Identification of Novel Somatic Mutations, Regions of Recurrent Loss of Heterozygosity (LOH) and Significant Clonal Evolution From Diagnosis to Relapse in Childhood AML Determined by Exome Capture Sequencing – an NCI/COG Target AML Study

Abstract 123

Comprehensive cataloguing of somatic changes in acute myeloid leukemia (AML) has revealed novel molecular pathways that contribute to the development and progression of the disease, however many lack prognostic significance. The challenge that lies ahead is to understand how these molecular changes relate to transcription and protein deregulation to influence treatment responses and outcomes. Also, some mutations found in adult AML are detected at lower frequency or not at all in pediatric AML.

To better define the genomic profile of somatic mutations in childhood AML and evaluate the extent of clonal evolution from diagnosis to relapse, we performed whole exome sequencing in matched trios of specimens (diagnostic, germline and relapse) from 20 children with AML who lacked known karyotypic high-risk features. Candidate variants were verified by 454 sequencing. In total, 364 somatic variants were successfully verified, of which 263 were non-silent mutations (127 at diagnosis and 136 at relapse). Of the 195 unique nonsynonymous mutations present in 180 genes, mutations in 6 genes were detected in more than one patient, including ETV6, KIT, KRAS, NRAS in 2 patients and TET2 and WT1 in 3 patients. A polyphen prediction estimated 72% of the missense and nonsense mutations as possibly and/or probably damaging. Mutations identified in single patients included known genes implicated in leukemias (e.g., GATA2, IKZF1), as well novel mutations in genes not previously implicated in AML.

Prevalence and clinical significance of ETV6 mutations were assessed in a large cohort of pediatric AML. The entire coding sequence of ETV6 gene was sequenced in 180 cases of pediatric AML. ETV6 mutations, including missense (N=5), Indels (N=4) or splice site mutations (N=1) were detected in 6% of the patients without karyotypic or molecular risk factors. Those with and without ETV6 mutations had an overall survival of 33% and 71%, respectively (p=0.006) with a corresponding relapse rate of 80% and 29% (p=0.004).

We examined the clonal evolution of mutations from diagnosis to relapse by comparing the mutation profile at diagnosis to that in relapse. In the discovery phase, at diagnosis, 234 somatic mutations in 202 genes were detected, of which 174 were non-silent events, including 36 (21%) indels, 126 (72%) missense, 11 (6%) nonsense mutations and 1 (1%) splice site mutation. At relapse, 350 mutations in 309 genes were detected, of which only 112 mutations were detectable at diagnosis (29%). In verification phase, 71 mutations in 70 genes were identified only in diagnostic specimens, and 105 mutations in 104 genes were detected in relapse only, with an overlap of 192 variants, suggesting significant molecular evolution and loss or gain of genomic events from diagnosis to relapse. With secondary targeted deep sequencing the overlap of mutations present at diagnosis and relapse increased to 52%. Comparison of allele fractions between diagnosis and relapse revealed a significant enrichment of the variant from diagnosis to relapse with evolution of a minor clone at diagnosis to a more dominant presence at relapse. It also highlights that in a substantial subset of patients, novel genomic events may emerge, leading to disease relapse.

In addition, analysis of the allele fractions of SNPs in the normal and tumors of these patients allowed us to detect regions of recurrent loss of heterozygosity (LOH). Six of 20 patients exhibited LOH in one or more regions. The most common region of LOH, found in 4 patients (20%) was the q-arm of chromosome 13 (3 with FLT3/ITD), including 10 genes with germ line variation across 4 patients, and in two patients the q-arm of chromosome 16 was involved. Two of the 4 patients had LOH only in the relapse specimen.

This study highlights the potential impact of applying novel sequencing approaches to pediatric AML and not only identifies clinically significant somatic mutations, but defines genomic complexity in childhood AML by demonstrating significant clonal evolution from diagnosis to relapse. Mutations emerging at relapse may represent initially rare, chemotherapy-resistant events that cooperate with other mutations and lead to adverse outcome. Identifying mutations that may be therapeutically targeted early in the course of therapy may provide improved outcomes for patients.