Amygdalobacter indicium gen. nov., sp. nov., and Amygdalobacter nucleatus sp. nov., gen. nov.: novel bacteria from the family Oscillospiraceae isolated from the female genital tract.

Four obligately anaerobic Gram-positive bacteria representing one novel genus and two novel species were isolated from the female genital tract. Both novel species, designated UPII 610-JT and KA00274T, and an additional isolate of each species were characterized utilizing biochemical, genotypic and phylogenetic analyses. All strains were non-motile and non-spore forming, asaccharolytic, non-cellulolytic and indole-negative coccobacilli. Fatty acid methyl ester analysis for UPII 610-JT and KA00274T and additional isolates revealed C16 : 0, C18 : 0, C18:1ω9c and C18:2ω6,9c to be the major fatty acids for both species. UPII 610-JT had a 16S rRNA gene sequence similarity of 99.4 % to an uncultured clone sequence (AY724740) designated as Bacterial Vaginosis Associated Bacterium 2 (BVAB2). KA00274T had a 16S rRNA gene sequence similarity of 96.5 % to UPII 610-JT. Whole genomic DNA mol% G+C content was 42.2 and 39.3 % for UPII 610-JT and KA00274T, respectively. Phylogenetic analyses indicate these isolates represent a novel genus and two novel species within the Oscillospiraceae family. We propose the names Amygdalobacter indicium gen. nov., sp. nov., for UPII 610-JT representing the type strain of this species (=DSM 112989T, =ATCC TSD-274T) and Amygdalobacter nucleatus gen. nov., sp. nov., for KA00274T representing the type strain of this species (=DSM 112988T, =ATCC TSD-275T).

Bacterial Communities Associated With Abnormal Nugent Score in Postmenopausal Versus Premenopausal Women.

The Nugent score is the reference standard for bacterial vaginosis (BV) diagnosis but has not been validated in postmenopausal women. We compared relative abundances from 16S ribosomal RNA gene sequencing of vaginal microbiota with Nugent score in cohorts of premenopausal (n = 220) and postmenopausal (n = 144) women. In premenopausal women, 33 taxa were significantly correlated with Nugent score, including the classic BV-associated taxa Gardnerella, Atopobium, Sneathia, Megasphaera, and Prevotella. In postmenopausal women, 11 taxa were significantly associated with Nugent score, including Prevotella but no other BV-associated genera. High Nugent scores should not be used to infer BV in postmenopausal women.

Pathologic findings and clinical outcomes in women undergoing risk-reducing surgery to prevent ovarian and fallopian tube carcinoma: A large prospective single institution experience.

OBJECTIVES: Risk-reducing salpingo-oophorectomy (RRSO) is recommended for women at increased risk of ovarian, fallopian tube (FT), and peritoneal carcinoma (collectively OC). We describe rates of occult neoplasia in the largest single-institution prospective cohort of women undergoing RRSO, including those with mutations in non-BRCA homologous repair (HRR) genes. METHODS: Participants undergoing RRSO enrolled in a prospective tissue bank between 1999 and 2017. Ovaries and FTs were serially sectioned in all cases. Participants had OC susceptibility gene mutations or a family history suggesting OC risk. Analyses were completed in Stata IC 15.1. RESULTS: Of 644 women, 194 (30.1%) had mutations in BRCA1, 177 (27.5%) BRCA2, 27 (4.2%) other HRR genes, and 15 (2.3%) Lynch Syndrome-associated genes. Seventeen (2.6%) had occult neoplasms at RRSO, 15/17 (88.2%) in the FT. Of BRCA1 carriers, 14/194 (7.2%) had occult neoplasia, 8/194 (4.1%) invasive. One PALB2 and two BRCA2 carriers had intraepithelial FT neoplasms. Occult neoplasm occurred more frequently in BRCA1/2 carriers ≥45 years of age (6.5% vs 2.2%, chi square, p = .04), and 211/371 (56.9%) BRCA1/2 carriers had surgery after guideline-recommended ages. Four in 8 (50%) invasive and 2/9 (22%) intraepithelial neoplasms had positive pelvic washings. None with intraepithelial neoplasms developed recurrence or peritoneal carcinoma. CONCLUSIONS: BRCA1 carriers have the highest risk of occult neoplasia at RRSO, and the frequency increased with age. Women with BRCA1/2 mutations often have RRSO beyond recommended ages. One PALB2 carrier had FT intraepithelial neoplasia, a novel finding. Serial sectioning is critical to identifying occult neoplasia and should be performed for all risk-reducing surgeries.

