Cancer Genomics - Proteomics

Clonal Relationship Between Lichen Sclerosus, Differentiated Vulvar Intra-epithelial Neoplasia and Non HPV-related Vulvar Squamous Cell Carcinoma

Differentiated vulvar intraepithelial neoplasia (dVIN) and lichen sclerosus (LS) can give rise to vulvar squamous cell carcinoma (VSCC), but genetic evidence is currently still limited. We aimed to determine genetic abnormalities in VSCC and backtrack these abnormalities in the dVIN and LS lesions. DNA from VSCC and patient-matched dVIN and LS samples of twelve patients was collected. High-resolution genome-wide copy number analysis was performed and subsequently, we sequenced TP53. Copy number alterations were identified in all VSCC samples. One dVIN lesion presented with three copy number alterations that were preserved in the paired VSCC sample. Targeted sequencing of TP53 identified mutations in five VSCCs. All five mutations were traced back in the dVIN (n=5) or the LS (n=1) with frequencies ranging from 3-19%. Our data provide genetic evidence for a clonal relationship between VSCC and dVIN or LS.

CRY1Regulates Chemoresistance in Association WithNANOGby Inhibiting Apoptosisvia STAT3Pathway in Patients With Cervical Cancer

Cryptochrome 1 (CRY1), a core circadian gene, modulates circadian rhythm and carcinogenesis. Here, we investigated the role of CRY1 and its correlation with NANOG, a stem cell transcription factor, in cervical cancer. Immunohistochemistry with tissue microarray was performed to evaluate CRY1 and NANOG expression in cervical cancer tissues, and their functional roles were assessed in cervical cancer cell lines. CRY1 or NANOG was significantly over-expressed in cervical cancer tissues. Notably, combined over-expression of CRY1 and NANOG was correlated with a significantly poor OS and DFS and showed a stronger predictive value for chemoradiation response than each single protein. Furthermore, siCRY1 induced apoptosis, decreased NANOG expression, suppressed STAT3 signalling, and activated p53 signalling in cervical cancer cell lines. CRY1 and NANOG over-expression serves as a strong predictive biomarker for prognosis and chemoradiation response, and may be a new therapeutic target in patients with cervical cancer.

Restoration of ARID1A Protein in ARID1A-deficient Clear Cell Carcinoma of the Ovary Attenuates Reactivity to Cytotoxic T Lymphocytes

Clear cell carcinoma is a prevalent histological type of ovarian cancer in East Asia, particularly in Japan, known for its resistance to chemotherapeutic agents and poor prognosis. ARID1A gene mutations, commonly found in ovarian clear cell carcinoma (OCCC), contribute to its pathogenesis. Recent data revealed that the ARID1A mutation is related to better outcomes of cancer immunotherapy. Thus, this study aimed to investigate the immunotherapy treatment susceptibility of OCCC bearing ARID1A mutations. Expression of ARID1A was analyzed using western blotting in ovarian cancer cell lines. OCCC cell lines JHOC-9 and RMG-V were engineered to overexpress NY-ESO-1, HLA-A*02:01, and ARID1A. Sensitivity to chemotherapy and T cell receptor-transduced T (TCR-T) cells specific for NY-ESO-1 was assessed in ARID1A-restored cells compared to ARID1A-deficient wild-type cells. JHOC-9 cells and RMG-V cells showed no expression of ARID1A protein. Overexpression of ARID1A in JHOC-9 and RMG-V cells did not impact sensitivity to gemcitabine. While ARID1A overexpression decreased sensitivity to cisplatin in RMG-V cells, it had no such effect in JHOC-9 cells. ARID1A overexpression reduced the reactivity of NY-ESO-1-specific TCR-T cells, as observed by the IFNγ ESLIPOT assay. Cancer immunotherapy is an effective approach to target ARID1A-deficient clear cell carcinoma of the ovary.

Deregulated circRNAs in Epithelial Ovarian Cancer With Activity in PreclinicalIn VivoModels: Identification of Targets and New Modalities for Therapeutic Intervention

Epithelial ovarian cancer (EOC) is associated with a dismal prognosis due to development of resistance to chemotherapy and metastasis in the peritoneal cavity and distant organs. In order to identify new targets and treatment modalities we searched the literature for up- and and down-regulated circRNAs with efficacy in preclinical EOC-related in vivo systems. Our search yielded circRNAs falling into the following categories: cisplatin and paclitaxel resistance, transmembrane receptors, secreted factors, transcription factors, RNA splicing and processing factors, RAS pathway-related components, proteolysis and cell-cycle regulation, signaling-related proteins, and circRNAs regulating proteins in additional categories. These findings can be potentially translated by validation and manipulation of the corresponding targets, inhibition of circRNAs with antisense oligonucleotides (ASO), small interfering RNAs (siRNA) or small hairpin RNA (shRNA) or by reconstituting their activity.

Multisite Tumor Sampling Reveals Extensive Heterogeneity of Tumor and Host Immune Response in Ovarian Cancer

Ovarian cancer (OVCA) is characterized by genomic/molecular intra-patient heterogeneity (IPH). Tissue histology and morphological features are surrogates of the underlying genomic/molecular contexture. We assessed the morphological IPH of OVCA tumor compartments and of lymphocytic infiltrates in multiple matched samples per patient. We examined 294 hematoxylin & eosin (H&E) OVCA tumor whole sections from 70 treatment-naïve patients who had undergone cytoreductive surgery. We assessed morphological subtypes as immunoreactive (IR), solid - proliferative (SD), papilloglandular (PG), and mesenchymal transition (MT); subtype load per patient; stromal tumor-infiltrating lymphocyte (sTIL) density as average per sample; and, as maximal sTIL values (max-TILs) among all samples per patient, ovaries and implants. Among all 294 tumor sections, the most frequent primary morphological subtype was PG (n=150, 51.0%), followed by MT (71, 24.1%), SD (48, 16.3%) and IR (15, 5.1%). Subtype combinations were observed in 67/294 sections (22.8%) and IPH in 48/70 patients (68.6%). PG prevailed in ovaries (p<0.001), SD and MT in implants (p=0.023 and p<0.001, respectively). sTILs were higher in SD compared to non-SD (p=0.019) and lower in PG, respectively (p<0.001). sTIL density was higher in implants than in ovaries (p<0.001). Higher max-TILs were associated with stage IV disease (p=0.043), upper abdominal dissemination (p=0.024), endometrioid histology (p=0.013), and grade 3 tumors (p=0.021). Favorable prognosticators were higher max-TILs per patient (PFS, OS) and higher SD-load (PFS). Clinically relevant morphological and host immune-response IPH appear to be the norm in OVCA. This may complicate efforts to decipher sensitivity of the tumor to certain treatment modalities from a single pre-operative biopsy.

