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The idiopathic inflammatory myopathies (IIM) are heterogeneous diseases, thought to be initiated by immune activation in genetically predisposed individuals. In this study we imputed variants from the Immunochip array using a large reference panel to fine-map associations and identify novel associations in IIM.We analysed 2,565 Caucasian IIM samples collected through the Myositis Genetics Consortium (MYOGEN) and 10,260 ethnically-matched controls. We imputed 1,648,116 variants from the Immunochip array using the Haplotype Reference Consortium panel and conducted association analysis on IIM, and clinical and serological subgroups.The human leukocyte antigen (HLA) locus was consistently the most significantly associated region. Four non-HLA regions reached genome-wide significance, three in the whole IIM cohort (SDK2 and LINC00924 - both novel, and STAT4), with evidence of independent variants in STAT4, and NAB1 in the polymyositis (PM) subgroup. We also found suggestive evidence of association with loci previously associated with other autoimmune rheumatic diseases (TEC and LTBR). We identified more significant associations than those previously reported in IIM, for STAT4 and DGKQ in the total cohort, for NAB1 and FAM167A-BLK loci in PM, and CCR5 in inclusion body myositis. We found enrichment of variants among DNase I hypersensitivity sites and histone marks associated with active transcription within blood cells.We report novel and strong associations in IIM and PM, and localise signals to single genes and immune cell types. This article is protected by copyright. All rights reserved.

Lead author Steven Gullberg reports with colleagues on a new joint initiative to acknowledge and respect Indigenous sensitivities where observatories have been built upon sacred grounds

Can a previously defined relationship between sperm capacitation and the probability of a man generating pregnancy within three cycles, prospectively predict male fertility in diverse clinical settings? A second study asked, what is the prevalence of impaired sperm fertilizing ability in men questioning their fertility (MQF), and does this relate to traditional semen analysis metrics?In the multicentric, prospective observational study, data (n = 128; six clinics) were analysed to test a published relationship between the percentage of fertilization-competent, capacitated spermatozoa (Cap-Score) and probability of generating pregnancy (PGP) within three cycles of intrauterine insemination. Logistic regression of total pregnancy outcomes (n = 252) assessed fit. In the cohort comparison, Cap-Scores of MQF (n = 2155; 22 clinics) were compared with those of 76 fertile men.New outcomes (n = 128) were rank-ordered by Cap-Score and divided into quintiles (25-26 per group); chi-squared testing revealed no difference between predicted and observed pregnancies (P = 0.809). Total outcomes (n = 252; 128 new + 124 previous) were pooled and the model recalculated, yielding an improved fit (P0.001). Applying the Akaike information criterion found that the optimal model used Cap-Score alone. Cap-Scores were performed on 2155 men (with semen analysis data available for 1948). To compare fertilizing ability, men were binned by PGP (≤19%, 20-29%, 30-39%, 40-49%, 50-59%, ≥60%). Distributions of PGP and the corresponding Cap-Scores were significantly lower in MQF versus fertile men (P0.001). Notably, 64% of MQF with normal volume, concentration and motility (757/1183) had PGP of 39% or less (Cap-Scores ≤31), versus 25% of fertile men.Sperm capacitation prospectively predicted male fertility. Impaired capacitation affects many MQF with normal semen analysis results, informing diagnosis versus idiopathic infertility.

Perinatal mood and anxiety disorders (PMADs) impact multiple domains of maternal and child well-being. Estimates of postpartum depressive symptoms range from 6.5% to 12.9% and from 8.6% to 9.9% for postpartum anxiety. We sought to identify the role social support could play in mitigating PMADs.The data are drawn from the Listening to Mothers in California survey; results are representative of women who gave birth in 2016 in a California hospital. The Patient Health Questionnaire-4 was used to assess total symptoms of PMADs and anxiety and depressive symptoms individually. Two questions adapted from the Medical Outcomes Study Social Support Survey were used to assess emotional, practical, and functional (combined) social support. After exclusions for missing data related to PMADs or social support, we analyzed data from 2,372 women.At the time of survey administration (mean 5.7 months after birth), 7.0% of respondents reported elevated PMAD symptoms and 45.9% reported that they always received functional social support. In multivariable analysis, controlling for demographic and pregnancy-related factors and prenatal anxiety and depressive symptoms, women who reported consistent support had a prevalence of elevated PMAD symptoms one-half that of those who did not (adjusted odds ratio, 0.50; 95% confidence interval, 0.34-0.74).This study suggests that consistent social support serves as a robust protective factor against postpartum symptoms of PMADs. Because many predictors of PMADs are not modifiable, social support stands out as an important target for programmatic intervention, particularly in light of increased isolation related to the COVID-19 pandemic.

