Elucidation of Additional Mutations By Next-Generation Sequencing Is of Clinical Significance in Patients with Rare MPNs and MDS/MPN Overlap Syndromes
Authors: Kirschner Martin MJ , Schemionek Mirle , Begemann Matthias , Isfort Susanne , Feldberg Kristina , Brümmendorf Tim H. , Koschmieder Steffen 
Published: October 2019

Abstract
Introduction:
Recently, next-generation sequencing (NGS) has revolutionized the molecular characterization and understanding of several hematologic entities, including myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS)/MPN overlap syndromes. Nevertheless, the frequency and clinical impact of the mutations detected by NGS, remain largely unclear, especially in rare MPN which were analyzed in this study.
Methods:
Thus, we established a novel amplicon-based NGS panel, comprising genes that are known to be recurrently mutated in MPN and/or MDS/MPN. Hot spot regions or all exons of the following 32 genes were chosen: ABL, ASXL1, BARD, CALR, CBL, CEBPA, CHEK2, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NFE2, NRAS, PDGFRA, PTPN11, RUNX1, SETBP1, SF3A1, SF3B1, SH3B2 (LNK), SRSF2, TCF12, TET2, TP53, U2AF1. After establishing this panel, peripheral blood samples of 19 patients, which were diagnosed with CMML(10), aCML(2), MPNu(1), MDS/MPNu or other MPN(6), were analyzed on a MiSeq® illumina sequencer. Variants were only analyzed if the absolute coverage at each SNV site was >50 reads, and the absolute coverage of the mutant allele was 10 or more reads and its relative coverage exceeded 10%.
Results:
Mean coverage of the run was 1516 reads with good Phred-score quality parameters (>84% of called bases with Q-score >= 30). In 300 bidirectional cycles, a yield of nearly seven gigabases of sequencing data was reached. One out of 19 analyzed patients was excluded from analysis due to insufficient DNA quality. In 89% of the samples(16/18), mutations were detected which had not previously been known to be present in these patients. TET2 (50%, 9/18) and SETBP1 mutations were the most common (44%, 8/18). As expected, TET2 mutations were spread over the entire gene and SETBP1 mutations were restricted to the known hot spot region (exon 4, c.2602-c.2620). Additionally, CSF3R mutations were detected in 22% (4/18) of patients. Epigenetic regulator genes were also affected as EZH2 mutations were detected in 17% (3/18), ASXL1 mutations in 39% (7/18) and IDH1/2 mutations were found in 6% (1/18) of all samples, whereas DNMT3A mutations were not present. Further mutations were found in the following genes: CBL (11%), ETV6 (6%), JAK2V617F (6%), KRAS (11%), NRAS (11%), PTPN11 (6%), SH2B3 (6%) and SRSF2 (11%). Besides previously known mutations, several novel variants could be detected. All but one patient harbored more than one of these mutations.
Furthermore, clinical correlates and morphologic and cytogenetic subtypes of each patient were available to associate with the NGS data of individual patients. For example, the one patient with a solitary NRAS c.35G>A (amino acid: p.G12D) mutation showed the most aggressive clinical course in our cohort with transformation to AML only 7 months after first diagnosis of CMML. Moreover, CSF3R mutations have been shown to confer sensitivity to ruxolitinib and may thus open up new avenues of treatment for our patients.
Conclusion:
In a cohort of unclassified MPN and rare MDS/MPN subtypes, NGS is a powerful tool to characterize samples more extensively. Our data suggests that a more comprehensive understanding of the mutational spectrum may have important clinical impact in individual patients, including diagnosis, prognosis, and more specific treatment.

Disclosures
Isfort: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; BMS: Honoraria; Mundipharma: Other: Travel; Amgen: Other: Travel; Hexal: Other: Travel. Brümmendorf:Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.