Prognostic Significance of Transient Receptor Potential Vanilloid Type 1 (TRPV1) and Phosphatase and Tension Homolog (PTEN) in Epithelial Ovarian Cancer

Transient receptor potential vanilloid type 1 (TRPV1) has been studied in human malignancies, but has not been studied in epithelial ovarian cancer (EOC). We, therefore, investigated the significance of TRPV1 and correlation with phosphatase and tension homolog (PTEN) in EOC. Immunohistochemical analyses for TRPV1 and PTEN were performed using a tissue microarray. Moreover, the role of TRPV1 in cell growth was assessed in a EOC cell line. High TRPV1 expression and the combination of high TRPV1 and low PTEN expression were an independent prognostic factor for overall survival and disease-free survival. In vitro results demonstrated that knockdown of TRPV1 was associated with decreased cell viability and colony formation. There is a strong association between TRPV1 and PTEN and the combination of TRPV1 and PTEN is a strong indicator of prognostic predictor in EOC.

The Role of Circulating Adiponectin and SNP276G&gt;T at ADIPOQ Gene in BRCA-mutant Women

Environmental factors may influence the lifetime risk of cancer (penetrance) in women with a BRCA mutation. In 89 BRCA-mutant women, affected or unaffected by breast/ovarian cancer, we explored serum levels of adipokines and their relation with the polymorphism SNP276G>T as modulators of BRCA penetrance. Affected women had significantly lower adiponectin than healthy women. Affected women with rs1501299 TT had significantly lower adiponectin and higher leptin than GT and GG genotypes. GT genotype was significantly associated with the disease status [odds ratio (OR)=3.24, 95% confidence interval (95% CI)=1.03-10.17]. Women in the lower tertile of serum adiponectin had a RR of BRCA-associated cancer of 2.80, 95% CI=1.1-7.1 (p for trend=0.03) compared with women in the higher tertile. In the SNP rs1501299 the T allele was significantly associated with lower serum levels of adiponectin in affected women, suggesting that the T allele might be related to cancer.

Invasive Stratified Mucin-producing Carcinoma (ISMC) of the Uterine Cervix: Clinicopathological and Molecular Characteristics With Special Emphasis on the First Description of Consistent Programmed Death-ligand 1 (PD-L1) Over-expression

Invasive stratified mucin-producing carcinoma (ISMC) of the uterine cervix has been reported to be more aggressive than other subtypes of endocervical adenocarcinoma. We investigated the clinicopathological and molecular characteristics of eight ISMCs. We reviewed the electronic medical records and pathology slides of eight patients with ISMC and conducted programmed death-ligand 1 (PD-L1) immunostaining and targeted sequencing. The patients were between 31 and 54 years. Six tumors were pure ISMCs, and two showed co-existing squamous cell carcinoma and usual-type endocervical adenocarcinoma. Lymph node metastases were detected in three cases. Three patients developed distant metastases to the adnexa, lungs, inguinal lymph nodes, and small intestine. Two patients experienced disease progression, and three developed postoperative local recurrences. All tumors showed PD-L1 over-expression, with a mean combined positive score of 73.8 (range=30-100). One tumor harbored erb-b2 receptor tyrosine kinase 2 amplification. ISMC of the uterine cervix exhibits a high risk of recurrence, metastasis, and resistance to chemoradiation therapy. PD-L1 over-expression was consistently observed in all ISMCs. This finding raises the possibility that patients with ISMC may benefit from PD-L1 immunotherapy.

Genetic Analysis in Anal and Cervical Cancer: Exploratory Findings About Radioresistance in the ProfiLER Database

This study aimed to describe genomic alterations on squamous cell cervical and anal carcinomas. From 2013 to 2019, 3,269 patients were included in the molecular screening ProfiLER trial. Only patients with non-metastatic cervical or anal cancer, and those initially treated with radiotherapy in a curative intent were selected. Genetic analyses were performed by next generation sequencing (NGS). Genomic alterations were observed in most patients: 5 patients out of 15 (33.3%) had at least one mutation on NGS and 4 out of 15 (26.7%) had at least one aberration of the number of copies of genes in the comparative genomic hybridation (CGH) analysis. The most common mutated gene was PIK3CA. All omic approaches must be integrated in the locally advanced cancer setting by new clinical trial design to develop two routes in the treatment strategy: intensification or de-escalation treatment strategy according to omic markers.

