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Background: There are 2 known pathways for tumorigenesis of vulvar squamous cell carcinoma-a human papillomavirus-dependent pathway characterized by p16 overexpression and a human papillomavirus-independent pathway linked to lichen sclerosus, characterized by TP53 mutation. A correlation of human papillomavirus dependency with a favorable prognosis has been proposed., Objective: The objective of the study was to further understand the role of human papillomavirus and p53 status in vulvar squamous cell carcinoma and characterize its clinical relevance., Study Design: The Arbeitsgemeinschaft Gynaecological Oncology Chemo and Radiotherapy in Epithelial Vulvar Cancer-1 study is a retrospective cohort study of 1618 patients with primary vulvar squamous cell carcinoma Fédération Internationale de Gynécologie et d'Obstétrique stage ≥1B treated at 29 gynecologic cancer centers in Germany between 1998 and 2008. For this translational substudy, formalin-fixed paraffin-embedded tissue was collected. A tissue microarray was constructed (n=652 samples); p16 and p53 expression was determined by immunohistochemistry. Human papillomavirus status and subtype were analyzed by polymerase chain reaction., Results: p16 immunohistochemistry was positive in 166 of 550 tumors (30.2%); p53 staining in 187 of 597 tumors (31.3%). Only tumors with available information regarding p16 and p53 immunohistochemistry and without p53 silent expression pattern were further analyzed (n=411); 3 groups were defined: p53+ (n=163), p16+/p53- (n=132), and p16-/p53- (n=116). Human papillomavirus DNA was detected in 85.6% of p16+/p53- tumors; human papillomavirus-16 was the most common subtype (86.3%). Patients with p16+ tumors were younger (64 vs 72 years for p53+, respectively, 69 years for p16-/p53- tumors; P<.0001) and showed lower rates of lymph-node involvement (28.0% vs 42.3% for p53+, respectively, 30.2% for p16-/p53- tumors; P=.050). Notably, 2-year-disease-free and overall survival rates were significantly different among the groups: disease-free survival, 47.1% (p53+), 60.2% (p16-/p53-), and 63.9% (p16+/p53-) (P<.001); overall survival, 70.4% (p53+), 75.4% (p16-/p53-), and 82.5% (p16+/p53-) (P=.002). In multivariate analysis, the p16+/p53- phenotype showed a consistently improved prognosis compared with the other groups (hazard ratio, 0.66; 95% confidence interval, 0.44-0.99; P=.042)., Conclusion: p16 overexpression is associated with an improved prognosis whereas p53 positivity is linked to an adverse outcome. Our data support the hypothesis of a clinically relevant third subgroup of vulvar squamous cell carcinoma with a p53-/p16- phenotype showing an intermediate prognosis that needs to be further characterized., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: To date, no predictive or prognostic molecular biomarkers except BRCA mutations are clinically established for epithelial ovarian cancer (EOC) despite being the deadliest gynecological malignancy. Aim of this biomarker study was the analysis of DNA methylation biomarkers for their prognostic value independent from clinical variables in a heterogeneous cohort of 203 EOC patients from two university medical centers., Results: The marker combination CAMK2N1/RUNX3 exhibited a significant prognostic value for progression-free (PFS) and overall survival (OS) of sporadic platinum-sensitive EOC (n = 188) both in univariate Kaplan-Meier (LogRank p < 0.05) and multivariate Cox regression analysis (p < 0.05; hazard ratio HR = 1.587). KRT86 methylation showed a prognostic value only in univariate analysis because of an association with FIGO staging (Fisher's exact test p < 0.01). Thus, it may represent a marker for EOC staging. Dichotomous prognostic values were observed for KATNAL2 methylation depending on BRCA aberrations. KATNAL2 methylation exhibited a negative prognostic value for PFS in sporadic EOC patients without BRCA1 methylation (HR 1.591, p = 0.012) but positive prognostic value in sporadic EOC with BRCA1 methylation (HR 0.332, p = 0.04) or BRCA-mutated EOC (HR 0.620, n.s.)., Conclusion: The retrospective analysis of 188 sporadic platinum-sensitive EOC proved an independent prognostic value of the methylation marker combination CAMK2N1/RUNX3 for PFS and OS. If validated prospectively this combination may identify EOC patients with worse prognosis after standard therapy potentially benefiting from intensive follow-up, maintenance therapies or inclusion in therapeutic studies. The dichotomous prognostic value of KATNAL2 should be validated in larger sample sets of EOC.

During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells ("metastatic specific"). Among the up-regulated "metastatic specific" genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a "metastatic specific" upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.

