Microbial signatures and continuum in endometrial cancer and benign patients.

BACKGROUND: Endometrial cancer is a multifactorial disease with inflammatory, metabolic and potentially microbial cues involved in disease pathogenesis. The endometrial cancer microbiome has been poorly characterised so far and studies have often overestimated bacterial biomass due to lack of integration of appropriate contamination controls. There is also a scarcity of evidence on the functionality of microbial microenvironments in endometrial cancer. This work addresses that knowledge gap by interrogating the genuine, contamination-free microbial signatures in the female genital tract and rectum of women with endometrial cancer and the mechanistic role of microbiome on carcinogenic processes. RESULTS: Here we sampled different regions of the reproductive tract (vagina, cervix, endometrium, fallopian tubes and ovaries) and rectum of 61 patients (37 endometrial cancer; 24 benign controls). We performed 16S rRNA gene sequencing of the V1-V2 hypervariable regions and qPCR of the 16S rRNA gene to qualitatively and quantitatively assess microbial communities and used 3D benign and endometrial cancer organoids to evaluate the effect of microbial products of L. crispatus, which was found depleted in endometrial cancer patients following primary analysis, on endometrial cell proliferation and inflammation. We found that the upper genital tract of a subset of women with and without endometrial cancer harbour microbiota quantitatively and compositionally distinguishable from background contaminants. Endometrial cancer was associated with reduced cervicovaginal and rectal bacterial load together with depletion of Lactobacillus species relative abundance, including L. crispatus, increased bacterial diversity and enrichment of Porphyromonas, Prevotella, Peptoniphilus and Anaerococcus in the lower genital tract and endometrium. Treatment of benign and malignant endometrial organoids with L. crispatus conditioned media exerted an anti-proliferative effect at high concentrations but had minimal impact on cytokine and chemokine profiles. CONCLUSIONS: Our findings provide evidence that the upper female reproductive tract of some women contains detectable levels of bacteria, the composition of which is associated with endometrial cancer. Whether this is a cause or consequence of cancer pathophysiology and what is the functional significance of this finding remain to be elucidated to guide future screening tools and microbiome-based therapeutics. Video Abstract.

mBodyMap: a curated database for microbes across human body and their associations with health and diseases.

mBodyMap is a curated database for microbes across the human body and their associations with health and diseases. Its primary aim is to promote the reusability of human-associated metagenomic data and assist with the identification of disease-associated microbes by consistently annotating the microbial contents of collected samples using state-of-the-art toolsets and manually curating the meta-data of corresponding human hosts. mBodyMap organizes collected samples based on their association with human diseases and body sites to enable cross-dataset integration and comparison. To help users find microbes of interest and visualize and compare their distributions and abundances/prevalence within different body sites and various diseases, the mBodyMap database is equipped with an intuitive interface and extensive graphical representations of the collected data. So far, it contains a total of 63 148 runs, including 14 401 metagenomes and 48 747 amplicons related to health and 56 human diseases, from within 22 human body sites across 136 projects. Also available in the database are pre-computed abundances and prevalence of 6247 species (belonging to 1645 genera) stratified by body sites and diseases. mBodyMap can be accessed at: https://mbodymap.microbiome.cloud.

Characterization of the endometrial, cervicovaginal and anorectal microbiota in post-menopausal women with endometrioid and serous endometrial cancers.

OBJECTIVE: To characterize the microbiota of postmenopausal women undergoing hysterectomy for endometrioid (EAC) or uterine serous cancers (USC) compared to controls with non-malignant conditions. METHODS: Endometrial, cervicovaginal and anorectal microbial swabs were obtained from 35 postmenopausal women (10 controls, 14 EAC and 11 USC) undergoing hysterectomy. Extracted DNA was PCR amplified using barcoded 16S rRNA gene V4 primers. Sequenced libraries were processed using QIIME2. Phyloseq was used to calculate α- and ß- diversity measures. Biomarkers associated with case status were identified using ANCOM after adjustment for patient age, race and BMI. PICRUSt was used to identify microbial pathways associated with case status. RESULTS: Beta-diversity of microbial communities across each niche was significantly different (R2 = 0.25, p < 0.001). Alpha-diversity of the uterine microbiome was reduced in USC (Chao1, p = 0.004 and Fisher, p = 0.007) compared to EAC. Biomarkers from the three anatomical sites allowed samples to be clustered into two distinct clades that distinguished controls from USC cases (p = 0.042). The USC group was defined by 13 bacterial taxa across the three sites (W-stat>10, FDR<0.05) including depletion of cervicovaginal Lactobacillus and elevation of uterine Pseudomonas. PICRUSTt analysis revealed highly significant differences between the USC-associated clades within the cervicovaginal and uterine microbiota. CONCLUSIONS: The microbial diversity of anatomic niches in postmenopausal women with EAC and USC is different compared to controls. Multiple bacteria are associated with USC case status including elevated levels of cervicovaginal Lactobacillus, depletion of uterine Pseudomonas, and substantially different functional potentials identified within cervicovaginal and uterine niches.