GEMO: Ligue Nationale Contre le Cancer; the Association “Le cancer du sein, parlons‐en!” Award, Canadian Institutes of Health Research for the "CIHR Team in Familial Risks of Breast Cancer" program and French National Institute of Cancer (INCa). Cancer Research UK Grants. Grant Numbers: C1287/A11990, C1287/A10118. University of California Los Angeles: Jonsson Comprehensive Cancer Center Foundation; Breast Cancer Research Foundation. Mayo Clinic: NIH, NCI Specialized Program of Research Excellence (SPORE), and a grant from the Breast Cancer Research Foundation. Grant Numbers: CA176785, CA192393, CA116201, CA116167. NCI: Intramural Research Program of the US National Cancer Institute, NIH, and by support services contracts NO2‐CP‐11019‐50, N02‐CP‐21013‐63 and N02‐CP‐65504 with Westat, Inc, Rockville, MD. Grant Numbers: N02‐CP‐21013‐63, N02‐CP‐65504, NO2‐CP‐11019‐50. National Cancer Institute. Grant Numbers: RC4CA153828, 5U54CA156732‐07, R25CA112486. FPGMX: Mutua Madrilena Foundation (FMMA). Grant Number: FISPI05/2275. Memorial Sloane Kettering Cancer Center: Breast Cancer Research Foundation, Robert and Kate Niehaus Clinical Cancer Genetics Initiative, Andrew Sabin Research Fund and a Cancer Center Support Grant/Core Grant (P30 CA008748). Grant Number: P30 CA008748. Fox Chase Cancer Center: University of Kansas Cancer Center and Kansas Bioscience Authority Eminent Scholar Program and by Chancellors Distinguished Chair in Biomedical Sciences Professorship. Grant Numbers: R01 CA214545, P30 CA168524, R0 1CA140323. BCFR ‐ all: CA164920 from National Cancer Institute. The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the Breast Cancer Family Registry (BCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government or the BCFR. University of Pennsylvania: National Institutes of Health (NIH) (R01‐CA102776 and R01‐CA083855; Breast Cancer Research Foundation; Susan G. Komen Foundation for the cure, Basser Research Center for BRCA. UPITT/MWH: Hackers for Hope Pittsburgh. VFCTG: Victorian Cancer Agency, Cancer Australia, National Breast Cancer Foundation. Grant Numbers: R01‐CA102776, R01‐CA083855. HRBCP: Hong Kong Sanatorium and Hospital, Dr Ellen Li Charitable Foundation, Kerry Group Kuok Foundation, National Institute of Health1R 03CA130065, and North California Cancer Center. Grant Number: 03CA130065. BMBSA is supported by Cancer Association of South Africa. University California San Francisco: UCSF Cancer Risk Program and Helen Diller Family Comprehensive Cancer Center. CIMBA: CIMBA data management and data analysis were supported by Cancer Research – UK grants C12292/A20861, C12292/A11174. ACA is a Cancer Research ‐UK Senior Cancer Research Fellow. GCT and ABS are NHMRC Research Fellows. iCOGS: European Community's Seventh Framework Programme under grant agreement n° 223175 (HEALTH‐F2‐2009‐223175) (COGS), Cancer Research UK (C1287/A10118, C1287/A 10710, C12292/A11174, C1281/A12014, C5047/A8384, C5047/A15007, C5047/A10692, C8197/A16565), National Beth Israel Deaconess Medical Center is supported by Breast Cancer Research Foundation. UKFOCR: Cancer Research UK. GEORGETOWN: Non‐Therapeutic Subject Registry Shared Resource at Georgetown University (NIH/NCI grant P30‐CA051008), Fisher Center for Hereditary Cancer and Clinical Genomics Research, and Swing Fore the Cure. Grant Number: P30‐CA051008.

Single-cell sequencing (SCS) is a powerful new tool that applies Next Generation Sequencing at the cellular level. SCS has revolutionized our understanding of tumor heterogeneity and the tumor microenvironment, immune infiltration, cancer stem cells (CSCs), circulating tumor cells (CTCs), and clonal evolution. The following chapter highlights the current literature on SCS in genitourinary (GU) malignancies and discusses future applications of SCS technology. The renal cell carcinoma (RCC) section highlights the use of SCS in characterizing the initial cells driving tumorigenesis, the intercellular mutational landscape of RCC, intratumoral heterogeneity (ITH) between primary and metastatic lesions, and genes driving RCC cancer stem cells (CSCs). The bladder cancer section will also illustrate molecular drivers of bladder cancer stem cells (BCSCs), SCS use in reconstructing tumor developmental history and underlying subclones, and understanding the effect of cisplatin on intratumoral heterogeneity in vitro and potential mechanisms behind platinum resistance. The final section featuring prostate cancer will discuss how SCS can be used to identify the cellular origins of benign prostatic hyperplasia and prostate cancer, the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, and the use of SCS in CTCs to understand chemotherapy resistance and gene expression changes after androgen deprivation therapy (ADT). The studies listed in this chapter illustrate many translational applications of SCS in GU malignancies, including diagnostic, prognostic, and treatment-related approaches. The ability of SCS to resolve intratumor heterogeneity and better define the genomic landscape of tumors and CTCs will be fundamental in the new era of precision-based care.

Single-cell sequencing (SCS) is a powerful new tool that applies Next Generation Sequencing at the cellular level. SCS has revolutionized our understanding of tumor heterogeneity and the tumor microenvironment, immune infiltration, cancer stem cells (CSCs), circulating tumor cells (CTCs), and clonal evolution. The following chapter highlights the current literature on SCS in genitourinary (GU) malignancies and discusses future applications of SCS technology. The renal cell carcinoma (RCC) section highlights the use of SCS in characterizing the initial cells driving tumorigenesis, the intercellular mutational landscape of RCC, intratumoral heterogeneity (ITH) between primary and metastatic lesions, and genes driving RCC cancer stem cells (CSCs). The bladder cancer section will also illustrate molecular drivers of bladder cancer stem cells (BCSCs), SCS use in reconstructing tumor developmental history and underlying subclones, and understanding the effect of cisplatin on intratumoral heterogeneity in vitro and potential mechanisms behind platinum resistance. The final section featuring prostate cancer will discuss how SCS can be used to identify the cellular origins of benign prostatic hyperplasia and prostate cancer, the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, and the use of SCS in CTCs to understand chemotherapy resistance and gene expression changes after androgen deprivation therapy (ADT). The studies listed in this chapter illustrate many translational applications of SCS in GU malignancies, including diagnostic, prognostic, and treatment-related approaches. The ability of SCS to resolve intratumor heterogeneity and better define the genomic landscape of tumors and CTCs will be fundamental in the new era of precision-based care.

