First-Line Immune Combination Therapies for Nonclear Cell Versus Clear Cell Metastatic Renal Cell Carcinoma: Real-World Multicenter Data From Germany.

INTRODUCTION: The aim was to compare treatment outcomes of clear cell metastatic renal cell carcinoma (ccmRCC) versus non-ccmRCC (nccmRCC) patients who received first-line immune combination therapies. MATERIALS AND METHODS: Within our retrospective multi-institutional consecutive database of eight tertiary-care centers, we identified mRCC patients treated with first-line immune combination therapies between 11/2017 and 12/2022. Using log-rank analysis and multivariable Cox regression, we tested for differences in overall survival (OS) and progression-free survival (PFS) of nccmRCC versus ccmRCC patients. Covariables consisted of age at diagnosis, sex, International Metastatic Renal Cell Carcinoma Database Consortium risk groups, Eastern Cooperative Oncology Group status, and sarcomatoid feature. RESULTS: Of 289 study patients, 39 (13%) patients harbored nccmRCC. Median OS was 37 months versus not reached for ccmRCC versus nccmRCC patients (P = .6). Median PFS was 13 versus 15 months (P = .9). Multivariable Cox regression models did not identify nccmRCC as an independent predictor of higher overall mortality in mRCC patients (hazard ratio [HR]: 1.23; P = .6) or a higher progression rate (HR: 1.0; P = 1.0). CONCLUSION: In our real-world multi-institutional study, no differences in OS and PFS between ccmRCC and nccmRCC patients receiving first-line immune combination treatment were observed, even after adjustment for important patient and tumor characteristics. More prospective trials in nccmRCC patients are needed.

Identification of novel snoRNA-based biomarkers for clear cell renal cell carcinoma from urine-derived extracellular vesicles.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC with high rates of metastasis. Targeted therapies such as tyrosine kinase and checkpoint inhibitors have improved treatment success, but therapy-related side effects and tumor recurrence remain a challenge. As a result, ccRCC still have a high mortality rate. Early detection before metastasis has great potential to improve outcomes, but no suitable biomarker specific for ccRCC is available so far. Therefore, molecular biomarkers derived from body fluids have been investigated over the past decade. Among them, RNAs from urine-derived extracellular vesicles (EVs) are very promising. METHODS: RNA was extracted from urine-derived EVs from a cohort of 78 subjects (54 ccRCC patients, 24 urolithiasis controls). RNA-seq was performed on the discovery cohort, a subset of the whole cohort (47 ccRCC, 16 urolithiasis). Reads were then mapped to the genome, and expression was quantified based on 100 nt long contiguous genomic regions. Cluster analysis and differential region expression analysis were performed with adjustment for age and gender. The candidate biomarkers were validated by qPCR in the entire cohort. Receiver operating characteristic, area under the curve and odds ratios were used to evaluate the diagnostic potential of the models. RESULTS: An initial cluster analysis of RNA-seq expression data showed separation by the subjects' gender, but not by tumor status. Therefore, the following analyses were done, adjusting for gender and age. The regions differentially expressed between ccRCC and urolithiasis patients mainly overlapped with small nucleolar RNAs (snoRNAs). The differential expression of four snoRNAs (SNORD99, SNORD22, SNORD26, SNORA50C) was validated by quantitative PCR. Confounder-adjusted regression models were then used to classify the validation cohort into ccRCC and tumor-free subjects. Corresponding accuracies ranged from 0.654 to 0.744. Models combining multiple genes and the risk factors obesity and hypertension showed improved diagnostic performance with an accuracy of up to 0.811 for SNORD99 and SNORA50C (p = 0.0091). CONCLUSIONS: Our study uncovered four previously unrecognized snoRNA biomarkers from urine-derived EVs, advancing the search for a robust, easy-to-use ccRCC screening method.

Early kinetics of C-reactive protein as prognosticator for survival in a real-world cohort of patients with metastatic renal cell cancer under first-line therapy with immune checkpoint inhibitors.

PURPOSE: This study investigated the prognostic potential of baseline C-reactive protein (CRP) levels and early CRP kinetics in a real-world cohort of patients with metastatic renal cell carcinoma (mRCC) under first-line (1L) therapy with immune checkpoint inhibitors (CPI). METHODS/PATIENTS: Analyses were performed retrospectively in a cohort of 61 mRCC patients under CPI-based 1L therapy. Patients were stratified based on baseline CRP (< 10 vs ≥ 10 mg/l) and CRP change within the initial three months of CPI therapy (normal: baseline < 10 mg/l, normalized: baseline ≥ 10 mg/l and nadir < 10 mg/l, non-normalized: baseline and nadir ≥ 10 mg/l). Finally, the association of baseline CRP and CRP change with progression-free (PFS) and overall survival (OS) was evaluated. RESULTS: Baseline CRP was not significantly associated with both PFS (p = 0.666) and OS (p = 0.143). Following stratification according to early CRP kinetics, 23, 25 and 13 patients exhibited normal, normalized and non-normalized CRP levels, respectively. Patients with normal and normalized CRP had a markedly prolonged PFS (p = 0.091) and OS (p = 0.008) compared to patients with non-normalized CRP. Consequently, significantly better PFS (p = 0.031) and OS (p = 0.002) were observed for the combined normal-normalized group. In multivariate analysis including ECOG and IMDC risk, normalized CRP kinetics alone or in combination with the normal group was identified as significant independent risk factor for OS, whereas a statistical trend was observed for PFS. CONCLUSIONS: The present study emphasizes the prognostic potential of early CRP kinetics in CPI-treated mRCC. As a standard laboratory parameter, CRP can be easily implemented into clinical routine to facilitate therapy monitoring.

Early CRP kinetics to predict long-term efficacy of first-line immune-checkpoint inhibition combination therapies in metastatic renal cell carcinoma: an updated multicentre real-world experience applying different CRP kinetics definitions.

Objectives: Although biomarkers predicting therapy response in first-line metastatic renal carcinoma (mRCC) therapy remain to be defined, C-reactive protein (CRP) kinetics have recently been associated with immunotherapy (IO) response. Here, we aimed to assess the predictive and prognostic power of two contemporary CRP kinetics definitions in a large, real-world first-line mRCC cohort. Methods: Metastatic renal carcinoma patients treated with IO-based first-line therapy within 5 years were retrospectively included in this multicentre study. According to Fukuda et al., patients were defined as 'CRP flare-responder', 'CRP responder' and 'non-CRP responder'; according to Ishihara et al., patients were defined as 'normal', 'normalised' and 'non-normalised' based on their early CRP kinetics. Patient and tumor characteristics were compared, and treatment outcome was measured by overall (OS) and progression-free survival (PFS), including multivariable Cox regression analyses. Results: Out of 316 mRCC patients, 227 (72%) were assigned to CRP groups according to Fukuda. Both CRP flare- (HR [Hazard ratio]: 0.59) and CRP responders (HR: 0.52) had a longer PFS, but not OS, than non-CRP responders. According to Ishihara, 276 (87%) patients were assigned to the respective groups, and both normal and normalised patients had a significantly longer PFS and OS, compared with non-normalised group. Conclusion: Different early CRP kinetics may predict therapy response in first-line mRCC therapy in a large real-world cohort. However, further research regarding the optimal timing and frequency of measurement is needed.

Bevacizumab May Differentially Improve Prognosis of Advanced Ovarian Cancer Patients with Low Expression of VEGF-A165b, an Antiangiogenic VEGF-A Splice Variant.

PURPOSE: The identification of a robust IHC marker to predict the response to antiangiogenic bevacizumab in ovarian cancer is of high clinical interest. VEGF-A, the molecular target of bevacizumab, is expressed as multiple isoforms with pro- or antiangiogenic properties, of which VEGF-A165b is the most dominant antiangiogenic isoform. The balance of VEGF-A isoforms is closely related to the angiogenic capacity of a tumor and may define its vulnerability to antiangiogenic therapy. We investigated whether the expression of VEGF-A165b could be related to the effect of bevacizumab in advanced ovarian cancer patients. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tissues from 413 patients of the ICON7 multicenter phase III trial, treated with standard platinum-based chemotherapy with or without bevacizumab, were probed for VEGF-A165b expression by IHC. RESULTS: In patients with low VEGF-A165b expression, the addition of bevacizumab to standard platinum-based chemotherapy significantly improved progression-free (HR: 0.727; 95% CI, 0.538-0.984; P = 0.039) and overall survival (HR: 0.662; 95% CI, 0.458-0.958; P = 0.029). Multivariate analysis showed that the addition of bevacizumab in low VEGF-A165b-expressing patients conferred significant improvements in progression-free survival (HR: 0.610; 95% CI, 0.446-0.834; P = 0.002) and overall survival (HR: 0.527; 95% CI, 0.359-0.775; P = 0.001), independently from established risk factors. CONCLUSIONS: We demonstrate for the first time that bevacizumab may differentially improve the prognosis of advanced ovarian cancer patients with low expression of VEGF-A165b, an antiangiogenic VEGF-A splice variant. We envision that this novel biomarker could be implemented into routine diagnostics and may have direct clinical implications for guiding bevacizumab-related treatment decisions in advanced ovarian cancer patients.

Acquired resistance to irradiation or docetaxel is not associated with cross-resistance to cisplatin in prostate cancer cell lines.

PURPOSE: Platinum chemotherapy can be considered to treat metastatic castration-resistant prostate cancer (mCRPC) with features of neuroendocrine differentiation. However, platinum compounds are generally only applied after the failure of multiple prior-line treatment options. This study investigated whether acquired resistance against ionizing radiation or docetaxel chemotherapy-two commonly applied treatment modalities in prostate cancer-influences the cisplatin (CDDP) tolerance in mCRPC cell line models. METHODS: Age-matched parental as well as radio- or docetaxel-resistant DU145 and PC-3 cell lines were treated with CDDP and their sensitivity was assessed by measurements of growth rates, viability, apoptosis, metabolic activity and colony formation ability. RESULTS: The data suggest that docetaxel resistance does not influence CDDP tolerance in all tested docetaxel-resistant cell lines. Radio-resistance was associated with sensitization to CDDP in PC-3, but not in DU145 cells. In general, DU145 cells tolerated higher CDDP concentrations than PC-3 cells regardless of acquired resistances. Furthermore, non-age-matched treatment-naïve PC-3 cells exhibited significantly different CDDP tolerances. CONCLUSION: Like patients, different mCRPC cell lines exhibit significant variability regarding CDDP tolerance. The presented in vitro data suggest that previous radiation treatment may be associated with a moderate sensitization to CDDP in an isogenic and age-matched setting. Therefore, previous radiotherapy or docetaxel chemotherapy might be no contraindication against initiation of platinum chemotherapy in selected mCRPC patients.

Comprehensive Evaluation of Multiple Approaches Targeting ABCB1 to Resensitize Docetaxel-Resistant Prostate Cancer Cell Lines.

Docetaxel (DTX) is a mainstay in the treatment of metastatic prostate cancer. Failure of DTX therapy is often associated with multidrug resistance caused by overexpression of efflux membrane transporters of the ABC family such as the glycoprotein ABCB1. This study investigated multiple approaches targeting ABCB1 to resensitize DTX-resistant (DTXR) prostate cancer cell lines. In DU145 DTXR and PC-3 DTXR cells as well as age-matched parental controls, the expression of selected ABC transporters was analyzed by quantitative PCR, Western blot, flow cytometry and immunofluorescence. ABCB1 effluxing activity was studied using the fluorescent ABCB1 substrate rhodamine 123. The influence of ABCB1 inhibitors (elacridar, tariquidar), ABCB1-specific siRNA and inhibition of post-translational glycosylation on DTX tolerance was assessed by cell viability and colony formation assays. In DTXR cells, only ABCB1 was highly upregulated, which was accompanied by a strong effluxing activity and additional post-translational glycosylation of ABCB1. Pharmacological inhibition and siRNA-mediated knockdown of ABCB1 completely resensitized DTXR cells to DTX. Inhibition of glycosylation with tunicamycin affected DTX resistance partially in DU145 DTXR cells, which was accompanied by a slight intracellular accumulation and decreased effluxing activity of ABCB1. In conclusion, DTX resistance can be reversed by various strategies with small molecule inhibitors representing the most promising and feasible approach.

Targeted Quantification of Carbon Metabolites Identifies Metabolic Progression Markers and an Undiagnosed Case of SDH-Deficient Clear Cell Renal Cell Carcinoma in a German Cohort.

Renal cell carcinoma (RCC) is among the 10 most common cancer entities and can be categorised into distinct subtypes by differential expression of Krebs cycle genes. We investigated the predictive value of several targeted metabolites with regards to tumour stages and patient survival in an unselected cohort of 420 RCCs. Unsupervised hierarchical clustering of metabolite ratios identified two main clusters separated by α-ketoglutarate (α-KG) levels and sub-clusters with differential levels of the oncometabolite 2-hydroxyglutarate (2HG). Sub-clusters characterised by high 2HG were enriched in higher tumour stages, suggesting metabolite profiles might be suitable predictors of tumour stage or survival. Bootstrap forest models based on single metabolite signatures showed that lactate, 2HG, citrate, aspartate, asparagine, and glutamine better predicted the cancer-specific survival (CSS) of clear cell RCC patients, whereas succinate and α-ketoglutarate were better CSS predictors for papillary RCC patients. Additionally, this assay identifies rare cases of tumours with SDHx mutations, which are caused predominantly by germline mutations and which predispose to development of different neoplasms. Hence, analysis of selected metabolites should be further evaluated for potential utility in liquid biopsies, which can be obtained using less invasive methods and potentially facilitate disease monitoring for both patients and caregivers.

Serum miRNAs Support the Indication for MRI-Ultrasound Fusion-Guided Biopsy of the Prostate in Patients with Low-PI-RADS Lesions.

Multiparametric MRI (mpMRI) and targeted biopsy of the prostate enhance the tumor detection rate. However, the prediction of clinically significant prostate cancer (PCa) is still limited. Our study tested the additional value of serum levels of selected miRNAs in combination with clinical and mpMRI information for PCa prediction and classification. A total of 289 patients underwent targeted mpMRI-ultrasound fusion-guided prostate biopsy complemented by systematic biopsy. Serum miRNA levels of miRNAs (miR-141, miR-375, miR-21-5p, miR-320b, miR-210-3p, let-7c, and miR-486) were determined by quantitative PCR. Detection of any PCa and of significant PCa were the outcome variables. The patient age, pre-biopsy PSA level, previous biopsy procedure, PI-RADS score, and serum miRNA levels were covariates for regularized binary logistic regression models. The addition of miRNA expression of miR-486 and let-7c to the baseline model, containing only clinical parameters, increased the predictive accuracy. Particularly in patients with PI-RADS ≤3, we determined a sensitivity for detecting significant PCa (Gleason score ≥ 7a corresponding to Grade group ≥2) of 95.2%, and an NPV for absence of significant PCa of 97.1%. This accuracy could be useful to support patient counseling in selected cases.

Soluble Neuropilin-1 is an independent marker of poor prognosis in early breast cancer.

BACKGROUND: Neuropilin-1 (NRP-1) is a transmembrane protein that acts as a multifunctional non-tyrosine kinase receptor with an established role in development and immunity. NRP-1 also regulates tumor biology, and high expression levels of tissue NRP-1 have been associated with a poor prognosis. Recently, ELISA-based quantification of soluble NRP-1 (sNRP-1) has become available, but little is known about the prognostic value of sNRP-1 in malignancies. MATERIALS AND METHODS: We measured sNRP-1 in the serum of 509 patients with primary early breast cancer (BC) at the time of diagnosis using ELISA. RESULTS: Mean serum values of sNRP-1 were 1.88 ± 0.52 nmol/l (= 130.83 ± 36.24 ng/ml). SNRP-1 levels weakly correlated with age, and were higher in peri- and postmenopausal patients compared to premenopausal patients, respectively (p < 0.0001). Low levels of sNRP-1 were associated with a significant survival benefit compared to high sNRP-1 levels at baseline (p = 0.005; HR 1.94; 95%CI 1.23-3.06). These findings remained significant after adjustment for tumor stage including lymph node involvement, grading, hormone receptor, HER2 status, and age (p = 0.022; HR 1.78; 95%CI 1.09-2.91). CONCLUSION: Our findings warrant further investigations into the prognostic and therapeutic potential of sNRP-1 in BC.

Systemic Triple Therapy in Metastatic Hormone-Sensitive Prostate Cancer (mHSPC): Ready for Prime Time or Still to Be Explored?

For decades, mono androgen deprivation therapy (ADT) has been the gold standard for metastatic hormone-sensitive prostate cancer (mHSPC) treatment. Several studies have been published within the last seven years demonstrating a significant survival advantage by combination treatment with standard ADT plus docetaxel or androgen receptor-axis-targeted therapy (ARAT) compared to ADT monotherapy. As a result, overall survival can be prolonged by at least 18 months. Recently published congress data of the PEACE-1 study suggests that in the future, triple therapy might be the new gold standard. In addition to this study, which has shown that triple treatment with standard ADT plus docetaxel plus abiraterone is superior to standard ADT plus docetaxel, several other phase III triple therapy studies are currently ongoing. The different modes of action that are investigated reach from AR-targeting over mitotic inhibition and immunotherapy to PARP and AKT inhibition. In this review we will explore if triple therapy has the potential to be the new standard for mHSPC treatment in the near future.

Evaluation of MicroRNAs as Non-Invasive Diagnostic Markers in Urinary Cells from Patients with Suspected Prostate Cancer.

Currently used tumor markers for early diagnosis of prostate cancer (PCa) are often lacking sufficient specificity and sensitivity. Therefore, the diagnostic potential of selected microRNAs in comparison to serum PSA levels and PSA density (PSAD) was explored. A panel of 12 PCa-associated microRNAs was quantified by qPCR in urinary sediments from 50 patients with suspected PCa undergoing prostate biopsy, whereupon PCa was detected in 26 patients. Receiver operating characteristic (ROC) curve analyses revealed a potential for non-invasive urine-based PCa detection for miR-16 (AUC = 0.744, p = 0.012; accuracy = 76%) and miR-195 (AUC = 0.729, p = 0.017; accuracy = 70%). While serum PSA showed an insufficient diagnostic value (AUC = 0.564, p = 0.656; accuracy = 50%) in the present cohort, PSAD displayed an adequate diagnostic performance (AUC = 0.708, p = 0.031; accuracy = 70%). Noteworthy, the combination of PSAD with the best candidates miR-16 and miR-195 either individually or simultaneously improved the diagnostic power (AUC = 0.801-0.849, p < 0.05; accuracy = 76-90%). In the sub-group of patients with PSA ≤ 10 ng/mL (n = 34), an inadequate diagnostic power of PSAD alone (AUC = 0.595, p = 0.524; accuracy = 68%) was markedly surpassed by miR-16 and miR-195 individually as well as by their combination with PSAD (AUC = 0.772-0.882, p < 0.05; accuracy = 74-85%). These findings further highlight the potential of urinary microRNAs as molecular markers with high clinical performance. Overall, these results need to be validated in a larger patient cohort.

ProstaTrend-A Multivariable Prognostic RNA Expression Score for Aggressive Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is the most prevalent solid cancer among men in Western Countries. The clinical behavior of localized PCa is highly variable. Some cancers are aggressive leading to death, while others can even be monitored safely. Hence, there is a high clinical need for precise biomarkers for identification of aggressive disease in addition to established clinical parameters. OBJECTIVE: To develop an RNA expression-based score for the prediction of PCa prognosis that facilitates clinical decision making. DESIGN, SETTING, AND PARTICIPANTS: We assessed 233 tissue specimens of PCa patients with long-term follow-up data from fresh-frozen radical prostatectomies (RPs), from formalin-fixed and paraffin-embedded RP specimens and biopsies by transcriptome-wide next-generation sequencing and customized expression microarrays. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We applied Cox proportional hazard models to the cohorts from different platforms and specimen types. Evidence from these models was combined by fixed-effect meta-analysis to identify genes predictive of the time to death of disease (DoD). Genes were combined by a weighted median approach into a prognostic score called ProstaTrend and transferred for the prediction of biochemical recurrence (BCR) after RP in an independent cohort of The Cancer Genome Atlas (TCGA). RESULTS AND LIMITATIONS: ProstaTrend comprising ∼1400 genes was significantly associated with DoD in the training cohort of PCa patients treated by RP (leave-one-out cross-validation, Cox regression: p=2e-09) and with BCR in the TCGA validation cohort (Cox regression: p=3e-06). The prognostic impact persisted after multivariable Cox regression analysis adjusting for Gleason grading group (GG) ≥3 and resection status (p=0.001; DoD, training cohort) and for GG≥3, pathological stage ≥T3, and resection state (p=0.037; BCR, validation cohort). CONCLUSIONS: ProstaTrend is a transcriptome-based score that predicts DoD and BCR in cohorts of PCa patients treated with RP. PATIENT SUMMARY: ProstaTrend provides molecular patient risk stratification after radical prostatectomy.

Urinary MicroRNAs as Potential Markers for Non-Invasive Diagnosis of Bladder Cancer.

Currently, voided urine cytology (VUC) serves as the gold standard for the detection of bladder cancer (BCa) in urine. Despite its high specificity, VUC has shortcomings in terms of sensitivity. Therefore, alternative biomarkers are being searched, which might overcome these disadvantages as a useful adjunct to VUC. The aim of this study was to evaluate the diagnostic potential of the urinary levels of selected microRNAs (miRs), which might represent such alternative biomarkers due to their BCa-specific expression. Expression levels of nine BCa-associated microRNAs (miR-21, -96, -125b, -126, -145, -183, -205, -210, -221) were assessed by quantitative PCR in urine sediments from 104 patients with primary BCa and 46 control subjects. Receiver operating characteristic (ROC) curve analyses revealed a diagnostic potential for miR-96, -125b, -126, -145, -183, and -221 with area under the curve (AUC) values between 0.605 and 0.772. The combination of the four best candidates resulted in sensitivity, specificity, positive and negative predictive values (NPV), and accuracy of 73.1%, 95.7%, 97.4%, 61.1%, and 80.0%, respectively. Combined with VUC, sensitivity and NPV could be increased by nearly 8%, each surpassing the performance of VUC alone. The present findings suggested a diagnostic potential of miR-125b, -145, -183, and -221 in combination with VUC for non-invasive detection of BCa in urine.

High serum levels of periostin are associated with a poor survival in breast cancer.

PURPOSE: Periostin is a secreted extracellular matrix protein, which was originally described in osteoblasts. It supports osteoblastic differentiation and bone formation and has been implicated in the pathogenesis of several human malignancies, including breast cancer. However, little is known about the prognostic value of serum periostin levels in breast cancer. METHODS: In this study, we analyzed serum levels of periostin in a cohort of 509 primary, non-metastatic breast cancer patients. Disseminated tumor cell (DTC) status was determined using bone marrow aspirates obtained from the anterior iliac crests. Periostin levels were stratified according to several clinical parameters and Pearson correlation analyses were performed. Kaplan-Meier survival curves were assessed by using the log-rank (Mantel-Cox) test. To identify prognostic factors, multivariate Cox regression analyses were used. RESULTS: Mean serum levels of periostin were 505 ± 179 pmol/l. In older patients (> 60 years), periostin serum levels were significantly increased compared to younger patients (540 ± 184 pmol/l vs. 469 ± 167 pmol/l; p < 0.0001) and age was positively correlated with periostin expression (p < 0.0001). When stratifying the cohort according to periostin serum concentrations, the overall and breast cancer-specific mortality were significantly higher in those patients with high serum periostin (above median) compared to those with low periostin during a mean follow-up of 8.5 years (17.7% vs. 11.4% breast cancer-specific death; p = 0.03; hazard ratio 1.65). Periostin was confirmed to be an independent prognostic marker for breast cancer-specific survival (p = 0.017; hazard ratio 1.79). No significant differences in serum periostin were observed when stratifying the patients according to their DTC status. CONCLUSIONS: Our findings emphasize the relevance of periostin in breast cancer and reveal serum periostin as a potential marker for disease prediction, independent on the presence of micrometastases.

Diphenhydramine increases the therapeutic window for platinum drugs by simultaneously sensitizing tumor cells and protecting normal cells.

