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.

Autologous Stem Cell Collection after Daratumumab, Bortezomib, Thalidomide, and Dexamethasone versus Bortezomib, Cyclophosphamide, and Dexamethasone in Newly Diagnosed Multiple Myeloma.

Introduction: In transplant-eligible, newly diagnosed multiple myeloma (NDMM) patients, autologous peripheral blood stem cell (PBSC) collection is usually pursued after induction therapy. While induction regimens are constantly refined regarding response, their impact on PBSC collection is not fully studied. The inclusion of the anti-CD38 antibody daratumumab into induction therapy significantly improved outcomes for patients with NDMM, e.g., as part of the daratumumab, bortezomib, thalidomide, and dexamethasone (Dara-VTD) protocol. Preliminary data from the phase 3 CASSIOPEIA study proved the efficacy of Dara-VTD. While overall PBSC collection upon addition of daratumumab was reduced in the study population, more detailed analyses on the impact are missing. Methods: We here report on PBSC mobilization and collection metrics in n = 119 patients with NDMM who underwent induction therapy with bortezomib, cyclophosphamide, and dexamethasone (VCD, n = 61) or Dara-VTD (n = 58). Results: Patient characteristics were well balanced between groups. The Dara-VTD group showed improved response parameters with 66% of patients reaching at least very good partial response versus 54% in the VCD group. Dara-VTD patients exhibited inferior mobilization metrics such as peripheral blood CD34+ cell count at the first leukapheresis (LP) session (65 vs. 106/µL, p = 0.001), median number of LP sessions (2 vs. 1, p = 0.001), and PBSC collection at first LP (5.5 vs. 8.3 × 106/kg body weight [bw], p = 0.001). Utilization of plerixafor was slightly higher after Dara-VTD (33% vs. 21% of patients, p = 0.143). The overall PBSC collection result was significantly lower after Dara-VTD (8.4 vs. 9.6 × 106/kg bw, p = 0.026). 78% and 85% of patients successfully collected 3 transplants with ≥2 × 106 CD34+ cells/kg bw in the Dara-VTD and the VCD groups, respectively. Conclusion: In summary, Dara-VTD, possibly due to both anti-CD38 antibody and thalidomide exposure, imposes a limitation on PBSC collection which can be only partly overcome by utilization of plerixafor.

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.

PARP inhibition in prostate cancer.

Defects in DNA damage repair genes are more common in prostate cancer than previously thought. These alterations provide an opportunity for precision oncology approaches and a number of studies have now shown that PARP inhibitors can have significant antitumor activity in men with DNA damage repair-deficient metastatic castration-resistant prostate cancer. This review summarizes the key clinical trials related to the use of PARP inhibitors in prostate cancer. Besides clinical outcomes, toxicity, and PARP inhibitor resistance, the role of different DNA repair genes in the response to PARP inhibition will be discussed.

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.

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.

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.

Patients Resistant Against PSMA-Targeting α-Radiation Therapy Often Harbor Mutations in DNA Damage-Repair-Associated Genes.

Prostate-specific membrane antigen (PSMA)-targeting α-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer. There is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The aim of this work was to characterize PSMA-TAT-nonresponding lesions by targeted next-generation sequencing. Methods: Of 60 patients treated with 225Ac-PSMA-617, we identified 10 patients who presented with a poor response despite sufficient tumor uptake in PSMA PET/CT. We were able to perform CT-guided biopsies with histologic validation of the nonresponding lesions in 7 of these nonresponding patients. Specimens were analyzed by targeted next-generation sequencing interrogating 37 DNA damage-repair-associated genes. Results: In the 7 tumor samples analyzed, we found a total of 15 whole-gene deletions, deleterious or presumably deleterious mutations affecting TP53 (n = 3), CHEK2 (n = 2), ATM (n = 2), and BRCA1, BRCA2, PALB2, MSH2, MSH6, NBN, FANCB, and PMS1 (n = 1 each). The average number of deleterious or presumably deleterious mutations was 2.2 (range, 0-6) per patient. In addition, several variants of unknown significance in ATM, BRCA1, MSH2, SLX4, ERCC, and various FANC genes were detected. Conclusion: Patients with resistance to PSMA-TAT despite PSMA positivity frequently harbor mutations in DNA damage-repair and checkpoint genes. Although the causal role of these alterations in the patient outcome remains to be determined, our findings encourage future studies combining PSMA-TAT and DNA damage-repair-targeting agents such as poly(ADP-ribose)-polymerase inhibitors.

Rearranged ERG confers robustness to prostate cancer cells by subverting the function of p53.

