Kathy Agnew

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.

Germline and Somatic Mutations in Homologous Recombination Genes Predict Platinum Response and Survival in Ovarian, Fallopian Tube, and Peritoneal Carcinomas

Purpose: Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination DNA repair genes is uncertain. Experimental Design: Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the homologous recombination pathway. Results: Thirty-one percent of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 homologous recombination genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Nonserous ovarian carcinomas had similar rates of homologous recombination mutations to serous carcinomas (28% vs. 31%, P = 0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic homologous recombination mutations was highly predictive of primary platinum sensitivity (P = 0.0002) and improved overall survival (P = 0.0006), with a median overall survival of 66 months in germline homologous recombination mutation carriers, 59 months in cases with a somatic homologous recombination mutation, and 41 months for cases without a homologous recombination mutation. Conclusions: Germline or somatic mutations in homologous recombination genes are present in almost one third of ovarian carcinomas, including both serous and nonserous histologies. Somatic BRCA1/2 mutations and mutations in other homologous recombination genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of homologous recombination mutations in nonserous carcinomas supports their inclusion in PARP inhibitor clinical trials. Clin Cancer Res; 20(3); 764–75. ©2013 AACR.

Temporal Variability of Human Vaginal Bacteria and Relationship with Bacterial Vaginosis

Background Little is known about short-term bacterial fluctuations in the human vagina. This study used PCR to assess the variability in concentrations of key vaginal bacteria in healthy women and the immediate response to antibiotic treatment in women with bacterial vaginosis (BV). Methodology/Principal Findings Twenty-two women assessed for BV using Amsels criteria were evaluated daily for 7 or 14 days, then at 2, 3 and 4 weeks, using a panel of 11 bacterium-specific quantitative PCR assays. Participants with BV were treated with 5 days of intravaginal metronidazole. Participants without BV had vaginal biotas dominated by lactobacilli, whose levels fluctuated with menses. With onset of menstruation, quantities of Lactobacillus jensenii and Lactobacillus crispatus decreased and were found to be inversely related to Gardnerella vaginalis concentrations (p<0.001). Women with BV had a variety of fastidious bacteria whose concentrations dropped below detection thresholds 1–5 days after starting metronidazole. Recurrent BV was characterized by initial profound decreases of BV-associated bacteria after treatment followed by subsequent increases at relapse. Conclusions/Significance The microbiota of the human vagina can be highly dynamic. Healthy women are colonized with Lactobacillus species, but levels can change dramatically over a month. Marked increases in G. vaginalis were observed during menses. Participants with BV have diverse communities of fastidious bacteria that are depleted by vaginal metronidazole therapy. Women with recurrent BV initially respond to antibiotic treatment with steep declines in bacterial concentrations, but these bacteria later reemerge, suggesting that antibiotic resistance in these bacteria is not an important factor mediating BV recurrence.

Characterization of Vaginal Flora and Bacterial Vaginosis in Women Who Have Sex with Women

Bacterial vaginosis (BV) may be common among women who report having sex with women (WSW) and frequently occurs in both members of monogamous couples. The results of Gram staining of a vaginal smear were consistent with BV in 81 (25%) and intermediate in 37 (11%) of 326 WSW included in this study. Lactobacilli were detected in 64% of subjects, and 42% of subjects had H(2)O(2)-producing strains. BV was associated with a higher lifetime number of female sex partners, failure to always clean an insertive sex toy before use, and oral-anal sex with female partners. Neither recent douching nor sexual practices with male partners were associated with BV. Vaginal smears from each partner were concordant in 55 (95%) of 58 monogamous couples; BV was present in both partners in 16 couples (28%) (P<.001, compared with expected distribution). BV was common among subjects who did not douche, who did not have concurrent sex with male partners, or who did not have a new sex partner, which suggests that other risk factors for BV exist. These data support the hypothesis that sexual exchange of vaginal secretions is a possible mechanism for acquisition of BV.