Ovarian cancer remains a highly lethal disease due to its late clinical presentation and lack of reliable early biomarkers. Protein-based diagnostic markers have presented limitations in identifying ovarian cancer. We tested the potential of phospholipids as markers of ovarian cancer by utilizing inter-related regulation of phospholipids, a unique property that allows the use of ratios between phospholipid species for quantitation. High-performance liquid chromatography mass spectrometry was used to measure phospholipid, lysophospholipid, and sphingophospholipid content in plasma from patients with benign ovarian masses, patients with ovarian cancer, and controls. We applied both absolute and relative phospholipid ratios for quantitation. Receiver operating characteristic analysis was performed to test the sensitivity and specificity. We found that utilization of ratios between phospholipid species greatly outperformed absolute quantitation in the identification of ovarian cancer. Of the phospholipids analyzed, species in phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) were found to have great biomarker potential. LPC(20:4)/LPC(18:0) carried the greatest capacity to differentiate cancer from control, SM(d18:1/24:1)/SM(d18:1/22:0) to differentiate benign from cancer, and PC(18:0/20:4)/PC(18:0/18:1) to differentiate benign from control. These results demonstrate the potential of plasma phospholipids as a novel marker of ovarian cancer by utilizing the unique characteristics of phospholipids to further enhance the diagnostic power.

Airway dysbiosis in premature infants may be associated with bronchopulmonary dysplasia (BPD). Early oropharyngeal colostrum (OPC) administration alters the oral microbiome, which may impact the lung microbiome. We aim to compare the oral and tracheal microbiota during the first week of life, and to determine whether early OPC administration affects microbial diversity or leukocyte inflammatory activity in the lung.Intubated premature infants (The oral and tracheal microbiota were significantly different on DOL 3, but the tracheal microbiome on DOL 7 was more similar to the oral from DOL 3. Tracheal bacterial diversity decreased from DOL 3 to DOL 7. Longer time to first OPC administration tended to be associated with lower bacterial diversity in the airways.The tracheal microbiome in intubated premature infants in the first week is likely determined, in part, by the composition of the oral microbiome. Bacterial diversity in intubated babies decreases during the first week of life, a pattern that could be consistent with risk for BPD. Decreased bacterial diversity and increased inflammatory activity in the lung may also be associated with delayed administration of OPC.

Background: Triple Negative Breast Cancer (TNBC) is a subset of breast cancer that is difficult to treat clinically and is characterized by being estrogen receptor (ER) negative, progesterone receptor (PR) negative, and does not overexpress human epidermal growth factor receptor 2 (HER2). Patients with TNBC tend to have a worse prognosis than other breast cancer subtypes. Methods: We obtained fifteen TNBC sample FFPE tissue blocks with corresponding plasma samples. All samples were from primary tumors; seven samples having metastasized, four samples that had not metastasized and four samples with unknown metastatic status. The total RNA was isolated from FFPE blocks using the RecoverAll Total Nucleic Acid Isolation Protocol. miRNA from plasma was isolated using Ambion's mirVANA kit. The plasma and tissue miRNAs were evaluated using the QuantStudio qPCR platform, capturing ˜750 miRNAs. The mRNA was processed using the TruSeq RNA Access kit and sequenced on the Illumina NextSeq platform. Analysis of the miRNA and mRNA individually was performed using limma and DESeq2 packages, respectively. Gene enrichment analysis of the mRNA expression was done using the GAGE package on KEGG pathways while the integrative analysis was done with sparse Canonical Correlation Analysis (sCCA) using the PMA pacakge. Results: Analysis of plasma miRNA had four miRNAs with a significant difference in raw p-value (p < 0.05) between metastatic and non-metastatic TNBC; miRNA 708, 483-3p, 518f, and 766; in the tissue there were fifteen miRNA with p < 0.05, with one miR-872 still having significance after adjusting for multiple testing. mRNA had 33 genes being significant after multiple testing correction with several immune KEGG pathways being downregulated in metastatic samples (adjusted p < 0.05). The integrative analysis revealed five microRNA (miR-216, miR-127, miR-370, miR-382, and miR-487b) and 312 gene modules enriched in integrin (Fisher p < 10^-5) and extracellular matrix (Fisher p < 10^-6) signaling. Conclusions: One of the circulating plasma miRNAs, miR483-3p, has been found to promote tumorigenesis, while miR581f and miR766 have not been reported in cancer to date. Further investigation into these miRNA could provide a feasible biomarker. The downregulation of immune pathways observed within the metastatic TNBC subjects implies immune evasion is of particular importance for metastasis and a targeted immunotherapy may be a viable treatment option. The integrative analysis of the miRNA and mRNA showed an enrichment in pathways previously linked to increased proliferation and chemoresistance, with an increased signal compared to either miRNA or mRNA alone. Citation Format: Shih AJ, Korsunsky I, Guttadauria B, Bhuiya T, Liew A, Khalili H, Gregersen PK, Lee AT. Integrative analysis of miRNA and mRNA expression in metastatic versus non-metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-09.