Quantitative Proteomic Analysis of Cervical Cancer Tissues Identifies Proteins Associated With Cancer Progression

To date, several proteomics studies in cervical cancer (CC) have focused mainly on squamous cervical cancer (SCC). Our study aimed to discover and clarify differences in SCC and CAD that may provide valuable information for the identification of proteins involved in tumor progression, in CC as a whole, or specific for SCC or CAD. Total protein extracts from 15 individual samples corresponding to 5 different CC tissue types were compared with a non-cancerous control group using bidimensional liquid chromatography-mass spectrometry (2D LC-MS/MS), isobaric tags for relative and absolute quantitation (ITRAQ), principal component analysis (PCA) and gene set enrichment analysis (GSEA). A total of 622 statistically significant different proteins were detected. Exocytosis-related proteins were the most over-represented, accounting for 25% of the identified and quantified proteins. Based on the experimental results, reticulocalbin 3 (RCN3) and Ras-related protein Rab-14 (RAB14) were chosen for further downstream in vitro and vivo analyses. RCN3 was overexpressed in all CC tissues compared to the control and RAB14 was overexpressed in squamous cervical cancer (SCC) compared to invasive cervical adenocarcinoma (CAD). In the tumor xenograft experiment, RAB14 protein expression was positively correlated with increased tumor size. In addition, RCN3-expressing HeLa cells induced a discrete size increment compared to control, at day 47 after inoculation. RAB14 and RCN3 are suggested as potential biomarkers and therapeutic targets in the treatment of CC.

hTERT Protein Expression in Cytoplasm and Nucleus and its Association With HPV Infection in Patients With Cervical Cancer

Few studies have analyzed the association between human telomerase reverse transcriptase (hTERT) protein expression (nuclear and cytoplasmic localization), hTERT methylation status, and human papillomavirus (HPV) genotype infection in cervical cancer. One hundred seventy-three patients with cervical cancer were analyzed. hTERT protein expression was detected by immunohistochemistry. hTERT DNA methylation analysis was performed using a PCR-RLB-hTERT assay, targeting two regions of the hTERT promoter. Type specific HPV infection was detected by using GP5+/GP6+PCR-RLB. hTERT protein expression was found in both cytoplasm and nucleus (78.0% of the samples showed a cytoplasmic localization and 79.8% had a nuclear localization). A statistically significant association was found between alpha 9 and 7 HPV species with a non-methylation pattern of the hTERT promoter and between these species and high expression of hTERT protein with nuclear localization. hTERT protein is found in both the nucleus and cytoplasm of patients with cervical cancer and confirm the relationship between the non-methylated status of hTERT promoter and some HPV species as well as the relationship between these species and hTERT protein expression.

In VivoEffects of Conditioned Medium from Human Uterine Cervical Stem Cells in an Ovarian Cancer Xenograft Mouse Model

Ovarian cancer is the most lethal of all gynecological cancers, despite advances in surgical techniques and medical treatments. During the last years, therapies based on mesenchymal stem cells and particularly their secretome (conditioned medium, CM) have emerged as promising treatments for various types of tumors. In the present study, we evaluated the in vivo antitumor effect of human uterine cervical stem cell conditioned medium (hUCESC-CM) after intraperitoneal administration in an ovarian cancer mouse model. We found that intraperitoneal injection of hUCESC-CM in immunodeficient mice, injected fifty days previously with the human ovarian adenocarcinoma SKOV-3 cell line, significantly reduced abdominal tumor growth, and significantly increased overall survival, compared to control mice. hUCESC-CM could be an alternative approach to intraperitoneal treatment of ovarian cancer, either administered alone and/or with conventional chemotherapy.

Suppressing Expression of SERPINE1/PAI1 Through Activation of GPER1 Reduces Progression of Vulvar Carcinoma

The serine proteinase inhibitor 1 (SERPINE1) gene codes for the plasminogen activator inhibitor 1 (PAI1) protein and is thought to play a tumor supportive role in various cancers. In this work we aimed to uncover the role PAI1 plays in the proliferation, migration, and invasion of vulvar cancer (VC), and define the protein's function as an oncogene or tumor suppressor. Through treatment with an agonist (G1) and antagonist (G36) of G-coupled estrogen receptor 1 (GPER1), an upstream regulator of SERPINE1 expression, and a forward transfection knockdown protocol, the expression of SERPINE1/PAI1 in VC cells was altered. The effects these altered SERPINE1/PAI1 levels had on tumor cell functions were then examined. Proliferation was analyzed using the resazurin assay, while migration was studied via the gap closure assay. Through colony- and tumor sphere- formation assays clonogenicity was tested, and western blots showed protein expression. In A431 VC cells, when the levels of PAI1 were reduced via knockdown or treatment with G1, migration, proliferation, and colony growth was reduced. Treatment with G36 increased expression of PAI1 and increased migration and colony size in CAL39 cells. Based on the findings in this study, suppressing PAI1 expression in VC cells appears to reduce their progression and tumorigenic potential. Therefore, PAI1 could possibly function as an oncogene in VC. GPER1 appears to be a suitable target for suppressing PAI1 in VC.

The Clinical and Genetic Landscape of Hereditary Cancer: Experience from a Single Clinical Diagnostic Laboratory

Kinase D-interacting Substrate of 220 kDa Is Overexpressed in Gastric Cancer and Associated With Local Invasion

Mesonephric-like Adenocarcinoma of the Uterine Corpus: Comprehensive Analyses of Clinicopathological, Molecular, and Prognostic Characteristics With Retrospective Review of 237 Endometrial Carcinoma Cases

Uterine mesonephric-like adenocarcinoma (MLA) is a rare malignant tumor of the female genital tract. We reviewed 237 endometrial carcinoma cases and investigated the clinicopathological and molecular characteristics of uterine MLA. We found that 3.0% (7/237) of the endometrial carcinoma cases were MLAs. Compared to endometrial endometrioid carcinoma, MLA showed larger tumor size, deeper myometrial invasion, increasingly advanced-stage disease, and more frequent lymphovascular space invasion. All MLAs exhibited architectural diversity, compactly aggregated small tubules, eosinophilic intraluminal secretions, overlapped and angulated nuclei, scant cytoplasm, and presence of spindle cells. All the MLAs expressed at least two mesonephric markers. All except one MLA harbored activating Kirsten rat sarcoma viral oncogene homolog mutations. All patients with MLA developed postoperative metastases. MLA had the lowest progression-free survival rate among different histological types of endometrial carcinoma. Uterine MLA is a highly aggressive gynecological malignancy, showing unique morphological and molecular features, frequent recurrences and metastases, as well as poor prognosis.

