Claudin18.2 as a potential therapeutic target for primary ovarian mucinous carcinomas and metastatic ovarian mucinous carcinomas from upper gastrointestinal primary tumours.

BACKGROUND: The vast majority of ovarian mucinous carcinomas are metastatic tumours derived from nonovarian primary cancers, typically gastrointestinal neoplasms. Therapy targeting claudin18.2 might be used in gastric, gastroesophageal junction and pancreatic cancers with high expression of claudin18.2. In this study, we aimed to profile the expression of claudin18.2 in primary ovarian mucinous carcinoma (POMC) and metastatic gastrointestinal mucinous carcinoma (MGMC). METHODS: Immunohistochemistry was used to detect claudin 18.2 expression in whole tissue sections of ovarian mucinous carcinomas, including 32 POMCs and 44 MGMCs, 23 of which were derived from upper gastrointestinal primary tumours and 21 of which were derived from lower gastrointestinal primary tumours. Immunohistochemical studies for claudin18.2, SATB2, PAX8, CK7 and CK20 were performed in all 76 cases. RESULTS: Among 76 primary and metastatic mucinous carcinomas, claudin18.2 was expressed in 56.6% (43/76) of cases. MGMCs from the upper gastrointestinal tract, including 22 derived from primary stomach tumours and one derived from a pancreas tumour, were positive for claudin 18.2 in 69.5% (16/23) of cases. MGMCs from the lower gastrointestinal tract, including 10 derived from primary appendiceal cancer and 11 derived from colorectal cancers, showed no claudin18.2 expression (0/21). The expression rate of claudin18.2 in primary ovarian mucinous neoplasms, including 22 primary ovarian mucinous carcinomas and 10 primary ovarian borderline mucinous tumours, was 84.4% (27/32). The common immunophenotypic characteristics of POMCs, upper gastrointestinal tract-derived MGMCs, and lower gastrointestinal tract-derived MGMCs were claudin18.2 + /PAX8 + /SATB2- (17/32), claudin18.2 + /PAX8-/SATB2- (16/23) and claudin18.2-/PAX8-/SATB2 + (19/21), respectively. CONCLUSION: Claudin18.2 is highly expressed in POMCs and MGMCs derived from upper gastrointestinal tract primary tumours; therefore, claudin18.2-targeted therapy might serve as a potential therapeutic strategy for POMCs and MGMCs from the upper gastrointestinal tract.

miRNA-34a decreases ovarian cancer cell proliferation and chemoresistance by targeting HDAC1.

This study aimed to explore the roles of miRNA-34a (miR-34a) in ovarian cancer (OC) cells and uncover possible mechanisms. The proliferation of OC cells was measured with an MTT assay and soft agar colony formation assay. TargetScan analysis, real-time PCR, and a luciferase reporter assay were used to demonstrate the downstream target of miR-34a in OC cells. HDAC1 expression levels were detected by immunoblot analysis. miR-34a inhibited the proliferation of SKOV3 and OVCA433 cells and enhanced cisplatin sensitivity in cisplatin-resistant SKOV3cp cells. The results of TargetScan analysis, real-time PCR, and luciferase reporter assay confirmed that miR-34a downregulated HDAC1 expression by directly targeting the 3'-UTR of HDAC1 mRNA. The overexpression of HDAC1 decreased cisplatin sensitivity and promoted proliferation in OC cells. MTT assay and soft agar colony formation assay showed that HDAC1 overexpression blocked the suppressive effects of miR-34a on SKOV3 cell proliferation. In addition, treatment with the miR-34a mimic partially recovered the cisplatin sensitivity of SKOV3cp cells, whereas HDAC1 overexpression blocked the above phenomena caused by treatment with the miR-34a mimic. miR-34a exhibited suppressive effects on OC cells via directly binding and downregulating HDAC1 expression, which subsequently decreased the resistance to cisplatin and suppressed proliferation in OC cells.

High expression of SLC7A1 in high-grade serous ovarian cancer promotes tumor progression and is involved in MAPK/ERK pathway and EMT.

