ALBERT

Description: Methotrexate is a major component in every treatment protocol for childhood acute lymphoblastic leukemia (ALL). Both beneficial and detrimental effects of methotrexate in ALL have been clearly related to methotrexate plasma pharmacokinetics, which vary substantially among patients. However the genetic basis of such variability remains largely unknown. Herein, we surveyed 600,000 germline single nucleotide polymorphisms (SNPs) to determine how inherited genetic variation affects the disposition of methotrexate among 434 children with ALL who received 3014 courses of methotrexate at 2 to 5 g/m2. Adjusting for age, race, gender and methotrexate regimen, the most significant SNPs associated with methotrexate clearance were annotated to a plausible gene, the organic anion transporter polypeptide, SLCO1B1. The three top SNPs included rs11045879 (P = 1.7 × 10−10), rs4149081 (P = 1.7 × 10−9), and rs2900478 (P = 2.8 × 10−8). Linkage disequilibrium (LD) was observed among these three SLCO1B1 SNPs (r2=1) and with a known functional polymorphism in SLCO1B1, T521C (rs4149056, r2 = 0.86). The top two SLCO1B1 SNPs rs11045879 and rs4149081 were further validated (P = 0.018 and P = 0.017) in an independent cohort of 206 patients with ALL. Additional SNPs annotated to SLCO1B1 were identified and further validated. In a stepwise multiple linear regression analysis, SLCO1B1 genetic variation remained significant and explained clearance variability comparable to that of other non-genetic factors including treatment regimen. SNPs in SLCO1B1 were also associated with methotrexate-related gastrointestinal toxicity (P= 0.03 to 0.0005, odds ratio 8.3 to 16.4). In summary, we have identified a candidate gene, SLCO1B1, which is strongly associated with the pharmacokinetics of methotrexate, an anticancer drug with a low therapeutic index in multiple treatment regimens. Although SLCO1B1 is widely recognized as having a strong impact on the disposition of many drugs in clinical use, based on in vitro data, it was not thought to have a major role in methotrexate transport or disposition. Our study demonstrates proof of principle that genome-wide tools in clinical pharmacologic problems can lead to the discovery of important and novel pharmacogenetic links between inherited genomic variation and drug response in humans.

Description: 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. Disclosures: No relevant conflicts of interest to declare.

Description: High-throughput genome-wide studies have identified significant associations between inherited genetic variations and complex diseases. To delineate the heterogeneous nature of pediatric acute lymphoblastic leukemia (ALL), we performed a genome-wide interrogation of approximately 600K single nucleotide polymorphisms (SNPs) in 470 Caucasian children (using 〉 99% European ancestry genetic markers to define race) with newly diagnosed ALL to identify the contribution of germline SNP genotypes to ALL subtype. Our patient population included children enrolled on St Jude Children’s Research Hospital Total XIIIB and Total XV ALL protocols and the COG9906 ALL protocol. The study cohort included 47 cases with the t(12;21)/TEL-AML1 (aka ETV6/RUNX1) translocation, 116 with hyperdiploidy 〉 50 chromosomes, 46 with T-lineage ALL, and 139 with non-hyperdiploid B-lineage ALL (without known translocations). Using genotype data from the Affymetrix 100K and 500K Mapping Array sets and logistic regression analyses, we found that only patients with TEL-AML1-positive ALL demonstrated a distinct germline SNP signature (p = 0.043, 1000 permutations) compared to patients with other ALL subtypes. We identified 1,046 germline SNPs whose allele frequencies discriminated TEL-AML1 ALL from non-TEL-AML1 ALL (p 〈 0.005). For example, the odds ratio associated with the risk of TEL-AML1 vs. other ALL subtypes for the T vs. C allele at the ITPR2 SNP (rs12814812) was 3.5 (p=0.000019), and that for the G vs. A allele at the IL26 SNP (rs11570906) was 3.1 (p = 0.000111). These 1,046 SNPs represent 224 unique genes. Among these 1,046 SNPs, 28 cis SNPs were significantly associated with the mRNA expression of 19 genes in diagnostic leukemic lymphoblasts. Of these 19 genes, the gene expression of the growth factor, ANGPT2;, coagulation factor, F13A1; and the enzyme, AGA, differed significantly between TEL-AML1-positive and negative cases. We also identified one intronic SNP in each of the translocation target genes (RUNX1 and ETV6) whose genotype frequencies differed between TEL-AML1-positive and negative cases (p = 0.01 and 0.03 respectively). Conversely, SNPs in high linkage disequilibrium with previously hypothesized risk alleles in MTHFR and NQO1 did not differ in allele frequencies between the two subgroups (p 〉 0.05). It is well recognized that TEL-AML1-positive ALL represents a fourth of childhood cases but is exceedingly rare in adults, and hence, it is not surprising that the TEL-AML1-positive ALL subtype may be more likely to be affected by inherited genomic variability compared to other subtypes. We conclude that inherited genetic variation may contribute to risk of or pathogenesis of the TEL-AML1 subtype of childhood ALL.

