BAP1 and PTEN mutations shape the immunological landscape of clear cell renal cell carcinoma and reveal the intertumoral heterogeneity of T cell suppression: a proof-of-concept study.

Clear cell renal cell carcinoma (ccRCC) is an immunologically vulnerable tumor entity, and immune checkpoint inhibitors are now widely used to treat patients with advanced disease. Whether and to what extent immune responses in ccRCC are shaped by genetic alterations, however, is only beginning to emerge. In this proof-of-concept study, we performed a detailed correlative analysis of the mutational and immunological landscapes in a series of 23 consecutive kidney cancer patients. We discovered that a high infiltration with CD8 + T cells was not dependent on the number of driver mutations but rather on the presence of specific mutational events, namely pathogenic mutations in PTEN or BAP1. This observation encouraged us to compare mechanisms of T cell suppression in the context of four different genetic patterns, i.e., the presence of multiple drivers, a PTEN or BAP1 mutation, or the absence of detectable driver mutations. We found that ccRCCs harboring a PTEN or BAP1 mutation showed the lowest level of Granzyme B positive tumor-infiltrating lymphocytes (TILs). A multiplex immunofluorescence analysis revealed a significant number of CD8 + TILs in the vicinity of CD68 + macrophages/monocytes in the context of a BAP1 mutation but not in the context of a PTEN mutation. In line with this finding, direct interactions between CD8 + TILs and CD163 + M2-polarized macrophages were found in BAP1-mutated ccRCC but not in tumors with other mutational patterns. While an absence of driver mutations was associated with more CD8 + TILs in the vicinity of FOXP3 + Tregs and CD68 + monocytes/macrophages, the presence of multiple driver mutations was, to our surprise, not found to be strongly associated with immunosuppressive mechanisms. Our results highlight the role of genetic alterations in shaping the immunological landscape of ccRCC. We discovered a remarkable heterogeneity of mechanisms that can lead to T cell suppression, which supports the need for personalized immune oncological approaches.

Senescent Tumor Cells Are Frequently Present at the Invasion Front: Implications for Improving Disease Control in Patients with Locally Advanced Prostate Cancer.

INTRODUCTION: Local tumor invasion is a critical factor for the outcome of men with prostate cancer. In particular, seminal vesicle invasion (SVI) has been reported to be associated with a more unfavorable prognosis. A better understanding of the functional state of invading prostate cancer cells is crucial to develop novel therapeutic strategies for patients with locally advanced disease. METHODS: The prognostic impact of local tumor progression was ascertained in over 1,000 men with prostate cancer. Prostate cancer specimens were stained by double-immunohistochemistry for the proliferation marker Ki-67 and the senescence marker p16INK4A. The migratory properties of senescent prostate cancer cells were analyzed in vitro using a wound healing assay and immunofluorescence microscopy for p16INK4A. RESULTS: We confirm the notion that patients with SVI have a more unfavorable prognosis than patients with extraprostatic extension alone. Surprisingly, we found that the tumor invasion front frequently harbors p16INK4A-positive and Ki-67-negative, i.e., senescent, tumor cells. While the intraprostatic tumor periphery was a hotspot for both proliferation and expression of p16INK4A, the area of SVI showed less proliferative activity but was at the same time a hotspot of cells with increased nuclear p16INK4A expression. Senescence was associated with an accelerated migration of prostate cancer cells in vitro. CONCLUSION: This proof-of-concept study shows that invading prostate cancer cells frequently show signs of cellular senescence. This finding may open new avenues for neoadjuvant and adjuvant treatment concepts in men with locally advanced prostate cancer.

Interleukin-2 and Interferon-α for Advanced Renal Cell Carcinoma: Patient Outcomes, Sexual Dimorphism of Responses, and Multimodal Treatment Approaches over a 30-Year Period.