Our previous studies have shown that upregulation of SLC7A1 in epithelial ovarian cancer (EOC) tumor cells significantly increases cancer cell proliferation, migration, and cisplatin resistance; however, the molecular mechanism by which SLC7A1 functions in EOC remains unknown. In later studies, we found that SLC7A1 is also highly expressed in the interstitial portion of high-grade serous ovarian cancer (HGSOC), but the significance of this high expression in the interstitial remains unclear. Here, we showed the Interstitial high expression of SLC7A1 in HGSOC by immunohistochemistry. SLC7A1 enriched in cancer-associated fibroblasts (CAFs) was upregulated by TGF-ß1. Transwell assay, scratch assay, cck8 assay and cell adhesion assay showed that SLC7A1 highly expressed in CAFs promoted tumor cells invasion, migration and metastasis in vitro. The effect of SLC7A1 on MAPK and EMT pathway proteins in ovarian cancer (OC) was verified by RNA sequencing and western blotting. Overexpression of SLC7A1 in OC is involved in MAPK/ ERK pathway and EMT. In general, in HGSOC, CAFs overexpressing SLC7A1 supported the migration and invasion of tumor cells; SLC7A1 is highly expressed in ovarian cancer and is involved in ERK phosphorylation and EMT signaling in MAPK signaling pathway. This suggests that SLC7A1 may be a potential therapeutic target for OC metastasis.

LRRC superfamily expression in stromal cells predicts the clinical prognosis and platinum resistance of ovarian cancer.

BACKGROUND: Leucine-rich repeat sequence domains are known to mediate protein‒protein interactions. Recently, some studies showed that members of the leucine rich repeat containing (LRRC) protein superfamily may become new targets for the diagnosis and treatment of tumours. However, it is not known whether any of the LRRC superfamily genes is expressed in the stroma of ovarian cancer (OC) and is associated with prognosis. METHODS: The clinical data and transcriptional profiles of OC patients from the public databases TCGA (n = 427), GTEx (n = 88) and GEO (GSE40266 and GSE40595) were analysed by R software. A nomogram model was also generated through R. An online public database was used for auxiliary analysis of prognosis, immune infiltration and protein‒protein interaction (PPI) networks. Immunohistochemistry and qPCR were performed to determine the protein and mRNA levels of genes in high-grade serous ovarian cancer (HGSC) tissues of participants and the MRC-5 cell line induced by TGF-ß. RESULTS: LRRC15 and LRRC32 were identified as differentially expressed genes from the LRRC superfamily by GEO transcriptome analysis. PPI network analysis suggested that they were most enriched in TGF-ß signalling. The TCGA-GTEx analysis results showed that only LRRC15 was highly expressed in both cancer-associated fibroblasts (CAFs) and the tumour stroma of OC and was related to clinical prognosis. Based on this, we developed a nomogram model to predict the incidence of adverse outcomes in OC. Moreover, LRRC15 was positively correlated with CAF infiltration and negatively correlated with CD8 + T-cell infiltration. As a single indicator, LRRC15 had the highest accuracy (AUC = 0.920) in predicting the outcome of primary platinum resistance. CONCLUSIONS: The LRRC superfamily is related to the TGF-ß pathway in the microenvironment of OC. LRRC15, as a stromal biomarker, can predict the clinical prognosis of HGSC and promote the immunosuppressive microenvironment. LRRC15 may be a potential therapeutic target for reversing primary resistance in OC.

Membranous and nuclear staining of CLDN18 in HPV-independent and HPV-associated endocervical adenocarcinomas.

OBJECTIVES: A classification system for endocervical adenocarcinoma (ECA) based on high-risk human papillomavirus (HPV) status has been established; however, the immunohistochemical markers distinguishing HPV-independent and HPV-associated ECAs have not been fully described. Here, we aimed to characterize ECA immunopathological features. METHODS: We evaluated the immunohistochemical profile of CLDN18, CDX2, PAX8, p16, p53, and CEA in 60 ECAs comprising 10 HPV-independent ECAs and 50 HPV-associated ECAs. Both the membranous and nuclear expression levels of CLDN18 were analyzed. RESULTS: Membranous CLDN18 (CLDN18 [M]) was found to be expressed in the mucinous epithelium of all HPV-independent ECAs, including eight gastric-type ECAs (G-ECAs), one endometrioid ECA, and one clear cell ECA, but no nuclear CLDN18 (CLDN18 [N]) expression was detected in HPV-independent ECAs. Among HPV-associated ECAs, CLDN18 (M) expression levels in intestinal-type (I-ECAs) and usual-type ECAs (U-ECAs) were significantly different from those in invasive stratified mucin-producing (iSMILE) carcinomas (p = 0.036). Positive CLDN18 (M) staining was present in 55.6% (5/9) of intestinal-type and 39.4% (13/33) of usual-type ECAs and was not present in iSMILE ECAs. Silva pattern C cancers expressed higher levels of CLDN18 (M) than Silva pattern A and B cancers (p = 0.004), whereas the CLDN18 (N) expression levels in cancers showing Silva pattern A were significantly higher than those in cancers exhibiting Silva patterns B and C (p < 0.001). CONCLUSION: Membranous CLDN18 is expressed in ECAs and is particularly frequently expressed in HPV-independent ECAs, and membranous CLDN18 expression has potential as a therapeutic target. Nuclear staining of CLDN18 is a new immunohistochemical marker for diagnosing Silva pattern A HPV-associated ECAs and is associated with a good prognosis. Further studies should investigate the therapeutic and prognostic significance of membranous and nuclear CLDN18 expression and develop a related test that can be implemented in the clinical evaluation of ECAs.

