Mutation Analysis in 200 Cases of Newly Diagnosed Myelodysplastic Syndrome By Next Generation Sequencing: Association with Established Prognostic Variables and IPSS-R

INTRODUCTION

Myelodysplastic syndrome (MDS) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed MDS cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and the Revised International Prognostic Scoring System (IPSS-R).

MATERIALS AND METHODS

We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 200 newly diagnosed treatment na•ve MDS patients presenting at a single institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for MDS per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis.  CEBPA mutation analysis was performed by PCR followed by Sanger sequencing on 186 patients.

RESULTS

Median age was 67 years. Patients included 139 males (69.5%) and 61 females (30.5%). Hematologic parameters are as follows [median (range)]: Hb 9.6 g/dL (5-16.7), platelets 75 K/μL (5-652), WBC: 2.8 K/μL (0.4-20.8), ANC 1.3 K/μL (0.0 -12.0), AMC 0.2 K/μL (0.0-3). Bone marrow (BM) blasts [median (range)] were 4% (0-19). Of 192 patients with cytogenetic analysis performed, 65 (33.85%) had diploid karyotype, 53 (27.6%) had one, 21 (10.93%) had two, 13 (6.77%) had three, 40 (20.83%) had > three abnormalities. IPSS-R risk categorization of the 200 cases is as follows: very low (17 cases, 8.5%), low (46, 23%) intermediate (42, 21%), high (47, 23.5%), very high (48, 24%). Mutations identified by NGS are as detailed in Table 1. Of the 4 patients with FLT mutations detected, the breakdown is as follows: FLT3 ITD (3, 75%), FLT3 D835 (1, 25%), FLT3, ITD + D835 (0, 0%). CEBPA mutation was detected in 12 of 186 (6.45%) cases assessed. CEBPA was detected in 12 (6.45%). Sixty three (31.5%) cases had no mutations detected in the genes analyzed by NGS or PCR, 80 (40%) had mutations in one, 42 (21%) had mutations in two, 8 (4%) in three and 7 (3.5%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. No association was found between frequency of any particular mutation and the IPSS-R score.

CONCLUSIONS:

MDS is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC and BM blast percentage. With longer follow-up, we could use this data to refine IPSS-R.

Table 1

	Number of cases	% cases
TP53	46	23
TET2	33	16.5
RUNX1	27	13.5
ASXL1	25	12.5
DNMT3A	17	8.5
EZH2	12	6
IDH2	8	4
IDH1	7	3.5
NRAS	7	3.5
JAK2	5	2.5
FLT3	4	2
PTPN11	3	1.5
EGFR	2	1
MPL	2	1
WT1	2	1
GATA2	1	0.5
KIT	1	0.5
KRAS	1	0.5
MYD88	1	0.5
NPM1	1	0.5
BRAF	1	0.5

Table 2

	Mutated genes	p value
WBC	ASXL1	<0.042
AEC	TET2	<0.016
BM blast %	RUNX1, CEBPA	<0.008, p<0.02
BM myelocyte %	TP53, TET2, RUNX1, DNMT3A	<0.014, <0.014, <0.015, <0.038

AEC: absolute eosinophil count, BM: bone marrow