Influence of the Normal Menstrual Cycle on Vaginal Tissue, Discharge, and Microflora

The objective of this study was to examine genital tissue, vaginal fluid, and vaginal microbial flora at 3 phases of the menstrual cycle in asymptomatic women. Vaginal examinations were performed 3 times in 74 women: at the menstrual phase (days 1-5), the preovulatory phase (days 7-12), and the postovulatory phase (days 19-24). Flora of 50 women without bacterial vaginosis (BV) was analyzed separately from flora of 24 women with BV. The volume of vaginal discharge increased and the amount of cervical mucus decreased over the menstrual cycle. Among subjects without BV, the rate of recovery of any Lactobacillus changed little (range, 82% to 98%; P = .2); however, a small increase occurred in the rate of recovery of heavy (3+ to 4+ semiquantitative) growth of Lactobacillus over the menstrual cycle (P = .04). A linear decrease occurred in the rate of recovery of heavy growth of any non-Lactobacillus species, from 72% at days 1-5 to 40% at days 19-24 (P = .002). A linear decrease also occurred in the rate of recovery of Prevotella species, from 56% on days 1-5 to 28% on days 19-24 (P =. 007), while a small linear increase occurred in the rate of recovery of Bacteroides fragilis (P=.05). Among subjects with BV, the only significant change was an increase in the rate of recovery of Lactobacillus, from 33% at days 1-5 to 54% at days 19-24 (P = .008). Among all subjects, the rate of recovery of heavy growth of Lactobacillus increased over the menstrual cycle and, in contrast, the concentration of non-Lactobacillus species tended to be higher at menses, which is evidence that the vaginal flora becomes less stable at this time.

Maternal stress is associated with bacterial vaginosis in human pregnancy.

Objectives: Maternal infection, particularly bacterial vaginosis (BV) in pregnancy, is one of the leading causes of adverse perinatal outcomes. The determinants of individual differences in susceptibility, or vulnerability, to maternal infections are poorly understood. This study examines whether chronic maternal stress predisposes women to infection during pregnancy, and if so, whether the effects of chronic stress on infection are independent of other established risk factors. Methods: We conducted a cross-sectional, clinical prevalence study of chronic maternal stress and BV status in a sample of 454 pregnant women at 14.3 ± 0.3 weeks gestation (±SEM). BV was diagnosed by Gram-stain of vaginal fluid samples; chronic maternal stress was assessed using the Cohen Perceived Stress Scale. Other established risk factors for BV, including maternal age, race/ethnicity, marital status, SES, and behaviors related to feminine hygiene, sexual practices, and substance use, were measured using a structured interview. Results: Of the 454 women enrolled in this study, 224 (49%) were BV positive (Nugent score 7–10), 64 (14%) had intermediate vaginal flora (Nugent score 4–6), and 166 (37%) were BV negative (Nugent score 0–3). BV+ women had significantly higher chronic stress levels than BV− women (24.6 ± 0.5 vs. 22.2 ± 0.6 units (±SEM), respectively; t = 3.19; p < .01). Maternal sociodemographic variables (African-American race/ethnicity) and behavioral characteristics (vaginal douching, number of lifetime sexual partners, and use of illicit drugs) also were significantly associated with the presence of BV. After controlling for the effects of these variables, using a multivariable logistic regression model, chronic maternal stress remained a significant and independent predictor of BV status. Women in the moderate-stress group (third quartile) and high-stress (fourth quartile) group were 2.3 times (95% CI = 1.2–4.3) and 2.2 times (95% CI = 1.1–4.2) more likely to be BV+ than women in the low-stress group (bottom quartile). Conclusions: High levels of chronic stress during pregnancy are associated with bacterial vaginosis. The effect of chronic maternal stress is independent of the effects of other established sociodemographic and behavioral risk factors for BV.

