Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies

Locally confined prostate cancer: Studies on the frequency, significance and therapy of occult disseminated tumor cells and the effect of endocrine treatment on the primary tumor

HabilitationsschriftNach alleiniger operativer Therapie des klinisch lokal begrenzten Prostatakarzinoms ( kommt es in ca. 30% der Patienten zu einem lokalen und/oder systemischen Tumorprogress. Die endokrine Therapie stellt die wirksamste systemische Therapie des Prostatakarzinoms dar. Sie wird primär im fortgeschrittenen Stadium, also bei hoher Tumorlast und zeitlich begrenzte Wirkdauer unter nicht kurativer Intention eingesetzt. Ein vorgezogener, präoperativer Einsatz der endokrinen Therapie bei geringerer Tumorlast könnte das antitumoröse Potential dieser Therapie möglicherweise besser nutzen und hierbei die lokale Resektion in sano verbessern als auch eventuell gleichzeitig vorhandene okkulte Tumorzelldisseminate, als mögliche Vorläufer späterer Metastasen eliminieren.Die vorgelegte Habilitationsschrift fast die eigenen Untersuchungsergebnisse zur okkulten Tumorzelldissemination des Prostatakarzinoms, zur Wirksamkeit zeitlich unterschiedlicher neoadjuvanter endokriner Therapiekonzepte auf den Primärtumor, als auch auf Tumorzelldisseminate im Knochenmark, sowie zur prognostischen Relevanz der erhobenen Befunde zusammen und diskutiert sie kritisch im Lichte der hierzu vorliegenden Literatur.Following primary surgical treatment of clinically organ confined prostate cancer up to 30% of the patients will develop signs of tumor progression / relapse. Endocrine therapy represents the most effective form of systemic treatment of the disease. It is usually applied in advanced stages of the disease, corresponding to a high tumor burden. Although initially being highly effective in this setting its therapeutic benefit is of limited duration. Initiation of endocrine treatment at an earlier stage with a lower tumor burden followed by radical prostatectomy is therefore a promising concept to more effectively take advantage of the anticancer effect of endocrine treatment possibly resulting in improved complete surgical resection rates and elimination ofalready disseminated tumor cells. The investigations presented and discussed in this postdoctoral thesis focus on occult tumor cell dissemination of prostate cancer, on the therapeutic effect of neoadjuvant endocrine treatment on the primary tumor and its disseminates in the bone marrow as well as on the prognostic significance of the findings

Leydig Cell Tumor in a Patient with 46,XX Disorder of Sex Development (DSD), Ovotesticular: A Case Report and a Review of the Literature

Disorder of sex development (DSD) is a rare condition with atypical development of chromosomal, gonadal, or anatomical sex. It is classified in different subgroups based on the patient’s karyotype, gonadal dysgenesis, and the appearance of the internal and external genitalia. Within the subgroups, the risk for developing neoplasms varies a lot. Here, we report the case of a 41-year-old patient with disorder of sex development, showing a 46,XX karyotype with an ovotestis and the simultaneous manifestation of a Leydig cell tumor in the ovotestis. The patient initially presented with infertility, and a suspicious lesion of the left testicle was noted on MRI-Scan. Upon resection, a Leydig cell tumor and an ovotestis were diagnosed. Nongerm call tumors are rare in patients with DSD. We report a nongerm cell tumor in a patient with 46,XX DSD, ovotesticular. This shows that although 46,XX DSD, ovotesticular is known to have a low potential for germ cell neoplasia, nongerm cell tumors can develop and should be into account for the management of those patients

The peri- and intratumoral immune cell infiltrate and PD-L1 status in invasive squamous cell carcinomas of the penis

Introduction: Penile carcinomas are rare tumors throughout Europe. Therefore, little attention is drawn to this disease. That makes it important to study tumor-associated key metrics and relate these to known data on penile neoplasias. Materials and methods: A cohort of 60 well-defined penile invasive carcinomas with known human papillomavirus (HPV) infection status was investigated. Data on tumor type, grading and staging were recorded. Additionally, data on the peri- and intratumoral immune cell infiltrate in a semiquanititave manner applying an HE stain were assessed. Results: Our study showed a significant correlation of immune cell infiltrate and pT stage with overall survival. Therefore, in a subset of tumors, PD-L1 staining was applied. For tumor proportion score (TPS), 26 of 30 samples (87%) were scored >0%. For the immune cell score (IC), 28 of 30 samples (93%) were defined as >0% and for CPS, 29 of 30 samples (97%) scored >0. PD-L1 expression was not associated with overall survival. Conclusion: PD-L1 is expressed in penile carcinomas, providing a rationale for targeted therapy with checkpoint inhibitors. We were able to show that immune reaction appears to be prognostically relevant. These data enhance the need for further studies on the immune cell infiltrate in penile neoplasias and show that PD-L1 expression is existent in our cohort, which may be a potential target for checkpoint inhibitor therapy