Mesonephric-like Carcinosarcoma of the Uterine Corpus: Clinicopathological, Molecular and Prognostic Characteristics in Comparison With Uterine Mesonephric-like Adenocarcinoma and Conventional Endometrial Carcinosarcoma

This study aimed to investigate the clinicopathological, prognostic and molecular characteristics of uterine mesonephric-like carcinosarcoma (MLCS). We collected clinical, pathological, and genetic information from 12 MLCS patients, and analyzed their differences from mesonephric-like adenocarcinoma (MLA) and conventional endometrial carcinosarcoma (CECS). The epithelial component was exclusively MLA in all MLCS cases. Metastatic and recurrent tumors consisted predominantly or exclusively of MLA in the majority of MLCS cases. Patients with MLCS and MLA presented with more advanced-stage disease than those with CECS. They also exhibited post-treatment recurrence and lung metastases more frequently than CECS. Disease-free survival rates of MLCS and MLA were shorter than those of CECS. Tumor protein 53 gene mutations were detected in four MLCS cases. The predominance or exclusive presence of MLA in metastatic and recurrent tumors highlights the possibility that MLA may determine the clinical outcomes of patients with MLCS. Further studies are required to provide direct molecular evidence of the monoclonal origin of uterine MLCS.

Genomic Features of Cervical Cancer in Okinawa, Japan: Preliminary Findings From 23 Patients

Regional differences in genomic mutation profiles of uterine cervical cancer have been reported. Japanese people are divided into two genetic background clusters, originating from mainland Japan and Okinawa. Okinawa is an island prefecture surrounded by the sea, located more than 800 km from the southernmost point of mainland Japan. No studies have examined gene mutation profiles of cervical cancer in Okinawa. This study aimed to investigate the mutation profile of cervical cancer in Okinawa. Twenty-three patients with biopsy-proven squamous cell carcinoma and adenocarcinoma of the intact uterine cervix who were treated with definitive radiotherapy were analyzed. Genomic DNA was extracted from fresh frozen tissue samples collected by tumor biopsy prior to treatment. Variants of 224 cancer-related genes were identified using next-generation sequencing. A total of 29 gene mutations were observed in 16 patients, including nine genes mutated in multiple samples: Gene mutation profiles of cervical cancer in Okinawa may differ from those in other regions.

RFC3 Knockdown Decreases Cervical Cancer Cell Proliferation, Migration and Invasion

Replication factor C subunit 3 (RFC3) is a critical component of the replication factor C complex, which is essential for DNA replication and repair. Recent studies have highlighted the RFC3's significance in various cancer types. Herein, we aimed to elucidate its biological role in cervical cancer. Cervical cancer cells were transfected with RFC3 or control siRNA. Cell viability was assessed using the MTT assay over a 4-day period and its clonogenic potential was determined using colony formation assays. Flow cytometry analysis was performed to evaluate cell cycle distribution. Transwell migration and invasion assays were performed to assess the migration and invasion abilities of cervical cancer cells. RFC3 knockdown significantly inhibited cell proliferation, induced cell-cycle arrest, and decreased migration and invasion in HeLa and ME-180 cells compared to control siRNA-transfected cells. The crucial role of RFC3 in cervical cancer progression is highlighted. RFC3 knockdown resulted in decreased cervical cancer cell proliferation, migration and invasion, suggesting its potential as a therapeutic target in cervical cancer.

G Protein-coupled Estrogen Receptor 1 (GPER1) Regulates Expression ofSERPINE1/PAI-1 and Inhibits Tumorigenic Potential of Cervical Squamous Cell Carcinoma CellsIn Vitro

G protein-coupled estrogen receptor 1 (GPER1) appears to play a tumor-suppressive role in cervical squamous cell carcinoma (CSCC)GPER1 suppression leads to significantly increased expression of serpin family E member 1 (SERPINE1)/protein plasminogen activator inhibitor type 1 (PAI-1). The question arises, what role does SERPINE1/PAI-1 play in GPER1-dependent tumorigenic potential of CSCC. SiHa and C33A CSCC cells were treated with GPER1 agonist G1 or antagonist G36. SERPINE1/PAI-1 expression was suppressed by RNAi and success was confirmed by RT-qPCR. Protein expression of PAI-1 was quantified by Western blot. Viability was analyzed using resazurin assay, while migration was investigated using gap closure. Colony and tumor sphere formation were used to test clonogenicity. After G1 treatment, viability of SiHa and C33A cells remained unchanged. Cell migration was dose-dependently reduced. SiHa and C33A cells formed significantly fewer and smaller colonies as well as spheroids. Furthermore, treatment with G1 led to decreased expression of SERPINE1/PAI-1, while blockade of GPER1 with G36 resulted in significantly increased SERPINE1/PAI-1 expression. After suppression of SERPINE1/PAI-1 in SiHa cells using RNAi, cell viability remained unaffected; however, significantly smaller colonies were formed, and fewer and smaller spheroids were developed. Cell migration remained unaffected. Activation of GPER1 reduces clonogenicity and migration of CSCC cells and suppresses expression of SERPINE1/PAI-1. Suppression of SERPINE1/PAI-1 in CSCC cells reduces tumorigenic potential. GPER1 may be a suitable target for suppression of SERPINE1/PAI-1 in CSCC. However, SERPINE1/PAI-1 does not appear to be the decisive factor for GPER1-regulated cell migration.

