Johannes Giedl

Prognostic Value of Perinodal Lymphovascular Invasion Following Radical Cystectomy for Lymph Node-positive Urothelial Carcinoma

BACKGROUND Metastasis of urothelial carcinoma of the bladder (UCB) into regional lymph nodes (LNs) is a key prognosticator for cancer-specific survival (CSS) after radical cystectomy (RC). Perinodal lymphovascular invasion (pnLVI) has not yet been defined. OBJECTIVE To assess the prognostic value of histopathologic prognostic factors, especially pnLVI, on survival. DESIGN, SETTING, AND PARTICIPANTS A total of 598 patients were included in a prospective multicentre study after RC for UCB without distant metastasis and neoadjuvant and/or adjuvant chemotherapy. En bloc resection and histopathologic evaluation of regional LNs were performed based on a prospective protocol. The final study group comprised 158 patients with positive LNs (26.4%). INTERVENTION Histopathologic analysis was performed based on prospectively defined morphologic criteria of LN metastases. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Multivariable Cox proportional hazard regression models determined prognostic impact of clinical and histopathologic variables (age, gender, tumour stage, surgical margin status, pN, diameter of LN metastasis, LN density [LND], extranodal extension [ENE], pnLVI) on CSS. The median follow-up was 20 mo (interquartile range: 11-38). RESULTS AND LIMITATIONS Thirty-one percent of patients were staged pN1, and 69% were staged pN2/3. ENE and pnLVI was present in 52% and 39%, respectively. CSS rates after 1 yr, 3 yr, and 5 yr were 77%, 44%, and 27%, respectively. Five-year CSS rates in patients with and without pnLVI were 16% and 34% (p<0.001), respectively. PN stage, maximum diameter of LN metastasis, LND, and ENE had no independent influence on CSS. In the multivariable Cox model, the only parameters that were significant for CSS were pnLVI (hazard ratio: 2.47; p=0.003) and pT stage. However, pnLVI demonstrated only a minimal gain in predictive accuracy (0.1%; p=0.856), and the incremental accuracy of prediction is of uncertain clinical value. CONCLUSIONS We present the first explorative study on the prognostic impact of pnLVI. In contrast to other parameters that show the extent of LN metastasis, pnLVI is an independent prognosticator for CSS.

Low Frequency of Epigenetic Events in Urothelial Tumors in Young Patients

PURPOSE Bladder urothelial cell carcinoma is uncommon in young patients. We recently reported a series of tumors in patients younger than 20 years at diagnosis and performed exhaustive genetic screening for molecular alterations. Few events typical of bladder urothelial cell carcinoma were detected. Since many carcinogenic events occur at the epigenetic rather than the genetic level, we analyzed the same tumors for alterations in DNA hypermethylation. We compared our findings with those in tumors in older patients with similar pathological profiles. MATERIALS AND METHODS We analyzed 76 bladder urothelial cell carcinomas from 3 groups stratified by age at diagnosis, including less than 19, 20 to 45 and greater than 46 years (median 78), and matched for low grade and nonmuscle invasive stage. We used quantified methyl specific polymerase chain reaction to investigate promoter methylation for 8 tumor suppressor genes implicated in urothelial carcinogenesis. RESULTS Tumors in the youngest age group had the lowest incidence of global hypermethylation compared to the other tumors with a methyl index of 37.5% vs 62.5% and 50%, respectively (ANOVA p = 0.009). When individual loci were analyzed, younger patients had a significantly lower rate and concentration of methylation at APC, Bcl2, MGMT and E-cadherin promoters than in the older groups (p <0.05). Few differences were present between the 2 older cohorts but the APC and MGMT methylation concentration increased with age. CONCLUSIONS Urothelial tumors in patients younger than 19 years have a low rate of epigenetic alteration. Tumors in patients older than 20 years have epigenetic profiles similar to those of tumors in patients within the typical bladder urothelial cell carcinoma age range.

The analysis of mononuclear cell infiltrations in colorectal adenocarcinoma

SummaryMononuclear cell (MNC) populations in tissue specimens from 11 colorectal adenocarcinomas and 1 mucinous adenocarcinoma were analyzed, applying different monoclonal antibodies (moAB). Tumor epithelium could be characterized by MoAB VEP9, predominantly binding to mucus secreted by the tumor cells. In approximately 30%–50%, the tumor epithelium also reactdd in a patchy pattern with the MoABs OKT10 and OKIa, which define in the peripheral blood activated and HLA-DR expressing cells. T-lymphocytes, as defined by the MoAB 9.6, and T-lymphocyte subpopulations, as characterized by the MoABs OKT4 and OKT8, were found in the intratumoral stroma and in peritumoral inflammatory areas. Quantifying the relative amounts of mononuclear cell subpopulations in the different stroma compartments, a predominance of OKT10- and OKIa-defined and MoAB-S39-reactive cells was observed. NK cells, as labelled by the MoAB Leu-7, were only demonstrated singularly in different areas of the stroma. Nonlymphoid structures such as vessel walls were shown to express antigens recognized by the MoABs OKIa, OKT10, and Leu-7. In some instances, nerve structures were labelled by the MoABs OKIa, OKT10, S39, and Leu-7. Using semiquantitative analysis, no correlation could be obtained between the intratumoral and peritumoral infiltration with T-lymphocytes, T-lymphocyte subpopulations and monocytic cells, and histopathological tumor grading and staging.