An Alternate Diagnostic Algorithm for the Diagnosis of Intraepithelial Fallopian Tube Lesions.

Intraepithelial fallopian tube neoplasia is thought to be a precursor lesion to high-grade serous carcinoma of the Müllerian adnexae, particularly in women with BRCA1 or BRCA2 mutations. This association has led to recommendations to assess fallopian tubes for intraepithelial atypia. However, the diagnostic reproducibility of a diagnosis of intraepithelial neoplasia is unclear. In this study, 2 gynecologic pathologists independently evaluated sections of fallopian tubes from a sample of women (N=198, 623 slides) undergoing salpingectomy. A total of 101 (54%) women were undergoing risk-reducing salpingo-oophorectomy. Pathologists were blinded to patient histories and prior diagnoses. Pathologists rendered one of three diagnoses for each slide: "negative for fallopian tube intraepithelial neoplasia (FTIN)," "indeterminate for FTIN," or "definite for FTIN." Cases that were considered by histology definite for FTIN or suspicious for FTIN were stained with p53 and Ki67. Pathologists agreed on the diagnosis of "definite for FTIN" 61.5% of the time. There was no agreement on any cases for the diagnosis of "indeterminate for FTIN." Fifteen "indeterminate for FTIN" and 12 "definite for FTIN" cases were stained with p53 and Ki67. Two of the "indeterminate" cases (13%) had p53-positive foci. Five of the "definite" cases had p53-positive foci. In 3 of the other 8 "definite" cases, there was obvious carcinoma present, but the carcinoma did not stain with p53, suggesting a possible null phenotype. We propose that immunostains should only be used to aid in the diagnosis of FTIN in cases with indeterminate histology. The use of p53 immunohistochemistry in cases that were considered "definite for FTIN" by histology was minimally helpful, and in fact often served to further confuse the diagnosis.

Tumor infiltrating lymphocytes and homologous recombination deficiency are independently associated with improved survival in ovarian carcinoma.

OBJECTIVE: The presence of tumor infiltrating lymphocytes (TIL) and defects in homologous recombination (HR) are each important prognostic factors in ovarian carcinoma (OC). We characterized the association between HR deficiency (HRD) and the presence of TILs in a cohort of OC patients and the relative contribution to overall survival. METHODS: Patients with carcinoma of the ovary, fallopian tube, or peritoneum were prospectively enrolled. Malignant neoplasm and serum samples were collected. Immunohistochemistry for CD3+ T cells and CD68+ tumor associated macrophages (TAMs) was performed on specimens collected at primary surgery. Damaging germline and somatic mutations in genes in the HR-mediated repair (HRR) pathway were identified using BROCA sequencing. HRD was defined as a damaging mutation in one of 12 genes in the HRR pathway or promoter hypermethylation in BRCA1 or RAD51C. RESULTS: Ninety-eight of 250 patients included in the analysis had HRD OC (39.2%). HRD OC were enriched for CD3+ TILs and CD68+ TAMs. High CD3+ TIL was present in 65.3% of HRD OC compared to 43.4% of non-HRD OC (P = 0.001). High CD68+ TAM was present in 66.3% of HRD OC compared to 50.7% of non-HRD OC (P = 0.015). Patients with HRD OC and high CD3+ TILs had the longest median overall survival compared to non-HRD OC with low CD3+ TILs (70.9 vs. 35.8 months, adjusted HR 0.38, 95% CI (0.25-0.59)). CONCLUSIONS: Patients that have both CD3+ TILs and HRD OC are afforded the greatest improvement in overall survival. This finding may have therapeutic implications for OC patients treated with emerging immunotherapies.

