Comprehensive Mutational Profiling In Myelodysplastic Syndromes Treated With Decitabine and Tretinoin
Authors: Shih Alan H. , Devlin Sean M. , Dolezal Emily K. , Knapp Kristina M. , Wang Kai , Sanford Eric M. , Brennan Tim , Yelensky Roman , Nahas Michelle , Otto Geoff A , Lipson Doron , Stephens Philip J , Miller Vincent , van den Brink Marcel R.M. , Levine Ross L. , Nimer Stephen , Klimek Virginia M. 
Published: October 2019

Abstract
Although DNA methyltransferase Inhibitor (DNMTI) therapy demonstrates efficacy in patients (pts) with Myelodysplastic Syndromes (MDS), to date there are no clinical or genomic markers to select patients most likely to benefit from DNMTI therapy. Two recent retrospective studies have suggested TET2 mutations (muts) are associated with a higher response rate to 5-azacytidine (5-aza) and decitabine (DAC). We investigated the efficacy of DAC (20 mg/m2/day x 5 days) plus tretinoin (ATRA) given on days 10-19 of a 28-day schedule in MDS pts in a phase I/II study. We comprehensively analyzed pretreatment samples from 23 pts (15M,8F; median age 67, range, 44-78) treated on this study for alterations in known oncogenes to identify potential correlations between mut profile and clinical outcomes. The median follow-up was 41 mos (range, 10-66). IPSS cytogenetics (CG) risk categories included Good (n=8), Intermediate (n=5), and High risk (n=8) pts (2 karyotype failures). The overall IPSS risk categories were Int-1 (n=3), Int-2 (n=16), and High (n=4). Of 23 pts, 16 responded, including 2 CRs, 9 mCR+/-HI, 1 PR, and 4 HI.
Genomic DNA and total RNA were isolated from all pt samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >584X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The median number of muts detected was 2 (range, 0-6), with 22/23 (96%) pts having at least 1 mut. The number of muts detected for 14 PB samples with ≤ 1% blasts was 2 (range, 0-6) compared to 4 muts (range, 1-5) for 8 pts with > 1% PB or BM blasts (unknown source for 1 sample).
The most common mut identified in this cohort was TP53 (35%). Previous studies have shown that complex CGs and mut TP53 are strong poor prognostic indicators. Six pts with complex CGs and either mut or deleted TP53 had a median of 0 (range 0-2) additional muts, whereas pts with non-complex CGs who were wild-type (WT) TP53 (n=12) had a median of 3 (range 1-6) muts in other disease alleles (p=0.006). We identified mutually exclusive recurrent muts in RNA splicing factors including SRSF2 (26%), SF3B1 (17%), and ZRSR2 (9%), and recurrent muts in RUNX1 (22%), IDH1/2 (22%), TET2 (17%), ASXL1 (17%), PTPN11 (13%), CEBPA (13%), STAG2 (9%), and KRAS (9%). Thirteen additional muts were detected in single pts, and we identified novel candidate muts in DNA repair pathways (ATM, ATR, FANCA BRCA2), and in the recently described tumor suppressor FAT3. We also hypothesized that prognostication of pts with normal karyotype (NK) MDS may be aided by molecular stratification, as we and others have shown in AML. Of note, all 5 pts with IDH1/2 muts occurred in pts with NK, and in 4/5 cases they were associated with concomitant SRSF2 muts that were previously shown to be associated with adverse outcomes.
We identified TET2 muts in 4 pts who were also ASXL1 WT. Notably, all 4 TET2 mutated/ASXL1 WT pts responded to DAC/ATRA therapy. The other 12 responders included 7 pts with TP53 mut/loss +/- other muts. No other muts were associated with a differential response to DAC/ATRA in this trial cohort. The strong association between TET2 muts and DAC/ATRA response, and more detailed mutational profiling of the 12 TET2 WT pts who responded to DAC/ATRA therapy is being investigated in additional pt samples and will be presented in more detail. Pts with TP53 muts showed a trend toward a worse survival which was not statistically significant; however, no other individual mut was associated with a differential survival in this small cohort.

Conclusions
The use of comprehensive mutational profiling employed in this study allowed us to identify muts in 25 genes in MDS pts, and allowed us to show that the overall mut frequency was higher in pts with a NK and without TP53 muts, thereby providing evidence for alternate disease initiation events and pathogenesis in these two genomically defined MDS subsets. All pts with mutated TET2 responded to DAC/ATRA, suggesting that pts with muts in TET2 are the best candidates for novel therapeutic trials which include DNMTI therapy.

Disclosures:
Wang: Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc.: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.