Differential expression analysis identifies a prognostically significant extracellular matrix-enriched gene signature in hyaluronan-positive clear cell renal cell carcinoma.

Hyaluronan (HA) accumulation in clear cell renal cell carcinoma (ccRCC) is associated with poor prognosis; however, its biology and role in tumorigenesis are unknown. RNA sequencing of 48 HA-positive and 48 HA-negative formalin-fixed paraffin-embedded (FFPE) samples was performed to identify differentially expressed genes (DEG). The DEGs were subjected to pathway and gene enrichment analyses. The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) data and DEGs were used for the cluster analysis. In total, 129 DEGs were identified. HA-positive tumors exhibited enhanced expression of genes related to extracellular matrix (ECM) organization and ECM receptor interaction pathways. Gene set enrichment analysis showed that epithelial-mesenchymal transition-associated genes were highly enriched in the HA-positive phenotype. A protein-protein interaction network was constructed, and 17 hub genes were discovered. Heatmap analysis of TCGA-KIRC data identified two prognostic clusters corresponding to HA-positive and HA-negative phenotypes. These clusters were used to verify the expression levels and conduct survival analysis of the hub genes, 11 of which were linked to poor prognosis. These findings enhance our understanding of hyaluronan in ccRCC.

Comparison of different 2D muscle indexes measured at the level of the 3rd lumbar vertebra in survival prediction in patients with renal cell carcinoma.

BACKGROUND: Low computed tomography (CT)-determined muscle mass, commonly determined with height-adjusted muscle indexes (MIs), predicts worse survival in several cancers and has been suggested as a prognostic assessment tool. Although several MIs measured at the level of the 3rd lumbar vertebra (L3) are commonly used, it remains unestablished how different L3-determined MIs perform in survival prognostication compared to each other. The objective of this study was to investigate the performance of different MIs for survival prognostication in renal cell carcinoma (RCC). METHODS: We retrospectively enrolled 214 consecutive patients with RCC. We determined three L3-MIs (psoas muscle index (PMI), psoas muscle index and erector spinae index (PMI+ESI), and whole skeletal muscle index (SMI)) from preoperative CT scans. Categorization of those with low and normal muscle mass was based on the Youden Index sex-specific MI cut-offs. We determined sensitivity, specificity, and accuracy metrics for predicting 1-year, 5-year, and overall survival (OS) using Cox regression models. RESULTS: Low PMI, PMI+ESI, and SMI significantly predicted decreased 1-year, 5-year, and OS in uni- and multivariate models. PMI+ESI and SMI were more accurate than PMI in males, and PMI and PMI+ESI were more accurate than SMI in females in the prediction of 1-year survival. However, there were no differences in accuracies between MIs in 5-year and OS prediction. INTERPRETATION: PMI+ESI performed well overall in short-term prognostication, but there were no differences between the MIs in long-term prognostication. We recommend the use of PMI+ESI for muscle evaluation, particularly when SMI cannot be evaluated.

Nationwide analysis of survival after radical cystectomy for bladder cancer in Finland.

BACKGROUND: Population-based survival results after radical cystectomy (RC) are limited. Our objective was to report short and long-term survival results after RC for bladder cancer from Finland in a population-based setting. MATERIALS AND METHODS: The Finnish National Cystectomy Database containing retrospectively collected essential RC data covering the years 2005-2017 was combined with the survival data from the Finnish Cancer Registry. Kaplan-Meier plots were used to estimate survival and the survival graphs were illustrated according to the final pathological staging. Centers were divided according to operational volume, and the results were then compared using Pearsons's Chi-squared test. RESULTS: A total of 2047 patients were included in the study. 30-, and 90-day mortality was 1.3%, and 3.8%, respectively. The OS of the entire RC population at 5- and 10 years was 66% and 55%, and CSS was 74% and 72%, respectively. Center volume did not significantly associate with surgical mortality or long-term survival. The 5- and 10-year OS according to pT-category was 87% and 74% for pT0, 85% and 69% for pTa-pTis-pT1, 70% and 58% for pT2, 50% and 42% for pT3 and 41% and 30% for pT4. The corresponding 5- and 10-year CSS rates were 96% and 93% for pT0, 91% and 90% for pTa-pTis-pT1, 78% and 75% for pT2, 56% and 55% for pT3 and 47% and 44% for pT4. The 5- and 10-year OS rates in patients with no lymph node metastases (pN-) were 74% and 62%, and CSS 82% and 80%, respectively. If lymph nodes were positive (pN+), the corresponding OS rates were 44% and 34% and CSS 49% and 48%, respectively. CONCLUSION: RC survival results have improved in contemporary series and are associated with the pTNM-status. The nationwide results from Finland demonstrate outcome comparable to high volume single-center series.