Item does not contain fulltext OBJECTIVE: In many rheumatoid arthritis (RA) patients, disease is controlled with anti-tumor necrosis factor (anti-TNF) biologic therapies. However, in a significant number of patients, the disease fails to respond to anti-TNF therapy. We undertook the present study to examine the hypothesis that rare and low-frequency genetic variants might influence response to anti-TNF treatment. METHODS: We sequenced the coding region of 750 genes in 1,094 RA patients of European ancestry who were treated with anti-TNF. After quality control, 690 genes were included in the analysis. We applied single-variant association and gene-based association tests to identify variants associated with anti-TNF treatment response. In addition, given the key mechanistic role of TNF, we performed gene set analyses of 27 TNF pathway genes. RESULTS: We identified 14,420 functional variants, of which 6,934 were predicted as nonsynonymous 2,136 of which were further predicted to be "damaging." Despite the fact that the study was well powered, no single variant or gene showed study-wide significant association with change in the outcome measures disease activity or European League Against Rheumatism response. Intriguingly, we observed 3 genes, of 27 with nominal signals of association (P < 0.05), that were involved in the TNF signaling pathway. However, when we performed a rigorous gene set enrichment analysis based on association P value ranking, we observed no evidence of enrichment of association at genes involved in the TNF pathway (Penrichment = 0.15, based on phenotype permutations). CONCLUSION: Our findings suggest that rare and low-frequency protein-coding variants in TNF signaling pathway genes or other genes do not contribute substantially to anti-TNF treatment response in patients with RA.

ObjectivesIdiopathic inflammatory myopathies (IIM) are a spectrum of rare autoimmune diseases characterised clinically by muscle weakness and heterogeneous systemic organ involvement. The strongest genetic risk is within the major histocompatibility complex (MHC). Since autoantibody presence defines specific clinical subgroups of IIM, we aimed to correlate serotype and genotype, to identify novel risk variants in the MHC region that co-occur with IIM autoantibodies.MethodsWe collected available autoantibody data in our cohort of 2582 Caucasian patients with IIM. High resolution human leucocyte antigen (HLA) alleles and corresponding amino acid sequences were imputed using SNP2HLA from existing genotyping data and tested for association with 12 autoantibody subgroups.ResultsWe report associations with eight autoantibodies reaching our study-wide significance level of p–5. Associations with the 8.1 ancestral haplotype were found with anti-Jo-1 (HLA-B*08:01, p=2.28×10–53 and HLA-DRB1*03:01, p=3.25×10–9), anti-PM/Scl (HLA-DQB1*02:01, p=1.47×10–26) and anti-cN1A autoantibodies (HLA-DRB1*03:01, p=1.40×10–11). Associations independent of this haplotype were found with anti-Mi-2 (HLA-DRB1*07:01, p=4.92×10–13) and anti-HMGCR autoantibodies (HLA-DRB1*11, p=5.09×10–6). Amino acid positions may be more strongly associated than classical HLA associations; for example with anti-Jo-1 autoantibodies and position 74 of HLA-DRB1 (p=3.47×10–64) and position 9 of HLA-B (p=7.03×10–11). We report novel genetic associations with HLA-DQB1 anti-TIF1 autoantibodies and identify haplotypes that may differ between adult-onset and juvenile-onset patients with these autoantibodies.ConclusionsThese findings provide new insights regarding the functional consequences of genetic polymorphisms within the MHC. As autoantibodies in IIM correlate with specific clinical features of disease, understanding genetic risk underlying development of autoantibody profiles has implications for future research.

Patients that dialyze in the nursing home setting are known to be an especially at-risk vulnerable population compared to the overall ESRD population. This is likely due to the nursing home dialysis patients' advanced age, multiple co-morbidities, and frailty, requiring skilled nursing support. These challenges often result in worse outcomes compared to the overall end-stage renal disease (ESRD) population but few studies have investigated interventions to improve health outcomes in this population. Previously, we reported results from a study using a large epidemiological database of patients from an independent nursing home dialysis provider, showing that patients treated with daily home hemodialysis had improved outcomes compared to patients treated with conventional dialysis. One limitation of the previous study was that the timeframe for the two comparison groups was different; therefore, the results could have been due to over-all improvements in care over time unrelated to the modality of dialysis: To address this as well as expand on the previous analysis, the objective of the present study was to compare outcomes in ESRD patients in the nursing home setting treated with daily home hemodialysis versus con- ventional three-day-a-week (TIW) hemodialysis using an updated database, specifically assessing patients treated during a concurrent time: frame. Health status was evaluated for 6,314 patients (n=4,778 conventional, n=1,902 daily home hemodialysis; 2006 to November 2015 for conventional; 2011 to November 2015 for daily home hemodialysis). Analyses included monthly mortality rates, Kaplan-Meier survival analysis, and laboratory values. In the "Compared to the conventional dialysis population, daily HHD patients had similar or lower incident mortality rates." analysis of patients treated during the concurrent timeframe, median overall survival was 36 months with daily home hemodialysis versus 21 months with conventional dialysis (P=0.0025). These results were similar to the analysis of all patients regardless of timeframe. Compared to the conventional dialysis population, daily home hemodialysis patients had similar or lower incident mortality rates. Survival rates were higher at 3 months (89% vs 82%), 6 months (84% vs 73%), and 12 months (74% vs 62%) in the daily home hemodialy- sis population compared to conventional dialysis population. Monthly mean albumin was consistent over time in the daily home hemodialysis population but gradually increased in the conventional dialysis population. Hemoglobin values were consistently lower over the follow-up period in the daily home hemodialysis population and ferritin values were similar in both populations. These results confirm and extend previous findings that daily home hemodialysis is associated with improved patient outcomes compared to conventional hemodialysis. Although difficult to conduct practically, a prospective randomized outcomes study evaluating daily home hemodialysis versus conventional TIW dialysis would be valuable in informing the standard of dialysis care in this population.

