5q Deletions in MDS and MPN: The Accompanying Molecular Mutations Determine the Phenotype but TP53 Is Frequently Mutated in All Entities: MPN, MDS and MPN/MDS Overlap
Authors: Haferlach Claudia , Meggendorfer Manja , Schnittger Susanne , Zenger Melanie , Kern Wolfgang , Haferlach Torsten 
Published: October 2019

Abstract
Background: 5q deletions are recurrent cytogenetic abnormalities. They occur either as the sole abnormality or accompanied by additional chromosome aberrations in MDS, MPN and MDS/MPN.
Aim: To determine 1. the frequency of 5q deletions in MPN, MDS and MDS/MPN, 2. the spectrum of accompanying molecular mutations and their impact on the phenotype.
Patients and Methods: Out of 6,373 MPN, 11,398 MDS, and 1,107 MDS/MPN with available cytogenetics 97, 1,869, and 37 cases with 5q deletion were identified. From these we selected all cases with 5q deletion and MPN or MDS/MPN with available material for molecular studies. These were 24 MPN (15 in chronic phase, 8 in accelerated and 1 in blastic phase) and 19 MDS/MPN. For comparison we selected 48 MDS with isolated del(5q) as defined by the WHO classification 2008. Thus, in total 91 cases with 5q deletion were further investigated by a myeloid gene panel containing: 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. With the exception of RUNX1 which was sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT), all remainder genes were studied using a combination of a microdroplet-based assay (RainDance, Billerica, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA).
For all 91 patients data from chromosome banding analysis and for 89 patients array CGH (4 x 180K microarray slides, Agilent Technologies, Santa Clara, CA) was available.
Results: The frequency of 5q deletion was higher in MDS (16.4% of all MDS and 37.8% of MDS with aberrant karyotype) as compared to MDS/MPN (3.3%/11.6%) and MPN (1.5%/7.1%). The proportion of del(5q) cases showing a complex karyotype was comparable between entities (MDS: 41.4%, MDS/MPN: 45.9%, MPN: 43.3%). Based on array CGH data the size of the 5q deletion was determined for each of the 89 patients analyzed and varied in MPN between 15.5 and 124.9 Mb (median: 58.0 Mb), in MDS 25.8-86.5 Mb (71.0 Mb), and in MDS/MPN 28.4-118.8 Mb (67.2 Mb). The minimal deleted region for the three subsets was identified between genomic position 129,680.693 and 138,585.833, 129,723.966 and 135,062.049, and 130.858.633 and 138,676.848, respectively, and thus were overlapping.
Sequencing revealed overall 78 mutations in 91 patients. In detail, in only 13 patients (14.3%) no mutation was detected, while 44 (48.4%) showed one, 25 (27.5%) two, 7 (7.7%) three, and 2 (2.2%) four mutations, respectively. The mean number of mutations per patient was 1.6 in MDS/MPN, 1.5 in MPN, and 1.2 in MDS. The highest mutation frequency in all subgroups was observed for TP53: total cohort: 30.8% (MPN: 25.0%, MDS: 33.3%, MDS/MPN: 31.6%). Further, frequencies were 13.2% for SF3B1 and TET2 each and 11% for ASXL1 in the total cohort. For these 3 genes no differences in mutation frequency were observed between MPN, MDS and MDS/MPN (SF3B1: 8.3%, 18.8%, 5.3%; TET2: 12.5%, 16.7%, 5.3%; ASXL1: 12.5%, 8.3%, 15.8%). Of note, ASXL1 mutations were mutually exclusive of mutations in TP53, SF3B1 and DNMT3A. JAK2V617F mutations were present in the total cohort in 28.6% of cases and, as expected, were significantly more frequent in MPN (58.3%) and MDS/MPN (47.4%) than in MDS (6.3%; for both comparisons p<0.0001). DNMT3A mutations were detected overall in 18.7% and were most frequent in MDS (27.1%) compared to MDS/MPN (10.5%) and MPN (8.3%) (p=0.034). Thus, the majority of DNMT3A mutations was detected in MDS (13/17 (76.5%)). Patients with mutations in ASXL1, JAK2, TET2, and TP53 showed at least one additional molecular mutation in 90%, 68.2%, 50%, and 50% of patients, respectively. Mutation frequencies in the total cohort below 5% were found for BCOR (1.1%), CBL (2.2%), EZH2 (3.3%), FLT3-TKD (1.1%), GATA2 (1.1%), KRAS (1.1%), MPL (2.2%), NRAS (2.2%), RUNX1 (1.1%), SRSF2 (2.2%), and U2AF1 (2.2%). No mutations were observed in ETV6, GATA1, IDH1, IDH2, KIT, NPM1, PHF6, WT1.
Conclusions: 1. The size of the 5q deletion and the commonly deleted region largely overlapped between MDS, MPN and MDS/MPN. 2. 85.7% of all patients with 5q deletion and either MPN, MDS or MDS/MPN harbored at least one molecular mutation. 3. TP53 is frequently mutated in patients with del(5q) irrespective of the morphological subtype. 4. A mutation frequency above 10% was also observed for JAK2, SF3B1, TET2, and ASXL1. 5. While JAK2V617F is most frequent in MPN and MDS/MPN, DNMT3A mutations occur predominantly in MDS.

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