Frequency of TERT Promoter Mutations in Prostate Cancer

Objective: Recently, recurrent mutations within the core promoter of the human telomerase reverse transcriptase (TERT) gene generating consensus binding sites for ETS transcription factor family members were described in melanomas and other malignancies (e.g. bladder cancer, hepatocellular carcinoma). These mutations were discussed as early drivers for malignant transformation. In prostate cancer (PrCa) TERT expression has been associated with a poor prognosis and higher risk for disease recurrence. The underlying mechanisms for high TERT expression in PrCa have still not been clarified. To date, data on TERT promoter mutation analysis in PrCa are sparse. Therefore, we performed sequence analysis of the core promoter region of the TERT gene in an unselected cohort of prostate tumors. Methods: Sections from 167 formalin-fixed, paraffin-embedded and cryopreserved prostate tumors were microdissected and used for DNA isolation. The mutation hotspot region within the TERT core promoter (-260 to +60) was analyzed by direct Sanger sequencing or SNaPshot analysis. Results: All cases were analyzed successfully. Mutations within the core promoter of the TERT gene were not detected in any of the cases with all tumors exhibiting a wild-type sequence. Conclusion:TERT core promoter mutations reported from several other malignancies were not detected in our unselected cohort of PrCa. These data indicate that alterations within the core promoter of the TERT gene do not play an important role in prostate carcinogenesis.

Loss of MTUS1/ATIP expression is associated with adverse outcome in advanced bladder carcinomas: data from a retrospective study

BackgroundSeventy percent of all bladder tumours tend to recur and need intensive surveillance, and a subset of tumours progress to muscle-invasive and metastatic disease. However, it is still difficult to find the adequate treatment for every individual patient as it is a very heterogeneous disease and reliable biomarkers are still missing. In our study we searched for new target genes in the critical chromosomal region 8p and investigated the potential tumour suppressor gene candidate MTUS1/ATIP in bladder cancer.MethodsMTUS1 was identified to be the most promising deleted target gene at 8p in aCGH analysis with 19 papillary bladder tumours. A correlation with bladder cancer was further validated using immunohistochemistry of 85 papillary and 236 advanced bladder tumours and in functional experiments. Kaplan-Meier analysis and multivariate Cox-regression addressed overall survival (OS) and disease-specific survival (DSS) as a function of MTUS1/ATIP expression. Bivariate correlations investigated associations between MTUS1/ATIP expression, patient characteristics and histopathology. MTUS1 expression was analysed in cell lines and overexpressed in RT112, where impact on viability, proliferation and migration was measured.ResultsMTUS1 protein expression was lost in almost 50% of all papillary and advanced bladder cancers. Survival, however, was only influenced in advanced carcinomas, where loss of MTUS1 was associated with adverse OS and DSS. In this cohort, there was also a significant correlation of MTUS1 expression and histological subtype: positive expression was detected in all micropapillary tumours and aberrant nuclear staining was detected in a subset of plasmocytoid urothelial carcinomas. MTUS1 was expressed in all investigated bladder cell lines and overexpression in RT112 led to significantly decreased viability.ConclusionsMTUS1 is a tumour suppressor gene in cultured bladder cancer cells and in advanced bladder tumours. It might represent one new target gene at chromosome 8p and can be used as an independent prognostic factor for advanced bladder cancer patients. The limitation of the study is the retrospective data analysis. Thus, findings should be validated with a prospective advanced bladder tumour cohort.

TERT Core Promotor Mutations in Early-Onset Bladder Cancer

Activating mutations in the core promoter of the TERT gene have been described in many different tumor entities. In vitro models showed a two- to fourfold increase in transcriptional activity of the TERT promoter through creation of a consensus binding motif for Ets/TCF transcription factors caused by these mutations. TERT core promoter mutations are the most common mutations in bladder cancer with a frequency between 55.6% and 82.8% described so far, and are independent of stage and grade. Since limited data on molecular alterations of early-onset bladder tumors exists, we assessed the frequency of TERT core promoter mutations in early-onset bladder cancer. Two cohorts of bladder tumors (early-onset patient group; n=144 (age of onset of disease ≤45 years); unselected, consecutive group; n=125) were examined for TERT core promoter mutations. After microdissection and extraction of DNA the corresponding hotspot regions in the TERT core promoter were examined by Sanger-sequencing or a SNaPshot approach. A significantly lower frequency of TERT core promoter mutations was found in tumors from the early-onset cohort compared to the consecutive cohort (57.6% vs. 84.8%, p<0.001). Among the early-onset cohort cases younger than the cohorts median age of 39 years at disease onset showed a significantly reduced number of TERT promoter mutations (31/67, 46,3%) than cases aged between 39 and 45 years (52/77, 67.5%; p=0.012). This association was not found in the consecutive cases. Mutation status was independent of tumor stage and grade. We conclude that in tumors from early-onset bladder cancer patients TERT core promoter mutations are not as frequent as in bladder tumors from consecutive cases, but seem to play an important role there as well. In patients below 39 years of age TERT core promoter mutations are a more infrequent event, suggesting different mechanisms of tumorigenesis in these young patients.

