Tumor heterogeneity of fibroblast growth factor receptor 3 (FGFR3) mutations in invasive bladder cancer: implications for perioperative anti-FGFR3 treatment.

BACKGROUND: Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response. PATIENTS AND METHODS: We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201). RESULTS: We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type. CONCLUSIONS: FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting.

Prediction of neo-adjuvant chemotherapy response in bladder cancer: the impact of clinical parameters and routine biomarkers.

PURPOSE: To investigate the role of clinical parameters and immunohistochemical (IHC) biomarkers in their feasibility to predict the effect of neo-adjuvant chemotherapy (NAC) in patients with muscle-invasive urothelial bladder cancer (MIBC). MATERIALS AND METHODS: The first 76 consecutive patients with MIBC treated with NAC and radical cystectomy in two University hospitals in Finland between 2008 and 2013 were chosen for this study. After excluding patients with non-urothelial cancer, less than two cycles of chemotherapy, no tissue material for IHC analysis or non-muscle-invasive bladder cancer in re-review, 59 patients were included in the final analysis. A tissue microarray block was constructed from the transurethral resection samples and IHC stainings of Ki-67, p53, Her-2 and EGFR were made. The correlations between histological features in transurethral resection samples and immune-histochemical stainings were calculated. The associations of clinicopathological parameters and IHC stainings with NAC response were evaluated. Factors affecting survival were estimated. RESULTS: The complete response rate after NAC was 44%. A higher number of chemotherapy cycles was associated with better response to neo-adjuvant chemotherapy. No response to neo-adjuvant chemotherapy and female gender was associated with decreased cancer-specific survival. The IHC stainings used failed to show an association with neo-adjuvant chemotherapy response and overall or cancer specific survival. CONCLUSIONS: Patients who do not respond to neo-adjuvant chemotherapy do significantly worse than responders. This study could not find clinical tools to distinguish responders from non-responders. Further studies preferably with larger cohorts addressing this issue are warranted to improve the selection of patients for neo-adjuvant chemotherapy.

SPCG-15: a prospective randomized study comparing primary radical prostatectomy and primary radiotherapy plus androgen deprivation therapy for locally advanced prostate cancer.

OBJECTIVE: To describe study design and procedures for a prospective randomized trial investigating whether radical prostatectomy (RP) ± radiation improves cause-specific survival in comparison with primary radiation treatment (RT) and androgen deprivation treatment (ADT) in patients with locally advanced prostate cancer (LAPC). MATERIALS AND METHODS: SPCG-15 is a prospective, multi-centre, open randomized phase III trial. Patients are randomized to either standard (RT + ADT) or experimental (RP with extended pelvic lymph-node dissection and with addition of adjuvant or salvage RT and/or ADT if deemed necessary) treatment. Each centre follows guidelines regarding the timing and dosing of postoperative RT and adjuvant treatment such as ADT The primary endpoint is cause-specific survival. Secondary endpoints include metastasis-free and overall survival, quality-of-life, functional outcomes and health-services requirements. Each subject will be followed up for a minimum of 10 years. RESULTS: Twenty-three centres in Denmark, Finland, Norway and Sweden, well established in performing RP and RT for prostate cancer participated. Each country's sites were coordinated by national coordinating investigators and sub-investigators for urology and oncology. Almost 400 men have been randomized of the stipulated 1200, with an increasing rate of accrual. CONCLUSIONS: The SPCG-15 trial aims to compare the two curatively intended techniques supplying new knowledge to support future decisions in treatment strategies for patients with LAPC The Scandinavian healthcare context is well suited for performing multi-centre long-term prospective randomized clinical trials. Similar care protocols and a history of entirely tax-funded healthcare facilitate joint trials.

Hypoxia Marker GLUT-1 (Glucose Transporter 1) is an Independent Prognostic Factor for Survival in Bladder Cancer Patients Treated with Radical Cystectomy.