OBJECTIVE: ERG rearrangements are frequent and early events in prostate cancer. The functional role of rearranged ERG, however, is still incompletely understood. ERG rearrangements are maintained during prostate cancer progression suggesting that they may confer a selective advantage. The molecular basis of this notion is the subject of this study. METHODS: A variety of immunological methods were used to characterize the effects of rearranged ERG on p53. Consequences of an overexpression of N-terminally deleted ERG on p53 function were interrogated by measuring apoptosis and cellular senescence in the presence or absence of exogenous DNA damage. Effects of N-terminally deleted ERG on the transactivation function of p53 were analyzed by qRT-PCR. RESULTS: We show that overexpression of ERG leads to an increased basal level of DNA damage and a stabilization of p53 that involves a sequestration of its E3 ubiquitin ligase, MDM2, into nucleoli. A higher p53 expression was also observed in vivo in an ERG-overexpressing prostatic intraepithelial neoplasia mouse model. The correlation between ERG and p53 expression was corroborated in 163 patients with prostate cancer. ERG overexpression was found to inhibit both apoptosis and cellular senescence induced by exogenous DNA damage. Mechanistically, this protective effect of ERG involved an abrogation of the DNA damage-induced expression of p53 target genes. CONCLUSIONS: By protecting tumor cells from the antiproliferative consequences of genotoxic stress, ERG may allow the survival and proliferation of genomically unstable tumor cells. Targeting ERG may therefore represent a promising strategy to suppress such adverse features during prostate cancer progression.

High prevalence of DNA damage repair gene defects and TP53 alterations in men with treatment-naïve metastatic prostate cancer -Results from a prospective pilot study using a 37 gene panel.

BACKGROUND: Defects in DNA damage repair genes characterize a subset of men with prostate cancer and provide an attractive opportunity for precision oncology approaches. The prevalence of such perturbations in newly diagnosed, treatment-naïve patients with a high risk for lethal disease outcome, however, has not been sufficiently explored. PATIENTS AND METHODS: Prostate cancer specimens from 67 men with newly diagnosed early onset, localized high-risk/locally advanced or metastatic prostate cancer were included in this prospective pilot study. Tumor samples, including 30 prostate biopsies, were analyzed by targeted next generation sequencing using a formalin-fixed, paraffin-embedded tissue-optimized 37 DNA damage repair and checkpoint gene panel. RESULTS: The drop-out rate due to an insufficient quantity of DNA was 4.5% (3 of 67 patients). In the remaining 64 patients, the rate of pathogenic DNA damage repair gene mutations was 26.6%. The highest rate of pathogenic DNA damage repair and checkpoint gene mutations was found in men with treatment-naïve metastatic prostate cancer (38.9%). In addition, a high number of likely pathogenic mutations and gene deletions were detected. Altogether, one or more pathogenic mutation, likely pathogenic mutation or gene deletion affected 43 of 64 patients (67.2%) including 29 of 36 patients (80.6%) with treatment-naïve metastatic prostate cancer. Men with metastatic prostate cancer showed a high prevalence of alterations in TP53 (36.1%). CONCLUSIONS: This pilot study demonstrates the feasibility, performance and clinical relevance of somatic targeted next generation sequencing using a unique 37 DNA damage repair and checkpoint gene panel under routine conditions. Our results indicate that this approach can detect actionable DNA repair gene alterations, uncommon mutations as well as mutations associated with therapy resistance in a high number of patients, in particular patients with treatment-naïve metastatic prostate cancer.

Clinical characteristics, treatment outcomes and potential novel therapeutic options for patients with neuroendocrine carcinoma of the prostate.

BACKGROUND: Neuroendocrine carcinomas of the prostate (NEPCs) are rare tumors with poor prognosis. While platinum and etoposide-based chemotherapy regimens (PE) are commonly applied in first-line for advanced disease, evidence for second-line therapy and beyond is very limited. METHODS: Retrospective analysis of all patients with NEPCs including mixed differentiation with adenocarcinoma component and well differentiated neuroendocrine tumors (NETs, carcinoids) at two high-volume oncological centers between 12/2000 and 11/2017. RESULTS: Of 46 identified patients 39.1 % had a prior diagnosis of prostatic adenocarcinoma only, 43.5 % had a mixed differentiation at NEPC diagnosis, 67.4 % developed visceral metastases, 10.9 % showed paraneoplastic syndromes. Overall survival (OS) from NEPC diagnosis was 15.5 months, and significantly shorter in patients with a prior prostatic adenocarcinoma (5.4 vs. 32.7 months, p=0.005). 34 patients received palliative first-line systemic therapy with a median progression-free survival (PFS) of 6.6 months, mostly PE. Overall response rate (ORR) for PE was 48.1 %. 19 patients received second-line therapy, mostly with poor responses. Active regimens were topotecan (1 PR, 3 PD), enzalutamide (1 SD), abiraterone (1 SD), FOLFIRI (1 SD), and ipilimumab+nivolumab (1 PR). One patient with prostatic carcinoid was sequentially treated with octreotide, peptide receptor radionuclide therapy and everolimus, and survived for over 9 years. CONCLUSIONS: EP in first-line shows notable ORR, however limited PFS. For second-line therapy, topotecan, FOLFIRI, enzalutamide, abiraterone and immune checkpoint blockade are treatment options. Prostatic carcinoids can be treated in analogy to well differentiated gastrointestinal NETs.