SLC7A1 Overexpression Is Involved in Energy Metabolism Reprogramming to Induce Tumor Progression in Epithelial Ovarian Cancer and Is Associated with Immune-Infiltrating Cells.

Cationic amino acid transporters (SLC7A1/CAT1) are highly expressed in human ovarian cancer (OC) tissues. However, the specific biological functions and mechanisms involved remain unclear. We used bioinformatics analysis to explore SLC7A1 expression level, prognostic value, and tumor mutation burden (TMB) in ovarian cancer (OC) tissues. We performed in vitro experiments to identify the expression and biological function of SLC7A1 in epithelial ovarian cancer (EOC) tissues and cells. An amino acid autoanalyzer was used to detect the effect of SLC7A1 on amino acid metabolism in EOC cells. Finally, SLC7A1 in OC was evaluated for cell-to-cell signalling and immune infiltration using online databases. We found that increased SLC7A1 expression in EOC cells and tissues was associated with poorer survival outcomes (P < 0.05) but not with tumor stage or grade of OC (P > 0.05). SLC7A1 is involved in the transport of phenylalanine and arginine in EOC cells, and its knockdown reduced the proliferation and migration of EOC cells and the resistance of cells to cisplatin. Furthermore, the TIMER database indicated that SLC7A1 overexpression was significantly positively correlated with levels of CD4+ memory resting cells, CD8+ effector memory cells, M0 macrophages, and cancer-associated fibroblasts (CAFs) in OC (P < 0.05) and significantly negatively correlated with CD4+ memory-activated cells (P < 0.05). Cell immunofluorescence indicated that SLC7A1 overexpression may affect the distribution of immune-infiltrating lymphocytes in tumors by inhibiting the expression of CCL4. Therefore, we concluded that SLC7A1 is involved in the metabolic remodelling of amino acids in EOC to promote tumor development and cisplatin resistance and is related to the tumor-infiltrating immune microenvironment of OC. SLC7A1 is a biomarker for predicting EOC progression and cisplatin resistance and represents a promising target for EOC treatment.

PD-L1, PARP1, and MMRs as potential therapeutic biomarkers for neuroendocrine cervical cancer.

OBJECTIVE: Neuroendocrine cervical cancer (NECC) is a rare cervical cancer with high aggressivity that causes poor prognosis even in the early stage. Given other neuroendocrine carcinomas and other types of cervical cancer have been proved to have expression of programmed cell death protein 1 ligand 1(PD-L1) and poly ADP-ribose polymerase-1(PARP1), we would measure and analyze these proteins in this invasive cancer. The purpose of this study is to investigate the application value of PD-1/PD-L1 and PARP1 inhibitors in NECC. METHODS: The NECC cases in our center with formalin-fixed paraffin-embedded tissue blocks were collected, and immunohistochemical (IHC) staining of PD-L1, PARP1, Mismatch repair proteins (MMRs), and P53 was performed. Chi-square test was used to analyze associations between various protein expressions. We analyzed the efficacy of immunotherapy in a recent patient with secondary recurrence after two courses of chemotherapy. RESULTS: After rigorous screening, 20 cases were finally included. Three cases did not undergo surgical treatment because of their advanced stage. Twelve (60%) developed distant metastases or relapsed within five years, and most of them within two years. The positive rate of PD-L1 and PARP1 were 70% and 75% respectively. Among all the cases, microsatellite instability (MSI) was seen in six cases (30%) and abnormal p53 expression was in 15 patients (75%). PD-L1 was associated with PARP1 expression in the MSI subgroup. The patient treated with chemotherapy + VEGF inhibitor (VEGFi) + programmed cell death protein 1(PD-1) inhibitor had an excellent improvement in clinical symptoms, tumor markers, and mass size. CONCLUSION: The IHC results of PD-L1, PARP1, and MMRs suggested that NECC was the target of immunotargeted therapy. Our case confirmed that immune checkpoint therapy was effective in patients with PD-L1 positive and MMRs loss. Considering the clinical practicability, more cases should be collected, and effective biomarkers still need to be further searched.

