McDonnell, Shannon K.

Abstract Colorectal cancer (CRC) is estimated to be the third most common cancer in both men and women in the US in 2012, accounting for over 50,000 deaths. Familial CRC accounts for approximately 30% of all CRC, yet only 5% of familial CRCs can be attributed to mutations in known hereditary colon cancer (HCC) genes. Many families with a strong family history of CRC do not have mutations in known CRC genes. Finding the genes underpinning CRC in these families may improve screening for disease and allow the development of more targeted therapeutics. We completed targeted sequencing of over 2,000 genes and miRNAs in 1,500 cases of CRC from the Colon Cancer Family Registry (CCFR) in an effort to identify rare variants associated with CRC risk and to identify mutations in known CRC genes missed by conventional sequencing. Several categories of genes were included: 18 known HCC genes; 18 candidate HCC genes; 185 genes in previously reported CRC linkage peaks; 1,423 miRNAs; and 363 genes identified through whole exome sequencing of 40 affected cases in 16 families with hereditary CRC (n=2,007 total genes sequenced). Genomic DNA samples were indexed, then libraries were made using Agilent's SureSelect Targeted Enrichment kit. Samples were pooled (96 samples/pool), then each pool of samples was run on five lanes on an Illumina HiSeq2000 (average of 19.2 samples/lane). GenomeGPS, an in-house developed workflow, was used for read alignment, variant calling, and annotation. In early analyses of the first 161 cases, the mean number of mapped reads exceeded 20 million per sample and over 90% of the target region exceeded 40x coverage. A total of 26,484 SNPs were identified and passed quality filtering. Sequencing identified known mutations in MMR genes in positive control cases as well as mutations in HCC genes in previously untested cases. Analysis is currently on-going on the remaining samples and we will report results on the full set of 1500 cases at the meeting. Citation Format: Melissa S. DeRycke, Shanaka R. Gunawardena, Sumit Middha, Shannon K. McDonnell, Shaun M. Riska, Zachary C. Fogarty, Bruce W. Eckloff, Daniel J. Schaid, Ellen L. Goode, Stephen N. Thibodeau. Targeted sequencing to identify rare variants in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2548. doi:10.1158/1538-7445.AM2013-2548

Purpose Liver injury is a serious adverse event leading to permanent discontinuation of lapatinib in affected patients. This study aimed to validate previously associated major histocompatibility complex (MHC) variants as predictors of risk of liver injury by using a large, randomized, placebo-controlled trial of lapatinib in human epidermal growth factor receptor 2–positive, early-stage breast cancer (Tykerb Evaluation After Chemotherapy [TEACH]: Lapatinib Versus Placebo In Women With Early-Stage Breast Cancer). Patients and Methods The frequency of ALT elevation cases was compared among four MHC variants in 1,194 patients randomly assigned to lapatinib. Cumulative ALT elevation time courses during treatment were also compared between carriers and noncarriers of specified MHC variants. Results In lapatinib-treated patients, there was a significant difference in ALT case incidence between HLA carriers and noncarriers. The highly correlated alleles HLA-DRB1*07:01 and HLA-DQA1*02:01 (study frequency, 22.4%) were associated with ALT elevation (odds ratio, 14) between cases (n = 37) and controls (n = 1,071). These associations strengthened at higher ALT elevation thresholds and in Hy's Law cases. In lapatinib-treated patients, the overall risk for National Cancer Institute-Common Terminology Criteria for Adverse Events grade 3 ALT elevation (> 5× upper limit of normal) was 2.1%; HLA allele carriers had an increased risk of 7.7%; in noncarriers, risk was reduced to 0.5%, comparable to ALT elevation for all patients receiving placebo. The increase in ALT case incidence in the lapatinib arm showed no evidence of plateau during 1 year of lapatinib treatment. Conclusion These results validate HLA-DRB1*07:01 allele carriage as a predictor of increased risk of lapatinib-induced liver injury and implicate an immune pathology. The HLA association could support clinical management of patients experiencing hepatotoxicity during lapatinib treatment.

ABSTRACTSearching for rare genetic variants associated with complex diseases can be facilitated by enriching for diseased carriers of rare variants by sampling cases from pedigrees enriched for disease, possibly with related or unrelated controls. This strategy, however, complicates analyses because of shared genetic ancestry, as well as linkage disequilibrium among genetic markers. To overcome these problems, we developed broad classes of “burden” statistics and kernel statistics, extending commonly used methods for unrelated case‐control data to allow for known pedigree relationships, for autosomes and the X chromosome. Furthermore, by replacing pedigree‐based genetic correlation matrices with estimates of genetic relationships based on large‐scale genomic data, our methods can be used to account for population‐structured data. By simulations, we show that the type I error rates of our developed methods are near the asymptotic nominal levels, allowing rapid computation of P‐values. Our simulations also show that a linear weighted kernel statistic is generally more powerful than a weighted “burden” statistic. Because the proposed statistics are rapid to compute, they can be readily used for large‐scale screening of the association of genomic sequence data with disease status.