Megasphaera lornae sp. nov., Megasphaera hutchinsoni sp. nov., and Megasphaera vaginalis sp. nov.: novel bacteria isolated from the female genital tract.

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6T, KA00182T and BV3C16-1T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera. UPII 199-6T and KA00182T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182T had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6T, KA00182T and BV3C16-1T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C12 : 0, C16 : 0, C16 : 0 dimethyl acetal (DMA) and summed feature 5 (C15 : 0 DMA and/or C14 : 0 3-OH) in UPII 199-6T; C16 : 0 and C16 : 1 cis 9 in KA00182T; C12 : 0; C14 : 0 3-OH; and summed feature 5 in BV3C16-1T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6T, KA00182T and BV3C16-1T represent novel species within the genus Megasphaera. We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6T representing the type strain of this species (=DSM 111201T=ATCC TSD-205T), Megasphaera hutchinsoni sp. nov. for KA00182T representing the type strain of this species (=DSM 111202T=ATCC TSD-206T) and Megasphaera vaginalis sp. nov. for BV3C16-1T representing the type strain of this species (=DSM 111203T=ATCC TSD-207T).

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.

Combined symptom index and second-generation multivariate biomarker test for prediction of ovarian cancer in patients with an adnexal mass.

OBJECTIVE: To assess the performance of a symptom index (SI) and multivariate biomarker panel in the identification of ovarian cancer in women presenting for surgery with an adnexal mass. STUDY DESIGN: Prospective study of patients seen at a tertiary medical center. Following consent, patients completed an SI and preoperative serum was collected for individual markers (CA 125) and a second-generation FDA-cleared biomarker test (MIA2G). Results for the SI and MIA2G were correlated with operative findings and surgical pathology. Logistic regression modeling was performed to assess the interaction of the SI with MIA2G to determine the risk of malignancy (ROM). RESULTS: Of the 218 patients enrolled, the mean age was 53.6 years (range 18-86). One-hundred and forty-seven patients (67.4%) were postmenopausal. Sixty-four patients (29.4%) had epithelial ovarian cancer or fallopian tube cancer (EOC/FTC) and 17 (7.8%) had borderline ovarian tumors. A positive SI or MIA2G correctly identified 96.1% of patients with EOC/FTC. Using logistic regression, we found that both SI and MIA2G score were significantly associated with ROM (p < 0.001). In a simulation with disease prevalence set at 5%, patients with a negative SI and a MIA2G score of 6 had a ROM of 1.8% whereas patients with the same MIA2G and positive SI had a 10.5% ROM, nearly a 6-fold higher risk. CONCLUSIONS: The combination of a patient-reported symptom index and refined biomarker panel allows for improved accuracy in the assessment for ovarian cancer in patients with an adnexal mass. This strategy could offer a personalized approach to addressing ROM to triage patients with an adnexal mass to appropriate care.

Clinical characteristics and outcomes of patients with BRCA1 or RAD51C methylated versus mutated ovarian carcinoma.

OBJECTIVE: In ovarian carcinoma, mutations in homologous recombination DNA repair (HRR) genes, including BRCA1 and RAD51C, are associated with increased survival and specific clinical features. Promoter hypermethylation is another mechanism of reducing gene expression. We assessed whether BRCA1 and RAD51C promoter hypermethylation is associated with similar survival and clinical characteristics. METHODS: Promoter methylation of BRCA1 and RAD51C was evaluated using methylation-sensitive PCR in 332 primary ovarian carcinomas. Damaging germline and somatic mutations in 16 HRR genes were identified using BROCA sequencing. RESULTS: BRCA1 methylation was detected in 22 carcinomas (6.6%) and RAD51C methylation in 9 carcinomas (2.7%). These small numbers limited the power to detect differences in survival and platinum sensitivity. Mutations in one or more HRR genes were found in 95 carcinomas (29%). Methylation of BRCA1 or RAD51C was mutually exclusive with mutations in these genes (P=0.001). Patients whose carcinomas had BRCA1 methylation (57.7years±2.5) or BRCA1 mutations (54.1years±1.4) were younger than those without (63.3years±0.8; P=0.029, P<0.0001). BRCA1 methylation and germline BRCA1 mutation were associated with high grade serous (HGS) histology (P=0.045, P=0.001). BRCA1 mutations were associated with increased sensitivity to platinum chemotherapy while BRCA1 methylation was not (P=0.034, P=0.803). Unlike HRR mutations, methylation was not associated with improved overall survival compared to cases without methylation or mutation. CONCLUSIONS: Patients with BRCA1-methylated carcinomas share clinical characteristics with patients with BRCA1-mutated carcinomas including younger age and predominantly HGS histology. However, unlike mutation, RAD51C and BRCA1 methylation were not associated with improved survival or greater sensitivity to platinum chemotherapy.

Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.

BACKGROUND: Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin-6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease. OBJECTIVE: The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development. STUDY DESIGN: We obtained fetal cardiac tissue after necropsy from a well-described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin-6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum. RESULTS: Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin-6 and -8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5-fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled-related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05). CONCLUSION: Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long-term effects on cardiac function.

Combining a symptom index, CA125 and HE4 (triple screen) to detect ovarian cancer in women with a pelvic mass.

OBJECTIVES: To assess a simple algorithm of CA125, HE4 and Symptom Index to predict ovarian cancer in women with a pelvic mass. METHODS: This was a prospective study of women referred to a gynecologic oncology clinic for surgical evaluation of a pelvic mass. Preoperatively, women completed a SI and had serum markers drawn. Results were correlated with pathology. A triple screen was considered positive if at least 2 of the 3 markers were abnormal (positive SI, CA125≥35U/mL, HE4≥140pmol/L). RESULTS: 218 patients enrolled in the study. 66 patients (30%) had ovarian or fallopian tube cancer (97% epithelial), 124 (57%) had benign masses, 17 (8%) had borderline tumors, and 11 (5%) had metastatic disease. The SI, CA125 and HE4 were positive in 87.9%, 74.2% and 60.6% of ovarian cancer patients, respectively. Of the 112 women with a positive SI 58 (52%) had ovarian cancer and 75 (67%) had non-benign masses. Excluding borderline and metastatic cancers the sensitivity of the triple screen was 79%; specificity 91%, PPV 83% and NPV 89%. CA125 alone had a sensitivity, specificity, PPV and NPV of 79%, 76%, 63% and 87% respectively. Requiring only one of the three tests to be abnormal resulted in a sensitivity of 97% but specificity dropped to 50%. CONCLUSIONS: An algorithm using SI, CA125 and HE4 has good performance statistics for predicting cancer in women with pelvic masses. The triple screen has higher specificity and PPV than CA125 alone but similar sensitivity and NPV for predicting ovarian cancer.

Evaluation of a Validated Biomarker Test in Combination With a Symptom Index to Predict Ovarian Malignancy.

OBJECTIVE: This study aimed to evaluate the predictive ability of a multivariate biomarker test in combination with a symptom index (SI) to identify ovarian cancer in a cohort of women planning to undergo surgery for a pelvic mass. METHODS: This was a prospective study of patients seen at a tertiary care medical center. Following consent, patients completed an SI and preoperative serum was collected for a Food and Drug Administration-cleared multivariate biomarker test [multivariate index assay (MIA)]. Results for the SI and MIA were correlated with operative findings and surgical pathology. RESULTS: Of 218 patients enrolled, 124 (56.9%) had benign disease and 94 (43.1%) had borderline tumors or carcinomas. Sixty-six patients had a primary ovarian or fallopian tube cancer. The median age of patients enrolled in this study was 54 years (interquartile range, 44-63 years), of whom 148 (67.9%) were postmenopausal. More than a third (36.3%) of patients with benign masses was accurately identified as low risk by MIA and SI. The sensitivity and negative predictive value (NPV) of the SI relative to primary ovarian cancer was 87.9% (95% CI, 77.9%-93.7%) and 91.6% (95% CI, 84.3%-95.7%), respectively. The sensitivity and NPV of CA125 was 75.4% (95% CI, 63.7%-84.2%) and 86.4% (95% CI, 79.1%-91.5%), respectively, and the sensitivity and NPV of the MIA were 93.9% (95% CI, 85.4%-97.6%) and 94.5% (95% CI, 94.5%-100%), respectively. The overall sensitivity for the combination of MIA plus SI was 100% (66/66; 95% CI, 94.5%-100%), and specificity was 36.3% (45/124; 95% CI, 28.4%-45.0%), with an NPV of 100% (95% CI, 92.1%-100%). CONCLUSIONS: The addition of a patient-reported SI, which captures subjective symptoms in an objective manner, improved the sensitivity of MIA across all stages and subtypes of ovarian cancer.

