Estimation of the Multiple Testing Burden for Genomewide Association Studies of Common Variants

Genomewide association studies are an exciting strategy in genetics, recently becoming feasible. While pioneering studies are being underway, it is already clear that the analytic issue of determining the significance of results presents a challenge to the wide community of researchers. Rather than each study engaging in independent evaluation, there is need in the community to have standards set for whole-genome significance. 
We have therefore undertaken the task of developing such standards, based on data collected with collaborators in the International Haplotype Map Consortium. We report an estimated burden of a million independent tests genomewide in Europeans, and twice that number in Africans. We further identify the sensitivity of the testing burden to the required significance level, with implications to staged design of association studies.
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Proxy genotypes and phenotypes for human genetics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008.Includes bibliographical references.Genetic mapping by association is an unbiased approach to discover genes and pathways influencing disease traits and response to drugs and environmental exposures. There are two key obstacles to mapping in humans: (1) The full sequence of study subjects cannot yet be obtained; and (2) There are substantial limits to the phenotypes that can be safely elicited or measured. Geneticists thus rely on practically measurable sets of genotypes to proxy for the sequence and human in-vitro models that proxy for in-vivo genetics and physiology while allowing for perturbation and characterization in high throughput. This thesis presents the development of one important class of proxy genotypes, those that capture most common genetic variation, as well as an evaluation and refinement of proxy phenotypes offered by one commonly used in-vitro model, the lymphoblastoid cell-line.Capturing common human genetic variation for genome-wide association studies requires genotyping a feasible subset of proxy (or "tag") SNPs. We investigated selection and analysis of tag SNPs, examined the relationship between investment in genotyping and statistical power, and evaluated whether power is compromised when tags are selected from an incomplete resource such as HapMap. We demonstrate an efficient haplotypebased tagging approach and other methods that dramatically increase tagging efficiency. Examining all observed haplotypes for association increases power to detect rare causal alleles, while reducing power for common alleles. Power is robust to completeness of the reference panel and holds across demographically related groups.Lymphoblastoid cell lines (LCLs) are being developed into an in-vitro model where genetics of human gene expression, drug response, and other traits can be studied under controlled conditions. However, the impact of the immortalization process, the relative influence of non-genetic factors, and reproducibility of measured traits are not yet understood.(cont.) We addressed these questions while mapping loci for response to chemotherapy and found that traits in LCLs are subject to substantial confounders and are only modestly reproducible in independent experiments. Despite this, RNA expression of many genes is affected by genetic variation and predicts response to drugs; integrating SNPs, RNA, and drug response can identify novel pharmacogenetic variation mediated by RNA.by Roman Yelensky.Ph.D

Multiple gene aberrations and breast cancer: lessons from super-responders.

BackgroundThe presence of multiple molecular aberrations in patients with breast cancer may correlate with worse outcomes.Case presentationsWe performed in-depth molecular analysis of patients with estrogen receptor-positive, HER2-negative, hormone therapy-refractory breast cancer, who achieved partial or complete responses when treated with anastrozole and everolimus. Tumors were analyzed using a targeted next generation sequencing (NGS) assay in a Clinical Laboratory Improvement Amendments laboratory. Genomic libraries were captured for 3,230 exons in 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer and sequenced to high coverage. Patients received anastrozole (1 g PO daily) and everolimus (5 or 10 mg PO daily). Thirty-two patients with breast cancer were treated on study and 5 (16 %) achieved a partial or complete response. Primary breast tissue was available for NGS testing in three of the responders (partial response with progression free survival of 11 and 14 months, respectively; complete response with progression free survival of 9+ months). The following molecular aberrations were observed: PTEN loss by immunohistochemistry, CCDN1 and FGFR1 amplifications, and PRKDC re-arrangement (NGS) (patient #1); PIK3CA and PIK3R1 mutations, and CCDN1, FGFR1, MYC amplifications (patient #2); TP53 mutation, CCNE1, IRS2 and MCL1 amplifications (patient #3). Some (but not all) of these aberrations converge on the PI3K/AKT/mTOR pathway, perhaps accounting for response.ConclusionsPatients with estrogen receptor-positive breast cancer can achieve significant responses on a combination of anastrozole and everolimus, even in the presence of multiple molecular aberrations. Further study of next generation sequencing-profiled tumors for convergence and resistance pathways is warranted

Next generation sequencing of exceptional responders with BRAF-mutant melanoma: implications for sensitivity and resistance.

BackgroundPatients with BRAF mutation-positive advanced melanoma respond well to matched therapy with BRAF or MEK inhibitors, but often quickly develop resistance.MethodsTumor tissue from ten patients with advanced BRAF mutation-positive melanoma who achieved partial response (PR) or complete response (CR) on BRAF and/or MEK inhibitors was analyzed using next generation sequencing (NGS) assay. Genomic libraries were captured for 3230 exons in 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer and sequenced to average median depth of 734X with 99% of bases covered >100X.ResultsThree of the ten patients (median number of prior therapies = 2) attained prolonged CR (duration = 23.6+ to 28.7+ months); seven patients achieved either a PR or a short-lived CR. One patient who achieved CR ongoing at 28.7+ months and had tissue available close to the time of initiating BRAF inhibitor therapy had only a BRAF mutation. Abnormalities in addition to BRAF mutation found in other patients included: mutations in NRAS, APC and NF1; amplifications in BRAF, aurora kinase A, MYC, MITF and MET; deletions in CDKN2A/B and PAX5; and, alterations in RB1 and ATM. Heterogeneity between patients and molecular evolution within patients was noted.ConclusionNGS identified potentially actionable DNA alterations that could account for resistance in patients with BRAF mutation-positive advanced melanoma who achieved a PR or CR but whose tumors later progressed. A subset of patients with advanced melanoma may harbor only a BRAF mutation and achieve a durable CR on BRAF pathway inhibitors

Anastrozole and everolimus in advanced gynecologic and breast malignancies: activity and molecular alterations in the PI3K/AKT/mTOR pathway.

