, PHF6 mutations were nearly exclusively present in secondary AML (11 AML-MRC, 6 t-AML and only 1 de novo AML). In contrast to their rarity in de novo AML, PHF6 mutations were identified in 11% (11/88) of AML-MRC and 7.1% (6/84) of t-AML. Interestingly, T-cell markers (cytoplasmic CD3, CD2, CD5, CD7) were often expressed on the blasts of secondary AML cases regardless of PHF6 mutational status (56%, 9/16 vs. unmutated 51%, 36/70). In comparison to unmutated cases, the blasts were often negative for CD33 (69%, 11/16, vs 28%, 20/70, p=0.004 by Fisher's exact test) and CD38 (44%, 7/16, vs. 17%, 12/70, p=0.04 by Fisher's exact test) in PHF6 mutated AML, suggestive of a stemness phenotype with aberrant T-cell differentiation.

In conclusion, PHF6 mutations are the most common somatic alteration identified in MPAL and define a subgroup of MPAL with altered T-cell differentiation and inferior outcome. Amongst AML, PHF6 mutations are almost exclusively seen in secondary AML and are also associated with T-cell antigen expression and a more primitive stem/progenitor immunophenotype. PHF6 mutations co-exist with RUNX1 mutations but are mutually exclusive to TP53 mutations. Notably, PHF6 mutations may provide a genetic and functional link between MPALs with poor outcome and secondary AML and provide insights into shared mechanisms of transformation. RNA sequencing studies on the sorted blast population are ongoing and will be presented at the ASH meeting.

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