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

BACKGROUND: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. CONCLUSIONS: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

Neoplastic cellularity is associated with clinical and molecular features of high-grade serous ovarian carcinoma.

OBJECTIVE: Most molecular analyses of high-grade serous ovarian, peritoneal and fallopian tube carcinomas (HGSC) require ≥70% tumor (neoplastic) cell nuclei. We characterized the distribution of the percentage of neoplastic nuclei (PNN) in a large cohort of HGSC and correlated PNN with clinical outcomes to determine the fraction of cases outside this range and whether this cut-off introduces selection bias. METHODS: Subjects were prospectively enrolled and normal and neoplastic tissues were snap-frozen. All subjects had grade 2 to 3 HGSC. Subjects that received neoadjuvant chemotherapy were excluded. PNN was determined by estimating the fraction of neoplastic nuclei relative to non-neoplastic nuclei on a representative hematoxylin and eosin stained frozen section from the primary neoplasm. Germline BRCA mutation status was determined with Sanger or BROCA sequencing. RESULTS: PNN was <70% in 101 (33%) of 306 cases. PNN was significantly higher among subjects without optimal cytoreduction (P=0.018). 55 subjects had germline BRCA1/BRCA2 mutations. HGSC associated with BRCA2 but not BRCA1 mutations had significantly lower PNN compared to HGSC in non-carriers (54% vs. 70%, P=0.018). Overall survival was not significantly different between subjects with <70% or ≥70% PNN (median survival 51.8 vs. 46.6months, P=0.858). CONCLUSIONS: One-third of HGSC has PNN <70%. Higher PNN is associated with suboptimal cytoreduction, while lower PNN is associated with inherited BRCA2 mutations. Our findings suggest a nonrandom distribution of PNN that may reflect cancer biology. Further studies exploring the stromal microenvironment are needed. Molecular analyses of HGSC selected for high PNN exclude a significant fraction of patients.

Genetic characterization of early onset ovarian carcinoma.

OBJECTIVE: Ovarian carcinoma (OC) is rare in young women and the fraction of early onset OC attributable to inherited mutations in known OC genes is uncertain. We sought to characterize the fraction of OC that is heritable in women diagnosed with ovarian, fallopian tube, or peritoneal carcinoma at forty years of age or younger. METHODS: We sequenced germline DNA from forty-seven women diagnosed with OC at age 40 or younger ascertained through a gynecologic oncology tissue bank or referred from outside providers using BROCA, a targeted capture and massively parallel sequencing platform that can detect all mutation classes. We evaluated 11 genes associated with ovarian carcinoma (BARD1, BRCA1, BRCA2, BRIP1, MLH1, MSH2, MSH6, PALB2, PMS2, RAD51D, and RAD51C) and additional candidate genes in DNA repair (ATM, BAP1, CHEK2, MRE11A, NBN, PTEN, TP53). We counted only clearly damaging mutations. RESULTS: Damaging mutations in OC genes were identified in 13 of 47 (28%) subjects, of which 10 (77%) occurred in BRCA1 and one each occurred in BRCA2, MSH2, and RAD51D. Women with a strong family history were no more likely to have an OC gene mutation (8/17, 47%) than those without a strong family history (9/30, 30%, P=0.35). Additionally, damaging mutations in non-OC genes were identified, one in NBN and one in CHEK2. CONCLUSIONS: A high proportion of young women with invasive OC have mutations in BRCA1, and a smaller fraction have mutations in other known OC genes. Family history was not associated with mutation status in these early onset cases.