Description: Abstract 1287 The NCI-directed TARGET AML initiative has provided an opportunity to evaluate the genomic, transcriptomic and epigenomic patterns of large numbers of pediatric patients with newly diagnosed and relapsed AML with the goal of identifying distinctive changes in pathways that are prognostic or can be therapeutically targeted. Genomic sequencing has revealed an important, but limited number of gene mutations, suggesting that epigenetic alterations may be required to explain the development and biological behavior of AML. We have analyzed genome-wide promoter methylation patterns using Illumina Infinium Methylation 27 arrays on 200 paired diagnostic (Dx) and remission (Rm) cases plus 47 case-matched relapsed (Rl) samples. Marked differences in DNA methylation were observed in the AML compared to remission bone marrows. DNA methylation patterns could discriminate specific types of cytogenetically characterized AML, but no subset of specific methylation changes were identified that predicted outcome across the entire cohort irrespective of AML subtype. Due to the strong correlations of methylation pattern with cytogenetically defined subtypes, such a global signature for outcome would be expected to require either a very strong signal or very large sample size to detect a prognostic, methylation signature that was able to validated in heterogeneous cohorts. We therefore evaluated prognostic methylation features within each cytogenetic group. Using a linear model and empirical Bayes statistic, selective groups of high quality methylation probe signals revealed an association with survival that independently predicted outcome within each cytogenetic subtype. Using a top-scoring pair (TSP) algorithm for each karyotype subtype, five different TSPs each for Inv16, MLL, t(8;21) and normal karyotype subgroups were identified that predicted outcome with an accuracy of 100% except for the t(8;21) subtype in which accuracy of predicting outcome was 87%. We used an alternative analytical approach to determine whether specific epigenetic promoter patterns were associated with diagnosis and/or relapse AML compared to remission bone marrows across all subtypes. These patterns could potentially be considered to be essential for AML development and/or progression. We divided patient data into balanced training and test sets. Using a novel pathway analysis based on outlier statistics, we identified methylation changes independent of cytogenetic subtype in gene promoters associated with specific cellular pathways. Cellular pathways of potential interest characterized by preferentially hypermethylated promoters of genes in the AML diagnostic samples compared to remission samples included Hedgehog, extracellular matrix receptor interactions, cell adhesion molecules, (NCAM/CAM) and phospholipase pathways. Those gene sets that showed hypomethylation in the AML diagnostic samples were selectively associated with cytochrome P450, tyrosine, and arachidonic acid metabolism. We generated a 10 gene signature from genes for two of the most significant pathways, i.e., the Hedgehog and cytochrome P450 metabolism pathways, using TSP and applied this TSP signature using a Fisher's exact test for both Dx vs Rm and Rl vs Rm samples (Table). Test Set Dx v Rm Call Dx Call Rm Test Rl v Rm Call Rl Call Rm True Dx 93 3 True Rl 24 1 True Rm 19 77 True Rm 7 18 OR 120, p 〈 2.2 × 10−16 OR 55, p 〈 8.1 × 10−7 The confirmation of the signature in the independent test set validated the significance of the Hedgehog and P450 pathways in newly diagnosed and relapsed pediatric AML. These data demonstrate the unique contribution of epigenetic changes as a prognostic indicator in subgroups of AML. Importantly, outlier analysis followed by pathway-based interrogation identified several key gene sets that are selectively epigenetically altered in an otherwise uncharacterized subset of pediatric AML. Of note, the hypermethylation and hypomethylation of gene promoters characterizing the Hedgehog and of promoters of cytochrome P450 pathways respectively was highly associated with AML diagnosis and relapse samples compared to remission marrows, suggesting a specific role for these pathways in the physiology and maintenance of pediatric AML. Work in progress is examining samples for mutations, gene copy number, expression and functional consequences of the pathways that are selectively altered by epigenetic mechanisms. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 125 Despite identification of genomic alterations in AML, clinically actionable variants are limited. Further, mutations identified in adult AML are less frequent or not found in childhood AML. TARGET AML initiative, a collaboration between National Cancer Institute Office of Cancer Genomics (NCI/OCG) and Children's Oncology Group (COG) provided resources to perform multi-platform analysis of a large number of pediatric AML cases in order to identify therapeutic targets in childhood AML. As part of this effort, diagnostic, remission and relapse specimens from a large cohort of children with AML were subjected to whole genome sequencing (WGS). In addition, all specimens underwent characterization by expression profiling, SNP genotyping, methylation profiling and miRNA sequencing. Specimens from a subset of patients also underwent whole exome sequencing (WES) and whole transcript sequencing (RNA Seq). Here we present data from an interim analysis of the data that identified clinically significant, novel somatic mutations, deletions and translocations involving the ETV6 gene in pediatric patients with AML. Non-silent somatic mutations of ETV6, including missense, splice site mutations and indels were identified by WGS and WES. Large deletions involving ETV6 gene were identified by WGS and SNP/CGH arrays, and novel fusion transcripts (cryptic translocations) involving ETV6 were identified by WGS and RNA Seq. We present the prevalence and clinical significance of genomic alterations of ETV6 that were identified as part of TARGET AML study. WGS and WES data were available from 54 and 22 pediatric AML patients, respectively. In these 76 selected cases, somatic mutations in ETV6 were identified and verified in 3 patients. Frequency validation was performed by sequencing of the entire coding region of the ETV6 gene in diagnostic specimens from 180 children with AML. Somatic mutations of ETV6 gene were identified in 6% of patients, of which none had cytogenetic or molecular risk features. Those with somatic ETV6 mutations had an overall survival (OS) of 20% vs. 76% for those without mutations (p=0.002) with a corresponding relapse risk (RR) of 80% vs. 29% (p=0.004), demonstrating the potential impact of ETV6 mutations in pediatric AML. Deletions involving ETV6 gene (del ETV6) were detected in 3 patients by WGS and confirmed by SNP genotyping array and by fluorescent in situ hybridization (FISH). Subsequent interrogation of the SNP genotyping data identified deletions of 12p13 involving ETV6 in 19/242 patients (8%) ranging from 0.25 Mb to 〉20 Mb. Those with del ETV6 lacked cytogenetic (−7, or −5/del5q) or molecular (FLT3/ITD) high-risk features, but 50% of those with del ETV6 had core binding factor (CBF) AML. All patients with del ETV6 achieved a morphologic complete remission (CR) and had no evidence of minimal residual disease at the end of induction chemotherapy. Relapse risk for patients with and without del ETV6 was 63% vs. 45% (p=0.3) with a corresponding disease-free survival (DFS) of 32% vs. 53% (p=0.2). Due to the high prevalence of CBF AML in patients with del ETV6, we inquired whether this alteration might impact outcome in patients with CBF AML. In patients with CBF AML, del ETV6 was associated with adverse outcome, where CBF patients with and without del ETV6 had an event-free survival of 0% vs. 63%, respectively (p=0.002). Of the CBF AML patients who achieved an initial CR, those with compared to those without a del ETV6 had a RR of 88% (vs. 38%, p=0.08) with a DFS of 0% (vs. 61%, p=0.009) respectively. Cryptic ETV6 translocations involving at least two distinct translocation partners represented a third class of ETV6 alterations identified by WGS and RNA Seq. FISH evaluation of select patients was available and identified 16 patients with positive FISH for ETV6 variants. Those who had ETV6 alterations by FISH had a median age at diagnosis of 3.7 years. Evaluation of remission induction rate as well as post remission outcome demonstrated that all but 3 of these 16 patients either failed to achieve a CR (N=4) or relapsed after an initial remission (82% failure rate). These data identified varying genomic alterations of ETV6 by whole genome sequencing that identify a significant proportion of pediatric AML cases with adverse outcome who may be targeted for altered therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Recurrent somatic mutations in MAP2K1 were identified in 33% of LCH lesions with wild-type BRAF. The mutant MAPK kinase 1 proteins activate ERK. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in vitro was dependent on the specific LCH mutation.