Irreversible electroporation (IRE) : standardization of terminology and reporting criteria for analysis and comparison

BACKGROUND: Irreversible electroporation (IRE) as newer ablation modality has been introduced and its clinical niche is under investigation. At present just one IRE system has been approved for clinical use and is currently commercially available (NanoKnife® system). In 2014, the International Working Group on Image-Guided Tumor Ablation updated the recommendation about standardization of terms and reporting criteria for image-guided tumor ablation. The IRE method is not covered in detail. But the non-thermal IRE method and the NanoKnife System differ fundamentally from established ablations techniques, especially thermal approaches, e.g. radio frequency ablation (RFA). MATERIAL AND METHODS: As numerous publications on IRE with varying terminology exist so far - with numbers continuously increasing - standardized terms and reporting criteria of IRE are needed urgently. The use of standardized terminology may then allow for a better inter-study comparison of the methodology applied as well as results achieved. RESULTS: Thus, the main objective of this document is to supplement the updated recommendation for image-guided tumor ablation by outlining a standardized set of terminology for the IRE procedure with the NanoKnife Sytem as well as address essential clinical and technical informations that should be provided when reporting on IRE tumor ablation. CONCLUSIONS: We emphasize that the usage of all above recommended reporting criteria and terms can make IRE ablation reports comparable and provide treatment transparency to assess the current value of IRE and provide further development

Optimizing identification of consensus molecular subtypes in muscle-invasive bladder cancer: a comparison of two sequencing methods and gene sets using FFPE specimens

Abstract Background Molecular subtypes predict prognosis in muscle-invasive bladder cancer (MIBC) and are explored as predictive markers. To provide a common base for molecular subtyping and facilitate clinical applications, a consensus classification has been developed. However, methods to determine consensus molecular subtypes require validation, particularly when FFPE specimens are used. Here, we aimed to evaluate two gene expression analysis methods on FFPE samples and to compare reduced gene sets to classify tumors into molecular subtypes. Methods RNA was isolated from FFPE blocks of 15 MIBC patients. Massive analysis of 3’ cDNA ends (MACE) and the HTG transcriptome panel (HTP) were used to retrieve gene expression. We used normalized, log2-transformed data to call consensus and TCGA subtypes with the consensusMIBC package for R using all available genes, a 68-gene panel (ESSEN1), and a 48-gene panel (ESSEN2). Results Fifteen MACE-samples and 14 HTP-samples were available for molecular subtyping. The 14 samples were classified as Ba/Sq in 7 (50%), LumP in 2 (14.3%), LumU in 1 (7.1%), LumNS in 1 (7.1%), stroma-rich in 2 (14.3%) and NE-like in 1 (7.1%) case based on MACE- or HTP-derived transcriptome data. Consensus subtypes were concordant in 71% (10/14) of cases when comparing MACE with HTP data. Four cases with aberrant subtypes had a stroma-rich molecular subtype with either method. The overlap of the molecular consensus subtypes with the reduced ESSEN1 and ESSEN2 panels were 86% and 100%, respectively, with HTP data and 86% with MACE data. Conclusion Determination of consensus molecular subtypes of MIBC from FFPE samples is feasible using various RNA sequencing methods. Inconsistent classification mainly involves the stroma-rich molecular subtype, which may be the consequence of sample heterogeneity with (stroma)-cell sampling bias and highlights the limitations of bulk RNA-based subclassification. Classification is still reliable when analysis is reduced to selected genes

CK5/6 and GATA3 defined phenotypes of muscle-invasive bladder cancer: impact in adjuvant chemotherapy and molecular subtyping of negative cases