Objective B lymphoid kinase (BLK) is associated with rheumatoid arthritis (RA) and several other B cell–associated autoimmune disorders. BLK risk variants are consistently associated with reduced BLK expression, but the mechanisms by which reduced expression alters human B cell function to confer autoimmune disease susceptibility are unknown. This study was undertaken to characterize the BLK risk haplotype and to determine associated B cell functional phenotypes involved in autoimmunity. Methods The BLK risk haplotype association with RA (determined using whole-genome sequencing data) was confirmed in 2,526 RA cases and 2,134 controls. Peripheral blood mononuclear cells (PBMCs) from RA patients, healthy adults, and umbilical cord blood were used to study B cell functional phenotypes associated with the BLK risk genotype. Association of the BLK haplotype with B cell phenotypes was analyzed using cell culture and flow cytometry. Results Two insertion/deletions were found on the RA risk haplotype in BLK, and the reduction in BLK expression associated with the risk haplotype was confirmed in primary B lymphocytes. Carriers of the RA-associated haplotype had evidence of lower basal B cell receptor (BCR) signaling activity, yet their B cells were hyperactivatable, with enhanced up-regulation of CD86 after BCR crosslinking and greater T cell stimulatory capacity. The number of isotype-switched memory B cells was also significantly increased in subjects carrying the risk haplotype. Conclusion A major mechanism underlying the BLK association with autoimmune disease involves lowered thresholds for BCR signaling, enhanced B cell–T cell interactions, and altered patterns of isotype switching.

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations.

Background Endometriosis is a chronic and underdiagnosed disease which affects 5–10% of women of childbearing age and is characterized by growth of endometrial tissue outside of the uterus, most often in the peritoneal cavity. Delay in diagnosis is a major problem for management of this disorder, and treatment is often not initiated until the disease has progressed for many years. Although the exact etiology of endometriosis remains unknown, retrograde menstruation is recognized as a common underlying factor leading to the deposit of menstrual effluent (ME) into the peritoneal cavity. Differences in the cellular biology and genetics of the cells within ME are therefore likely to explain why endometriosis develops in only a subset of women. Methods Patients with and without endometriosis were consented to provide ME. ME was analyzed by flow cytometry for CD45- and CD45+ cell populations or used to isolate stromal fibroblast cells. ME-derived stromal fibroblast cells were assessed using decidualization assays following the addition of cAMP and IGFBP-1 concentrations in the culture supernatants were measured by ELISA. In addition, RNA was collected and analyzed by RNA-Seq and qPCR for markers of decidualization and to identify differentially expressed genes in ME-derived stromal fibroblast cells obtained from controls and subjects with endometriosis (±cAMP). Results Flow cytometry analysis of cell subsets within the CD45+ fraction of ME revealed a significant decrease in the number of uterine NK cells in endometriosis patients compared with controls (p

Objective To establish whether the analysis of whole blood gene expression can be useful in predicting or monitoring response to anti-TNF therapy in RA.

More than 100 risk loci for rheumatoid arthritis (RA) have been identified in individuals of European and Asian descent, but the genetic basis for RA in African Americans is less well understood. We genotyped 610 African Americans with autoantibody-positive RA and 933 African American controls on the Immunochip (iChip) array. Using multivariable regression, we evaluated the association between iChip markers and the risk of RA and radiographic severity. The single nucleotide polymorphism (SNP) rs1964995 (odds ratio = 1.97, p = 1.28 × 10−15) near HLA-DRB1 was the most strongly associated risk SNP for RA susceptibility; SNPs in AFF3, TNFSF11 and TNFSF18 loci were suggestively associated (10−4 < p < 3.1 × 10−6). Trans-ethnic fine mapping of AFF3 identified a 90% credible set containing previously studied variants, including rs9653442, rs7608424 and rs6712515, as well as the novel candidate variant rs11681966; several of these likely influence AFF3 gene expression level. Variants in TNFRSF9, CTLA4, IL2RA, C5/TRAF1 and ETS1 — but no variants within the major histocompatibility complex — were associated with RA radiographic severity. Conditional regression and pairwise linkage disequilibrium (LD) analyses suggest that additional pathogenic variants may be found in ETS1 and IL2RA beyond those found in other ethnicities. In summary, we used the dense genotyping of the iChip array and the unique LD structure of African Americans to validate known risk loci for RA susceptibility and radiographic severity, and to better characterize the associations of AFF3, ETS1 and IL2RA.

We fine-mapped 76 rheumatoid arthritis (RA) and type 1 diabetes (T1D) loci outside of the MHC. After sequencing 799 1kb regulatory (H3K4me3) regions within these loci in 568 individuals, we observed accurate imputation for 89% of common variants. We fine-mapped1,2 these loci in RA (11,475 cases, 15,870 controls)3, T1D (9,334 cases and 11,111 controls) 4 and combined datasets. We reduced the number of potential causal variants to ≤5 in 8 RA and 11 T1D loci. We identified causal missense variants in five loci (DNASE1L3, SIRPG, PTPN22, SH2B3 and TYK2) and likely causal non-coding variants in six loci (MEG3, TNFAIP3, CD28/CTLA4, ANKRD55, IL2RA, REL/PUS10). Functional analysis confirmed allele specific binding and differential enhancer activity for three variants: the CD28/CTLA4 rs117701653 SNP, the TNFAIP3 rs35926684 indel, and the MEG3 rs34552516 indel. This study demonstrates the potential for dense genotyping and imputation to pinpoint missense and non-coding causal alleles.