An Evaluation of the Fecal Microbiome in Lynch Syndrome.

PURPOSE: There is limited data regarding the fecal microbiome findings in patients with Lynch syndrome. We aimed to study the fecal micobiome of patients with Lynch syndrome with and without cancer. METHODS: We performed an observational study comparing the fecal microbiome of patients with Lynch syndrome (LS) with cancer with those without cancer. We included subjects older than 18 years with LS and excluded those with a history of colectomy or inflammatory bowel disease. We analyzed their fecal microbiome by 16S ribosomal subunit PCR amplification and performed comparative analyses. RESULTS: Eight patients were included: 3 of these with LS and cancer (LS-C) and 5 patients with LS and no cancer (LS-NC). We found non-significant differences at the phyla and genera level between the LS-C and LS-NC groups. At the phyla level, LS-C patients had a higher percentage of Bacteroidetes (42.2% vs. 28.5%; P = 0.068) and Verrucomicrobia (0.644% vs 0.0007%; P = 0.10), and a lower percentage of Firmicutes (48.3% vs. 65.4%; P = 0.078). At the genus level, LS-C patients had a higher rate of Akkermania (0.766% vs. 0.001%; P = 0.11). LS-C patients with endometrial cancer had a higher rate of Bacteroides (37.4% vs 17.3%; P = 0.10). LS-C patients had a lower rate of Pseudobutyrvibrio (0.74% vs. 2.71%; P = 0.10). CONCLUSIONS: The fecal microbiome of LS patients with extraintestinal cancer differs that of LS patients without cancer. Further studies are needed to explore microbiome changes in these high risk patients.

Dysbiosis of the endometrial microbiota and its association with inflammatory cytokines in endometrial cancer.

The underlying molecular mechanisms involved in the pathogenesis of endometrial cancer (EC) are still not well understood. Our goal was to investigate the composition of the endometrial microbiota and the association with inflammatory cytokines in EC. Endometrial microbiota profiles of women with EC (n = 25) and benign uterine lesions (BUL, n = 25) were assessed by 16S ribosomal RNA gene amplicon sequencing. The expression levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-17 (IL-17) mRNA and protein in the endometrial tissues of the two groups were determined by real-time quantitative polymerase chain reaction and Western blot, respectively. There were significant differences in alpha diversity based on the observed operational taxonomic units (P = .002), Pielou evenness (P = .001), and Shannon index (P < .001) between EC and BUL groups. Significant differences were also found in Bray-Curtis (P = .001) and unweighted UniFrac (P = .001) beta diversity measures between the two groups. At the genus level, Micrococcus was more abundant in the EC group. Pseudoramibacter_Eubacterium, Rhodobacter, Vogesella, Bilophila, Rheinheimera, and Megamonas were enriched in the BUL group. There were no differences in IL-8 and IL-17 protein levels between the two groups, except IL-6 protein levels. However, the mRNA expression levels of IL-6, IL-8, and IL-17 were significantly different. Moreover, the relative abundances of Micrococcus was positively correlated with IL-6, and IL-17 mRNA levels. In conclusion, our results suggested that dysbiosis of endometrial microbiota and the inflammatory cytokines were associated with Micrococcus in EC patients, which might be useful for exploration of the mechanism between the endometrial microbiota and inflammatory responses in future studies.

Prospects of NSAIDs administration as double-edged agents against endometrial cancer and pathological species of the uterine microbiome.