Association of Matrix Metalloproteinase-2 Promoter Genotypes With Leiomyoma Risk

The role of matrix metalloproteinase-2 (MMP-2) in leiomyoma pathogenesis has been suggested, but the association between Genotypic distributions of

G-Protein-coupled Estrogen Receptor 1 (GPER1) Overexpression Affects Aggressiveness of Cervical Carcinoma Cells Depending on Histological Entity

Cervical cancer (CC) is the fourth most common cancer in women worldwide. There are two main histological subtypes of CC: the more common cervical squamous cell carcinoma (CSCC) and the rarer cervical adenocarcinoma (CAC), which has a poorer prognosis. Unlike estrogen receptor (ER) α and ERβ, G-protein-coupled estrogen receptor 1 (GPER1) is recognized as a rapid mediator of cellular estrogenic action and tends to have tumor suppressive properties in CC. Since a clinical study showed that an elevated GPER1 expression is associated with a worse prognosis, we investigated the effects of stable GPER1 overexpression (GPER1-OE) on SiHa CSCC and HeLa CAC cells. SiHa CSCC and HeLa CAC cells with stable GPER1-OE were generated. GPER1-OE was tested by RT-qPCR, western blot and fluorescence-activated cell analysis (FACS). The effects of GPER1-OE on proliferation, migration, invasion, apoptosis and stem cell properties (colony and sphere formation) were then examined. Successful GPER1-OE in SiHa CSCC and HeLa CAC cells was confirmed. The cell characterization experiments showed that SiHa CSCC cells with stable GPER1-OE had faster proliferation and migration, and increased stem cell properties with larger and more numerous colonies and larger tumor spheres. In HeLa CAC cells, on the other hand, GPER1-OE resulted in slower cell proliferation, migration and invasion, reduced colony formation and tumor sphere formation. An increased rate of apoptosis was also observed. GPER1-OE resulted in a more aggressive tumor behavior of SiHa CSCC cells and a less aggressive tumor behavior of HeLa CAC cells, due to a different effect of GPER1 overexpression depending on the respective histological subtypes of CC. This underlines the need for personalized medicine and a precise differentiation of subtypes in CC-related research.

Expression of “Hallmarks of Cancer” Genes in Cervical Carcinoma Is Differentially Affected by GPER1 Overexpression Depending on Histologic Entity

Cervical cancer (CC) remains the fourth most common malignancy in women worldwide. Current treatments primarily consist of surgery and combined radiochemotherapy, while targeted therapies, as seen in other malignancies, remain underdeveloped. The G-protein-coupled estrogen receptor (GPER1) is implicated in various cancers and can differentially influence tumor behavior, though its precise role in CC remains unclear, with both tumor-promoting and tumor-suppressive effects reported. We previously explored the impact of stable GPER1 overexpression (OE) in CC cell lines, SiHa (cervical squamous cell carcinoma, CSCC) and HeLa (cervical adenocarcinoma, CAC), analyzing proliferation, migration, invasion, apoptosis, and stem cell properties. GPER1-OE enhanced tumorigenic properties in CSCC cells but demonstrated tumor-suppressive effects in CAC cells. To investigate the underlying mechanisms, we conducted next-generation sequencing (NGS) analyses, which supported our earlier findings. SiHa CSCC and HeLa CAC cells with stable GPER1-OE were generated. The effects of GPER1-OE on gene expression were then examined using next-generation sequencing (NGS) analyses. In CSCC cells, GPER1-OE upregulated genes involved in tumorigenic pathways, including epithelial-to-mesenchymal transition (EMT), mTOR-C1, Myc, p53, hypoxia, and angiogenesis signaling. In CAC cells, however, GPER1-OE downregulated these pathways, along with additional pathways such as KRAS, Hedgehog, TNFα ( The results highlight the divergent roles of GPER1-OE in CC cells, promoting oncogenesis in CSCC while exerting tumor-suppressive effects in CAC by modulating oncogenic signaling pathways.

Characterization of the Genomic Landscape in HPV-positive Cervical and Head and Neck Squamous Cell Carcinomas by Whole Genome Next Generation Sequencing

In this study, we provide a comprehensive characterization of HPV-positive primary cervical cancers (CC) and HPV-positive head and neck squamous cell carcinomas (HNSCC) through whole genome next-generation sequencing. Human papillomavirus (HPV) infection, recognized as a definitive human carcinogen, is increasingly acknowledged for its role in development of human cancers. HPV-driven cervical cancers are among the leading causes of cancer-related deaths worldwide, while HPV-driven head and neck cancers exhibit distinct biological and clinical characteristics. Recent data has provided convincing evidence that HPV-related cervical cancer, like HPV head and neck cancer also predict better outcomes, with viral integration patterns further predicting disease related outcomes. We designed an experimental study that encompasses four pairs of HPV-positive patient samples with controls, utilizing state-of-the-art Next Generation Sequencing (NGS) technology including whole genome sequencing, transcriptome sequencing and virus integration. Multiple mutated genes, including TTN, COL6A3, and FLNA, were identified shared between CC and HNSCC. Additionally, we observed a notable proportion of pathways affected by oncogenic alterations, particularly in the RTK-RAS and NOTCH pathways, in both CC and HNSCC. Furthermore, we discovered a shared down-regulation of the Hedgehog signaling pathway based on transcriptome expression analysis in KEGG. We also identified RUNX2 and TFPI as sites of virus integration, and upstream as well as downstream pathway modulators, and represent potential targets for therapeutic interventions. Overall, this study showed a thorough comparison between CC and HNSCC from multiple aspects, including gene variations, oncogenic pathways, KEGG enrichment and virus integration sites. However, further studies, which involve larger patient cohorts should be undertaken to further support these findings.