Broad-Spectrum Bacterial rDNA Polymerase Chain Reaction Assay for Detecting Amniotic Fluid Infection Among Women in Premature Labor

We amplified bacterial 16S rRNA encoding DNA (rDNA) with the polymerase chain reaction (PCR) to detect amniotic fluid infection in 69 women in premature labor whose membranes were intact. Bacterial rDNA was detected by PCR in samples from 15 (94%) of 16 patients with positive amniotic fluid cultures. Bacteria were detected by PCR in samples from 5 (36%) of 14 patients with negative cultures and elevated interleukin (IL)-6 levels vs. 1 (3%) of 39 patients with negative cultures and IL-6 levels of < or = 2,000 pg/mL (P < .01). The median amniotic fluid cytokine levels and the pregnancy outcomes were similar for patients with positive amniotic fluid cultures and those with negative cultures and positive rDNA PCR assays. The association between amniotic fluid infection and premature labor may be underestimated on the basis of amniotic fluid culture results. The broad-spectrum bacterial 16S rDNA PCR assay may prove useful for diagnosing amniotic fluid infection.

Vaginal Indicators of Amniotic Fluid Infection in Preterm Labor

Objective To determine whether vaginal interleukin-6, interleukin-8, neutrophils, bacterial vaginosis, and selected vaginal bacteria are predictors of amniotic fluid (AF) infection among women in preterm labor. Methods One hundred ninety-seven afebrile women in preterm labor with intact membranes had vaginal and AF samples collected for Gram stain, culture, and interleukin-8 and interleukin-6 determinations. Vaginal interleukin-6, interleukin-8, neutrophils, and vaginal flora were compared in women with positive and negative AF cultures. The negative AF culture group was subdivided according to AF interleukin-6 concentration. Logistic regression was used to examine the associations between vaginal cytokines and flora and AF infection or elevated AF interleukin-6. Results The vaginal interleukin-8 concentration and neutrophil count were significantly higher with both AF infection and elevated concentrations of AF interleukin-6 and interleukin-8. The vaginal interleukin-6 concentration was not associated with AF infection or high concentration of AF cytokines. Amniotic fluid infection was associated with bacterial vaginosis or intermediate vaginal flora by Gram stain, absence of hydrogen peroxide–producing Lactobacillus, and presence of vaginal Bacteroides ureolyticus and Fusobacterium. Vaginal interleukin-8 levels greater than 30 ng/mL had 80% sensitivity and a positive predictive value of 35%, and an abnormal vaginal Gram stain (more than five neutrophils per 400× field, bacterial vaginosis species, or intermediate flora) had 90% sensitivity and a positive predictive value of 27% to detect AF infection or elevated AF interleukin-6. Conclusion A high vaginal interleukin-8 concentration, abnormal vaginal Gram stain, absent hydrogen peroxide–producing Lactobacillus, and anaerobic vaginal flora were strongly associated with AF infection among women in preterm labor.

Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization

OBJECTIVE To assess the impact of individual bacteria isolated from the vagina and tip of the embryo transfer catheter on live-birth rates. DESIGN Prospective clinical study. SETTING Infertility outpatient clinic of a university hospital. PATIENT(S) Ninety-one women undergoing IVF-ET. INTERVENTION(S) Cultures were obtained from the vagina for aerobic and anaerobic bacteria at the time of both sonographic egg retrieval and embryo transfer and from the tip of the embryo transfer catheter. Doxycycline treatment was started after egg retrieval. MAIN OUTCOME MEASURE(S) The live birth of one or more neonates. RESULT(S) Doxycycline had no substantial impact on the recovery of individual vaginal bacteria or on bacterial vaginosis. An increase in live-birth rate was associated with the recovery of hydrogen peroxide-producing Lactobacillus from the vagina (P=0.01) and from the embryo transfer catheter (P=0.01). In contrast, a reduction in live-birth rate was associated with recovery of Streptococcus viridans (S. viridans) from the embryo transfer catheter tip (P=0.04). CONCLUSION(S) In the setting of IVF-ET, prophylactic doxycycline had little effect on vaginal bacteria. Specific bacteria recovered from the embryo transfer catheter appear associated with a detrimental or beneficial effect or with no effect on live-birth rates.

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.