Many types of cancers, including endometrial cancer, were found to have cyclooxygenase-2 (COX-2) overexpression. Because this enzyme belongs to the group of pro-inflammatory enzymes, so-called NSAIDs (non-steroidal anti-inflammatory drugs) directly inhibit its activity. An increasing number of reports on COX-2 involvement in cancer, as well as on the role of microbiota in abnormal metabolism and signaling of cells, forces the development of new NSAID types. Besides, NSAIDs can affect some bacteria, which are vaginal/endometrial microbiome members. The overgrowth of those species was found to be a major cause of some uterus diseases. Those infections can lead to chronic inflammatory response and suppress anti-tumorigenic cell pathways. The purpose of this review is to highlight the COX-2 enzyme role in endometrial cancer, the potential effect of the endometrial microbiome on COX-2 enzyme overexpression, and the prospects of NSAIDs use in terms of this type of cancer.

Postmenopause as a key factor in the composition of the Endometrial Cancer Microbiome (ECbiome).

Incidence rates for endometrial cancer (EC) are rising, particularly in postmenopausal and obese women. Previously, we showed that the uterine and vaginal microbiome distinguishes patients with EC from those without. Here, we sought to examine the impact of patient factors (such as menopause status, body mass index, and vaginal pH) in the microbiome in the absence of EC and how these might contribute to the microbiome signature in EC. We find that each factor independently alters the microbiome and identified postmenopausal status as the main driver of a polymicrobial network associated with EC (ECbiome). We identified Porphyromas somerae presence as the most predictive microbial marker of EC and we confirm this using targeted qPCR, which could be of use in detecting EC in high-risk, asymptomatic women. Given the established pathogenic behavior of P. somerae and accompanying network in tissue infections and ulcers, future investigation into their role in EC is warranted.

Potential contribution of the uterine microbiome in the development of endometrial cancer.

BACKGROUND: Endometrial cancer studies have led to a number of well-defined but mechanistically unconnected genetic and environmental risk factors. One of the emerging modulators between environmental triggers and genetic expression is the microbiome. We set out to inquire about the composition of the uterine microbiome and its putative role in endometrial cancer. METHODS: We undertook a study of the microbiome in samples taken from different locations along the female reproductive tract in patients with endometrial cancer (n = 17), patients with endometrial hyperplasia (endometrial cancer precursor, n = 4), and patients afflicted with benign uterine conditions (n = 10). Vaginal, cervical, Fallopian, ovarian, peritoneal, and urine samples were collected aseptically both in the operating room and the pathology laboratory. DNA extraction was followed by amplification and high-throughput next generation sequencing (MiSeq) of the 16S rDNA V3-V5 region to identify the microbiota present. Microbiota data were summarized using both α-diversity to reflect species richness and evenness within bacterial populations and ß-diversity to reflect the shared diversity between bacterial populations. Statistical significance was determined through the use of multiple testing, including the generalized mixed-effects model. RESULTS: The microbiome sequencing (16S rDNA V3-V5 region) revealed that the microbiomes of all organs (vagina, cervix, Fallopian tubes, and ovaries) are significantly correlated (p < 0.001) and that there is a structural microbiome shift in the cancer and hyperplasia cases, distinguishable from the benign cases (p = 0.01). Several taxa were found to be significantly enriched in samples belonging to the endometrial cancer cohort: Firmicutes (Anaerostipes, ph2, Dialister, Peptoniphilus, 1-68, Ruminococcus, and Anaerotruncus), Spirochaetes (Treponema), Actinobacteria (Atopobium), Bacteroidetes (Bacteroides and Porphyromonas), and Proteobacteria (Arthrospira). Of particular relevance, the simultaneous presence of Atopobium vaginae and an uncultured representative of the Porphyromonas sp. (99 % match to P. somerae) were found to be associated with disease status, especially if combined with a high vaginal pH (>4.5). CONCLUSIONS: Our results suggest that the detection of A. vaginae and the identified Porphyromonas sp. in the gynecologic tract combined with a high vaginal pH is statistically associated with the presence of endometrial cancer. Given the documented association of the identified microorganisms with other pathologies, these findings raise the possibility of a microbiome role in the manifestation, etiology, or progression of endometrial cancer that should be further investigated.

Neisseria gonorrhoeae breaches the apical junction of polarized epithelial cells for transmigration by activating EGFR.