Copy Number Variations (CNVs) Account for 10.8% of Pathogenic Variants in Patients Referred for Hereditary Cancer Testing

Germline copy number variation (CNV) is a type of genetic variant that predisposes significantly to inherited cancers. Today, next-generation sequencing (NGS) technologies have contributed to multi gene panel analysis in clinical practice. A total of 2,163 patients were screened for cancer susceptibility, using a solution-based capture method. A panel of 52 genes was used for targeted NGS. The capture-based approach enables computational analysis of CNVs from NGS data. We studied the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and of the non-commercial tool panelcn.MOPS. Additionally, we tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA). Pathogenic/likely pathogenic variants (P/LP) were identified in 464 samples (21.5%). CNV accounts for 10.8% (50/464) of pathogenic variants, referring to deletion/duplication of one or more exons of a gene. In patients with breast and ovarian cancer, CNVs accounted for 10.2% and 6.8% of pathogenic variants, respectively. In colorectal cancer patients, CNV accounted for 28.6% of pathogenic/likely pathogenic variants. In silico CNV detection tools provide a viable and cost-effective method to identify CNVs from NGS experiments. CNVs constitute a substantial percentage of P/LP variants, since they represent up to one of every ten P/LP findings identified by NGS multigene analysis; therefore, their evaluation is highly recommended to improve the diagnostic yield of hereditary cancer analysis.

A Multiplex Biomarker Assay Improves the Prediction of Survival in Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC) is usually diagnosed in advanced stages and has a high mortality rate. In this study, we used the proximity extension assay from Olink Proteomics to search for new plasma protein biomarkers to predict overall survival (OS) in patients with EOC. Peripheral blood samples were obtained preoperatively from 116 EOC patients undergoing primary debulking surgery: 28 early EOC cases (FIGO stage I-II) and 88 advanced EOC cases (FIGO stage III-IV). Proteins were measured using the Olink Oncology II and Inflammation panels. In total, 177 unique protein biomarkers were analysed. Cross-validation and LASSO regression were combined to select prediction models for OS. The model including age and the three-biomarker combination of neurotrophin-3 (NT-3)+transmembrane glycoprotein NMB (GPNMB)+mesothelin (MSLN) predicted worse OS with AUC=0.79 (p=0.004). Adding cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) to the model further improved performance (AUC=0.83; p=0.003). In a postoperative model including age and stage (III+IV vs. I+II), the three-biomarker panel of chemokine (C-C motif) ligand 28 (CCL28)+T-cell leukaemia/lymphoma protein 1A (TCL1A)+GPNMB improved the prediction of OS (from AUC=0.83 to AUC=0.90; p=0.05). In the postoperative model including age and dichotomized stage (III vs. I+II), the biomarkers CCL28 and GPNMB1 improved the prediction of OS (AUC=0.86; p<0.001). The combination of high levels of both CA125 and HE4 predicted worse survival (p=0.05). In this explorative study evaluating the performance of plasma protein biomarkers in predicting OS, we found that adding biomarkers, especially NT-3, to the panel improved the prediction of OS.

Loss of E-cadherin Activates EGFR-MEK/ERK Signaling, Promoting Cervical Cancer Progression

This study investigated the relationship between E-cadherin down-regulation and enhanced pERK1/2 signaling in cervical cancer, evaluated their combined prognostic impact, and explored potential therapeutic targets. We analyzed 188 cervical cancer specimens and 300 normal cervical tissue samples using tissue microarray and immunohistochemistry. Small interfering RNA transfection and western blotting were used to study molecular interactions in cervical cancer cell lines. We observed a significant inverse correlation between E-cadherin and pERK1/2 expression, as well as poor disease-free survival and overall survival. Additionally, molecular analysis indicated that E-cadherin silencing enhanced ERK signaling and promoted cancer cell proliferation. The findings suggest that E-cadherin and pERK1/2 are crucial biomarkers for cervical cancer prognosis and their interaction provides a potential target for therapeutic interventions. Further studies are recommended to explore these pathways in the clinical setting.

SNHG3/WISP2 Axis Promotes Hela Cell Migration and InvasionviaActivating Wnt/β-Catenin Signaling

Cervical cancer (CC) poses a significant threat to women's health and has a relatively poor prognosis due to local invasion and metastasis. It is, therefore, crucial to elucidate the molecular mechanisms of CC metastasis. SNHG3 has been implicated in various tumor metastasis processes, but its involvement in CC has not been thoroughly studied. Our study aimed to investigate the role of SNHG3 in metastasis and elucidate its underlying mechanisms in CC. LncRNA SNHG3 expression in CC tissues was analyzed using TCGA and GSE27469 databases. Normal cervical epithelial cells and CC cell lines were used to detect mRNA expression of SNHG3 via quantitative reverse transcription polymerase chain reaction (qRT-PCR). With RNA interference (RNAi) technology, antisense oligonucleotides (ASO) can act on HeLa cells to knockdown target gene expression. The influence of SNHG3 on cell migration and invasion were determined by wound healing and transwell assays. Transcriptome sequencing (RNA-seq) was used to seek abnormally expressed genes between SNHG3 knockdown cells and control cells. The expressions of epithelial-mesenchymal transition (EMT) and Wnt/β-catenin signaling related proteins were detected using western blot. SNHG3 was obviously up-regulated in CC tissues and cell lines, and ectopic expression of SNHG3 was associated with lymph node metastasis of CC. Knockdown of SNHG3 significantly inhibited cell migration and invasion in CC. Further molecular mechanism studies showed that SNHG3 knockdown could down-regulate the expression of WNT1 Inducible Signaling Pathway Protein 2 (WISP2) so as to inhibit the activation of the Wnt/β-catenin signaling pathway, and regulated the expression of EMT-related markers, that promoted the protein expression of E-cadherin, as well as decreased the expression of N-cadherin and vimentin. SNHG3 appears to exert a pro-metastatic effect in CC, as evidenced by inhibition of cell migration and invasion upon SNHG3 knockdown. EMT also appears to be attenuated. Of interest is the down-regulation of WISP2 following SNHG3 knockdown leads to the inactivation of the Wnt/β-catenin signaling pathway.