Purpose: The RAS family comprises three proto-oncogenes (H-RAS, K-RAS, and N-RAS) and is among the most widely studied of oncogenes. The present study aimed at investigating the clinical relevance of mRNA levels of the three isoforms in a large group of breast cancer patients with a long-term follow-up., Methods: 198 previously untreated patients were enrolled in the study. mRNA levels of K-RAS, H-RAS, and N-RAS were measured using microarray (Affymetrix HG-U133A)., Results: Elevated H-RAS levels were found significantly more frequently in patients with larger (p = 0.021) and ER-positive tumors (p = 0.048), while elevated K-RAS levels were associated with nodal positivity (p = 0.001) and HER2-positivity (p = 0.010). Patients with high N-RAS mRNA levels were more likely to be diagnosed with triple-negativity (p < 0.001) and higher grading (p = 0.001). Patients with high K-RAS levels were more likely to show an elevated H-RAS (p = 0.003). After a median follow-up of 183 months, patients with high N-RAS expression had significantly reduced overall survival (OS) compared with patients with low N-RAS (mean: 146.9 vs. 211.0 months; median 169.3 vs. not reached; p = 0.009). In patients with non-metastatic disease at the time of tissue sampling, mean disease-free survival (DFS) was 150.1 months for patients with high N-RAS versus 227.7 months with low N-RAS; median DFS was not reached (p = 0.004). The expression of H-RAS and K-RAS was not associated with DFS/OS. In the multivariable analysis, distant metastasis, HER2 positivity, and elevated N-RAS mRNA levels independently predicted reduced OS, while nodal status, HER2 status, and N-RAS predicted reduced DFS., Conclusions: Elevated N-RAS mRNA levels predict impaired clinical outcome; hypothetically, further exploration of the RAS signaling pathway might enable identifying potential targeted treatment strategies. The association between high N-RAS levels and the most aggressive among breast cancer subtypes, the triple-negative phenotype, for which targeted approaches are still lacking, underlines the need to further investigate the RAS family.

The underlying mechanisms of ovarian cancer (OvCa) dissemination are still poorly understood, and novel molecular markers for this cancer type are urgently needed. In search of adhesion molecules with prognostic relevance in OvCa, we compared tumors with good outcome (alive > 3 years) and those with poor outcome (dead < 2 years) within data from The Cancer Genome Atlas (TCGA). The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) turned out as the only gene with differential expression in these groups. In order to further investigation on its role in OvCa, we analyzed CEACAM1 mRNA levels extracted from TCGA microarray data ( n = 517) as well as CEACAM1 protein expression by Western blot analysis in a cohort of 242 tumor samples. Further, CEACAM1 localization in tumour tissue was evaluated by immunohistochemistry and CEACAM1 splice variants by RT-PCR in representative tumours. In Kaplan-Meier analysis, high CEACAM1 mRNA levels significantly correlated with longer survival ( p = 0.008). By Western blot analysis in the second cohort, similar associations of high CEACAM1 protein levels with longer recurrence-free survival (RFS, p = 0.035) and overall survival (OAS, p = 0.004) were observed. In multivariate Cox regression analysis including clinical prognostic parameters, CEACAM1 mRNA or protein expression turned out as independent prognostic markers. Stratified survival analysis showed that high CEACAM1 protein expression was prognostic in node-negative tumors ( p = 0.045 and p = 0.0002 for DFS and OAS) but lost prognostic significance in node-positive carcinomas. Similarly, high CEACAM1 mRNA expression did not show prognostic relevance in tumors with lymphatic invasion (L1) but was associated with longer survival in cases without lymphovascular involvement. Further analysis showed a predominance of 4S and 4L isoforms and mostly membraneous CEACAM1 localization in ovarian tumours. Our results suggest that CEACAM1 might be an independent favorable prognostic marker in OvCa, especially in the subgroup of patients with solely intraperitoneal metastasis.

Glycosylation of cellular proteins has important impact on their stability and functional properties, and glycan structures strongly influence cell adhesion. Many enzymes are involved in glycoconjugate synthesis and degradation, but there is only limited information about their role in breast cancer progression. Therefore, we retrieved RNA expression data of 202 glycosylation genes generated by microarray analysis (Affymetrix HG-U133A) in a cohort of 194 mammary carcinomas with long-term follow-up information. After univariate and multivariate Cox regression analysis, genes with independent prognostic value were identified. These were further analysed by Kaplan-Meier analysis and log-rank tests, and their prognostic value was validated in a second cohort of 200 tumour samples from patients without systemic therapy. In our first cohort, we identified 24 genes with independent prognostic value, coding for sixteen anabolic and eight catabolic enzymes. Functionally, these genes are involved in all important glycosylation pathways, namely O-glycosylation, N-glycosylation, O-fucosylation, synthesis of glycosaminoglycans and glycolipids. Eighteen genes also showed prognostic significance in chemotherapy-treated patients. In the second cohort, six of the 24 relevant genes were of prognostic significance (FUT1, FUCA1, POFUT1, MAN1A1, RPN1 and DPM1), whereas a trend was observed for three additional probesets (GCNT4, ST3GAL6 and UGCG). In a stratified analysis of molecular subtypes combining both cohorts, great differences appeared suggesting a predominant role of N-glycosylation in luminal cancers and O-glycosylation in triple-negative ones. Correlations of gene expression with metastases of various localizations point to a role of glycan structures in organ-specific metastatic spread. Our results indicate that various glycosylation reactions influence progression and metastasis of breast cancer and might thus represent potential therapeutic targets.

Identification and characterization of biomarkers in body fluids such as serum or urine serve as a basis for early detection of diseases, particularly of cancer. Performing 2-DE with subsequent MS analyses, conventional immunoblotting and immunohistochemistry we identified two proteins, orosomucoid (ORM) and human zinc-alpha(2)-glycoprotein (ZAG), which were increased in the urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. The highest amount of both proteins was found in invasive bladder cancer stages such as pT2-3. Immunohistochemical studies showed ORM in inflammatory cells but also in endothelial cells of blood vessels within or adjacent to the tumor area and in part of the tumor cells. ZAG was prominent in tumor cells at the tumor invasion front. Additionally, ZAG was localized at the luminal surface of normal urothelium, which switches to the basal side when a superficial papillary tumor was observed. These results show that we have been able to identify two new proteins that may be related to the development of superficial bladder cancer and to its switch to an invasive phenotype.