INTRODUCTION: Cytokine-based immunotherapy (IT) has been the mainstay of systemic treatment of advanced renal cell carcinoma (RCC) from the late 1980s until 2007. With the introduction of immune checkpoint inhibitors, a renaissance of immune oncological approaches is rapidly unfolding. MATERIALS AND METHODS: In the present study, we revisited survival outcomes, sexual dimorphism of treatment responses, and the relevance of multimodal treatment approaches over a 30-year period in 156 patients with advanced RCC treated with subcutaneous (s.c.) interleukin-2 (IL-2) and interferon-α (IFN-α) between 1990 and 2009. RESULTS: The median progression-free survival following the first IT was 5.8 months with a wide range from 0 to 197 months. The median overall survival (OS) was 25.8 months and the median cancer-specific survival after tumor nephrectomy was 24.6 months. A group of 29 patients (18.6%) and 11 patients (7.1%) survived longer than 5 and 10 years after surgery, respectively. A difference in the 5-year OS rate between male and female patients was detected (men, 21.6%; women, 11.1%). However, no sex-specific survival advantage was observed after 10 years. CONCLUSIONS: We provide evidence that IT with s.c. IL-2 and IFN-α played a vital role in long-term survivors either by inducing lasting complete remissions or as part of multimodal approaches that allowed patients to survive until novel therapies became available. The implications for current immune oncological treatment approaches are being discussed.

Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells.

Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel-Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1-actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC.

Microenvironment-Derived FGF-2 Stimulates Renal Cell Carcinoma Cell Proliferation through Modulation of p27Kip1: Implications for Spatial Niche Formation and Functional Intratumoral Heterogeneity.

BACKGROUND/OBJECTIVE: Clear cell renal cell carcinoma (ccRCC) is characterized by a high degree of functional intratumoral heterogeneity (ITH). This is highlighted by the finding that tumor cell proliferation and intracellular signaling occur preferentially in the tumor periphery. The driving forces for such a spatial organization are largely unknown. Herein, we investigate the role of the tumor microenvironment in the control of tumor cell proliferation and functional ITH. METHODS: Conditioned media (CM) derived from nonmalignant peritumoral kidney tissue were used to stimulate RCC cells in vitro. A neutralization assay was used to characterize the role of FGF-2 in the CM. The molecular mechanisms underlying the action of CM on RCC cells were investigated using immunoblotting, flow cytometry and immunofluorescence microscopy. Lastly, a series of ccRCCs were stained for Ki-67 and p27Kip1, and expression was analyzed in both tumor periphery and center. RESULTS: We show that CM derived from nonmalignant kidney cells adjacent to an RCC can downregulate the expression of the CDK inhibitor p27Kip1 through enhanced protein degradation in an FGF-2-dependent fashion. FGF-2 functions mainly through the PI3K/AKT pathway downstream of its receptors, and RCC cells with constitutively high AKT activity show not only an enhanced degradation of p27Kip1 through the Emi1-Skp2 axis, but also a subcellular mislocalization of p27Kip1 to the cytoplasmic compartment. Such a mislocalization was also detected in the tumor periphery in vivo suggesting that p27Kip1 plays an important role in shaping this spatial niche. CONCLUSIONS: Our results suggest that the tumor microenvironment is involved in shaping the tumor peripheral niche by stimulating the enhanced proliferation that is characteristic for this zone.

The BRCA2 mutation status shapes the immune phenotype of prostate cancer.

Defects in DNA damage repair caused by mutations in BRCA1/2, ATM or other genes have been shown to play an important role in the development and progression of prostate cancer. The influence of such mutations on anti-tumor immunity in prostate cancer, however, is largely unknown. To better understand the correlation between BRCA1/2 mutations and the immune phenotype in prostate cancer, we characterized the immune infiltrate of eight BRCA2-mutated tumors in comparison with eight BRCA1/2 wild-type patients by T-cell receptor sequencing and immunohistochemistry for CD45, CD4, CD8, FOXP3, and CD163. In addition, we analyzed seven prostate cancer biopsies that were either BRCA2 or ATM-mutated in comparison with wild-type tumors. Whereas in BRCA1/2 wild-type tumors, immune cells were found predominantly extratumorally, most BRCA2-mutated tumors including one biopsy showed a significantly increased intratumoral immune cell infiltration. The ratio of intratumoral to extratumoral immune cells was considerably higher in BRCA2-mutated tumors for all markers and reached statistical significance for CD4 (p = 0.007), CD8 (p = 0.006), and FOXP3 (p = 0.001). However, the intratumoral CD8 to FOXP3 ratio showed a trend to be lower in BRCA2-mutated tumors suggesting a more suppressed tumor immune microenvironment. Our findings provide a rationale for the future use of immune oncological approaches in BRCA2-mutated prostate cancer and may encourage efforts to target immunosuppressive T-cell populations to prime tumors for immunotherapy.