Neisseria gonorrhoeae initiates infection at the apical surface of columnar endocervical epithelial cells in the female reproductive tract. These cells provide a physical barrier against pathogens by forming continuous apical junctional complexes between neighbouring cells. This study examines the interaction of gonococci (GC) with polarized epithelial cells. We show that viable GC preferentially localize at the apical side of the cell-cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84. In GC-infected cells, continuous apical junctional complexes are disrupted, and the junction-associated protein ß-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites; however, overall cellular levels remain unchanged. This redistribution of junctional proteins is associated with a decrease in the 'fence' function of the apical junction but not its 'gate' function. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and ß-catenin, while inhibition of EGFR kinase activity significantly reduces both GC-induced ß-catenin redistribution and GC transmigration. Therefore, the gonococcus is capable of weakening the apical junction and polarity of epithelial cells by activating EGFR, which facilitates GC transmigration across the epithelium.

Risk of postoperative pelvic abscess in major gynecologic oncology surgery: one-year single-institution experience.

PURPOSE: This study was undertaken to evaluate risk factors for the occurrence of postoperative abscesses in a large single-institution series of gynecologic cancer patients undergoing major surgery. MATERIALS AND METHODS: Patients admitted to the Division of Gynecologic Oncology, Catholic University of Sacred Hearth, Rome, Italy, between January 2008 and February 2009, were retrospectively analyzed. The occurrence of pelvic abscesses was identified by sign and symptoms and confirmed with radiological and microbiological examinations. RESULTS: A total of 360 patients were analyzed for the study. Exenteration and use of fibrillar absorbable hemostat were significantly associated with the presence of postoperative abscesses (P < .0001) by multiple regression analysis, whereas operative time, type of surgery, lymphadenectomy, or other associated surgical procedures failed to result statistically relevant. CONCLUSIONS: The use of fibrillar oxidized regenerated cellulose as hemostatic agent may represent a risk factor for postoperative abscesses, especially when used during pelvic exenteration.

A first case of Streptococcus bovis bacteremia and peritonitis from endometrial cancer origin.

BACKGROUND: The most important clinical infections caused by Streptococcus Bovis are bacteremia and endocarditis. Usually, Streptococcus Bovis bacteremia has been described in association with bowel pathology. CASE REPORT: A 67-year-old woman with an history of endometrial cancer Ic was admitted with the suspicion of peritonitis at examination. At exploratory laparotomy, a total hysterectomy was performed and the abdomen was drained. Histology revealed an uterine adenocarcinoma staged IIIa with intramyometrial cocci accumulation. Streptococcus Bovis was isolated from the peritoneal fluid cultures and three haemocultures. CONCLUSION: Because we excluded bowel pathology and endocarditis, this is the first case of Streptococcus Bovis bacteremia from endometrial cancer origin.

Association of herpesvirus infection with the development of genital cancer.

Clinical observations and epidemiological studies on genital cancer have revealed an association with sexual behavior, thus motivating research into sexually transmitted agents which may be responsible for the neoplasia. In this study, we used the PCR technique to examine the presence of CMV, HSV and EBV viruses in 187 cases of human genital lesions and found that infection with CMV or HSV was associated with cervical cancer. When we stratified according to HPV status this association was found only for HPV-DNA-negative cases. These findings indicate that past infection with CMV or HSV could be interpreted as a surrogate marker of HPV infection. However, these viruses may play an important role themselves in cervical cancer.

Endometrial squamous cell carcinoma following whole pelvic radiation therapy: response to carboplatin.

A case of Stage IV endometrial squamous cell carcinoma occurring 8 years after a low anterior resection and whole pelvic radiation therapy for a Dukes D colon carcinoma is presented. Koilocytosis was present in the tumor. There was no evidence of human papillomavirus antigen or DNA in the tumor. The patient was treated with surgery followed by six cycles of carboplatin chemotherapy. At the completion of chemotherapy there was no clinical or radiological evidence of disease. The tumor recurred 9 months postchemotherapy and the patient died of disease 17 months postdiagnosis.

Endometrial bacterial flora detected in patients with uterine endometrial cancer.

Certain bacteria produce some carcinogens such as N-nitro compounds, n-butyric acid and n-valeric acid. From this point of view, the examination of intrauterine bacterial flora in patients with uterine endometrial cancer may provide important information. Twenty patients with the diagnosis of uterine endometrial cancer and 20 patients without complications other than myoma uteri were enrolled in the study. Enterobacteriaceae, Streptococcus agalactiae and anaerobic bacteria were mainly detected. The products of these bacteria might be considered to contribute to the initiation of endometrial carcinogenesis. Mixed abnormal flora between aerobic and anaerobic bacteria were detected in all patients with uterine endometrial cancer. It is suggested that uterine endometrial cancer provides favorable conditions for bacterial growth. Mixed abnormal bacterial flora also might influence the onset and growth of uterine endometrial cancer.