To define potentially causal variants for autoimmune disease, we fine-mapped1,2 76 rheumatoid arthritis (11,475 cases, 15,870 controls)3 and type 1 diabetes loci (9,334 cases, 11,111 controls)4. After sequencing 799 1-kilobase regulatory (H3K4me3) regions within these loci in 568 individuals, we observed accurate imputation for 89% of common variants. We defined credible sets of ≤5 causal variants at 5 rheumatoid arthritis and 10 type 1 diabetes loci. We identified potentially causal missense variants at DNASE1L3, PTPN22, SH2B3, and TYK2, and noncoding variants at MEG3, CD28–CTLA4, and IL2RA. We also identified potential candidate causal variants at SIRPG and TNFAIP3. Using functional assays, we confirmed allele-specific protein binding and differential enhancer activity for three variants: the CD28–CTLA4 rs117701653 SNP, MEG3 rs34552516 indel, and TNFAIP3 rs35926684 indel.

MicroRNAs have been established as key regulators of tumor gene expression and as prime biomarker candidates for clinical phenotypes in epithelial ovarian cancer (EOC). We analyzed the coexpression and regulatory structure of microRNAs and their co-localized gene targets in primary tumor tissue of 20 patients with advanced EOC in order to construct a regulatory signature for clinical prognosis. We performed an integrative analysis to identify two prognostic microRNA/mRNA coexpression modules, each enriched for consistent biological functions. One module, enriched for malignancy-related functions, was found to be upregulated in malignant versus benign samples. The second module, enriched for immune-related functions, was strongly correlated with imputed intratumoral immune infiltrates of T cells, natural killer cells, cytotoxic lymphocytes, and macrophages. We validated the prognostic relevance of the immunological module microRNAs in the publicly available The Cancer Genome Atlas data set. These findings provide novel functional roles for microRNAs in the progression of advanced EOC and possible prognostic signatures for survival.

The aim of this study was to identify genetic variants associated with rheumatoid arthritis (RA) risk in black South Africans. Black South African RA patients (n = 263) were compared with healthy controls (n = 374). Genotyping was performed using the Immunochip, and four-digit high-resolution human leukocyte antigen (HLA) typing was performed by DNA sequencing of exon 2. Standard quality control measures were implemented on the data. The strongest associations were in the intergenic region between the HLA-DRB1 and HLA-DQA1 loci. After conditioning on HLA-DRB1 alleles, the effect in the rest of the extended major histocompatibility (MHC) diminished. Non-HLA single nucleotide polymorphisms (SNPs) in the intergenic regions LOC389203|RBPJ, LOC100131131|IL1R1, KIAA1919|REV3L, LOC643749|TRAF3IP2, and SNPs in the intron and untranslated regions (UTR) of IRF1 and the intronic region of ICOS and KIAA1542 showed association with RA (p < 5 × 10−5). Of the SNPs previously associated with RA in Caucasians, one SNP, rs874040, locating to the intergenic region LOC389203|RBPJ was replicated in this study. None of the variants in the PTPN22 gene was significantly associated. The seropositive subgroups showed similar results to the overall cohort. The effects observed across the HLA region are most likely due to HLA-DRB1, and secondary effects in the extended MHC cannot be detected. Seven non-HLA loci are associated with RA in black South Africans. Similar to Caucasians, the intergenic region between LOC38920 and RBPJ is associated with RA in this population. The strong association of the R620W variant of the PTPN22 gene with RA in Caucasians was not replicated since this variant was monomorphic in our study, but other SNP variants of the PTPN22 gene were also not associated with RA in black South Africans, suggesting that this locus does not play a major role in RA in this population.

Increased detection of plasma lysophosphatidic acid (LPA) has been proposed as a potential diagnostic biomarker in ovarian cancer, but inconsistency exists in these reports. It has been shown that LPA can undergo an artificial increase during sample processing and analysis, which has not been accounted for in ovarian cancer research. The aim of this study is to provide a potential explanation about how the artificial increase in LPA may have interfered with previous LPA analysis in ovarian cancer research. Using an established LC-MS method, we measured LPA and other lysophospholipid levels in plasma obtained from three cohorts of patients: non-cancer controls, patients with benign ovarian tumors, and those with ovarian cancer. We did not find the LPA level to be higher in cancer samples. To understand this inconsistency, we observed that LPA content changed more significantly than other lysophospholipids as a function of plasma storage time while frozen. Additionally, only LPA was found to be adversely impacted by incubation time depending on the Ethylenediaminetetraacetic acid (EDTA) concentration used during blood drawing. We also show that the inhibition of autotaxin effectively prevented artificial LPA generation during incubation at room temperature. Our data suggests that the artificial changes in LPA content may contribute to the discrepancies reported in literature. Any future studies planning to measure plasma LPA should carefully design the study protocol to consider these confounding factors.