BackgroundSince PI3K/AKT/mTOR pathway activation diminishes the effects of hormone therapy, combining aromatase inhibitors (anatrozole) with mTOR inhibitors (everolimus) was investigated.Patients and methodsWe evaluated anastrozole and everolimus in 55 patients with metastatic estrogen (ER) and/or progesterone receptor (PR)-positive breast and gynecologic tumors. Endpoints were safety, antitumor activity and molecular correlates.ResultsFull doses of anastrozole (1 mg PO daily) and everolimus (10 mg PO daily) were well tolerated. Twelve of 50 evaluable patients (24%) (median = 3 prior therapies) achieved stable disease (SD) ≥ 6 months/partial response (PR)/complete response (CR) (n = 5 (10%) with PR/CR): 9 of 32 (28%) with breast cancer (n=5 (16%) with PR/CR); 2 of 10 (20%), ovarian cancer; and 1 of 6 (17%), endometrial cancer. Six of 22 patients (27%) with molecular alterations in the PI3K/AKT/mTOR pathway achieved SD ≥ 6 months/PR/CR. Six of 8 patients (75%) with SD ≥ 6 months/PR/CR with molecular testing demonstrated at least one alteration in the PI3K/AKT/mTOR pathway: mutations in PIK3CA (n=3) and AKT1 (n=1) or PTEN loss (n=3). All three responders (CR (n = 1); PR (n=2)) who had next generation sequencing demonstrated additional alterations: amplifications in CCNE1, IRS2, MCL1, CCND1, FGFR1 and MYC and a rearrangement in PRKDC.ConclusionsCombination anastrozole and everolimus is well tolerated at full approved doses, and is active in heavily-pretreated patients with ER and/or PR-positive breast, ovarian and endometrial cancers. Responses were observed in patients with multiple molecular aberrations. CLINICAL TRAILS INCLUDED: NCT01197170

Genomic Profiling of Advanced-Stage, Metaplastic Breast Carcinoma by Next-Generation Sequencing Reveals Frequent, Targetable Genomic Abnormalities and Potential New Treatment Options

Context.— Metastatic metaplastic breast carcinoma (MPBC) is an uncommon, but aggressive, tumor resistant to conventional chemotherapy. Objective.— To learn whether next-generation sequencing could identify potential targets of therapy for patients with relapsed and metastatic MPBC. Design.— Hybridization capture of 3769 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer was applied to a minimum of 50 ng of DNA extracted from 20 MPBC formalin-fixed, paraffin-embedded specimens and sequenced to high uniform coverage. Results.— The 20 patients with MPBC had a median age of 62 years (range, 42–86 years). There were 9 squamous (45%), 9 chondroid (45%), and 2 spindle cell (10%) MPBCs, all of which were high grade. Ninety-three genomic alterations were identified, (range, 1–11) with 19 of the 20 cases (95%) harboring an alteration that could potentially lead to a targeted treatment option. The most-common alterations were in TP53 (n = 69; 75%), PIK3CA (n = 37; 40%), MYC (n = 28; 30%), MLL2 (n = 28; 30%), PTEN (n = 23; 25%), CDKN2A/B (n = 19; 20%), CCND3 (n = 14; 15%), CCNE1 (n = 9; 10%), EGFR (n = 9; 10%), and KDM6A (n = 9; 10%); AKT3, CCND1, CCND2, CDK4, FBXW7, FGFR1, HRAS, NF1, PIK3R1, and SRC were each altered in a single case. All 16 MPBCs (100%) that were negative for ERBB2 (HER2) overexpression by immunohistochemistry and/or ERBB2 (HER2) amplification by fluorescence in situ hybridization were also uniformly (100%) negative for ERBB2 amplification by next-generation sequencing–based copy-number assessment. Conclusions.— Our results indicate that genomic profiling using next-generation sequencing can identify clinically meaningful alterations that have the potential to guide targeted treatment decisions in most patients with metastatic MPBC

Clinical actionability of comprehensive genomic profiling for management of rare or refractory cancers

Background. The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. Methods. A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. Results. Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0–10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. Conclusion. Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability. Implications for Practice: Identification of key factors that facilitate use of genomic tumor testing results and implementation of genomically guided therapy may lead to enhanced benefit for patients with rare or difficult to treat cancers. Clinical use of a targeted next-generation sequencing assay in the setting of an institutional molecular tumor board led to implementable clinical action in over one third of patients with rare and poor prognosis cancers. The major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access both on trial and off label. Approaches to increase actionability include early and serial sequencing in the clinical course and expanded access to genomically guided early phase clinical trials and targeted agents

Next-Generation Sequencing Reveals High Concordance of Recurrent Somatic Alterations Between Primary Tumor and Metastases From Patients With Non-Small-Cell Lung Cancer

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Genetic Analysis of Human Traits In Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype–phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance—i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.Molecular and Cellular Biolog

Comprehensive Genomic Profiling of Pancreatic Acinar Cell Carcinomas

significantly enriched for genomic alterations (GAs) causing inactivation of DNA repair genes (45%); these GAs have been associated with sensitivity to platinum-based therapies and PARP inhibitors. Collectively, these results identify potentially actionable GAs in the majority of PACCs, and provide a rationale for using personalized therapies in this disease. Statement of Significance PACC is genomically distinct from other pancreatic cancers. Fusions in RAF genes and mutually exclusive inactivation of DNA repair genes represent novel potential therapeutic targets that are altered in over two-thirds of these tumors