Identification of Novel Recurring Mutations in Relapsed Acute Lymphoblastic Leukemia.

Abstract 703

Relapse occurs across the spectrum of cytogenetic subtypes of acute lymphoblastic leukemia (ALL), and the biologic factors influencing risk of relapse are poorly understood. Previous studies have demonstrated substantial evolution in the complement of DNA copy number alterations from diagnosis to relapse, with the majority of cases acquiring new lesions at relapse that are not present at diagnosis, and also losing lesions present in the predominant clone at diagnosis. Moreover, fingerprinting and backtracking of deletions indicate a common ancestral origin of the diagnosis and relapse clones in most cases, suggesting that multiple, genetically distinct clones are present at diagnosis, and that specific genetic alterations influence risk of relapse. At present, detailed examination of DNA sequence variations in relapsed ALL has not been performed. In this study, we have performed genomic resequencing of 238 genes in leukemic samples obtained at diagnosis and relapse in 23 childhood ALL cases. The cohort comprised cases with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), MLL-rearrangement (N=3), BCR-ABL1 (N=3), and cases with low hyperdiploid, pseudodiploid, normal, and miscellaneous karyotypes (N=10). All samples had over 80% blasts or were flow sorted to high purity prior to DNA extraction. Whole genome amplification of DNA was performed prior to sequencing. We selected genes targeted by recurring copy number alterations in ALL, genes in key pathways targeted by genetic alterations in ALL (e.g. lymphoid development, tumor suppression, cell cycle regulation and apoptosis), known cancer genes and tyrosine kinases. The complete coding region of each gene was sequenced in both diagnosis and relapse sample in all cases. Validation of putative variants was performed by sequencing of matched normal DNA. 248 putative protein changing variations were identified. After removal of variants also identified in matched normal DNA, 55 variants in 32 genes were identified in 20 cases (mean 2.5 variants per case, range 0-5). Eleven genes were mutated in multiple patients (ASMTL, CREBBP, ERG, FLT3, KRAS, NF1, NRAS, PAX5, PTPN11, TP53 and TUSC3). We identified tumor-acquired variations in genes previously known to be mutated in acute leukemia, including ETV6 (1 case) JAK1 (1), NRAS (5), KRAS (2), NF1 (3) PTPN11 (2), PAX5 (2), FBXW7 (1), and TP53 (2). In addition, we identified recurring mutations in genes not previously known to be mutated in cancer, including the transcriptional regulators CREBBP (N=4), NCOR1 (N=2), the tumor suppressor candidate gene TUSC3 (N=2), and the ETS family transcription factor ERG (N=2). Single mutations were also identified in transcriptional regulators (THADA, SPI1 (PU.1), TCF4, TCF7L2), the histone gene HIST1H2BG, and additional genes also targeted by copy number alterations in ALL (ARMC2, ATP10A, PLEKHG1, STIM2). The patterns of evolution of sequence variations between diagnosis and relapse were similar to those previously reported for DNA copy number alterations. Four patients had identical sequence mutations at diagnosis and relapse. Twelve cases had the some sequence variations identified at both diagnosis and relapse, but either acquired additional mutations at relapse (N=9), lost mutations present at diagnosis (N=2), or both acquired new lesions at relapse and lost variants at diagnosis (N=1). An additional three cases had variants detected at either diagnosis or relapse with no commonality between the two samples. Notably, eight (35%) cases had lesions resulting in constitutive RAS activation at diagnosis or relapse (NRAS in 3 cases, FLT3 in 2, PTPN11 in 2, NF1 in 2, and KRAS in 1), with 5 cases harboring mutations at diagnosis only (NF1 in 2 cases, NRAS in 2, and FLT3 in 1), and three cases harboring mutations at relapse only (FLT1, PTPN11 and NRAS 1 case each). In several cases, relapse-acquired mutations were identified as minor subclones at diagnosis. These data have identified novel targets of somatic mutation in ALL, and suggest that sequence variation is important determinant of risk of relapse. Identification of mutations in multiple cases suggests that several of these variants are driver mutations. These findings also indicate that resequencing of the entire coding genome of relapsed ALL will be essential to identify all lesions influencing response to therapy.