BACKGROUND: Tumour hypoxia, which is frequent in many cancer types, is associated with treatment resistance and poor prognosis. The role of hypoxia in surgically treated bladder cancer (BC) is not well described. We studied the role of hypoxia in two independent series of urothelial bladder cancers treated with radical cystectomy. METHODS: 279 patients from the University Hospital Network (UHN), Toronto, Canada, and Turku University, Finland were studied. Hypoxia biomarkers (HIF1-α, CAIX, GLUT-1) and proliferation marker Ki-67 were analyzed with immunohistochemistry using defined tissue microarrays. Kaplan-Meier methods and Cox proportional hazards regression models were used to investigate prognostic role of the factors. RESULTS: In univariate analyses, strong GLUT-1 positivity and a high Ki-67 index were associated with poor survival. In multivariate model containing clinical prognostic variables, GLUT-1 was an independent prognostic factor associated with worse disease-specific survival (HR 2.9, 95% CI 0.7-12.6, Wald p = 0.15 in the Toronto cohort and HR 3.2, 95% CI 1.3-7.5, Wald p = 0.0085 in the Turku cohort). CONCLUSION: GLUT-1 is frequently upregulated and is an independent prognostic factor in surgically treated bladder cancer. Further studies are needed to evaluate the potential role of hypoxia-based and targeted therapies in hypoxic bladder tumours.

Lysophosphatidic acid and sphingosine-1-phosphate promote morphogenesis and block invasion of prostate cancer cells in three-dimensional organotypic models.

Normal prostate and some malignant prostate cancer (PrCa) cell lines undergo acinar differentiation and form spheroids in three-dimensional (3-D) organotypic culture. Acini formed by PC-3 and PC-3M, less pronounced also in other PrCa cell lines, spontaneously undergo an invasive switch, leading to the disintegration of epithelial structures and the basal lamina, and formation of invadopodia. This demonstrates the highly dynamic nature of epithelial plasticity, balancing epithelial-to-mesenchymal transition against metastable acinar differentiation. This study assessed the role of lipid metabolites on epithelial maturation. PC-3 cells completely failed to form acinar structures in delipidated serum. Adding back lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) rescued acinar morphogenesis and repressed invasion effectively. Blocking LPA receptor 1 (LPAR1) functions by siRNA (small interference RNA) or the specific LPAR1 inhibitor Ki16425 promoted invasion, while silencing of other G-protein-coupled receptors responsive to LPA or S1P mainly caused growth arrest or had no effects. The G-proteins Gα(12/13) and Gα(i) were identified as key mediators of LPA signalling via stimulation of RhoA and Rho kinases ROCK1 and 2, activating Rac1, while inhibition of adenylate cyclase and accumulation of cAMP may be secondary. Interfering with these pathways specifically impeded epithelial polarization in transformed cells. In contrast, blocking the same pathways in non-transformed, normal cells promoted differentiation. We conclude that LPA and LPAR1 effectively promote epithelial maturation and block invasion of PrCa cells in 3-D culture. The analysis of clinical transcriptome data confirmed reduced expression of LPAR1 in a subset of PrCa's. Our study demonstrates a metastasis-suppressor function for LPAR1 and Gα(12/13) signalling, regulating cell motility and invasion versus epithelial maturation.

Systematic knockdown of epigenetic enzymes identifies a novel histone demethylase PHF8 overexpressed in prostate cancer with an impact on cell proliferation, migration and invasion.

Our understanding of key epigenetic regulators involved in specific biological processes and cancers is still incomplete, despite great progress in genome-wide studies of the epigenome. Here, we carried out a systematic, genome-wide analysis of the functional significance of 615 epigenetic proteins in prostate cancer (PrCa) cells. We used the high-content cell-spot microarray technology and siRNA silencing of PrCa cell lines for functional screening of cell proliferation, survival, androgen receptor (AR) expression, histone methylation and acetylation. Our study highlights subsets of epigenetic enzymes influencing different cancer cell phenotypes. Plant homeo domain (PHD) finger proteins have a key role in cell survival and histone methylation, whereas histone deacetylases were primarily involved in regulating AR expression. In contrast, JumonjiC-domain (JmjC) containing histone lysine demethylases (KDMs) mainly had an impact on cell proliferation. Our results show that the KDMs JARID1B, PHF8, KDM3A, KDM3B and KDM4A were highly expressed in clinical PrCa samples. The PHD-finger protein 8 (PHF8), a transcriptional coactivator with both PHD- and JmjC-domains, was moderately to strongly expressed in 80% of clinical PrCa samples, whereas 76% of normal and benign samples were negative or only showed weak PHF8 expression. Strong PHF8 expression correlated significantly with high Gleason grade and was borderline significant for poor prognosis. The results of functional PHF8 knockdown implicate a role in cell migration and invasion, as shown by cell motility and 3-D invasion assays. Our study suggests that various cellular phenotypes are regulated by distinct subsets of epigenetic enzymes. Proteins interpreting and modifying histone methylation, such as JmjC-domain and particularly PHD-finger proteins like PHF8, are activated in subsets of PrCa's and promote cancer relevant phenotypes.