Molecular tumor heterogeneity in muscle invasive bladder cancer: Biomarkers, subtypes, and implications for therapy.

BACKGROUND: Despite years of slow progress, muscle invasive bladder cancer (MIBC) is finally entering the era of molecularly guided targeted therapy. However, tumor heterogeneity is high in MIBC and may impact treatment response and resistance. The objective of this review is to dissect recent insights into inter- and intratumor heterogeneity (ITH) in MIBC, with emphasis on the clinical implications of this heterogeneity for biomarker-driven strategies and the development of new therapies. METHODS: A nonsystematic review was performed in PubMed and EMBASE using the terms "tumor heterogeneity" and "bladder cancer." RESULTS: Intertumor heterogeneity, as reflected by different clinical phenotypes in different patients, has been partially explained with next generation sequencing and other molecular profiling technologies. RNA-based molecular subtyping, for example, provides a classification of MIBC into distinct categories that can be used for further molecular analysis, biomarker discovery, risk stratification, and treatment selection. Molecular subtyping and specific genomic alterations, especially in DNA damage repair genes, may help explain why some patients respond better to systemic chemotherapy and immunotherapy. Conversely, spatial and temporal ITH threaten to confound attempts to target specific molecular lesions since not all tumor cells within a patient may carry the relevant lesion. Improved understanding and management of ITH is required for the most effective use of biomarker-driven targeted therapies. CONCLUSION: Strategies to assess and overcome intertumor and ITH in MIBC will be critical steps toward realizing the objectives of precision oncology. Novel techniques such as analysis of circulating tumor DNA and single cell sequencing are likely to revolutionize our understanding of tumor heterogeneity.

Cellular hyaluronan is associated with a poor prognosis in renal cell carcinoma.

PURPOSE: Hyaluronan, a major glycosaminoglycan of the extracellular matrix, can act as an oncogenic component of the tumor microenvironment in many human malignancies. We characterized the hyaluronan content of renal cell carcinomas (RCCs) and investigated its correlations with clinicopathological parameters and patient survival. PATIENTS AND METHODS: This retrospective study included data from 316 patients that had undergone surgery for RCC in Kuopio University Hospital in 2000 to 2013. The hyaluronan content of surgical tumor samples were histochemically stained with a biotinylated hyaluronan-specific affinity probe. The amount of tumor infiltrating lymphocytes was evaluated in each tumor. Kaplan-Meier and univariate and multivariate Cox-regression analyses were performed to estimate the impact of hyaluronan content on overall survival, disease-specific survival, and metastasis-free survival. RESULTS: Detectable cellular hyaluronan was associated with higher tumor grades and the presence of tumor infiltrating lymphocytes. Cellular hyaluronan identified a prognostically unfavourable subgroup among low-grade carcinomas. Multivariate analyses showed that measurable cellular hyaluronan was an independent negative prognostic factor for overall survival (hazard ratio [HR] 1.4; 95% confidence interval [CI]: 1.02-2.0; P = 0.039), Disease-specific survival (HR 2.07; 95% CI: 1.2-3.3; P = 0.002), and metastasis-free survival (HR 2.45; 95% CI: 1.37-4.4; P = 0.003). CONCLUSIONS: Cellular hyaluronan was significantly associated with unfavourable features and a poor prognosis in RCC. Further studies are needed to investigate the biological mechanism underlying hyaluronan accumulation in RCC.

Current Clinical Trials in Non-muscle Invasive Bladder Cancer.

PURPOSE OF REVIEW: As our molecular understanding of bladder cancer continues to advance, more and more novel agents are entering clinical trials across the spectrum of bladder cancer stages. The clinical trial activity for non-muscle invasive bladder cancer (NMIBC) has been boosted further by the evolution of specific disease states that set more uniform inclusion criteria for clinical trial design. Here, we aimed to review the current clinical trials landscape in non-muscle invasive bladder cancer with respect to these disease states. RECENT FINDINGS: Most active clinical trials focus on high-risk NMIBC in either the BCG-naïve or BCG-unresponsive setting. Strict criteria to define the disease state and a clear pathway to drug registration have encouraged trials for patients with BCG-unresponsive NMIBC. The most promising potential breakthroughs for BCG-naïve patients include alternative BCG strains, immune-priming with intradermal BCG vaccination, and systemic immune checkpoint blockade. The latter therapy is also being actively investigated in multiple trials in BCG-unresponsive NMIBC, along with novel viral agents such as INSTILADRIN (nadofaragene firadenovec) and targeted agents such as oportuzumab monatox. After many years of relative stagnation, multiple new therapies currently under investigation in well-designed clinical trials appear poised for routine clinical implementation in the near future. These therapies should dramatically improve the outcome of patients with NMIBC. We can look forward to the challenges of biomarker-driven drug selection, optimal drug sequencing, and rational combination therapies.

A risk-stratified approach to the management of high-grade T1 bladder cancer.

PURPOSE OF REVIEW: A bladder-preserving approach for high-grade nonmuscle invasive bladder cancer that has invaded the lamina propria (T1HG) may result in increased recurrence, progression, and even death from bladder cancer in some patients. Initial radical cystectomy does have increased cancer-specific survival (CSS), but represents significant overtreatment for many patients. An evidence-based, risk-stratified approach is required to select patients for immediate radical cystectomy in order to improve CSS. RECENT FINDINGS: A restaging transurethral resection aids in optimal staging and treatment of T1HG. Intravesical Bacillus Calmette-Guerin induction followed by 3 years of maintenance is the standard adjuvant management. However, when very high-risk (hydronephrosis, abnormal bimanual examination, variant histology, lymphovascular invasion, or residual disease on re-resection, and Bacillus Calmette-Guerin failure or early recurrence) or multiple high-risk factors (concomitant CIS, size >3 cm, multifocality, unfavorable tumor location, extensive lamina propria invasion, and elderly) are present, the risk of progression often outweighs the risk associated with radical cystectomy. In these cases, an immediate radical cystectomy likely provides an improved opportunity for cure compared to a bladder-preserving strategy. SUMMARY: In order to increase the CSS of patients diagnosed with T1HG bladder cancer, an aggressive approach may benefit those with increased risk of progression.