Introduction and Objective: Identifying patients that benefit from cisplatin-based adjuvant chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC). The purpose of this study is to correlate “luminal” and “basal” type protein expression with histological subtypes, to investigate the prognostic impact on survival after adjuvant chemotherapy and to define molecular consensus subtypes of “double negative” patients (i.e., without expression of CK5/6 or GATA3). Materials and Methods: We performed immunohistochemical (IHC) analysis of CK5/6 and GATA3 for surrogate molecular subtyping in 181 MIBC samples. The mRNA expression profiles for molecular consensus classification were determined in CK5/6 and GATA3 (double) negative cases using a transcriptome panel with 19.398 mRNA targets (HTG Molecular Diagnostics). Data of 110 patients undergoing radical cystectomy were available for survival analysis. Results: The expression of CK5/6 correlated with squamous histological subtype (96%) and expression of GATA3 was associated with micropapillary histology (100%). In the multivariate Cox-regression model, patients receiving adjuvant chemotherapy had a significant survival benefit (hazard ratio [HR]: 0.19 95% confidence interval [CI]: 0.1–0.4, p < 0.001) and double-negative cases had decreased OS (HR: 4.07; 95% CI: 1.5–10.9, p = 0.005). Double negative cases were classified as NE-like (30%), stroma-rich (30%), and Ba/Sq (40%) consensus molecular subtypes and displaying different histological subtypes

Mri-fusion targeted vs. Systematic prostate biopsy–how does the biopsy technique affect gleason grade concordance and upgrading after radical prostatectomy?

Introduction: MRI-targeted biopsy (TB) increases overall prostate-cancer (PCa) detection-rates and decreases the risk of insignificant PCa detection. However, the impact of these findings on the definite pathology after radical prostatectomy (RP) is under debate. Materials and Methods: Between 01/2014 and 12/2018, 366 patients undergoing prostate biopsy and RP were retrospectively analyzed. The correlation between biopsy Gleason-score (highest Gleason-score in a core) and the RP Gleason-score in patients undergoing systematic biopsy (SB-group) (n = 221) or TB+SB (TB-group, n = 145) was tested using the ISUP Gleason-group grading (GGG, scale 1–5). Sub analyses focused on biopsy GGG 1 and GGG ≥ 2. Results: Proportions of biopsy GGG 1–5 in the SB-group and TB-group were 24.4, 37.6, 19, 10.9, 8.1% and 13.8, 43.4, 24.2, 13.8, 4.8%, respectively (p = 0.07). Biopsy and pathologic GGG were concordant in 108 of 221 (48.9%) in SB- and 74 of 145 (51.1%) in TB-group (p = 0.8). Gleason upgrading was recorded in 33.5 and 31.7% in SB- vs. TB-group (p = 0.8). Patients with biopsy GGG 1 undergoing RP showed an upgrading in 68.5%(37/54) in SB- and 75%(15/20) in TB-group (p = 0.8). In patients with biopsy GGG ≥ 2 concordance increased for both biopsy approaches (54.5 vs. 55.2% for SB- vs. TB-group, p = 0.9). Discussion: Irrespective of differences in PCa detection-rates between TB- and SB-groups, no significant differences in GGG concordance and upgrading between patients of both groups undergoing biopsy, followed by RP, were recorded. Concordance rates increased in men with biopsy GGG ≥ 2. TB seems to detect more patients with PCa without a difference in concordance with final pathology

Overexpression of KMT9α Is Associated with Aggressive Basal-like Muscle-Invasive Bladder Cancer

Muscle-invasive bladder cancer (MIBC) is associated with limited response rates to systemic therapy leading to a significant risk of recurrence and death. A recently discovered histone methyltransferase KMT9, acts as an epigenetic regulator of carcinogenesis in different tumor entities. In this study, we investigated the presence and association of histological and molecular subtypes and their impact on the survival of KMT9α in MIBC. We performed an immunohistochemical (IHC) analysis of KMT9α in 135 MIBC patients undergoing radical cystectomy. KMT9α was significantly overexpressed in the nucleus in MIBC compared to normal urothelium and low-grade urothelial cancer. Using the HTG transcriptome panel, we assessed mRNA expression profiles to determine molecular subtypes and identify differentially expressed genes. Patients with higher nuclear and nucleolar KMT9α expression showed basal/squamous urothelial cancer characteristics confirmed by IHC and differentially upregulated KRT14 expression. We identified a subset of patients with nucleolar expression of KMT9α, which was associated with an increased risk of death in uni- and multivariate analyses (HR 2.28, 95%CI 1.28–4.03, p = 0.005). In conclusion, basal-like MIBC and the squamous histological subtype are associated with high nuclear KMT9α expression. The association with poor survival makes it a potential target for the treatment of bladder cancer