631 Background: For most clear cell renal cell carcinoma (ccRCC) patients with early stage disease, surgical resection offers definitive cure. However, for the small percentage of tumors that metastasize, analyzing gene expression profiles from the primary site at the time of nephrectomy can serve as a model to understand the molecular aberrations behind a metastatic phenotype. Differences in gene expression profiles between patients with Stage I and II ccRCC who experience metastasis versus patients who maintain cure after surgery may help elucidate significant molecular targets and stratify patients at higher risk for metastasis. Methods: Nineteen Stage I and twenty Stage II ccRCC tumors preserved in FFPE blocks after nephrectomy were included in this study. Patients were matched for age, gender, tumor size and grade. In both stages, approximately half the patients that experienced metastasis within 5 years of surgery were part of the experimental group, whereas the control group had > 5 years of follow-up without evidence of disease. Extracted RNA for Stage I patients was sequenced using Illumina TruSeq RNA Access Library. Gene counts were assessed by ht-seq counts and differential expression using DESeq2. Significant genes found were validated in the Stage II group using RT-qPCR. Results: In the Stage I experimental group, statistically significant upregulation of several genes associated with unfavorable prognosis in RCC were found: COL1A1, NUMBL, and STEAP3. Random forest classification accurately separated Stage I control versus experimental patients based on expression of COL1A1. Affected genes were consistent with molecular changes seen in TCGA analysis. In the Stage II group, a double-blinded analysis correctly identified the clinical outcome for the majority of the patients using qPCR expression of COL1A1, NUMBL, and STEAP3. Conclusions: Differences in gene expression profiles harbored in the primary site of early stage ccRCC may be employed to predict patients at high risk for developing metastasis. Validating these findings in a larger study carries the potential to better understand mechanisms of metastasis and identify an at risk cohort of patients with early stage disease.

Background: Securing a diagnosis of ovarian cancer and establishing means to predict outcomes to therapeutics remain formidable clinical challenges. Early diagnosis is particularly important since survival rates are markedly improved if tumour is detected early. Methods: Comprehensive miRNA profiles were generated on presurgical plasma samples from 42 women with confirmed serous epithelial ovarian cancer, 36 women diagnosed with a benign neoplasm, and 23 comparably age-matched women with no known pelvic mass. Results: Twenty-two miRNAs were differentially expressed between healthy controls and the ovarian cancer group (P

Objective. To identify new genetic associations with juvenile and adult dermatomyositis (DM). Methods. We performed a genome-wide association study (GWAS) of adult and juvenile DM patients of European ancestry (n = 1178) and controls (n = 4724). To assess genetic overlap with other autoimmune disorders, we examined whether 141 single nucleotide polymorphisms (SNPs) outside the major histocompatibility complex (MHC) locus, and previously associated with autoimmune diseases, predispose to DM. Results. Compared to controls, patients with DM had a strong signal in the MHC region consisting of GWAS-level significance (P < 5x10(-8) ) at 80 genotyped SNPs. An analysis of 141 non-MHC SNPs previously associated with autoimmune diseases showed that three SNPs linked with three genes were associated with DM, with a false discovery rate (FDR) < 0.05. These genes were phospholipase C like 1 (PLCL1, rs6738825, FDR=0.00089), B lymphoid tyrosine kinase (BLK, rs2736340, FDR=0.00031), and chemokine (C-C motif) ligand 21 (CCL21, rs951005, FDR=0.0076). None of these genes was previously reported to be associated with DM. Conclusion. Our findings confirm the MHC as the major genetic region associated with DM and indicate that DM shares non-MHC genetic features with other autoimmune diseases, suggesting the presence of additional novel risk loci. This first identification of autoimmune disease genetic predispositions shared with DM may lead to enhanced understanding of pathogenesis and novel diagnostic and therapeutic approaches. � 2013 American College of Rheumatology.

There is no available targeted therapy for triple-negative or its more aggressive subtype, basal-like breast cancer. Multiple therapeutic strategies based on translational knowledge have not improved the treatment options for triple negative patients. As understanding of molecular pathways that drive tumor development is rapidly increasing, it is imperative to adapt our treatment strategies to perturbations in molecular pathways driving the malignant process. Basal-like breast cancers over-express EGFR (without mutations or EGFR gene amplifications) and have p53 mutations. While EGFR drives the malignant behavior in triple negative breast cancer (TNBC), anti-EGFR therapies have fallen short of the expected results in clinical trials. Here we bring evidence that the less than optimal results of the anti-EGFR therapies may be explained in part by the increased potency of the EGFR signaling due to increased endosomal recycling. The functional connection between EGFR and endosomal trafficking in TNBC is mutant p53 found in the most aggressive forms of TNBC. Mutant p53 acquires oncogenic functions and binds p63 protein, a member of p53 family with tumor suppressor activities. In the absence of functional p63 there is an upregulation of endosomal recycling EGFR and integrin to the membrane with increased proinvasive abilities of cancer cells. Blocking endosomal trafficking combined with anti-EGFR treatments may result in better clinical outcomes in TNBC.

Here we investigated different cell populations within ovarian cancer using single-cell RNA seq: fourteen samples from nine patients with differing grades (high grade, low grade and benign) as well as different origin sites (primary and metastatic tumor site, ovarian in origin and fallopian in origin). We were able to identify sixteen distinct cell populations with specific cells correlated to high grade tumors, low grade tumors, benign and one population unique to a patient with a breast cancer relapse. Furthermore the proportion of these populations changes from primary to metastatic in a shift from mainly epithelial cells to leukocytes with few cancer epithelial cells in the metastases. Differential gene expression shows myeloid lineage cells are the primary cell group expressing soluble factors in primary samples while fibroblasts do so in metastatic samples. The leukocytes that were captured did not seem to be suppressed through known pro-tumor cytokines from any of the cell populations. Single cell RNA-seq is necessary to de-tangle cellular heterogeneity for better understanding of ovarian cancer progression.

