Detection of Recurrent Mutations by Pooled Targeted Next-Generation Sequencing in MDS Patients Prior to Treatment with Hypomethylating Agents or Stem Cell Transplantation

The clinical heterogeneity observed in patients with myelodysplastic syndromes (MDS) is reflected in the wide range of molecular abnormalities than can be acquired by their diseased cells. Somatic mutations in specific genes have been associated with clinical manifestations of MDS, yet several of these genetic lesions have prognostic significance that is independent of the International Prognostic Scoring System (IPSS).¹ Some genetic abnormalities can predict response to treatment, but are found only in the minority of patients. Somatic mutations are more common than karyotype abnormalities and can be identified in over 75% patients with MDS.² One small study noted that mutations in TET2 appear to predict response to azacitidine.³ However, other mutations were not explored in this study. Similarly, certain karyotype abnormalities have been shown to predict outcomes in MDS patients undergoing stem cell transplantation, but somatic gene mutations have not been explored in this context.⁴ Here we report on gene mutations detected in over 200 MDS patients prior to treatment with hypomethylating agents (Cohort 1) and in 76 patients prior to undergoing stem cell transplantation (Cohort 2).

Cohort 1 is comprised of MDS patients treated with azacitidine or decitabine at the Dana-Farber Cancer Institute (n =48), the MD Anderson Cancer Center (n =109), or in the ADOPT clinical trial (n =83). A total of 47 patients received azacitidine, 164 received decitabine, and 29 patients received combination therapy. Bone marrow mononuclear cell DNA was obtained from patients prior to treatment with a hypomethylating agent. Responses were assessed using the International Working Group (IWG) Response Criteria for MDS revised in 2006.⁵ DNA was sheared, ligated to adaptors and barcodes, quantified, and then pooled prior to hybrid-capture enrichment of the target genomic regions.

Cohort 2 includes 76 patients with MDS who underwent stem cell transplantation at the Dana-Farber Cancer Institute between 2004 and 2009. Patients without a pretransplant biopsy sample or who died within 30 days of after transplant were excluded. Bone marrow mononuclear cell DNA was obtained from patients prior to transplant and enriched for target genomic regions using the HaloPlex PCR system.

For both cohorts, the target genomic region consisted of 74 genes including all genes reported to be mutated in MDS. Target enriched sample pools were sequenced on an Illumina HiSeq 2000. Sequence data was aligned and mutations called using the analysis pipeline at the Broad Institute.

Variants previously shown to be germline or present in databases of normal variants (dbSNP 132 or NHLBI Exome Sequencing Project) at a population frequency of 1% or more were discarded. Remaining variants were considered candidate somatic mutations. Candidate mutations were validated in tumor DNA by Sanger sequencing and examined in germline DNA when a matched sample was available.

Candidate mutations were identified in over 90% of patients in each cohort. The most frequently mutated genes in Cohort 1 were ASXL1 (39%), SF3B1 (24%), TET2 (23%), RUNX1 (19%), SRSF2 (18%), and DNMT3A (15%). The rate of complete response, partial response, or hematologic improvement (ORR) was 47% and did not differ between sites (ORR for DFCI, MD Anderson, and ADOPT were 42%, 45%, and 53% respectively). Nor was there a significant difference between hypomethylating agents (ORR for decitabine and azacitidine were 44% and 48% respectively).

The most frequently mutated genes in Cohort 2 were ASXL1 (25%), TP53 (22%), DNMT3A (17%), and RUNX1 (14%) with 37% of patients carrying a mutation in a splicing factor gene (SF3B1, U2AF1, SRSF2, or ZRSR2). The mutation profile in this higher prognostic risk group includes a high frequency of mutations in genes associated with a poor prognosis, particularly TP53. In contrast, the frequency of mutations in genes with a neutral or favorable association with prognosis (SF3B1 and TET2) was lower.

Analyses comparing mutation status, response to treatment, and overall survival are in progress for both cohorts.

Abnormalities in recurrently mutated genes can be found in most MDS patients using a pooled next-generation sequencing approach. Single genes are mutated frequently enough in our cohorts to allow us to determine if they can predict response to treatments such as hypomethylating agents and stem cell transplantation.

Bejar:Genoptix: Consultancy. Kantarjian:Genzyme: Research Funding. Ebert:Celgene: Consultancy; Genoptix: Consultancy.