Detection of human papilloma virus (HPV) infection in paraffin-embedded tissues of endometrial carcinoma.

Although human papilloma virus (HPV) associated lesions constitute a well recognized clinical entity in the female lower genital tract, namely vulva, vagina and cervix, few studies have demonstrated HPV infection in other genital sites, particularly in the ovary and uterine corpus. Recently, with the highly sensitive polymerase chain reaction (PCR) technique, HPV infections were found in an ovarian tumour and adenocarcinoma of the cervix. This prompted a retrospective analysis of HPV DNA in 22 cases of endometrial adenocarcinoma in order to investigate the possible carcinogenesis of HPV in the uterine corpus. In this study DNA extraction was performed from paraffinized cancerous tissues and the normal cervical counterpart. HPV 6, 11, 16 and 18 primers specific oligonucleotides were used in PCR to detect the presence of this oncogenic virus. HPV 16 DNA was found in 1 endometrial adenocarcinoma and 4 cervical tissues. Our result did not support the aetiological role of HPV in the carcinogenesis of endometrial carcinoma.

In vitro model of Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin.

Electron microscopy was used to examine Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin. A clinical H. ducreyi isolate (90-244) adhered in snake-like whorls to the surfaces of cervical carcinoma cells (HeLa 229), endometrial adenocarcinoma cells (HEC-1-B), and human neonatal foreskin fibroblast (HFF) cells. A prototype strain of H. ducreyi (CIP542) adhered in randomly organized clumps on the surfaces of HFF. Strain 90-244 entered HFF and HEC-1-B cells but did not enter HeLa cells. The H. ducreyi in the HFF cells at 2 h were partly surrounded by a membrane consistent with that of a phagocytic vacuole. At 2 h, strain CIP542 was found in interstitial spaces between the HFF cells and also in the cytoplasm of the cells. After 7 and 24 h, both strains of H. ducreyi were found in the large interstitial spaces between the HFF cells, in the cytoplasm, and extracellularly. This model of in vitro H. ducreyi infection of eukaryotic cells will allow for more specific study of factors that determine the virulence of H. ducreyi.

Detection of p53 gene mutations in human ovarian and endometrial cancers by polymerase chain reaction-single strand conformation polymorphism analysis.

The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162, 175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.

Human papillomavirus in benign and malignant ovarian and endometrial tissues.

Ovarian and endometrial cancer tissues were examined for the presence of human papillomavirus (HPV) and the results were compared with the findings in normal tissues by polymerase chain reaction. Putative DNA of HPV types 16 and 18 that target DNA sequences from paraffin-embedded tissues were amplified with paired oligonucleotide primers that encode the E6 gene of HPV. The amplified DNA sequences were then detected with Southern blot hybridization analysis. The HPV DNA sequences were detected in both benign (50% ovarian, 70% endometrial) and malignant ovarian (27.2%) and endometrial (37.5%) tissue samples. Interestingly, eight hepatoma samples were also analyzed as tissue controls. The results were negative in seven, but positive in one with repeated tests. The results suggest that the spread of HPV in the upper genital tract may not be uncommon. The explanation of a positive liver tissue study result will have to await further study.

[Demonstration and biological significance of viral and cellular oncogenes in uterine carcinomas]. / Nachweis und biologische Bedeutung von Viren und Onkogenen bei Uteruskarzinomen.

Premalignant and malignant lesions of the cervix uteri and endometrium were analyzed for the presence of human papillomavirus (HPV) DNA and c-myc or c-erbB2/neu oncogene expression. HPV DNA was detected by PCR in 100% of the cervical carcinomas and CIN lesions, but also in endometrial lesions (3/8 adenocarcinomas, two hyperplasias and one adenomyosis uteri). Myc-overexpression was found in 25-30% of the cervical carcinomas and severe dysplasias, but not in endometrial lesions. C-erbB2 was overexpressed in 4/11 endometrial carcinomas and 3/19 CIN3 lesions. The implications of these results are discussed.