Description: Abstract 571 Methotrexate is an antifolate chemotherapeutic drug commonly used to treat cancer, including acute lymphoblastic leukemia (ALL). Interindividual variation in clearance of methotrexate results in a vast range of exposure to the drug, which affects its clinical effectiveness and risk of toxicity. In a genome-wide association study of children with ALL, we identified the SLCO1B1 gene, encoding liver transporter OATP1B1, as harboring multiple common polymorphisms associated with methotrexate clearance (Trevino et al. J Clin Oncol, 27:5972-5978, 2009). Because these common polymorphisms could not account for the variation in methotrexate clearance entirely, we hypothesized that there may be rare variants in addition to the common polymorphisms in this gene that affect methotrexate disposition. To test this hypothesis, we sequenced SLCO1B1 exons in 699 pediatric leukemia patients, and compared the effects of common versus rare variants on methotrexate clearance (adjusting for clinical covariates). We identified 93 SNPs, 15 of which were non-synonymous (NS); 11 of the 15 NS SNPs were rare, with minor allele frequencies 〈 1%. We used four computational programs (SIFT, PMUT, SNPS3D, and Polyphen2) to predict whether the NS SNPs were damaging to the function of the protein. The 7 NS SNPs predicted to be functionally damaging (common or rare) were more likely to be found among patients with the lowest methotrexate clearance than patients with high clearance (p=2.4×10−8), including one SNP that was observed only once among nearly 1400 chromosomes. Four SLCO1B1 haplotypes were associated with reduced methotrexate clearance (p