Bioinformatics Analysis of Novel Targets for Treating Cervical Cancer by Immunotherapy Based on Immune Escape

Cervical cancer (CC) is a high-risk disease in women, and advanced CC can be difficult to treat even with surgery, radiotherapy, and chemotherapy. Hence, developing more effective treatment methods is imperative. Cancer cells undergo a renewal process to escape immune surveillance and then attack the immune system. However, the underlying mechanisms remain unclear. Currently, only one immunotherapy drug has been approved by the Food and Drug Administration for CC, thus indicating the need for and importance of identifying key targets related to immunotherapy. Data on CC and normal cervical tissue samples were downloaded from the National Center for Biotechnology Information database. Transcriptome Analysis Console software was used to analyze differentially expressed genes (DEGs) in two sample groups. These DEGs were uploaded to the DAVID online analysis platform to analyze biological processes for which they were enriched. Finally, Cytoscape was used to map protein interaction and hub gene analyses. A total of 165 up-regulated and 362 down-regulated genes were identified. Among them, 13 hub genes were analyzed in a protein-protein interaction network using the Cytoscape software. The genes were screened out based on the betweenness centrality value and average degree of all nodes. The hub genes were as follows: ANXA1, APOE, AR, C1QC, CALML5, CD47, CTSZ, HSP90AA1, HSP90B1, NOD2, THY1, TLR4, and VIM. We identified the following 12 microRNAs (miRNAs) that target the hub genes: hsa-miR-2110, hsa-miR-92a-2-5p, hsa-miR-520d-5p, hsa-miR-4514, hsa-miR-4692, hsa-miR-499b-5p, hsa-miR-5011-5p, hsa-miR-6847-5p, hsa-miR-8054, hsa-miR-642a-5p, hsa-miR-940, and hsa-miR-6893-5p. Using bioinformatics, we identified potential miRNAs that regulated the cancer-related genes and long noncoding RNAs (lncRNAs) that regulated these miRNAs. We further elucidated the mutual regulation of mRNAs, miRNAs, and lncRNAs involved in CC occurrence and development. These findings may have major applications in the treatment of CC by immunotherapy and the development of drugs against CC.

MiR-575 in Exosomes of Vaginal Discharge Is Downregulated in Ovarian Cancer Patients

Ovarian cancer is asymptomatic in its early stages, and often diagnosed at advanced stages, leading to a high recurrence rate. In recent years, exosomes have been shown to be useful for early-detection, prognosis prediction, and treatment of cancer. Although many studies of cancer-related exosomes using other bodily fluids have been reported, there are few studies examining vaginal discharge, but none related to ovarian cancer. In this study, we investigated a method for early-detection of ovarian cancer using vaginal discharge, which are physically close to the fallopian tubes, where ovarian cancer originates, and can be easily collected from outside the body. Vaginal discharge was collected from 30 patients with ovarian cancer and 29 patients with benign gynecological diseases, and exosomal miRNAs were extracted. Samples from each group were submitted to miRNA microarray in order to examine miRNAs with significant differences in expression levels. We further narrowed down the list to four miRNAs based on literature and microarray data and examined the expression levels of miRNAs in the malignant and benign groups by RT-qPCR. MiR-575 expression was significantly decreased in the malignant group compared to the benign group ( This is the first study to investigate exosomal miRNAs in vaginal discharge of ovarian cancer. Exosomal miR-575 in vaginal discharge may be used as a biomarker for ovarian cancer.

Preoperative ctDNA Levels Are Associated With Poor Overall Survival in Patients With Ovarian Cancer

Circulating tumor DNA (ctDNA), which is shed from cancer cells into the bloodstream, offers a potential minimally invasive approach for cancer diagnosis and monitoring. This research aimed to assess the preoperative ctDNA levels in ovarian tumors patients' plasma and establish correlations with clinicopathological parameters and patient prognosis. Tumor DNA was extracted from ovarian tumor tissue from 41 patients. Targeted sequencing using a panel of 127 genes recurrently mutated in cancer was performed to identify candidate somatic mutations in the tumor DNA. SAGAsafe digital PCR (dPCR) assays targeting the candidate mutations were used to measure ctDNA levels in patient plasma samples, obtained prior to surgery, to evaluate ctDNA levels in terms of mutant copy number/ml and variant allele frequency. Somatic mutations were found in 24 tumor samples, 17 of which were from ovarian cancer patients. The most frequently mutated gene was TP53. Preoperative plasma ctDNA levels were detected in 14 of the 24 patients. With higher stage, plasma ctDNA mutant concentration increased (p for trend <0.001). The overall survival of cancer patients with more than 10 ctDNA mutant copies/ml in plasma was significantly worse (p=0.008). Pre-operative ctDNA measurement in ovarian cancer patients' plasma holds promise as a predictive biomarker for tumor staging and prognosis.

TMED9Expression Level as a Biomarker of Epithelial Ovarian Cancer Progression and Prognosis

Transmembrane emp24 domain-containing protein 9 (TMED9) belongs to the TMED/p24 family that transports, modifies, and packs proteins and lipids into vesicles for delivery to specific locations and is important in innate immune signaling via the endoplasmic reticulum-Golgi cargo pathway. TMED9 has been implicated in various cancer types; however, its role in epithelial ovarian cancer (EOC) is unclear. In this study, we aimed to elucidate the role and clinical significance of TMED9 in EOC. mRNA and protein levels of TMED9 and their associations with clinicopathological features in EOCs were evaluated using RNA-sequencing and immunohistochemistry data. Functional studies assessing the tumorigenic role of TMED9 in EOC cell lines were also performed. The mRNA expression of TMED9 was up-regulated in EOC compared to that in normal ovarian epithelium. TMED9 protein expression increased in progression from normal ovarian epithelium to EOC (p<0.001). Moreover, high expression of TMED9 was associated with advanced stage, serous cell type and poor histological grade in EOC and demonstrated independent prognostic significance for both disease-free and overall survival. Further functional studies showed that TMED9 knockdown reduced migration, invasion, cell proliferation, and colony formation of EOC cells. Overall, our results support the use of TMED9 as a valuable prognostic biomarker and provide evidence for targeting of TMED9 as a novel strategy for EOC treatment.