APOBEC3A Expression in Penile Squamous Cell Carcinoma.

BACKGROUND: APOBECs (apolipoprotein B mRNA-editing catalytic polypeptides) are cytidine deaminases that have been implicated in the host defense against viruses by blocking viral replication. They have also been shown to play a role in genome hypermutation in several human cancers. An APOBEC3 hypermutation signature has been discovered in cervical cancer, which is intimately associated with infection by high-risk human papillomaviruses (HPVs). At the same time, HPV genomes themselves are subject to DNA editing by APOBECs. Similar to cervical cancer, a proportion of penile squamous cell carcinomas (SCCs) is etiologically driven by high-risk HPVs, but very little is known about the role of APOBECs in penile SCC development and progression. METHODS: A series of 34 penile SCCs was analyzed for the expression of APOBEC3A protein by immunohistochemistry. HPV genotyping was carried out using a bead-based multiplex hybridization assay preceded by BSGP5+6+ primer-based amplification. RESULTS: We found a frequent reduction of APOBEC3A protein expression in the invasive parts of the majority of HPV-negative penile SCCs. In contrast, the majority of HPV-positive penile SCCs retained APOBEC3A expression during malignant progression. CONCLUSION: Our results suggest that APOBEC3A expression is downregulated during progression towards invasiveness in HPV-negative penile SCC, but maintained in HPV-positive penile SCC. How high-risk HPV-infected tumor cells tolerate high APOBEC3A, which appears to exert tumor suppressive functions in HPV-negative penile SCCs, remains to be elucidated.

Digital spatial profiling identifies the tumor center as a topological niche in prostate cancer characterized by an upregulation of BAD.

Prostate cancer is characterized by a high degree of intratumoral heterogeneity. However, little is known about the spatial distribution of cancer cells with respect to specific functional characteristics and the formation of spatial niches. Here, we used digital spatial profiling (DSP) to investigate differences in protein expression in the tumor center versus the tumor periphery. Thirty-seven regions of interest were analyzed for the expression of 47 proteins, which included components of the PI3K-AKT, MAPK, and cell death signaling pathways as well as immune cell markers. A total of 1739 data points were collected from five patients. DSP identified the BCL-2 associated agonist of cell death (BAD) protein as the most significantly upregulated protein in the tumor center. BAD upregulation was confirmed by conventional immunohistochemistry, which furthermore showed a phosphorylation of BAD at serine 112 indicating its inactivation. Knockdown of BAD in prostate cancer cells in vitro led to decreased cell viability and colony growth. Clinically, high BAD expression was associated with a shorter time to biochemical recurrence in 158 mostly high-risk prostate cancer patients. Collectively, our results suggest that the tumor center is a topological niche with high BAD expression that may drive prostate cancer progression.

Immune landscape of renal cell carcinoma with metastasis to the pancreas.

INTRODUCTION: Clear cell Renal Cell Carcinoma (ccRCC) has a poor prognosis once metastatic. However, certain metastatic sites have been reported to have a different impact on the patient prognosis. For example, patients with pancreatic metastases have a much more favorable prognosis than those with metastases to other organs. The biological basis for this observation remains poorly understood. The aim of this study was to characterize the immune landscape of pancreatic metastases and the corresponding primary tumors in order to identify possible immunological features that correlate with disease biology. PATIENTS AND METHODS: A detailed assessment of immune cell populations was performed using a total of 1,700 microscopic images from ccRCCs from 11 patients, their corresponding pancreatic metastases and ccRCCs from 10 patients without pancreatic metastases. Tumor specimens were stained for CD45, CD8, CD163 and FOXP3 and the densities of the respective immune cells were assessed semiquantitatively in the intratumoral and extratumoral compartment. Multispectral imaging was performed in selected tumors. RESULTS: We found that pancreatic metastases show the lowest intratumoral infiltration with CD8+ cytotoxic T lymphocytes of all tumor specimens analyzed. The frequency of CD8+ lymphocytes was on 1.9 fold lower in pancreatic metastases (median density 8.3 cells per field of view [FOV] = 1.23 mm2) when compared to the corresponding primary tumor (15.6 cells per FOV, P = 0.0002) and more than 3-fold lower when compared to ccRCCs without pancreatic metastases (27.2 cells per FOV, P = 0.0012). There was also a significantly reduced intratumoral infiltration with immunosuppressive FOXP3+ lymphocytes in pancreatic metastases (2.6 cells per FOV, P = 0.009) and corresponding primary tumors (2 cells per FOV, P = 0.028) when compared to ccRCCs without pancreatic metastases (5.6 cells per FOV). CONCLUSIONS: In this proof-of-concept study, we show that pancreatic metastases of ccRCC present with unique immunological features including a low intratumoral density of CD8+ and FOXP3+ lymphocytes. The low counts of CD8+ and FOXP3+ lymphocytes may reflect less aggressive features of ccRCC with pancreatic metastasis that may result in a more favorable patient prognosis.