Hydrogen Peroxide-Producing Lactobacilli Are Associated With Lower Levels of Vaginal Interleukin-1ß, Independent of Bacterial Vaginosis.

BACKGROUND: The presence of hydrogen peroxide (H2O2)-producing lactobacilli in the vagina is associated with decreased rates of preterm birth and HIV acquisition. We hypothesize that this is due to immunomodulatory effects of these species. METHODS: Concentrations of interleukin (IL)-1ß, IL-6, IL-8, secretory leukocyte protease inhibitor, and human ß-defensin 2 were quantified from vaginal swabs from 4 groups of women: women with and without bacterial vaginosis (BV) by Nugent score, further stratified by detection of H2O2-producing lactobacilli by semiquantitative culture. Ten quantitative polymerase chain reaction assays characterized the presence and quantity of select Lactobacillus and BV-associated species in each group. Levels of immune markers and bacteria were compared between the 4 groups using analysis of variance, Kruskal-Wallis, Mann-Whitney U, or χ tests. RESULTS: Swabs from 110 women from 4 groups were included: 26 had a normal Nugent score (BV-), and no H2O2-producing lactobacilli detected (H2O2-); 47 were BV-, H2O2+; 27 BV+, H2O2-; and 10 BV+, H2O2+. The groups were similar in age, marital status, and reproductive history, but not ethnicity: the BV-, H2O2- group had more white participants (P = 0.02). In women with and without BV, IL-1ß was lower in the H2O2+ groups. Human ß-defensin 2 was lowest in BV+ H2O2- women and highest in BV-, H2O2-. Secretory leukocyte protease inhibitor was lower in women with BV and did not differ by the presence of H2O2-producing lactobacilli. In regression analysis, higher quantities of Lactobacillus crispatus were associated with lower quantities of IL-1ß. Detection and quantity of BV-associated species by quantitative polymerase chain reaction was significantly different between women with and without BV, but not between women with and without H2O2-producing lactobacilli within those groups. CONCLUSIONS: The presence of H2O2-producing lactobacilli is associated with lower levels of some vaginal proinflammatory cytokines, even in women with BV.

Detection of the HE4 protein in urine as a biomarker for ovarian neoplasms: Clinical correlates.

OBJECTIVES: To measure HE4 levels in urine from normal donors, patients with LMP tumors and ovarian cancer patients and to correlate levels with clinical factors in ovarian cancer patients. METHODS: Archived samples from controls, patients with LMP tumors and ovarian cancer were tested using commercial assays, including serially collected serum and urine samples from women treated for stage III/IV serous ovarian cancer. RESULTS: Five of 6 patients with stage I/II and 26 of 36 stage III/IV serous ovarian cancer patients had HE4-positive urines, similar to serum samples (4 of 5 stage I/II and 26 of 34 stage III/IV) when tested at the same level of specificity (95%). Urine HE4 was more sensitive in LMP tumors: 9 of 32 urines (28%) HE4-positive versus 3 of 68 sera (4.4%, p=0.002). Mean levels of serum CA125 and HE4 decreased comparably in patients during initial treatment regardless of their primary platinum response, but mean urine HE4 levels decreased only 7% in primary platinum resistant patients while decreasing 68% in those who were sensitive. By 7months after diagnosis, urine HE4 levels were higher in primary platinum resistant patients compared to those who proved to be sensitive (p=0.051) and persisted 12months after diagnosis (p=0.014). HE4 values in urine also became positive in advance of clinical recurrence in several women while serum HE4 and serum CA-125 remained normal. CONCLUSIONS: Measuring HE4 in urine complements serum assays for the detection of ovarian cancer and may allow identification of patients at high risk for primary platinum resistance.

Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.

