Somatic Mutation-Detecting Algorithm Enables Analysis of MDS Patients during Azacitidine Therapy
Authors: Stopka Tomas , Vargova Karin , Kulvait Vojtech , Dusilkova Nina , Jonasova Anna T. 
Published: October 2019

Abstract
Introduction: Somatic mutation detection in myelodysplastic syndrome (MDS) is very important in deciphering clonal pathogenesis of every patient and if determined correctly will become useful tool in followup studies such as testing individual susceptibility to epigenetic therapy with azacitidine (AZA). While some patients respond to AZA by restoring hematologic parameters, others progress to AML. Recent identification of quite heterogeneous sets of mutated genes (Bejar R et al. 2013) suggested that: patients with specific mutation pattern/s may respond to epigenetic therapy differently.
Aim: We herein set to determine mutation profiles of MDS cohort indicated to and treated by AZA and utilized TrueSight DNA amplicon NGS sequencing approach containing 54 genes all previously associated with MDS or AML.
Patients: We analyzed immunomagnetically CD3-depleted bone marrows of two MDS patients - AZA responders. First patient (male, 68y), was diagnosed with RAEB2, IPSS int-2, transfusion dependent (4 TU/Mo), intermediate cytogenetics (tri21). Following 4 cycles of AZA (75 mg/m2 s.c., 5+2) the patient responded by partial remission, and AZA was discontinued after 17 cycles. Twelve months after discontinuation he progressed and AZA was readministered for additional 3 cycles and the patient achieved again partial remission. Analyzed are samples after 11 (P394) and 20 (P1380) cycles of Vidaza. Second patient (female, 64y), was diagnosed with RAEB2, IPSS high, transfusion dependent (2 TU/Mo), favorable cytogenetics (46XX). Following 4 cycles of AZA (75 mg/m2 s.c., 5+2) she responded by hematology improvement and later by partial remission. Analyzed is a sample after 4 (P1510) cycles of Vidaza. As negative controls we used two normal donor bone marrows from 41y male and 32y female. As a positive control we also used: 1 MDS/AML cell line MOLM-13 with previously identified mutations of CBL and FLT3 (DSMZ; ACC 554).
Methods and approach: Samples were sequenced on Illumina MiSeq sequencer. The mapping was performed using Burrows-Wheeler Aligner algorithm. Illumina Somatic variant caller was used to identify mutations. Then we applied following filters on the data: sequencing coverage should be higher than 1000 per mutation (~80% data left), mutation should be heterozygous (~95% data left), mutation frequency should be higher than 10% (~10% data left), Illumina Somatic variant caller should flag the mutation as "PASS" (~50% data left), mutation should not be synonymous (~75% data left) and mutation should be exonic (~40% data left). These filters were also applied to find mutations in the two control samples. Those mutations which were identified also in the control samples were removed from the analysis of patient samples (~50% data left).
Results: the MDS/AML cell line MOLM-13 contained mutations (SNVs or InDels) in ABL1 (SNV/frequency=46.5%), ASXL1 (SNV/49.8%), CEBPA (In/47.9%), HRAS (SNV/54.5%), TET2 (SNV/49.7%), and as expected also in the genes encoding CBL (delta-exon8/52.4%) and FLT3 (ITD/50.6%). Patient' sample P1510 contained mutations in CBL (SNV/67.9%), CUX1 (SNV/51.8%), IKZF1 (2 different SNVs/41.9 and 50.7%), KDM6A (SNV/51.6%), SF3B1 (SNV/38.9%), and SMC3 (SNV/33.1%). Patient samples P394 and P1380 contained mutations in the ASXL1 (SNV/ 35.5% and 32.6% respectively), CUX1 (2x SNVs, first SNV/46.1->64.5%, second 48.4->57.9%), and IKZF1 (SNV, 50.4->44.5%) in similar frequencies in the sample before and after 2.5 years (including 9 cycles of AZA) suggesting limited genetic heterogeneity in this AZA-responding patient. Consequently, to gain more insight into how AZA modulates mutation pattern in MDS, we now analyze a set of fourty nine additional patients before and following at least 4 cycles on AZA treatment.
Conclusions: Our data support use of immunomagnetic CD3-depletion of bone marrow and addition of normal control samples in the sequencing of MDS patient samples and support this approach for testing genetic heterogeneity during MDS disease course upon AZA treatment.

Disclosures
Stopka: GAČR P305/12/1033 and UNCE 204021: Research Funding; Celgene: Research Funding; PersMed ltd.: Equity Ownership. Vargova:GAČR P305/12/1033 and P305/11/1745: Research Funding; UNCE 204021: Research Funding; PRVOUK P24/LF1/3: Research Funding. Kulvait:PersMed ltd.: Equity Ownership. Jonasova:PRVOUK P24/LF1/1: Research Funding; Celgene: Research Funding.