Good performance of p16/Ki-67 dual-stain cytology for detection and post-treatment surveillance of high-grade CIN/VAIN in a prospective, cross-sectional study.

BACKGROUND: The limited sensitivity of Papanicolaou (Pap) cytology and the low specificity of HPV testing in detecting cervical or vaginal lesions means that either precancers are missed or women without lesions are overtreated. To improve performance outcomes, p16/Ki-67 dual-stain cytology has been introduced as a useful biomarker. METHODS: A prospective, cross-sectional study was performed and included 599 patients. Clinical performance estimates of Pap cytology, HPV DNA assay, and p16/Ki-67 dual-stain cytology for the detection of CIN2+/VAIN2+ were determined and compared. RESULTS: The sensitivity and specificity of p16/Ki-67 dual-stain cytology in detecting histology proven CIN2+/VAIN2+ was 91.6% and 95.0%, respectively, while that of Pap cytology was 42.1% and 95.2%, respectively, and that of HPV DNA testing was 100% and 41.6%, respectively. Among the three tests, the AUC of p16/Ki-67 immunocytochemistry was the largest, both for detecting cervical lesions and vaginal lesions, at 0.932 and 0.966, respectively. Among women who were HPV 16/18 positive or 12-other hrHPV positive and Pap positive (≥ASCUS), dual staining reduced the number of unnecessary colposcopy referrals from 274 to 181. Among the women who were 12-other hrHPV positive and Pap negative, dual staining could prevent underdiagnosis in six patients with CIN2+/VAIN2+ when used as a triage marker. Dual staining also identified four women with high-grade lesions detected by diagnostic conization but with negative colposcopy-guided biopsy results. CONCLUSION: p16/Ki-67 dual staining may be a promising tool for predicting high-grade cervical and vaginal lesions.

Knockdown of enhancer of rudimentary homolog inhibits proliferation and metastasis in ovarian cancer by regulating epithelial-mesenchymal transition.

Ovarian cancer (OC) is the deadliest gynecological malignancy. The pathogenesis of molecular in epithelial ovarian cancer (EOC), main histological type of OC, has not been completely defined. Enhancer of rudimentary homolog (ERH) had been reported to participate in transcriptional regulation, mRNA splicing, DNA repair and DNA synthesis by binding a variety of proteins. In this study, immunohistochemical staining revealed that the protein expression of ERH was associated with histological type, lymph node metastasis and pathological grade in EOC patients. To verify the association of ERH with the prognosis of OC, a GSE microarray dataset was downloaded from the Gene Expression Omnibus (GEO) database. Survival analysis suggested that ERH may be associated with poor prognosis of OC. In addition, shRNA was used to knockdown the protein and mRNA expression levels of ERH in the OC cell line SKOV3. Inhibition of ERH expression slowed proliferation, promoted apoptosis and inhibited metastasis and invasion by regulating epithelial-mesenchymal transition (EMT) in SKOV3 cells. These results indicate that ERH protein promotes the development of OC and provides an experimental basis for ERH as the potential target for ovarian cancer treatment.

Emodin inhibits TGF-ß2 by activating the FOXD3/miR­199a axis in ovarian cancer cells in vitro.

