Molecular Screening In Blastic Plasmacytoid Dendritic Cell Neoplasms Reveals Mutations In TET2 and ASXL1 In The Majority Of Patients

Blastic plasmacytoid dendritic cell neoplasm is a rare and clinically aggressive disease with frequent involvement of skin and bone marrow. It derives from precursors of plasmacytoid dendritic cells and is classified according to WHO 2008 into the group of “Acute myeloid leukemia and related precursor neoplasms”. Cytogenetics frequently reveals complex karyotypes data on molecular mutations is scarce.

To characterize blastic plasmacytoid dendritic cell neoplasm on the molecular level applying a comprehensive next-generation sequencing panel targeting 26 genes commonly mutated in myeloid neoplasms.

We studied six patients with blastic plasmacytoid dendritic cell neoplasm (5 bone marrow, 1 peripheral blood; 1 female, 5 male). The median age was 71.7 yrs, (range: 58.0-81.9). Cytomorphologic assessment revealed the findings characteristic for blastic plasmacytoid dendritic cell neoplasm in all cases. Furthermore, by multiparameter flow cytometry all cases had the typical immunophenotype with strong expression of CD56, expression of CD4 and CD123 and lack of expression of most myeloid and lymphoid markers. The degree of bone marrow infiltration as quantified by multiparameter flow cytometry ranged from 3% to 83% (median: 41%). One patient had a complex karyotype (44,XY,der(2)t(1;2)(q12;p22),del(5)(q14q35),del(7)(q11q22),-13,-15), four had other aberrations (46,XX,t(1;9)(q23;q33); 46,XY,t(3;8)(p12;q21),der(17)t(1;17)(q24;p13); 46,XY,i(7)(q10); 46,XY,del(13)(q14q31)), and one had a normal karyotype. None of the cases had received anti-neoplastic therapy.

Mutational analysis was based on sensitive next-generation sequencing assays comprising in total 26 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. Targets of interest included either complete coding gene regions or hotspots. With the exception of RUNX1, which was sequenced on the 454 Life Sciences NGS platform (Branford, CT), all remainder genes were studied using a combination of a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA).

Strikingly, all six patients had TET2 mutations, two had one and the other four cases had two different TET2 mutations. As described for other hematologic malignancies, there was no hotspot observed and all cases displayed different mutations (p.Leu615Serfs*24 and p.Ser1648Tyrfs*35, p.Asn767Metfs*46, p.Cys1193Tyr and p.Cys1811*, p.Gln1138*, p.Ser657Hisfs*43 and p.Glu798*, p.Ala1344Glyfs*3 and p.His1380Tyr). Of the ten TET2 mutations five were frame-shift mutations, three were nonsense mutations and two were missense mutations. Missense mutations were located in the same conserved region. The mutational load ranged from 12% to 50% (median, 32%). In one case a follow-up analysis after three weeks revealed the same two TET2 mutations with increases of the mutational load from 23% to 44% and from 24% to 39%. Although the sample size of the present series is rather limited, this is the first time that at least almost all cases of a distinct malignant disease entity are reported to carry TET2 mutations. Furthermore, there was also a very high incidence of ASXL1 mutations with 3/6 patients being affected. All cases carried the common mutation p.Gly646Trpfs*12, with mutational loads of 23%, 16% and 29%. Further mutations included SRSF2 (p.Pro95Ala) and ETV6 (p.Ile140Tyrfs*14) in one case and SRSF2 (p.Pro95Arg) with no further mutation in another case, PHF6 (p.Glu293Lys) in one case, and WT1(p.Arg596His) in one case. No mutations in any of the other analyzed genes were found.

The pattern of mutations in genes that have been described in other myeloid malignancies clearly underlines the correct classification of blastic plasmacytoid dendritic cell neoplasm into a myeloid disease category. Based on the present series with TET2 mutations in all (6/6) patients with blastic plasmacytoid dendritic cell neoplasm, TET2 mutations have to be considered to play a central role in the pathogenesis of this malignant disease. Additional mutational analyses on extended patient cohorts including ASXL1 should aim at clarifying the frequencies of these mutations and their potential impact on diagnostic and possible therapeutic interventions.

Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kuznia:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.