Mutation Analysis in 276 Cases of Newly Diagnosed Acute Myeloid Leukemia By Next Generation Sequencing: Association with Established Prognostic Variables and MRC Risk Groups

INTRODUCTION

Acute myeloid leukemia (AML) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed AML cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and Medical Research Council (MRC) risk groups.

MATERIALS AND METHODS

We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 276 newly diagnosed treatment na•ve AML patients presenting at a single referral 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 AML 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 on 262 patients by PCR followed by Sanger sequencing. Cases were categorized as favorable, intermediate and adverse groups as per revised MRC cytogenetic risk group classification.

RESULTS

Median age was 67 years. Patients included 167 (60.5%) males and 109 (39.5%) females. 38 (14%) and 6 (2%) patients had prior diagnosis of myelodysplastic syndrome and myeloproliferative neoplasms respectively. Hematologic parameters are as follows [median (range)]: Hb 8.7 g/dL (2.8- 13.9), platelets 50.5 K/μL (1-1109), WBC 5.4 K/μL (0.4-620.4), ANC 0.9 K/μL (0-145.7), AMC 0.3 K/μL (0-98.1). Bone marrow (BM) blast % [median (range)] was 45.5% (5-96). LDH was 733 IU/dL (225-13156). Of 275 patients with cytogenetic analysis performed, 98 (35.64%) had diploid karyotype, 75 (27.27%) had one, 38 (13.82%) had two, 8 (2.91%) had three, 56 (20.36%) had > three abnormalities, 75 (27.27%) had monosomies and 62 (22.55%) had trisomies. Of 34 cases classified as AML with recurrent cytogenetic abnormalities per WHO 2008, 10 (3.64%) had t(8;21), 13 (4.73%) had inv(16), 1 (0.36%) had t(15;17), 3 (1.09%) had inv (3), 4 (1.45%) had t(9;11)(p22;q23) and 3 (1.09%) had t(6;9)(p23;q34). MRC risk categorization of the cases was as follows: favorable 24 (8.72%), intermediate 161 (58.55%) and adverse 90 (32.73%). Mutations identified by NGS are as detailed in Table 1. Of 56 patients with FLT3 mutations detected by PCR, the breakdown is as follows: FLT3 ITD (39, 14.13%), FLT3 D835 (16, 5.80%), FLT3, ITD + D835 (1, 0.36%). Of 262 patients assessed, CEBPA mutation was detected in 26 (9.92%). Thirty one (11.23%) cases had no mutations detected in the genes analyzed by NGS or PCR, 93 (33.70%) had mutations in one, 80 (28.98%) in two, 42 (15.22%) in three and 30 (10.87%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2.

CONCLUSIONS:

AML 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. With longer follow-up, we could use this data to refine prognostic models for AML.

Table 1

Genes	Number of Cases	Percentage of Cases
FLT3	61	22.10
NPM1	48	17.39
NRAS	48	17.39
DNMT3A	47	17.03
TP53	45	16.30
IDH2	40	14.49
IDH1	33	11.96
TET2	32	11.59
ASXL1	30	10.87
RUNX1	30	10.87
PTPN11	13	4.71
KRAS	11	3.99
KIT	8	2.90
WT1	8	2.90
GATA2	7	2.54
EZH2	6	2.17
JAK2	4	1.45
MPL	2	0.72
ABL1	1	0.36
EGFR	1	0.36
GATA1	1	0.36
IKZF2	1	0.36
MDM2	1	0.36
MLL	1	0.36
MYD88	1	0.36
NOTCH1	1	0.36

Table 2

	Mutated genes	p value
Hb	NRAS, NPM1	<0.05, <0.04
Platelets	TP53, IDH2	<0.03, <0.02
WBC	FLT3, NRAS, TP53	<0.05, <0.05, <0.05
AMC	NRAS, NPM1, TP53	<0.001, <0.02, <0.02
ABC	FLT3 NPM1	<0.049, <0.02
PB blast %	FLT3, NPM1, TP53, CEBPA	<0.000, <0.002, <0.005, <0.000
BM blast %	FLT3, NRAS, NPM1, TP53, IDH1, CEBPA	>0.000, <0.0000, <0.014, <0.004, <0.002, <0.012

AMC: absolute monocyte count, ABC: absolute basophil count, PB: peripheral blood, BM: bone marrow