Ovarian cancer is a highly metastatic malignancy and a leading cause of cancer-related death in postmenopausal women. Emodin is a natural anthraquinone isolated from several traditional Chinese medicines including Rhubarb and Polygonum cuspidatum. Recently, emodin was demonstrated to reduce the growth of human ovarian carcinoma cells. However, the mechanism remains unclear. In the present study, we identified that transforming growth factor (TGF)-ß2 was significantly affected by emodin treatment in A2780 cells using microarray analysis. MicroRNA (miR)-199a was predicted as a potential miRNA targeting TGF-ß2 by in silico prediction using TargetScan. The mRNA and protein levels of TGF-ß2 were both significantly reduced by miR-199a. Spearman's correlation analysis revealed a significant correlation between the expression level of miR-199a and TGF-ß2 in human ovarian cancer specimens. Silencing of miR-199a with miR-199a inhibitor significantly restored the reduction in TGF-ß2 expression induced by emodin. Additionally, cell viability and colony formation of A2780 cells were markedly inhibited by emodin treatment, which was mediated by miR-199a. We analyzed the primary mature miR-199a-1 and miR-199a-2 transcripts in A2780 cells treated with emodin or dimethyl sulfoxide (DMSO) and found that only pri-miR-199a-1 was regulated by emodin. A conserved binding site of Forkhead box D3 (FOXD3) was identified within pri-miR­199a-1. We further revealed that miR-199a expression was significantly regulated by FOXD3. Taken together, the present study demonstrated that emodin may directly promote FOXD3 expression and sequentially activates miR-199a, which in turn suppresses the expression of TGF-ß2 to reduce cell viability and colony formation of A2780 cells.

Expression and prognostic value of lactate dehydrogenase-A and -D subunits in human uterine myoma and uterine sarcoma.

OBJECTIVE: This study aimed to determine the expression of lactate dehydrogenase (LDH)-A and LDH-D in patients with uterine myoma, cellular leiomyoma (CLM), and uterine sarcoma and to evaluate their prognostic significance. METHODS: Protein expression levels of LDH-A and LDH-D were determined in tissue samples from 86 patients (26 uterine myoma, 10 CLM, 50 uterine sarcoma) by immunohistochemistry and their associations with clinicopathologic parameters and outcomes were analyzed in patients with uterine sarcoma. RESULTS: The positivity rates for LDH-A and LDH-D were significantly higher in patients with uterine sarcoma compared with those with uterine myoma or CLM (P < .05). Patients with uterine sarcoma were classified as having uterine leiomyosarcoma (LMS), malignant endometrial stromal sarcoma, and malignant mixed Mullerian tumor, with 5-year overall survival rates of 59%, 71%, and 29%, respectively (P < .05). Univariate analysis showed that patients younger than 50 years and with stage I-II had better clinical prognoses. LDH-A-positive LMS patients had a poorer prognosis than LDH-A-negative patients (P = .03). The median survival time of LDH-A-positive patients was 35 months. CONCLUSIONS: We demonstrated that LDH-D was expressed in patients with uterine sarcoma. Furthermore, the overexpressions of LDH-A and LDH-D in uterine sarcoma patients may contribute to further understanding of the mechanism of LDH in tumor metabolism in uterine sarcoma. Positive expression of LDH-A in patients with LMS may act as a potential prognostic biomarker in these patients.

Effects of neoadjuvant chemotherapy on patients with primary vaginal squamous cell carcinoma.

Vaginal cancer is a rare gynecological malignancy, mainly treated by radiotherapy and surgery. However, the effect of neoadjuvant chemotherapy on patients with vaginal cancer has not been extensively evaluated. The aim of the present study was to assess the feasibility and efficacy of irinotecan and cisplatin in the management of patients with vaginal squamous cell cancer (SCC). Two patients with International Federation of Obstetrics and Gynecology (FIGO) stage I and one patient with FIGO stage II vaginal SCC were treated with irinotecan (240 mg) and cisplatin (100 mg) every 3-4 weeks. The effect of chemotherapy after 2-4 courses was assessed and the next step of treatment was determined according to the outcome. In the present study, all 3 patients had complete remission after 2-4 courses of chemotherapy. In case 1, the patient received a total of 6 courses of chemotherapy and had no recurrence after 45 months of follow-up. In case 2, the patient received 4 courses of chemotherapy and partial vaginal resection, and had no recurrence after 48 months of follow-up. In case 3, the patient underwent laparoscopic radical surgery and peritoneal vaginoplasty after 2 courses of chemotherapy, and no residual tumors were identified in the resected tissues on postoperative pathological examination. Effective neoadjuvant chemotherapy may decrease the size of the tumor, induce tumor regression, or even achieve pathologically-confirmed complete tumor eradication. Thus, neoadjuvant chemotherapy with irinotecan combined with cisplatin is a feasible treatment for patients with early-stage vaginal SCC. In the present study, all the patients achieved good therapeutic results following chemotherapy.