Inactivating Mutations of RB1 and TP53 Correlate With Sarcomatous Histomorphology and Metastasis/Recurrence in Gastrointestinal Stromal Tumors

OBJECTIVES Loss-of-function mutations in TP53 and CDKN2A have been found at varying frequencies in gastrointestinal stromal tumors (GISTs), while no mutations of RB1 have been reported to date. The aim of the current study was to determine the mutation frequency of TP53, RB1, and CDKN2A in GISTs. METHODS A cohort of 83 primary untreated GISTs was analyzed for mutations in TP53, RB1, and CDKN2A by massive parallel sequencing. Tumors with mutations in TP53 and RB1 were analyzed by fluorescence in situ hybridization for the corresponding gene loci. RESULTS Two GISTs harbored inactivating mutations in RB1, and two other GISTs displayed inactivating mutations in TP53 All four tumors were KIT mutant high-risk tumors with highly cellular sarcomatous histomorphology and variable combinations of plump spindle cells to epithelioid highly atypical cells and high mitotic activity. Three of these patients developed recurrent or metastatic disease, while the fourth patient showed tumor rupture intraoperatively. The combined overall frequency of TP53 and RB1 mutations was 13% considering high-risk or malignant GISTs. CONCLUSIONS TP53 and RB1 mutations seem to be restricted to high-risk/malignant GISTs and occur at an equal although relatively low frequency.

Lack of evidence for frequent MED12 p.L1224F mutation in prostate tumours from Caucasian patients.

Recently mutations in the MED12 gene have been reported in 5.4% of prostate tumours from Caucasian patients analysed by exome sequencing (Barbieri CE, Baca SC, Lawrence MS, et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nature Genet 2012; 44: 685–689). In more than 70% of prostate tumours with MED12 mutation, a recurrent p.L1224F mutation in exon 26 was found. In order to validate this MED12 p.L1224F mutation, an unselected cohort of prostate tumours from Caucasian patients was analysed by Sanger sequencing. Overall, 223 prostate tumours and three lymph node metastases were analysed. The MED12 p.L1224F mutation could not be detected in any of the cases. So far, the recently reported MED12 p.L1224F mutation could not be validated in our unselected cohort of prostate tumours. Contrary to the findings of Barbieri et al, our data indicate either that the p.L1224F mutation in the MED12 gene plays no role in prostate carcinogenesis or that this alteration is only relevant in a small subgroup of tumours. Copyright

Clinical Course of Plasmacytoid Urothelial Carcinoma of the Upper Urinary Tract: A Case Report

Plasmacytoid urothelial carcinoma of the bladder represents a rare and aggressive variant of urothelial carcinoma, which is usually diagnosed at an advanced pathologic stage. Until now, no reports exist on this rare tumor type in the upper urinary tract. Herein, we present the first report on the clinical course of a metastatic plasmacytoid urothelial carcinoma of the renal pelvis and show its unfavorable outcome despite multimodal therapy.

Functional analyses and prognostic significance of SFRP1 expression in bladder cancer

PurposeWe previously showed that the Wnt-signaling antagonist SFRP1 (secreted frizzled-related protein 1) is a promising marker in bladder cancer. The aim of this study was to validate the prognostic role and analyze the functional significance of SFRP1.MethodsFour bladder cancer cell lines (RT112, RT4, J82 and BFTC905) and one urothelial cell line (UROtsa) were used for functional characterization of SFRP1 expression. Effects on viability, proliferation and wound healing were investigated, and canonical Wnt-pathway activity as well as Wnt-signaling target gene expression was analyzed. Additionally, tissue micro-arrays from two different bladder tumor cohorts were evaluated for SFRP1 expression, and associations with survival and histopathological parameters were analyzed.ResultsThe cell lines RT112, RT4, J82 and UROtsa showed SFRP1 expression. In BFTC905, SFRP1 expression was inhibited by promoter hypermethylation. Wnt-pathway activity was absent in all cell lines and independent from SFRP1 expression. RT112 and BFTC905 were used for further functional characterization. SFRP1 overexpression resulted in decreased viability and migration in BFTC905 cells. Knockdown of SFRP1 expression in RT112 cells resulted only in marginal effects. In bladder tumors, SFRP1 expression was associated with lower tumor grade, but not with progression in patients with papillary bladder cancer. SFRP1 expressing papillary bladder cancer tumors also demonstrated a tendency to longer overall survival.ConclusionsSFRP1 is reducing malignant potential of BFTC905 cells, but not by regulation of canonical Wnt-signaling pathway. Other pathways, like non-canonical Wnt or the MAPK pathway, could be activated via SFRP1-expression loss. In bladder tumors, SFRP1 has the potential to predict outcome for a subset of papillary bladder tumors.