The cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.

MicroRNAs (miRNAs) are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available.

Genome-wide association studies (GWASs) assess correlation between traits and DNA sequence variation using large numbers of genetic variants such as single nucleotide polymorphisms (SNPs) distributed across the genome. A GWAS produces many trait-SNP associations with low p-values, but few are replicated in subsequent studies. We sought to determine if characteristics of the genomic loci associated with a trait could be used to identify initial associations with a higher chance of replication in a second cohort. Data from the age-related eye disease study (AREDS) of 100,000 SNPs on 395 subjects with and 198 without age-related macular degeneration (AMD) were employed. Loci highly associated with AMD were characterized based on the distribution of genotypes, level of significance, and clustering of adjacent SNPs also associated with AMD suggesting linkage disequilibrium or multiple effects. Forty nine loci were highly associated with AMD, including 3 loci (CFH, C2/BF, LOC387715/HTRA1) already known to contain important genetic risks for AMD. One additional locus (C3) reported during the course of this study was identified and replicated in an additional study group. Tag-SNPs and haplotypes for each locus were evaluated for association with AMD in additional cohorts to account for population differences between discovery and replication subjects, but no additional clearly significant associations were identified. Relying on a significant genotype tests using a log-additive model would have excluded 57% of the non-replicated and none of the replicated loci, while use of other SNP features and clustering might have missed true associations.

Age-related bone loss in humans is associated with a decrease in bone formation relative to bone resorption, although the mechanisms for this impairment in bone formation with aging are not well understood. It is known that the precursors for the bone-forming osteoblasts reside in the mesenchymal cell population in bone marrow. Thus, in an effort to identify relevant genetic pathways that are altered with aging, we examined the gene expression and DNA methylation patterns from a highly enriched bone marrow mesenchymal cell population from young (mean age, 28.7 years) versus old (mean age, 73.3 years) women. Bone marrow mononuclear cells from these women were depleted of hematopoietic lineage (lin) and endothelial cells using a combination of magnetic- and fluorescence-activated cell sorting, yielding a previously characterized mesenchymal cell population (lin-/CD34-/CD31- cells) that is capable of osteoblast differentiation. Whole transcriptome RNA sequencing (RNAseq) of freshly isolated cells (without in vitro culture) identified 279 differentially expressed genes (p < 0.05, false discovery rate [q]< 0.10) between the young and old subjects. Pathway analysis revealed statistically significant (all p < 0.05) alterations in protein synthesis and degradation pathways, as well as mTOR, gap junction, calcium, melatonin and NFAT signaling pathways. Further, Reduced Representational Bisulphite sequencing (RRBS DNA methylation sequencing) revealed significant differences in methylation between the young and old subjects surrounding the promoters of 1528 target genes that also exhibited significant differences in gene expression by RNAseq. In summary, these studies provide novel insights into potential pathways affected by aging in a highly enriched human mesenchymal cell population analyzed without the confounding effects of in vitro culture. Specifically, our finding of alterations in several genes and pathways leading to impaired protein synthesis and turnover with aging in bone marrow mesenchymal cells points to the need for further studies examining how these changes, as well as the other alterations with aging that we identified, may contribute to the age-related impairment in osteoblast formation and/or function.

Clinical genetic testing is commercially available for rs61764370, an inherited variant residing in a KRAS 3' UTR microRNA binding site, based on suggested associations with increased ovarian and breast cancer risk as well as with survival time. However, prior studies, emphasizing particular subgroups, were relatively small. Therefore, we comprehensively evaluated ovarian and breast cancer risks as well as clinical outcome associated with rs61764370.

Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.

Ovarian cancer represents the most lethal tumor type among malignancies of the female reproductive system. Overall survival rates remain low. In this study, we identify the serine protease inhibitor Kazal type 1 (SPINK1) as a potential therapeutic target for a subset of ovarian cancers. We show that SPINK1 drives ovarian cancer cell proliferation through activation of epidermal growth factor receptor (EGFR) signaling, and that SPINK1 promotes resistance to anoikis through a distinct mechanism involving protease inhibition. In analyses of ovarian tumor specimens from a Mayo Clinic cohort of 490 patients, we further find that SPINK1 immunostaining represents an independent prognostic factor for poor survival, with the strongest association in patients with nonserous histological tumor subtypes (endometrioid, clear cell, and mucinous). This study provides novel insight into the fundamental processes underlying ovarian cancer progression, and also suggests new avenues for development of molecularly targeted therapies.

