Myeloid Neoplasms with MYC-Positive Double Minutes, a Specific Subgroup?

Background: Double minutes (dmin) are a rare cytogenetic finding in hematologic malignancies, mostly associated with acute myeloid leukemias. The acentric chromosomal structures usually represent the cytogenetic equivalent of the amplification of an oncogene, such as MYC. Due to their small size dmin are difficult to detect in chromosome banding analysis (CBA) and FISH analysis is required to identify the corresponding gene amplification. In literature several case reports described an association of the presence of MYC-positive dmin with an APL-like cytomorphology, however, larger studies comprehensively characterizing these cases are missing so far.

Aim: Characterization of 68 cases of myeloid neoplasms harboring MYC dmin with respect to cytogenetics, morphology, gene expression (GE) and gene mutations.

Patients and methods: The study enrolled 68 patients (pts) with myeloid neoplasia and MYC dmin. The diagnosis was established following WHO guidelines. Dmin and MYC amplification were assessed via CBA and FISH. Cases were screened for morphological features indicating a similarity to APL and/or suspected diagnosis of APL (APL-like). NGS of a 72 gene panel and WTS (50 Mio reads) were performed on NovaSeq instruments. Variant calling was performed with Pisces (minimum sensitivity: 3 %).

Results: The majority of pts were diagnosed with AML (52/68, 77 %, 7 of these secondary AML) or MDS/AML (3/68, 4 %). Other diagnoses comprised CMML (4/68, 6 %), MPN in acceleration or blast crisis, MDS/MPN overlap or MDS (3/68, 4 % each). APL-like cases amounted to 17/68 (25 %). Auer rods were detected in 24/68 pts (35 %) with a higher frequency in APL-like cases (p=0.007). Myeloperoxidase level was >75 % in 46/54 pts (85 %).

CBA revealed a complex karyotype (ck, ≥3 aberrations in addition to MYC dmin) in 21/68 pts (31 %), whereas 19/68 pts (28 %) presented with MYC dmin as the only cytogenetic aberration (MYC dmin only). Most common additional chromosomal gains were +4 (11/68, 16 %), +6 (9/68, 13 %) and +22 (8/68, 12 %), while most frequent chromosomal losses comprised 5q deletions (19/68, 28 %), 17p deletions (18/68, 27 %) and 9q deletions or -9 (16/68, 24 %).

Gene mutations (mut) most frequently occurred in TET2 (48/68, 71 %), TP53 (22/67, 33 %), U2AF1 (17/67, 25 %) and NRAS (12/66, 18 %). Almost all pts (66/68, 97 %) harbored at least one mutation in TET2, TP53 or U2AF1. TP53 mut were associated with ck (p<0.001) and were not detected in MYC dmin only pts (p<0.001). EZH2 mut were exclusively observed in cases with non-ck and 5/7 mut occurred in MYC dmin only pts (p=0.014). TET2 mut were observed more frequently in cases with non-ck (89 % vs. 29 %, p<0.001). Similarly, U2AF1 mut co-occurred with ck only in one case (p=0.013). In addition, U2AF1 showed significantly higher mutation frequency in APL-like cases (47 % vs. 18 %, p=0.025).

A comparison of the mutation frequencies of all MYC dmin cases to a cohort of AML from routine diagnostics (707 pts) revealed overrepresentation of mut in TP53 (33 % vs. 11 %, p=0.003), TET2 (71 % vs. 19 %, p<0.001), U2AF1 (25 % vs. 4 %, p<0.001) and EZH2 (11 % vs. 2 %, p<0.001) in the cohort of MYC dmin cases, whereas CEBPA, NPM1, FLT3, FLT3-ITD and WT1 mut were only detected in the AML cohort (10 %, p=0.004; 22 %, p<0.001; 8 %, p=0.011, 16 %, p<0.001 %; 8 %, p=0.017 respectively) and IDH2 mut occurred less frequently in MYC dmin cases (2 % vs. 9 %, p=0.022).

GE analysis of 40 MYC dmin pts revealed high expression of MPO compared to various AML subtypes, similar to APL (Figure (A)). A tSNE plot based on the top 5 % most variable genes demonstrated similar GE profiles within the MYC dmin cases compared to other AML cases (857 pts) (Figure (B)). A comparison of GE profiles within the MYC dmin cohort revealed differential GE between APL-like and all other MYC dmin cases (46 differentially expressed genes (DEG), adjusted p-value <0.05), as well as between ck and non-ck MYC dmin cases (106 DEG, adjusted p-value <0.05).

Conclusions:MYC dmin cases often show an APL-like cytomorphology, Auer rods and high levels of MPO expression. They are characterized by a high frequency of TET2, TP53 and U2AF1 mut as well as a relatively uniform GE profile. Mutation patterns and GE profiles allow further differentiation within MYC dmin cases. In cases with suspected APL after exclusion of a PML::RARA or other RARA fusions CBA and FISH for the detection of MYC dmin is helpful to identify this rare myeloid subtype which is described to be associated with an unfavorable prognosis.

Summerer:MLL Munich Leukemia Laboratory: Current Employment. Walter:MLL Munich Leukemia Laboratory: Current Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Current Employment. Haferlach:Munich Leukemia Laboratory: Current Employment, Other: Part ownership. Hutter:MLL Munich Leukemia Laboratory: Current Employment. Nadarajah:MLL Munich Leukemia Laboratory: Current Employment. Baer:MLL Munich Leukemia Laboratory: Current Employment. Stengel:MLL Munich Leukemia Laboratory: Current Employment. Kern:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership. Haferlach:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership.