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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.

Supplementary Figure 2A from ST14 Gene Variant and Decreased Matriptase Protein Expression Predict Poor Breast Cancer Survival

Supplementary Figure 3 from 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. Cancer Epidemiol Biomarkers Prev; 19(9); 2133–42. ©2010 AACR.

Supplementary Figure Legends from ST14 Gene Variant and Decreased Matriptase Protein Expression Predict Poor Breast Cancer Survival

BACKGROUND There is huge variation in Clavien-Dindo (CD) complication rates in urology. We sought to optimize the use of the CD system in kidney tumor surgery. METHODS We retrospectively analyzed 1,286 patients undergoing kidney tumor operations in 12 Finnish hospitals during 2016-2017. Primary CD assignments were made by site urologists. Data were centrally reviewed by two authors in consensus meetings. Consistency of the primary assignments was assessed by the number of cases requiring correction. Complication load was compared as different outcome rates between five university hospital regions. RESULTS The overall complication rate in primary data was 40% (517/1286) and varied significantly from 32 to 62% (p

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.

Introduction With a five-year overall survival of approximately 50%, there is an important clinical need to understand the molecular behavior of muscle-invasive bladder cancer (MIBC). FBXW7, a subunit of an E3-ubiquitin ligase complex, is a tumour suppressor gene. Mutations in FBWX7 have been described in an expanding number of human neoplasms, including 10% of MIBC, making it one of the most commonly mutated genes in MIBC. However, it has received little attention in MIBC up to now. We aimed to assess the role of FBXW7 as a tumour suppressor in MIBC. Methods We studied clinical implications of FBXW7 mutation and expression using The Cancer Genome Atlas (TCGA) database (N=405). FBXW7 was silenced in human bladder cancer cell lines with Crispr-Cas9 in order to assess the functional significance of modulating FBXW7 in vitro. Results Low FBXW7 mRNA expression significantly correlated with worse overall survival (OS) in TCGA, and Cox regression analysis demonstrated that it was an independent predictor of OS. FBXW7 mRNA expression was low in the luminal-infiltrated, luminal and basal/squamous subtypes compared to the luminal-papillary subtype. Furthermore, deletion and putative mutations of FBXW7 correlated with worse OS in TCGA. GSEA analysis showed that MYC targets were enriched by low FBXW7 expression or the deletion/putative mutations. Consistent with this, upregulation of MYC protein was detected in FBXW7 knock out cells, and this was associated with the upregulation of numerous cell cycle genes. Conclusions FBXW7 loss of function was significantly correlated with worse OS. Preliminary analysis suggests that this may be related to MYC accumulation and downstream cell cycle gene upregulation. MYC, which is a substrate for FBXW7, could potentially be targeted to attenuate MIBC growth.

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.

Introduction Intravesical bacillus Calmette-Guerin (BCG) is the standard adjuvant treatment for high-grade non-muscle invasive bladder cancer (NMIBC) after transurethral resection of the bladder tumor (TURBT). Despite this standard of care - 40% of patients experience a recurrence and - 15% progress to muscle invasive disease. Tumors with Th1-polarized lymphocytic infiltrate in the tumor immune microenvironment may have more favorable response to BCG compared to Th2- polarized tumors. Since basophils have a promoting role in Th2-polarization, we explored the association of blood basophils on the outcome of patients with high-grade NMIBC receiving BCG instillations. Methods Clinical data and pre-operative complete blood count (CBC) results from patients with high-grade NMIBC (Ta, CIS and T1) who received BCG instillations at the Vancouver Prostate Centre (VPC) during years 2011 – 2018 were collected. Absolute basophil count (ABC) was correlated with response to BCG, disease recurrence and progression to muscle invasive disease. Results A total of 174 patients with primary high grade NMIBC treated with BCG at VPC were identified. Pre-operative complete blood count (CBC) data were available from 161 patients. After the initiation of BCG therapy 40% of patients developed recurrent tumors and 13% progressed to muscle invasive disease. ABC was measurable (³ 0.1 × 103 cells /µl) in the blood of 33 (19%) patients of whom 19 (58%) recurred. A total of 95% of recurrent tumors with measurable ABC were considered BCG-unresponsive. In Kaplan-Meier analysis measurable ABC was associated with tumor recurrence (p = 0.024) and progression to muscle invasive disease (p = 0.00015). In multivariate cox-regression analysis measurable ABC was considered an independent prognostic factor for progression to muscle invasive disease (HR 2.9, 95% CI:1.038 – 8.535, p = 0.042). Conclusions Measurable blood absolute basophil count may have a prognostic impact for patients with high-grade NMIBC. Further studies with larger patient cohorts are needed to confirm these preliminary results.

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. Most active clinical trials focus on high-risk NMIBC in either the BCG-naive 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-naive 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.

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.

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 (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.

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.

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. Cancer Epidemiol Biomarkers Prev; 19(9); 2133–42. ©2010 AACR.

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)

Cutting the hepatic branch of the anterior vagus nerve makes laparoscopic fundoplication technically easier; however, there is little data about the effect of cutting the nerve on gallbladder function.One surgeon (MPä) preserved this nerve until March 2001 when he changed the technique. We investigated patients consecutively operated on before and after this date. A symptom questionnaire was returned by 59 patients, of whom 19 in both groups were successfully further investigated. The follow-up varied from 4 to 9 years postoperatively. The volume of the gallbladder was measured by ultrasonography before and after a fatty test meal. Alkaline phosphatase (ALP), alanine aminotransferase (ALAT), bilirubin, and amylase were determined from plasma.There was no difference in symptoms or use of antireflux medication between the groups. No difference was found in the levels of bilirubin, ALAT, or ALP. A mild elevation in plasma amylase was noted after nerve division (p = 0.041). The gallbladder ejection fraction did not differ between groups, but the fasting gallbladder volume was smaller when the nerve was cut (median 18.1 (range, 6-57.7) ml versus median 23.2 (range, 7.9-66.7) ml; p = 0.049). Both differences in plasma amylase and gallbladder fasting volume were clearer in male patients.Cutting the hepatic branch of the anterior vagus nerve during fundoplication may reduce the size of gallbladder, but it has no effect on the ejection fraction. No clinical significance has been noted to date. Prospective trials will be necessary to confirm these findings.

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.