Neoadjuvant Chemotherapy Does Not Increase the Morbidity of Radical Cystectomy: A 10-year Retrospective Nationwide Study.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is underutilized in the treatment of bladder cancer (BC). OBJECTIVE: To investigate the effect of NAC on the risk of surgical complications for radical cystectomy (RC) in a population-based setting. DESIGN, SETTING, AND PARTICIPANTS: All radical cystectomies performed in Finland during 2005-2014 were included in the study. Data were collected retrospectively using a web-based data collection platform. Complications were recorded for 90 d using the Clavien classification. Patients treated with NAC were compared to patients receiving RC alone using three cohorts and approaches: the entire cohort, a neoadjuvant period cohort, and a matched cohort. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: For all three cohorts, odds ratios (ORs) were estimated using simple binary logistic regression. In addition, a multivariable stratified logistic model with propensity score was used. For the matched cohort analysis, both univariate and adjusted analyses were carried out. RESULTS AND LIMITATIONS: During 2005-2014, 1427 RCs were performed in Finland, of which 1385 were included in the analyses. NAC was introduced in 2008, and 231 patients (16%) were assigned to NAC and 214 (15%) received two or more cycles of chemotherapy. Within 90 d, 61% of patients experienced complications and mortality was 4% (1.9% in the NAC group, and 4.4% in the RC-alone group). In simple binary logistic regression, NAC patients had significantly fewer complications, but this was not observed in multivariable or propensity score analyses. In the matched cohort analyses, no differences in complication rates could be observed. None of the analyses demonstrated higher complication rates in the NAC group. CONCLUSIONS: Our retrospective study reports on nationwide use of NAC for BC and demonstrates that NAC does not increase RC morbidity. PATIENT SUMMARY: Chemotherapy given before radical surgery does not increase severe postoperative complications in the treatment of bladder cancer.

Decreased hyaluronidase 1 expression is associated with early disease recurrence in human endometrial cancer.

OBJECTIVE: Hyaluronidases (HYAL1 and HYAL2) are key enzymes in the degradation of hyaluronan, and their expression has been altered in various cancer types. We previously showed that hyaluronan accumulation in endometrial carcinomas was correlated with decreased mRNA expression of the HYAL genes. In this study, we analyzed HYAL1 and HYAL2 protein expressions in normal and precancerous endometrial tissues and in endometrial carcinomas. We also investigated whether the protein levels were associated with clinicopathological factors, invasion, and disease recurrence. METHODS: A total of 343 tissue specimens from normal, atrophic, hypertrophic, and neoplastic endometria were analyzed immunohistochemically for HYAL1 and HYAL2 expressions. The results were correlated with clinicopathological factors, the expression of the epithelial-mesenchymal transition marker, E-cadherin, and disease recurrence. RESULTS: Reduced HYAL1 expression was associated with the progression of endometrial carcinomas towards higher grades and also with large tumor sizes, lymph node metastasis, and lymphovascular invasion. Reduced expression of both HYAL1 and HYAL2 was associated with deep myometrial invasion. HYAL2 expression was primarily constant in neoplastic tissues, but its expression was altered in different phases of the endometrial cycle. In addition, a reduction in HYAL1 expression was associated with the depletion of E-cadherin. In a multivariate analysis, reduced HYAL1 expression was an independent prognostic factor for early disease recurrence (HR 5.13, 95% CI: 1.131-23.270, p=0.034). CONCLUSIONS: This study showed that reduced HYAL1 expression was associated with endometrial carcinoma aggressiveness, which further supported the role of hyaluronan degradation in cancer progression.

Matriptase-2 gene (TMPRSS6) variants associate with breast cancer survival, and reduced expression is related to triple-negative breast cancer.

Matriptase-2 (TMPRSS6) has been identified as a breast cancer risk factor. Here, we examined relationships between TMPRSS6 genetic variations and breast cancer risk and survival, and determined the gene and protein expressions in breast tumors and assessed their clinical importance. Thirteen TMPRSS6 polymorphisms were genotyped in 462 invasive breast cancer cases and 458 controls. Gene expression was analyzed from 83 tumors and protein expression from 370 tumors. We then assessed the statistical significance of associations among genotypes, clinicopathological characteristics and survival. The TMPRSS6 variant rs2543519 was associated with breast cancer risk (p = 0.032). Multivariate analysis showed that four variants had effects on survival-rs2543519 (p = 0.017), rs2235324 (p = 0.038), rs14213212 (p = 0.044) and rs733655 (p = 0.021)-which were used to create a group variable that was associated with poorer prognosis correlating with more alleles related to reduced survival (p = 0.006; risk ratio, 2.375; 95% confidence interval, 1.287-4.382). Low gene expression was related to triple-negative breast cancer (p = 0.0001), and lower protein expression was detected in undifferentiated (p = 0.019), large (p = 0.014) and ductal or lobular tumors (p = 0.036). These results confirm the association of TMRRSS6 variants with breast cancer risk and survival. Matriptase-2 levels decrease with tumor progression, and lower gene expression is seen in poor-prognosis-related triple-negative breast cancers. Our study is the first to show that matriptase-2 gene variants are related to breast cancer prognosis, supporting matriptase-2 involvement in tumor development.

Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma.

BACKGROUND: Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity. METHODS: A total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry. RESULTS: The mRNA levels of HAS1-3 were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, HAS3 mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of HYAL1 mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. HYAL2 mRNA was also reduced in cancer (p = 0.02) and correlated with HYAL1 (r = 0.8, p = 0.0001). There was an inverse correlation between HYAL1 mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001). CONCLUSION: The results indicated that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for HASs were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.

ST14 gene variant and decreased matriptase protein expression predict poor breast cancer survival.

BACKGROUND: Matriptase plays a role in carcinogenesis, but the role of its genetic variation or that of the hepatocyte growth factor activator inhibitor-1 (HAI-1) has not been evaluated. This study aimed to examine the genetic variation of matriptase (ST14 gene) and HAI-1 (SPINT1 gene) in breast cancer risk and prognosis, to assess matriptase and HAI-1 gene and protein expression in breast tumors, and to identify their clinicopathologic correlations and prognostic significance. METHODS: Five single nucleotide polymorphisms in ST14 and three in SPINT1 were genotyped in 470 invasive breast cancer cases and 446 healthy controls. Gene expression analysis was done for 40 breast cancer samples. Protein expression was assessed by immunohistochemical analyses in 377 invasive breast tumors. The statistical significance of the associations among genotypes, clinicopathologic variables, and prognosis was assessed. RESULTS: The ST14 single nucleotide polymorphism rs704624 independently predicted breast cancer survival, a poor outcome associated with the minor allele (P = 0.001; risk ratio, 2.221; 95% confidence interval, 1.382-3.568). Moreover, ST14 gene expression levels were lower among the minor allele carriers (P = 0.009), and negative/low matriptase protein expression was independently predictive of poorer survival (P = 0.046; risk ratio, 1.554; 95% confidence interval, 1.008-2.396). CONCLUSIONS: The ST14 variant rs704624 and protein expression of matriptase have prognostic significance in breast cancer. This study adds to the evidence for the role of matriptase in breast cancer and has found new evidence for the genotypes having an impact in breast cancer. IMPACT: This is the first study showing that genetic variation in matriptase has clinical importance. The results encourage further study on the genetic variation affecting protein levels and function in type II transmembrane serine proteases.

Expression of hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in serous ovarian carcinomas: inverse correlation between HYAL1 and hyaluronan content.

BACKGROUND: Hyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1-3 immunoreactivity. METHODS: Normal ovaries (n = 5) and 34 serous epithelial ovarian tumors, divided into 4 groups: malignant grades 1+2 (n = 10); malignant grade 3 (n = 10); borderline (n = 4) and benign epithelial tumors (n = 10), were analyzed for mRNA by real-time RT-PCR and compared to hyaluronidase activity, hyaluronan staining, and HAS1-3 immunoreactivity in tissue sections of the same specimens. RESULTS: The levels of HAS2 and HAS3 mRNA (HAS1 was low or absent), were not consistently increased in the carcinomas, and were not significantly correlated with HAS protein or hyaluronan accumulation in individual samples. Instead, the median of HYAL1 mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of HYAL1, but not HYAL2, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between HYAL1 mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025). CONCLUSION: The results indicate that in serous epithelial ovarian malignancies HAS expression is not consistently elevated but HYAL1 expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words).