Transformation-specific matrix metalloproteinases (MMP)-7 and MMP-13 are expressed by tumour cells in epidermolysis bullosa-associated squamous cell carcinomas.

BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) have an increased risk of developing rapidly progressive and metastatic cutaneous squamous cell carcinomas (SCC). It is unclear why these SCC behave more aggressively than sporadic SCC. Matrix metalloproteinases (MMP) are a family of endopeptidases that contribute to growth, invasion and metastasis of SCC. The role of MMP in RDEB-associated SCC is not known. OBJECTIVES: To investigate the expression of MMP-7, MMP-13 and MMP-9 in RDEB-associated SCC in comparison with sporadic SCC and Bowen's disease. METHODS: Immunohistochemical analysis of 25 RDEB-associated SCC, 61 sporadic SCC and 28 sporadic lesions of Bowen's disease was carried out using monoclonal antibodies for MMP-7, MMP-9, MMP-13 and E-cadherin and syndecan-1. RESULTS: MMP-7 was detected in all RDEB-associated SCC, in tumour cells within the invasive edge, where E-cadherin and syndecan-1 were markedly diminished or absent. MMP-7 expression was also observed in 98% of sporadic SCC and in 68% of Bowen's diseases. MMP-7 staining was significantly stronger in RDEB-associated SCC than in sporadic SCC, and was most abundant in poorly differentiated tumours. MMP-13 was detected in tumour cells in 96% of RDEB-associated SCC and in all sporadic cutaneous SCC. MMP-9 was detected in the inflammatory cells in all SCC examined. CONCLUSIONS: These results identify MMP-7 and MMP-13 as tumour cell-specific markers for SCC progression and as potential therapeutic targets in RDEB-associated SCC. The pattern of immunolabelling suggests that MMP-7 may shed E-cadherin and syndecan-1 from the SCC cell surface.

Cyclin A and Ki-67 with DNA content in benign and malignant prostatic epithelial lesions.

OBJECTIVE: To evaluate the usefulness of immunohistochemical staining of cyclin A and Ki-67 together with DNA content in the classification of benign prostatic hyperplasia, prostatic intraepithelial neoplasm (PIN) and prostatic carcinoma foci and to compare these parameters with each other and with parameters obtained from conventional histopathology. STUDY DESIGN: We selected 37 carcinoma, 18 PIN and 8 hyperplastic foci from prostatectomies done during 1996 and 1997 at Turku University Central Hospital. Cyclin A and Ki-67 staining was assessed by immunohistochemistry and DNA content by image cytometry. RESULTS: The hyperplastic, PIN and carcinoma foci differed clearly in their 2.5c exceeding rates, image cytometric proliferation indices and staining indices for cyclin A and Ki-67. No significant differences were found between these histologic entities in their modal DNA ploidy values. In carcinomas, cyclin A and Ki-67 indices differed between low, intermediate and high Gleason and World Health Organization grading groups. Diploid and tetraploid carcinomas had similar cyclin A and Ki-67 indices, which differed from those of aneuploid carcinomas. CONCLUSION: The 2.5c exceeding rate and image cytometric proliferation index as well as the cyclin A and Ki-67 indices differed significantly between different types of prostatic lesions. Cyclin A and Ki-67 had good correlations with the histologic grade of carcinoma.