PD-L1 as a Urine Biomarker in Renal Cell Carcinoma-A Case Series and Proof-of-Concept Study.

BACKGROUND: Renal cell carcinoma (RCC) is among the most lethal urologic malignancies once metastatic. Current treatment approaches for metastatic RCC (mRCC) involve immune checkpoint inhibitors (ICIs) that target the PD-L1/PD-1 axis. High PD-L1 expression in tumor tissue has been identified as a negative prognostic factor in RCC. However, the role of PD-L1 as a liquid biomarker has not yet been fully explored. Herein, we analyze urine levels of PD-L1 in mRCC patients before and after either ICI therapy or surgical intervention, as well as in a series of patients with treatment-naïve RCC. PATIENTS AND METHODS: The mid-stream urine of patients with mRCC (n = 4) or treatment-naïve RCC, i.e., prior to surgery from two centers (cohort I, n = 49: cohort II, n = 29) was analyzed for PD-L1 by ELISA. The results from cohort I were compared to a control group consisting of patients treated for non-malignant urologic diseases (n = 31). In the mRCC group, urine PD-L1 levels were measured before and after tumor nephrectomy (n = 1) or before and after ICI therapy (n = 3). Exosomal PD-L1 in the urine was analyzed in selected patients by immunoblotting. RESULTS: A strong decrease in urine PD-L1 levels was found after tumor nephrectomy or following systemic treatment with ICIs. In patients with treatment-naïve RCC (cohort I), urine PD-L1 levels were significantly elevated in the RCC group in comparison to the control group (median 59 pg/mL vs. 25.7 pg/mL, p = 0.011). PD-L1 urine levels were found to be elevated, in particular, in low-grade RCCs in cohorts I and II. Exosomal PD-L1 was detected in the urine of a subset of patients. CONCLUSION: In this proof-of-concept study, we show that PD-L1 can be detected in the urine of RCC patients. Urine PD-L1 levels were found to correlate with the treatment response in mRCC patients and were significantly elevated in treatment-naïve RCC patients.

Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment.

Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide.

Long-Term Survival of Patients with Stage T1N0M1 Renal Cell Carcinoma.

Metastatic renal cell carcinoma (RCC) is among the most lethal urological malignancies. However, small, localized RCCs (≤7 cm, stage T1) have an excellent prognosis. There is a rare patient subgroup diagnosed with synchronous distant metastasis (T1N0M1), of which very little is known in terms of survival outcomes and underlying disease biology. Herein, we examined the long-term survival of 27 patients with clear cell RCC (ccRCC) stage T1N0M1 in comparison to 18 patients without metastases (T1N0M0). Tumor tissue was stained by immunohistochemistry for CD8+ tumor infiltrating lymphocytes (TILs). As expected, patients with stage T1N0M1 showed a significantly worse median cancer specific survival (CSS; 2.8 years) than patients with stage T1N0M0 (17.7 years; HR 0.077; 95% CI, 0.022-0.262). However, eight patients (29.6%) with ccRCC stage T1N0M1 survived over five years, and three of those patients (11.1%) survived over a decade. Some of these patients benefitted from an intensified, multimodal treatment including metastasis-directed therapy. The number of CD8+ TILs was substantially higher in stage T1N0M1 ccRCCs than in stage T1N0M0 ccRCCs, suggesting a more aggressive tumor biology. In conclusion, long-term survival is possible in patients with ccRCC stage T1N0M1, with some patients benefitting from an intensified, multimodal treatment approach.