Disruption in circadian gene expression, whether due to genetic variation or environmental factors (e.g., light at night, shiftwork), is associated with increased incidence of breast, prostate, gastrointestinal and hematologic cancers and gliomas. Circadian genes are highly expressed in the ovaries where they regulate ovulation; circadian disruption is associated with several ovarian cancer risk factors (e.g., endometriosis). However, no studies have examined variation in germline circadian genes as predictors of ovarian cancer risk and invasiveness. The goal of the current study was to examine single nucleotide polymorphisms (SNPs) in circadian genes BMAL1, CRY2, CSNK1E, NPAS2, PER3, REV1 and TIMELESS and downstream transcription factors KLF10 and SENP3 as predictors of risk of epithelial ovarian cancer (EOC) and histopathologic subtypes. The study included a test set of 3,761 EOC cases and 2,722 controls and a validation set of 44,308 samples including 18,174 (10,316 serous) cases and 26,134 controls from 43 studies participating in the Ovarian Cancer Association Consortium (OCAC). Analysis of genotype data from 36 genotyped SNPs and 4600 imputed SNPs indicated that the most significant association was rs117104877 in BMAL1 (OR = 0.79, 95% CI = 0.68-0.90, p = 5.59 × 10-4]. Functional analysis revealed a significant down regulation of BMAL1 expression following cMYC overexpression and increasing transformation in ovarian surface epithelial (OSE) cells as well as alternative splicing of BMAL1 exons in ovarian and granulosa cells. These results suggest that variation in circadian genes, and specifically BMAL1, may be associated with risk of ovarian cancer, likely through disruption of hormonal pathways.

Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10(-5)). For three cis-eQTL associations (P<1.4 × 10(-3), FDR<0.05) at 1p36 (CDC42), 1p34 (CDCA8) and 2q31 (HOXD9), we evaluate the functional role of each candidate by perturbing expression of each gene in HGSOC precursor cells. Overexpression of HOXD9 increases anchorage-independent growth, shortens population-doubling time and reduces contact inhibition. Chromosome conformation capture identifies an interaction between rs2857532 and the HOXD9 promoter, suggesting this SNP is a leading causal variant. Transcriptomic profiling after HOXD9 overexpression reveals enrichment of HGSOC risk variants within HOXD9 target genes (P=6 × 10(-10) for risk variants (P<10(-4)) within 10 kb of a HOXD9 target gene in ovarian cells), suggesting a broader role for this network in genetic susceptibility to HGSOC.

Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis.

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r(2) = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.

Common variants in 94 loci have been associated with breast cancer including 15 loci with genome-wide significant associations (P<5 × 10(-8)) with oestrogen receptor (ER)-negative breast cancer and BRCA1-associated breast cancer risk. In this study, to identify new ER-negative susceptibility loci, we performed a meta-analysis of 11 genome-wide association studies (GWAS) consisting of 4,939 ER-negative cases and 14,352 controls, combined with 7,333 ER-negative cases and 42,468 controls and 15,252 BRCA1 mutation carriers genotyped on the iCOGS array. We identify four previously unidentified loci including two loci at 13q22 near KLF5, a 2p23.2 locus near WDR43 and a 2q33 locus near PPIL3 that display genome-wide significant associations with ER-negative breast cancer. In addition, 19 known breast cancer risk loci have genome-wide significant associations and 40 had moderate associations (P<0.05) with ER-negative disease. Using functional and eQTL studies we implicate TRMT61B and WDR43 at 2p23.2 and PPIL3 at 2q33 in ER-negative breast cancer aetiology. All ER-negative loci combined account for ∼11% of familial relative risk for ER-negative disease and may contribute to improved ER-negative and BRCA1 breast cancer risk prediction.

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations.