Atypical Mesonephric Hyperplasia of the Uterus Harbors Pathogenic Mutation of Kirsten Rat Sarcoma 2 Viral Oncogene Homolog (KRAS) and Gain of Chromosome 1q

Mesonephric carcinoma (MNC) is a rare but notable entity of the female genital tract. While many researchers have acknowledged and studied MNC, much remains unknown on the characteristics of mesonephric remnant (MNR) or hyperplasia (MNH). There has not been any study examining the molecular features of MNR and MNH so far. The aim of this study was to investigate the clinicopathological and molecular characteristics of ten uterine mesonephric lesions, including two MNRs without atypia, four MNHs without atypia, and three MNHs with atypia. We reviewed the electronic medical records and all available slides of ten cases from multiple institutions. Targeted sequencing and array comparative genomic hybridization were performed. Three atypical MNHs displayed nuclear enlargement, mild-to-moderate nuclear pleomorphism, and nuclear membrane irregularity, and harbored pathogenic Kirsten rat sarcoma 2 viral oncogene homolograt sarcoma 2 viral oncogene homolog (KRAS) mutation. Two of those that co-existed with MNC harbored the same sequence alterations as each of their adjacent MNC. One of the three atypical MNHs harbored chromosome 1q gain. Atypical MNH is a potential premalignant lesion in which KRAS mutation and chromosome 1q gain play an important role in the early stage of mesonephric carcinogenesis.

Clinicopathological and Molecular Differences Between Gastric-type Mucinous Carcinoma and Usual-type Endocervical Adenocarcinoma of the Uterine Cervix

We investigated differences in the clinicopathological and molecular characteristics between gastric-type mucinous carcinoma (GMC) and usual-type endocervical adenocarcinoma (UEA). We collected the clinicopathological information and performed targeted genomic sequencing analysis. GMCs exhibited significantly deeper invasion depth, larger horizontal spread, more advanced stage, more frequent distant metastasis, and more frequent parametrial and vaginal extension. Disease-free survival time of GMC patients was significantly shorter than that of UEA patients. GMCs displayed mutant p53 immunostaining pattern, whereas UEAs exhibited p16 block positivity. GMCs harbored mutations in KRAS, TP53, NF1, CDKN2A, STK11, and ARID1A. One GMC exhibited MDM2 amplification. In contrast, UEAs harbored mutations in HRAS, PIK3CA, and BRCA2. Two UEAs were found to have novel TP53 mutations. GMC is associated with more aggressive behavior than UEA. Distinctive p53 and p16 immunostaining patterns enable differential diagnosis. GMC and UEA exhibit genetic heterogeneity with potentially actionable molecular alterations.

Long Noncoding RNA ANROC on the INK4 Locus Functions to Suppress Cell Proliferation

The INK4 locus encodes three important genes p15 Following ANROC silencing in cells by siRNA, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and cell cycle analysis using flow cytometry were performed. ANROC expression was decreased by oncogenic RAS signalling. ANROC knockdown enhanced HeLa cell proliferation and induced cyclin B1 mRNA, which promotes G2/M progression of the cell cycle. Furthermore, flow cytometric analysis revealed that ANROC knockdown increased the percentage of cells in the S and G ANROC functions to suppress cell cycle progression by suppressing cyclin B1 expression, thus inhibiting cell proliferation.

Effect of HPV 16 E6 Oncoprotein Variants on the Alterations of the Proteome of C33A Cells

The E6 genotypic variants of HPV 16 identified in lesions of women with cervical cancer (CC) in Southern of Mexico include the E-G350, AAa, AAc, E-C188/G350, and E-A176/G350, transcriptomic analysis cells transfected with those variants showed to induce differential expression of the host genes involved in the development of CC, the aim of this work was to understand how the over-expression of the E6 oncoprotein and its variants can induce molecular mechanisms that lead to more aggressive HPV 16 phenotypes in cervical cancer and which proteins could be associated with the process. Total extracts from C33A, C33A mock, C33A AAa, C33A E-C188/G350, C33A E-A176/G350, and C33A E-prototype cells were analyzed using 2D electrophoresis, PDQuest software and mass spectrometry, validation of results was performed through qPCR. Statistically significant differential expression of 122 spots was detected, 12 of the identified proteins were associated with metabolism and metabolic programming. Out of these CCT8, ENO and ALDH1A were further validated. CCT8 and ALDH1A were found to be over-expressed in C33A AAa and C33A E-A176/G350, compared to the E prototype. Both proteins could be associated with a most aggressive phenotype due to their relationship with metabolism, protein folding and stemness, mechanisms associated to E6 that could be useful in the design of new therapies.

Clinicopathological and Prognostic Values of Telomerase Reverse Transcriptase (TERT) Promoter Mutations in Ovarian Clear Cell Carcinoma for Predicting Tumor Recurrence, Platinum Resistance and Survival

A small subset of patients with ovarian clear cell carcinoma (OCCC) harbors telomerase reverse transcriptase promoter (TERTp) mutations. We aimed to analyze the clinicopathological and molecular characteristics of TERTp-mutant OCCC and investigate whether TERTp mutations are associated with the clinicopathological characteristics and outcomes of patients with OCCC. We included 11 OCCC cases in our study. Targeted sequencing was performed with a thorough review of pathology slides and electronic medical records. Eleven OCCCs harbored two hotspot TERTp mutations: c.1-146C>T (6/11) and c.1-124C>T (5/11). All patients (11/11) who underwent postoperative adjuvant chemotherapy experienced tumor recurrence, and eight of them were classified as platinum-resistant. TERTp-mutant OCCC showed significantly higher frequencies of postoperative recurrence and relapse within six months of chemotherapy. TERTp mutations significantly predicted disease-free survival (DFS) in patients with OCCC. We demonstrate that TERTp mutations have significant prognostic value for predicting tumor recurrence, platinum resistance, and worse DFS in patients with OCCC.