Digital Spatial Profiling Identifies the Tumor Periphery as a Highly Active Biological Niche in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by a high degree of intratumoral heterogeneity (ITH). Besides genomic ITH, there is considerable functional ITH, which encompasses spatial niches with distinct proliferative and signaling activities. The full extent of functional spatial heterogeneity in ccRCC is incompletely understood. In the present study, a total of 17 ccRCC tissue specimens from different sites (primary tumor, n = 11; local recurrence, n = 1; distant metastasis, n = 5) were analyzed using digital spatial profiling (DSP) of protein expression. A total of 128 regions of interest from the tumor periphery and tumor center were analyzed for the expression of 46 proteins, comprising three major signaling pathways as well as immune cell markers. Results were correlated to clinico-pathological variables. The differential expression of granzyme B was validated using conventional immunohistochemistry and was correlated to the cancer-specific patient survival. We found that a total of 37 proteins were differentially expressed between the tumor periphery and tumor center. Thirty-five of the proteins were upregulated in the tumor periphery compared to the center. These included proteins involved in cell proliferation, MAPK and PI3K/AKT signaling, apoptosis regulation, epithelial-to-mesenchymal transition, as well as immune cell markers. Among the most significantly upregulated proteins in the tumor periphery was granzyme B. Granzyme B upregulation in the tumor periphery correlated with a significantly reduced cancer-specific patient survival. In conclusion, this study highlights the unique cellular contexture of the tumor periphery in ccRCC. The correlation between granzyme B upregulation in the tumor periphery and patient survival suggests local selection pressure for aggressive tumor growth and disease progression. Our results underscore the potential of spatial biology for biomarker discovery in ccRCC and cancer in general.

Kidney Cancer Models for Pre-Clinical Drug Discovery: Challenges and Opportunities.

Renal cell carcinoma (RCC) is among the most lethal urological malignancies once metastatic. The introduction of immune checkpoint inhibitors has revolutionized the therapeutic landscape of metastatic RCC, nevertheless, a significant proportion of patients will experience disease progression. Novel treatment options are therefore still needed and in vitro and in vivo model systems are crucial to ultimately improve disease control. At the same time, RCC is characterized by a number of molecular and functional peculiarities that have the potential to limit the utility of pre-clinical model systems. This includes not only the well-known genomic intratumoral heterogeneity (ITH) of RCC but also a remarkable functional ITH that can be shaped by influences of the tumor microenvironment. Importantly, RCC is among the tumor entities, in which a high number of intratumoral cytotoxic T cells is associated with a poor prognosis. In fact, many of these T cells are exhausted, which represents a major challenge for modeling tumor-immune cell interactions. Lastly, pre-clinical drug development commonly relies on using phenotypic screening of 2D or 3D RCC cell culture models, however, the problem of "reverse engineering" can prevent the identification of the precise mode of action of drug candidates thus impeding their translation to the clinic. In conclusion, a holistic approach to model the complex "ecosystem RCC" will likely require not only a combination of model systems but also an integration of concepts and methods using artificial intelligence to further improve pre-clinical drug discovery.

Targeting the translational machinery in gastrointestinal stromal tumors (GIST): a new therapeutic vulnerability.