Overexpression of nuclear AR-V7 protein in primary prostate cancer is an independent negative prognostic marker in men with high-risk disease receiving adjuvant therapy.

BACKGROUND: Overexpression of the androgen receptor (AR) splice variant 7 (AR-V7) has recently been reported to be associated with resistance to antihormonal therapy. Herein, we address the question whether tumor cells with AR-V7 expression can be detected at the time of radical prostatectomy, that is, before long-term hormonal manipulation and castration resistance, and what the potential prognostic impact on the biochemical recurrence (BCR)-free survival may be. METHODS: An anti-AR-V7 antibody was first validated in a training set of prostate cancer specimens by a comparison of AR-V7 protein to AR-V7 mRNA expression. We then analyzed nuclear AR-V7 protein expression in the primary tumors and lymph node metastases from 163 predominantly high-risk patients (cohort I) as well as the primary tumors from patients of a second, consecutive patient cohort (n = 238, cohort II) not selected for any clinicopathological features. Staining results were correlated to patient characteristics and BCR-free patient survival. RESULTS: High nuclear AR-V7 protein expression was detected in approximately 30%-40% of patients in cohort I and II at the time of radical prostatectomy. High baseline expression of nuclear AR-V7 protein was associated with an unfavorable BCR-free survival in the high-risk patient cohort I but not in the unselected consecutive cohort II. Remarkably, AR-V7 was an independent negative prognostic factor in high-risk prostate cancer patients of cohort I who were selected to receive adjuvant treatment. CONCLUSIONS: Prostate cancer cells with high nuclear AR-V7 protein expression can be detected in a substantial proportion of tumors at the time of radical prostatectomy. The presence of AR-V7-positive tumor cells is associated with an unfavorable prognosis for BCR-free survival in a high-risk patient cohort including a subgroup of patients selected to receive adjuvant therapy, in which AR-V7 was an independent negative prognosticator. Overexpression of nuclear AR-V7 protein hence identifies a subset of tumors with remarkably aggressive growth characteristics among clinically and histologically high-risk patients at the time of radical prostatectomy.

Genomic features of renal cell carcinoma with venous tumor thrombus.

A venous tumor thrombus (VTT) is a potentially lethal complication of renal cell carcinoma (RCC) but virtually nothing is known about the underlying natural history. Based on our observation that venous thrombi contain significant numbers of viable tumor cells, we applied multiregion whole exome sequencing to a total of 37 primary tumor and VTT samples including normal tissue specimens from five consecutive patients. Our findings demonstrate mutational heterogeneity between primary tumor and VTT with 106 of 483 genes (22%) harboring functional SNVs and/or indels altered in either primary tumor or thrombus. Reconstruction of the clonal phylogeny showed clustering of tumor samples and VTT samples, respectively, in the majority of tumors. However, no new subclones were detected suggesting that pre-existing subclones of the primary tumor drive VTT formation. Importantly, we found several lines of evidence for "BRCAness" in a subset of tumors. These included mutations in genes that confer "BRCAness", a mutational signature and an increase of small indels. Re-analysis of SNV calls from the TCGA KIRC-US cohort confirmed a high frequency of the "BRCAness" mutational signature AC3 in clear cell RCC. Our findings warrant further pre-clinical experiments and may lead to novel personalized therapies for RCC patients.

Mutations in BRCA2 and taxane resistance in prostate cancer.

Mutations in BRCA1 or BRCA2 define a subset of prostate cancer patients. Herein, we address the question whether BRCA1/2 mutations have a predictive impact on chemotherapy with docetaxel, a widely used drug in patients with metastatic castration resistant prostate cancer (mCRPC). Fifty-three men treated with docetaxel for mCRPC were tested for somatic BRCA1/2 mutations of the primary tumor. In a subgroup of patients, BRCA1/2 protein expression was tested as a potential surrogate marker for BRCA1/2 inactivation. Eight of 53 patients (15.1%) harbored a deleterious BRCA2 mutation. No BRCA1 mutation was found. Patients with a BRCA2 mutation showed a response rate of 25% to docetaxel in comparison to 71.1% in men with wildtype BRCA2 (p = 0.019). While the time to develop castration resistance was similar in both subgroups, the overall survival was significantly shorter in patients harboring a BRCA2 mutation. No correlation between the BRCA1/2 protein expression and the response to docetaxel was found. While the presence of a BRCA2 mutation does not preclude a response to docetaxel, there is overall a significant correlation between BRCA2 inactivation and a poor response rate. Our results suggest that a close oncological monitoring of patients with BRCA2 mutations for taxane resistance is warranted.