Landscape of Subclonal Mutations in Myelodysplastic Syndromes (MDS) Allows for a Novel Hierarchy of Clonal Advantage By Combining Germline and Somatic Mutations
Authors: Nagata Yasunobu , Makishima Hideki , Radivoyevitch Tomas , Hirsch Cassandra M. , Przychodzen Bartlomiej P , Kuzmanovic Teodora , Li Samuel , Yoshida Kenichi , Yoshizato Tetsuichi , Adema Vera , Negoro Eiju , Clemente Michael J. , Hosono Naoko , Shiraishi Yuichi , Chiba Kenichi , Tanaka Hiroko , Mukherjee Sudipto , Nazha Aziz , Sole Francesc , Carraway Hetty E. , Miyano Satoru , LaFramboise Thomas , Sekeres Mikkael A. , Ogawa Seishi , Maciejewski Jaroslaw P. 
Published: October 2019

Abstract
Targeted and unbiased next generation sequencing (NGS) has contributed to a better understanding of the molecular pathogenesis of myeloid neoplasms, including MDS. Discovery efforts have identified novel classes of mutated genes, while deep NGS approaches have yielded a better appreciation of clonal hierarchy, inter-case variability and intra-tumor heterogeneity. MDS is a disease continuum characterized by a wide spectrum of often overlapping lesions that determine phenotype, while also serving as initiation and progression events. In addition to somatic lesions, germ line (GL) alterations can serve as bona fidenon-clonal ancestral events that play an underappreciated role in MDS pathogenesis. While some of these lesions are associated with childhood familial leukemia syndromes, others are unknown, and are likely characterized by a low/variable penetrance and delayed disease manifestation.
To delineate clonal dynamics in MDS, we sequenced whole exomes of 262 cases with primary MDS and related disorders. For validation and confirmation we also deep sequenced a cohort of 1,686 additional cases with a various type of myeloid malignancies. An extensive bioanalytic pipeline and confirmatory sequencing, including GL DNA analysis, was used to discriminate somatic vs. GL lesions and exclude sequencing artifacts. Initially we focused on driver somatic events in significantly mutated genes. All somatic mutations were subjected to clonal hierarchy analysis using variant allele frequencies (VAFs). In selected cases (n = 180), serial analyses were performed. Using VAF rankings of each event, a position within the clonal hierarchy was assigned; while each patient has a single dominant clone, some may have a founding chromosomal abnormality and others may have VAFs too close to distinguish, i.e. have co-dominant events. In general, multiple subclonal events are detected in each patient. For the purpose of this analysis we distinguished between 2 types of ancestral events: 1) driver non-clonal mutations (e.g., GL TP53, RUNX1, ETV6) and 2) predisposition non-clonal events (FA genes, telomerase genes, BRCA1/2). The latter do not influence the clonal architecture.
Based on average sequencing depth, 5,474 somatic mutations were identified: 241 (92%) were clonal dominant and 234 (89%) were sub-clonal (secondary) events. The median number of mutations in subclonal events per case was 13. The number of mutations in subclonal events was higher than that in events that were clonal dominant (4,881 vs 593). No genes were mutated in a purely dominant fashion and some genes were almost entirely subclonal, e.g., RAS and FLT3. For each dominant event, there is a frequent secondary lesion, e.g., dominant TET2 mutations are followed by subclonal second TET2 events, SRSF2 and ASXL1 lesions. Thus, novel relationships between dominant and subclone events were found, indicating the presence of invariant functional interactions among different mutations in MDS pathogenesis. In a confirmatory cohort studied by NGS targeted to a selected panel of significantly mutated genes, the number of subclonal events increased due to greater coverage and thus sensitivity. The spectrum of dominant events, however, should not differ as they are inherently associated with a high clonal burden. For examples, TP53 clonal mutations frequently co-occur with TP53 subclonal mutations (12%, p=.004), but are exclusive of STAG2 subclonal mutations. EZH2 clonal and ASXL1 secondary mutations also co-occur. Classifications of clonal and secondary events may have prognostic and diagnostic implications.
We identified a spectrum of novel predisposition and non-clonal driver variants by comparing to ethnically weighted control populations. Eight mutations (3%, 8/262 cases) in 3 genes (DDX41, TP53, and ELANE) were identified as driver non-clonal mutations because identical mutations were reported in familial leukemia syndromes, while 16 mutations (6%) in 3 genes (CSF3R, BRCA1, and RPL5) were identified as non-clonal predisposition events.
Detailed understanding of such clonal dynamics and complexity of clonal hierarchical complexity may have clinical significance, both for somatic mutations and for germline events. Increasing clonal burden of extracted genes associated with predictive prognostic impact should be prospectively validated in a more uniform and larger cohort of MDS cases.

Disclosures
Makishima: The Yasuda Medical Foundation: Research Funding. Mukherjee:Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Sole:Celgene: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Speakers Bureau.