OBJECTIVE: The objective of the study was to evaluate the upper genital tract (UGT) presence of vaginal bacterial species using sensitive molecular methods capable of detecting fastidious bacterial vaginosis (BV)-associated bacteria. STUDY DESIGN: Vaginal swabs were collected prior to hysterectomy. The excised uterus was sterilely opened and swabs collected from the endometrium and upper endocervix. DNA was tested in 11 quantitative polymerase chain reaction (PCR) assays for 12 bacterial species: Lactobacillus iners, L crispatus, L jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp, Prevotella spp, Leptotrichia/Sneathia, BVAB1, BVAB2, BVAB3, and a broad-range16S ribosomal ribonucleic acid gene assay. Endometrial fluid was tested with Luminex and an enzyme-linked immunosorbent assay for cytokines and defensins and tissue for gene expression of defensins and cathelicidin. RESULTS: We enrolled 58 women: mean aged 43±7 years, mostly white (n=46; 79%) and BV negative (n=43; 74%). By species-specific quantitative PCR, 55 (95%) had UGT colonization with at least 1 species (n=52) or were positive by 16S PCR (n=3). The most common species were L iners (45% UGT, 61% vagina), Prevotella spp (33% UGT, 76% vagina) and L crispatus (33% UGT, 56% vagina). Median quantities of bacteria in the UGT were lower than vaginal levels by 2-4 log10 ribosomal ribonucleic acid gene copies per swab. There were no differences in the endometrial inflammatory markers between women with no bacteria, Lactobacillus only, or any BV-associated species in the UGT. CONCLUSION: Our data suggest that the endometrial cavity is not sterile in most women undergoing hysterectomy and that the presence of low levels of bacteria in the uterus is not associated with significant inflammation.

Long-term effect of depot medroxyprogesterone acetate on vaginal microbiota, epithelial thickness and HIV target cells.

BACKGROUND: Depot medroxyprogesterone acetate (DMPA) has been linked to human immunodeficiency virus type 1 (HIV-1) acquisition. METHODS: Vaginal microbiota of women using DMPA for up to 2 years were cultured. Mucosal immune cell populations were measured by immunohistological staining. RESULTS: Over 12 months, the proportion with H2O2-positive lactobacilli decreased (n = 32; 53% vs 27%; P = .03). Median vaginal CD3(+) cells also decreased (n = 15; 355 vs 237 cells/mm(2); P = .03), as did CD3(+)CCR5(+) cells (195 vs 128 cells/mm(2); P = .04), HLA-DR(+) cells (130 vs 96 cells/mm(2); P = .27), and HLA-DR(+)CCR5(+) cells (18 vs 10 cells/mm(2); P = .33). CONCLUSIONS: DMPA contraception does not increase vaginal mucosal CCR5(+) HIV target cells but does decrease CD3(+) T lymphocytes and vaginal H2O2-producing lactobacilli.

Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing.

Inherited loss-of-function mutations in BRCA1 and BRCA2 and other tumor suppressor genes predispose to ovarian carcinomas, but the overall burden of disease due to inherited mutations is not known. Using targeted capture and massively parallel genomic sequencing, we screened for germ-line mutations in 21 tumor suppressor genes in genomic DNA from women with primary ovarian, peritoneal, or fallopian tube carcinoma. Subjects were consecutively enrolled at diagnosis and not selected for age or family history. All classes of mutations, including point mutations and large genomic deletions and insertions, were detected. Of 360 subjects, 24% carried germ-line loss-of-function mutations: 18% in BRCA1 or BRCA2 and 6% in BARD1, BRIP1, CHEK2, MRE11A, MSH6, NBN, PALB2, RAD50, RAD51C, or TP53. Six of these genes were not previously implicated in inherited ovarian carcinoma. Primary carcinomas were generally characterized by genomic loss of normal alleles of the mutant genes. Of women with inherited mutations, >30% had no family history of breast or ovarian carcinoma, and >35% were 60 y or older at diagnosis. More patients with ovarian carcinoma carry cancer-predisposing mutations and in more genes than previously appreciated. Comprehensive genetic testing for inherited carcinoma is warranted for all women with ovarian, peritoneal, or fallopian tube carcinoma, regardless of age or family history. Clinical genetic testing is currently done gene by gene, with each test costing thousands of dollars. In contrast, massively parallel sequencing allows such testing for many genes simultaneously at low cost.