Although KIT-mutant GISTs can be effectively treated with tyrosine kinase inhibitors (TKIs), many patients develop resistance to imatinib mesylate (IM) as well as the FDA-approved later-line agents sunitinib, regorafenib and ripretinib. Resistance mechanisms mainly involve secondary mutations in the KIT receptor tyrosine kinase gene indicating continued dependency on the KIT signaling pathway. The fact that the type of secondary mutation confers either sensitivity or resistance towards TKIs and the notion that secondary mutations exhibit intra- and intertumoral heterogeneity complicates the optimal choice of treatment in the imatinib-resistant setting. Therefore, new strategies that target KIT independently of its underlying mutations are urgently needed. Homoharringtonine (HHT) is a first-in-class inhibitor of protein biosynthesis and is FDA-approved for the treatment of chronic myeloid leukemia (CML) that is resistant to at least two TKIs. HHT has also shown activity in KIT-mutant mastocytosis models, which are intrinsically resistant to imatinib and most other TKIs. We hypothesized that HHT could be effective in GIST through downregulation of KIT expression and subsequent decrease of KIT activation and downstream signaling. Testing several GIST cell line models, HHT led to a significant reduction in nascent protein synthesis and was highly effective in the nanomolar range in IM-sensitive and IM-resistant GIST cell lines. HHT treatment resulted in a rapid and complete abolishment of KIT expression and activation, while KIT mRNA levels were minimally affected. The response to HHT involved induction of apoptosis as well as cell cycle arrest. The antitumor activity of HHT was confirmed in a GIST xenograft model. Taken together, inhibition of protein biosynthesis is a promising strategy to overcome TKI resistance in GIST.

Mutations in TP53 or DNA damage repair genes define poor prognostic subgroups in primary prostate cancer.

BACKGROUND: Mutations in DNA damage repair genes, in particular genes involved in homology-directed repair, define a subgroup of men with prostate cancer with a more unfavorable prognosis but a therapeutic vulnerability to PARP inhibition. In current practice, mutational testing of prostate cancer patients is commonly done late i.e., when the tumor is castration resistant. In addition, most sequencing panels do not include TP53, one of the most crucial tumor suppressor genes in human cancer. In this proof-of-concept study, we sought to extend the clinical use of these molecular markers by exploring the early prognostic impact of mutations in TP53 and DNA damage repair genes in men with primary, nonmetastatic prostate cancer undergoing radical prostatectomy (RPX). METHODS: Tumor specimens from a cohort of 68 RPX patients with intermediate (n = 11, 16.2%) or high-risk (n = 57, 83.8%) disease were analyzed by targeted next generation sequencing using a 37 DNA damage repair and checkpoint gene panel including TP53. Sequencing results were correlated to clinicopathologic variables as well as PSA persistence or time to PSA failure. In addition, the distribution of TP53 and DNA damage repair gene mutations was analyzed in three large publicly available datasets (TCGA, MSKCC and SU2C). RESULTS: Of 68 primary prostate cancers analyzed, 23 (33.8%) were found to harbor a mutation in either TP53 (n = 12, 17.6%) or a DNA damage repair gene (n = 11, 16.2%). The vast majority of these mutations (22 of 23, 95.7%) were detected in primary tumors from patients with high-risk features. These mutations were mutually exclusive in our cohort and additional data mining suggests an enrichment of DNA damage repair gene mutations in TP53 wild-type tumors. Mutations in either TP53 or a DNA damage repair gene were associated with a significantly worse prognosis after RPX. Importantly, the presence of TP53/DNA damage repair gene mutations was an independent risk factor for PSA failure or PSA persistence in multivariate Cox regression models. CONCLUSION: TP53 or DNA damage repair gene mutations are frequently detected in primary prostate cancer with high-risk features and define a subgroup of patients with an increased risk for PSA failure or persistence after RPX. The significant adverse impact of these alterations on patient prognosis may be exploited to identify men with prostate cancer who may benefit from a more intensified treatment.

miR-449a Repression Leads to Enhanced NOTCH Signaling in TMPRSS2:ERG Fusion Positive Prostate Cancer Cells.

About 50% of prostate cancer (PCa) tumors are TMPRSS2:ERG (T2E) fusion-positive (T2E+), but the role of T2E in PCa progression is not fully understood. We were interested in investigating epigenomic alterations associated with T2E+ PCa. Using different sequencing cohorts, we found several transcripts of the miR-449 cluster to be repressed in T2E+ PCa. This repression correlated strongly with enhanced expression of NOTCH and several of its target genes in TCGA and ICGC PCa RNA-seq data. We corroborated these findings using a cellular model with inducible T2E expression. Overexpression of miR-449a in vitro led to silencing of genes associated with NOTCH signaling (NOTCH1, HES1) and HDAC1. Interestingly, HDAC1 overexpression led to the repression of HES6, a negative regulator of the transcription factor HES1, the primary effector of NOTCH signaling, and promoted cell proliferation by repressing the cell cycle inhibitor p21. Inhibition of NOTCH as well as knockdown of HES1 reduced the oncogenic properties of PCa cell lines. Using tissue microarray analysis encompassing 533 human PCa cores, ERG-positive areas exhibited significantly increased HES1 expression. Taken together, our data suggest that an epigenomic regulatory network enhances NOTCH signaling and thereby contributes to the oncogenic properties of T2E+ PCa.