Sclerosing adenosis (SA), found in ¼ of benign breast disease (BBD) biopsies, is a histological feature characterized by lobulocentric proliferation of acini and stromal fibrosis and confers a two-fold increase in breast cancer risk compared to women in the general population. We evaluated a NanoString-based gene expression assay to model breast cancer risk using RNA derived from formalin-fixed, paraffin-embedded (FFPE) biopsies with SA.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Interest in preclinical drug development for ovarian cancer has stimulated development of patient-derived xenograft (PDX) or tumorgraft models. However, the unintended formation of human lymphoma in severe combined immunodeficiency (SCID) mice from Epstein-Barr virus (EBV)-infected human lymphocytes can be problematic. In this study, we have characterized ovarian cancer PDXs which developed human lymphomas and explore methods to suppress lymphoproliferative growth. Fresh human ovarian tumors from 568 patients were transplanted intraperitoneally in SCID mice. A subset of PDX models demonstrated atypical patterns of dissemination with mediastinal masses, hepatosplenomegaly, and CD45-positive lymphoblastic atypia without ovarian tumor engraftment. Expression of human CD20 but not CD3 supported a B-cell lineage, and EBV genomes were detected in all lymphoproliferative tumors. Immunophenotyping confirmed monoclonal gene rearrangements consistent with B-cell lymphoma, and global gene expression patterns correlated well with other human lymphomas. The ability of rituximab, an anti-CD20 antibody, to suppress human lymphoproliferation from a patient's ovarian tumor in SCID mice and prevent growth of an established lymphoma led to a practice change with a goal to reduce the incidence of lymphomas. A single dose of rituximab during the primary tumor heterotransplantation process reduced the incidence of CD45-positive cells in subsequent PDX lines from 86.3% (n = 117 without rituximab) to 5.6% (n = 160 with rituximab), and the lymphoma rate declined from 11.1% to 1.88%. Taken together, investigators utilizing PDX models for research should routinely monitor for lymphoproliferative tumors and consider implementing methods to suppress their growth.

Germline genetic variation has been suggested to influence the survival of breast cancer patients independently of tumor pathology. We have studied survival associations of genetic variants in two etiologically unique groups of breast cancer patients, the carriers of germline pathogenic variants in BRCA1 or BRCA2 genes. We found that rs57025206 was significantly associated with the overall survival, predicting higher mortality of BRCA1 carrier patients with estrogen receptor-negative breast cancer, with a hazard ratio 4.37 (95% confidence interval 3.03-6.30, P = 3.1 × 10-9). Multivariable analysis adjusted for tumor characteristics suggested that rs57025206 was an independent survival marker. In addition, our exploratory analyses suggest that the associations between genetic variants and breast cancer patient survival may depend on tumor biological subgroup and clinical patient characteristics.

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS.

Mucinous ovarian carcinoma (MOC) is a unique subtype of ovarian cancer with an uncertain etiology, including whether it genuinely arises at the ovary or is metastatic disease from other organs. In addition, the molecular drivers of invasive progression, high-grade and metastatic disease are poorly defined. We perform genetic analysis of MOC across all histological grades, including benign and borderline mucinous ovarian tumors, and compare these to tumors from other potential extra-ovarian sites of origin. Here we show that MOC is distinct from tumors from other sites and supports a progressive model of evolution from borderline precursors to high-grade invasive MOC. Key drivers of progression identified are TP53 mutation and copy number aberrations, including a notable amplicon on 9p13. High copy number aberration burden is associated with worse prognosis in MOC. Our data conclusively demonstrate that MOC arise from benign and borderline precursors at the ovary and are not extra-ovarian metastases.

Copy number variants (CNVs) have been implicated in many complex diseases. We examined whether inherited CNVs were associated with overall survival among women with invasive epithelial ovarian cancer. Germline DNA from 1,056 cases (494 deceased, average of 3.7 years follow-up) was interrogated with the Illumina 610 quad genome-wide array containing, after quality control exclusions, 581,903 single nucleotide polymorphisms (SNPs) and 17,917 CNV probes. Comprehensive analysis capitalized upon the strengths of three complementary approaches to CNV classification. First, to identify small CNVs, single markers were evaluated and, where associated with survival, consecutive markers were combined. Two chromosomal regions were associated with survival using this approach (14q31.3 rs2274736 p = 1.59 × 10(-6), p = 0.001; 22q13.31 rs2285164 p = 4.01 × 10(-5), p = 0.009), but were not significant after multiple testing correction. Second, to identify large CNVs, genome-wide segmentation was conducted to characterize chromosomal gains and losses, and association with survival was evaluated by segment. Four regions were associated with survival (1q21.3 loss p = 0.005, 5p14.1 loss p = 0.004, 9p23 loss p = 0.002, and 15q22.31 gain p = 0.002); however, again, after correcting for multiple testing, no regions were statistically significant, and none were in common with the single marker approach. Finally, to evaluate associations with general amounts of copy number changes across the genome, we estimated CNV burden based on genome-wide numbers of gains and losses; no associations with survival were observed (p > 0.40). Although CNVs that were not well-covered by the Illumina 610 quad array merit investigation, these data suggest no association between inherited CNVs and survival after ovarian cancer.