The intestinal microbiome plays an important role in human physiology. Next-generation sequencing technologies, knockout and gnotobiotic mouse models, fecal transplant data and epidemiologic studies have accelerated our understanding of microbiome abnormalities seen in immune diseases and malignancies. Dysbiosis is the disturbed microbiome ecology secondary to external pressures such as host diseases, medications, diet and genetic conditions often leading to abnormalities of the host immune system. Specifically dysbiosis has been shown to lower circulating lymphocytes, and increase neutrophil to lymphocyte ratio, a finding which has been associated with a decreased survival in women with breast cancers. Dysbiosis also plays a role in the recycling of estrogens via the entero-hepatic circulation, increasing estrogenic potency in the host, which is another leading cause of breast malignancy. Non-modifiable factors such as age and genetic mutations disrupt the microbiome, but modifiable factors such as diet may also lead to profound disruptions as well. A better understanding of dietary factors and how they disrupt the microbiome may lead to beneficial nutritional interventions for breast cancer patients.

Suboptimal cellular DNA repair capacity (DRC) has been shown to be associated with enhanced cancer risk, but genetic variants affecting the DRC phenotype have not been comprehensively investigated. In this study, with the available DRC phenotype data, we analyzed correlations between the DRC phenotype and genotypes detected by the Illumina 317K platform in 1,774 individuals of European ancestry from a Texas lung cancer genome-wide association study. The discovery phase was followed by a replication in an independent set of 1,374 cases and controls of European ancestry. We applied a generalized linear model with single nucleotide polymorphisms as predictors and DRC (a continuous variable) as the outcome. Covariates of age, sex, pack-years of smoking, DRC assay-related variables, and case–control status of the study participants were adjusted in the model. We validated that reduced DRC was associated with an increased risk of lung cancer in both independent datasets. Several suggestive loci that contributed to the DRC phenotype were defined in ERCC2/XPD, PHACTR2, and DUSP1. In summary, we determined that DRC is an independent risk factor for lung cancer, and we defined several genetic loci contributing to DRC phenotype. Cancer Res; 73(1); 256–64. ©2012 AACR.

Contains fulltext : 118482.pdf (Publisher’s version ) (Open Access) BACKGROUND: Treatment strategies blocking tumour necrosis factor (anti-TNF) have proven very successful in patients with rheumatoid arthritis (RA). However, a significant subset of patients does not respond for unknown reasons. Currently, there are no means of identifying these patients before treatment. This study was aimed at identifying genetic factors predicting anti-TNF treatment outcome in patients with RA using a genome-wide association approach. METHODS: We conducted a multistage, genome-wide association study with a primary analysis of 2 557 253 single-nucleotide polymorphisms (SNPs) in 882 patients with RA receiving anti-TNF therapy included through the Dutch Rheumatoid Arthritis Monitoring (DREAM) registry and the database of Apotheekzorg. Linear regression analysis of changes in the Disease Activity Score in 28 joints after 14 weeks of treatment was performed using an additive model. Markers with p

Background: Black women with triple negative breast cancer have 46% lower survival rates; this may be due to differences in tumor biology. We analyzed presurgical plasma microRNA of white (W) and black (B) women with TNBC enrolled in the breast ovarian tissue bank to detect if differences in the pre-surgical plasma microRNA could be detected in B patients relative to W patients relative to healthy controls. Aims: MicroRNA in pre-surgical plasma of TNBC W or B was compared to plasma from controls race and age matched controls to assess if differences in plasma and tumor microRNA may explain the survival disparity observed in B. We addressed 2 questions 1) are the patterns in the pre-surgical miRNA profiles different between W and B patients, differences that could explain outcomes discrepancy? 2) What are the miRs changes in patients W or B relative to healthy controls? Methods: Between 2004 and 2011 plasma miRNAs was measured before, after surgery, during and after chemotherapy in 73 surgical breast cancer patients and 11 age and race matched controls. We also analized miRs in the tumor and benign adjacent breast in 5 specimes. Samples were analyzed using qRT-PCR in the ABI's TaqMan OpenArray Panel for 750 miRs. Samples were spiked with ath-miR-159a and hsa-miR-320 most strongly correlated in its expression to ath-miR-159a through the Pearson correlation coefficient. 2-sample t-test was used for comparisons between means and ANOVA followed by post-hoc test to compare the mean response between subject factors of interest. All tests were 2-tailed; statistical significance was set at p < 0.05. Results: 44 W and 23 B, mean age at surgery 48 years (range 35–78) and 11 controls – mean age 44 years (range 35–67). Black TNBC patients did not express over 70 % of pre-surgical plasma miRs present in the W pre-surgical plasma. B over-expressed high levels of miR-1244, -190 and -638, which were not detected in any of the W patients (p < 0.005). W patients and controls expressed miRs10a, -190, -502-3p, -548, and -9* not detected in B TNBC patients. White patients over-expressed over 80% of plasma miRs present in controls, while black patients over-expressed only 30% of miRs present in controls (P < 0.005). Conclusions: Patterns of pre-surgical miR expression are different in B versus W patients with TNBC. In B the presence of TNBC leads to silence in circulating plasma miRs in comparison to W and controls. In W the presence of the tumor increases the miR “chatter”. Black patients have a different “communication” style between host-TNBC when analyzed by the response of plasma microRNA. This difference may call for different treatment protocols for patients. Specific treatment interventions, such as administration of chemotherapy before surgery, in an attempt to increase the microRNA levels in plasma may improve the outcomes of black patients with TNBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-13.