Detection of PD-L1 in the urine of patients with urothelial carcinoma of the bladder.

There are currently five programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) inhibitors approved for the treatment of locally advanced or metastatic urothelial carcinoma (UC) of the bladder. For platinum-ineligible patients, testing of tumor specimens for PD-L1 expression is required. However, scoring of PD-L1 immunohistochemistry is complex due to different antibodies used, the requirement to score expression in different cellular compartments and intratumoral heterogeneity. It can also be difficult to obtain and test longitudinal tumor samples, which would be desirable to monitor treatment responses and tumor evolution under treatment-induced selective pressure. In the present proof-of concept study, we provide evidence that PD-L1 can be detected in the urine of patients with non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Urine PD-L1 levels were significantly higher in NMIBC and MIBC patients when compared to patients with various non-malignant urological diseases. Further prospective and independent studies are required to assess the value of PD-L1 in the urine as a novel biomarker with potential for the early detection, prediction and therapeutic monitoring of patients with UC of the bladder.

Targeting the Proteasome in Advanced Renal Cell Carcinoma: Complexity and Limitations of Patient-Individualized Preclinical Drug Discovery.

BACKGROUND: Systemic treatment options for metastatic renal cell carcinoma (RCC) have significantly expanded in recent years. However, patients refractory to tyrosine kinase and immune checkpoint inhibitors still have limited treatment options and patient-individualized approaches are largely missing. PATIENTS AND METHODS: In vitro drug screening of tumor-derived short-term cultures obtained from seven patients with clear cell RCC was performed. For one patient, a patient-derived xenograft (PDX) mouse model was established for in vivo validation experiments. Drug effects were further investigated in established RCC cell lines. RESULTS: The proteasome inhibitor carfilzomib was among the top hits identified in three of four patients in which an in vitro drug screening could be performed successfully. Carfilzomib also showed significant acute and long-term cytotoxicity in established RCC cell lines. The in vivo antitumoral activity of carfilzomib was confirmed in a same-patient PDX model. The cytotoxicity of carfilzomib was found to correlate with the level of accumulation of ubiquitinated proteins. CONCLUSIONS: In this proof-of-concept study, we show that patient-individualized in vitro drug screening and preclinical validation is feasible. However, the fact that carfilzomib failed to deliver a clinical benefit in RCC patients in a recent phase II trial unrelated to the present study underscores the complexities and limitations of this strategy.

Antibody selection influences the detection of AR-V7 in primary prostate cancer.

BACKGROUND: The androgen receptor (AR) splice variant V7 (AR-V7) is an emerging marker to aid clinical decision-making in patients with castration-resistant prostate cancer (CRPC). A number of studies have shown that a subset of patients also express AR-V7 in the primary tumor. These findings have recently been challenged by a study showing that AR-V7 becomes only detectable in CRPC but is virtually absent in castration-naïve prostate cancer. METHODS: Herein, we directly compare the two relevant antibodies used for the immunodetection of AR-V7 in the conflicting studies (clones AG10008 and RM7) in a predominantly high-risk prostate cancer patient cohort with primary tumor specimens assembled in a tissue microarray (TMA). RESULTS: The overall rate of AR-V7 positive TMA cores was comparable (AG10008, 24.9%; RM7, 21%). However, the percentage agreement of identical staining intensities of positive cores was only 7%. In contrast, the percentage agreement of negative cores was 62.8%. In approximately 30% of the cores, the antibodies produced discordant staining intensities. Only one of the two antibody stainings (AG10008) conveyed prognostic information and was associated with a shorter progression-free patient survival. CONCLUSIONS: Our study underscores that nuclear AR-V7 expression can be detected in primary prostate cancer prior to long-term androgen deprivation and castration resistance. There are staining differences between the two antibodies in tumor tissue, for which we currently have no explanation. Clearly, improvements in the detection of functional AR-V7 in prostate cancer are urgently needed.