Molecular Analyses of MDS/MPN Overlap Entities According to WHO Classification Reveal a Distinct Molecular Pattern for MDS/MPN, Unclassifiable
Authors: Meggendorfer Manja , Haferlach Torsten , Jeromin Sabine , Haferlach Claudia , Kern Wolfgang , Schnittger Susanne 
Published: October 2019

Abstract
Introduction: The World Health Organization (WHO) classification defines myelodysplastic/myeloproliferative neoplasms (MDS/MPN) based on clinical, morphologic, and laboratory findings that show features of MDS and characteristics more consistent with MPN. This category includes atypical chronic myeloid leukemia (aCML), chronic myelomonocytic leukemia (CMML), MDS/MPN, unclassifiable (MDS/MPN, U), and refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). In recent years RARS-T, CMML, and also aCML were deciphered by several molecular studies, while MDS/MPN, U cases warrant closer investigations.
Aim: To comprehensively investigate mutations in 17 genes known to be mutated in aCML, CMML, MDS/MPN, U, and RARS-T and to define entity specific mutation patterns in comparison to cytogenetic and clinical data.
Patients and Methods: We investigated 179 patients diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO criteria: 35 patients were diagnosed as aCML, 58 as CMML, 39 as MDS/MPN, U, and 47 as RARS-T. All patients underwent mutation analyses by a gene panel containing: ASXL1, TET2, DNMT3A, SRSF2, SF3B1, U2AF1, JAK2, CALR, MPL, NRAS, KRAS, CBL, BRAF, CSF3R, RUNX1, SETBP1, and NPM1. Gene mutations were analyzed by Sanger sequencing, next generation sequencing, melting curve analyses, or gene scan. Cytogenetics was available in 172/179 cases and was grouped as normal karyotype (n=128, 74%) or aberrant karyotype (n=44, 26%).
Results: In the total cohort the most frequently mutated gene was ASXL1 (41%), followed by TET2 (40%), and the spliceosomal genes SF3B1 (31%) and SRSF2 (30%). Also frequently mutated were JAK2 (21%), NRAS (15%), RUNX1 (12%), and CBL (12%). All other investigated genes showed mutation frequencies below 10%.
There were no significant differences between the 4 entities regarding frequencies of aberrant karyotypes (14-37%) and no correlation of the number of molecular mutations (0-6/patient) with any specific karyotype.
Addressing the mutation patterns of these 4 entities showed that ASXL1 and TET2 are frequently mutated in all entities (19-60% and 26-53%, respectively), although significant differences between the entities exist (see figure): ASXL1 is less frequently mutated in RARS-T (19%) in comparison to aCML (60%; p<0.001) and CMML (52%; p=0.001), TET2 is more often mutated in CMML (53%) in comparison to MDS/MPN, U (26%; p=0.007) and RARS-T (32%; p=0.031). SRSF2 is more frequently mutated in CMML (53%) than in RARS-T (9%; p<0.001) and MDS/MPN, U (15%; p<0.001), SF3B1 is more often mutated in RARS-T (92%) than in all other entities (aCML: 11%, CMML: 5%, MDS/MPN, U: 13%; for all p<0.001). One important difference between aCML and CMML versus MDS/MPN, U and RARS-T was reflected by two different signaling pathways: i) JAK2/CALR/MPL (JAK/STAT pathway) were significantly more often affected in MDS/MPN, U (33%) and RARS-T (53%), (aCML: 9%, CMML: 7%; p<0.001). ii) NRAS/KRAS/CBL (RAS pathway) were more often mutated in aCML (37%) and CMML (52%), (MDS/MPN, U: 5%, RARS-T: 9%; p<0.001).
The MDS/MPN, U cohort included most patients with no mutation in any analyzed gene (11/39, 28%) in contrast to aCML (2/23, 6%), CMML (5/58, 9%), and RARS-T (0/47, 0%). Furthermore all these MDS/MPN, U patients with no gene mutation had a normal karyotype. Looking at co-ocurrences of gene mutations in MDS/MPN, U revealed that SRSF2 and TET2 mutations occur together more frequently (4/10 vs. 2/29 in TET2wt; p=0.028). Of notice, in MDS/MPN, U U2AF1 (18%) was the most frequently mutated spliceosomal gene which was only rarely mutated in the other entities (5%, p=0.015).
Conclusions: 1) ASXL1 and TET2 are the most frequently mutated genes found overall in MDS/MPN overlap. 2) SF3B1 mutations are specific for RARS-T. 3) SRSF2 is most frequently mutated in CMML, but also in aCML. 4) MDS/MPN, U is affected by mutations in all spliceosomal genes. 5) The JAK/STAT pathway is more often affected in MDS/MPN, U and RARS-T. 6) The RAS pathway is more often affected in aCML and CMML. 7) MDS/MPN, U shows a specific molecular pattern with characteristics reflecting a mixture of all other MDS/MPN entities.
Red: gene mutation, orange: gene mutations combined, light grey: no mutation/normal karyotype, black: aberrant karyotype, white: not analyzed.
Figure: Molecular abnormalities and cytogenetics in MDS/MPN entities. Figure:. Molecular abnormalities and cytogenetics in MDS/MPN entities.

Disclosures
Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.