Abstract Background: Colorectal cancer (CRC) in densely affected families without Lynch Syndrome may be due to mutations in undiscovered genetic loci. Familial linkage analyses have yielded disparate results; the use of exome sequencing in coding regions may identify novel segregating variants. Methods: We completed exome sequencing on 40 affected cases from 16 multicase pedigrees to identify novel loci. Variants shared among all sequenced cases within each family were identified and filtered to exclude common variants and single-nucleotide variants (SNV) predicted to be benign. Results: We identified 32 nonsense or splice-site SNVs, 375 missense SNVs, 1,394 synonymous or noncoding SNVs, and 50 indels in the 16 families. Of particular interest are two validated and replicated missense variants in CENPE and KIF23, which are both located within previously reported CRC linkage regions, on chromosomes 1 and 15, respectively. Conclusions: Whole-exome sequencing identified DNA variants in multiple genes. Additional sequencing of these genes in additional samples will further elucidate the role of variants in these regions in CRC susceptibility. Impact: Exome sequencing of familial CRC cases can identify novel rare variants that may influence disease risk. Cancer Epidemiol Biomarkers Prev; 22(7); 1239–51. ©2013 AACR.

Summary A recent meta‐analysis of three genome‐wide association studies of chronic lymphocytic leukaemia ( CLL ) identified two common variants at the 6p21.31 locus that are associated with CLL risk. To verify and further explore the association of these variants with other non‐ H odgkin lymphoma ( NHL ) subtypes, we genotyped 1196 CLL cases, 1699 NHL cases, and 2410 controls. We found significant associations between the 6p21.31 variants and CLL risk (rs210134: P = 0·01; rs210142: P = 6·8 × 10 −3 ). These variants also showed a trend towards association with some of the other NHL subtypes. Our results validate the prior work and support specific genetic pathways for risk among NHL subtypes.

Abstract Several platforms for genome sequencing and methods for exon enrichment are now available and continue to evolve quickly. In this study, we compared the performance of whole exome sequencing using two instruments, Illumina's GAIIx and HiSeq 2000, comparing single versus duplicate runs. Eight runs of eight samples each were performed with genomic DNA derived from whole blood and sample libraries prepared with the Agilent SureSelect capture array. One run was conducted in duplicate on the GAIIx, and, for some analyses, the combined data were used. We used TREAT (Targeted RE-sequencing and Annotation Tool), developed in-house for data analysis, including sequence alignment (MAQ, BWA), local re-alignment (GATK), variant calling (utilizing MAQ), annotation (SIFT and Seattle Seq), and visualization. Overall, duplicate runs on the GAIIx platform (compared to a single run) increased the number of total reads by approximately two-fold: from ∼66 M to ∼130 M. For both single and duplicate runs, ∼90% of reads mapped to the reference sequence, and ∼55% of reads mapped on-target. Of importance, the percent coverage of the target region increased substantially with duplicate runs; ∼72% of the target region was covered at 40-fold or more when run in duplicate compared to only ∼52% when run once. At 30x, 20x, and 10x, there was a 1.3- (62% v. 78%), 1.2- (72% v. 84%), and 1.1-fold (84% v. 91%) increase respectively, in the percent coverage of the target region for samples run twice. A similar number of filtered, on-target SNPs per sample (∼24 K) was found for both single and duplicate run analyses. However, a 1.3-fold increase in the number of on-target indels was seen in the duplicate run (∼1,200) compared to the single run (∼900). When the combined data for samples run twice on a GAIIx were compared to the samples run using a HiSeq, the total number of reads were similar (∼130 M), although several of the samples had substantially more reads on the HiSeq platform. Samples run on a HiSeq had an increase in the percent coverage of the target region at 40x (72% v. 81%), 30x (78% v. 86%), 20x (84% v. 91%) and 10x (91% v. 95%) compared to the two-run approach on a GAIIx. A similar number of filtered on-target SNPs (∼24 K/sample) and indels (∼1200) was found with the HiSeq compared to two runs on a GAIIx. In summary, our results demonstrate increases in the total number of reads and in overall coverage with the HiSeq 2000 instrument. The total number of SNPs and indel that mapped on target for the version 1 Agilent SureSelect capture array is ∼25 K and 1,200 per sample, respectively. As capture is of variable efficiency for individual runs, higher average coverage of the target region is necessary for sufficient coverage of poorly captured regions. Additional analyses comparing Agilent SureSelect versions 1 and 2 arrays are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2742. doi:10.1158/1538-7445.AM2011-2742

Abstract We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10−16). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.