Platinum-based compounds remain a well-established chemotherapy for cancer treatment despite their adverse effects which substantially restrict the therapeutic windows of the drugs. Both the cell type-specific toxicity and the clinical responsiveness of tumors have been associated with mechanisms that alter drug entry and export. We sought to identify pharmacological agents that promote cisplatin (CP) efficacy by augmenting the levels of drug-induced DNA lesions in malignant cells and simultaneously protecting normal tissues from accumulating such damage and from functional loss. Formation and persistence of platination products in the DNA of individual nuclei were measured in drug-exposed cell lines, in primary human tumor cells and in tissue sections using an immunocytochemical method. Using a mouse model of CP-induced toxicity, the antihistaminic drug diphenhydramine (DIPH) and two methylated derivatives decreased DNA platination in normal tissues and also ameliorated nephrotoxicity, ototoxicity, and neurotoxicity. In addition, DIPH sensitized multiple cancer cell types, particularly ovarian cancer cells, to CP by increasing intracellular uptake, DNA platination, and/or apoptosis in cell lines and in patient-derived primary tumor cells. Mechanistically, DIPH diminished transport capacity of CP efflux pumps MRP2, MRP3, and MRP5 particularly in its C2+C6 bimethylated form. Overall, we demonstrate that DIPH reduces side effects of platinum-based chemotherapy and simultaneously inhibits key mechanisms of platinum resistance. We propose that measuring DNA platination after ex vivo exposure may predict the responsiveness of individual tumors to DIPH-like modulators.

Neuropilin-2 is an independent prognostic factor for shorter cancer-specific survival in patients with acinar adenocarcinoma of the prostate.

Neuropilin-2 (NRP2) is a member of the neuropilin receptor family and known to regulate autophagy and mTORC2 signaling in prostate cancer (PCa). Our study investigated the association of immunohistochemical NRP2 expression with clinicopathological data in PCa patients. For this purpose, we generated a tissue microarray with prostate tissue specimens from 400 PCa patients treated by radical prostatectomy. We focused on patients with high-risk factors such as extraprostatic extension (pT ≥ 3), Gleason score ≥8 and/or the presence of regional lymph node metastases (pN1). Protein levels of NRP2, the vascular endothelial growth factor C (VEGFC) and oncogenic v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene as an indicator for TMPRSS2-ERG fusion was assessed in relation to the patients' outcome. NRP2 emerged as an independent prognostic factor for cancer-specific survival (CSS) (hazard ratio 2.360, 95% confidence interval = 1.2-4.8; p = 0.016). Moreover, the association between NRP2 expression and shorter CSS was also especially pronounced in patients at high risk for progression (log-rank test: p = 0.010). We evaluated the association between NRP2 and the TMPRSS2-ERG gene fusion status assessed by immunohistochemical nuclear ERG staining. However, ERG staining alone did not show any prognostic significance. NRP2 immunostaining is significantly associated with shorter CSS in ERG-negative tumors (log-rank test: p = 0.012). No prognostic impact of NRP2 expression on CSS was observed in ERG-positive tumors (log-rank test: p = 0.153). Our study identifies NRP2 as an important prognostic marker for a worse clinical outcome especially in patients with a high-risk PCa and in patients with ERG-negative PCa.

Linking NRP2 With EMT and Chemoradioresistance in Bladder Cancer.

Neuropilin-2 (NRP2) is a prognostic indicator for reduced survival in bladder cancer (BCa) patients. Together with its major ligand, vascular endothelial growth factor (VEGF)-C, NRP2 expression is a predictive factor for treatment outcome in response to radiochemotherapy in BCa patients who underwent transurethral resection. Therefore, we investigated the benefit of combining cisplatin-based chemotherapy with irradiation treatment in the BCa cell line RT112 exhibiting or lacking endogenous NRP2 expression in order to evaluate NRP2 as potential therapeutic target. We have identified a high correlation of NRP2 and the glioma-associated oncogene family zinc finger 2 (GLI2) transcripts in the cancer genome atlas (TCGA) cohort of BCa patients and a panel of 15 human BCa cell lines. Furthermore, we used in vitro BCa models to show the transforming growth factor-beta 1 (TGFß1)-dependent regulation of NRP2 and GLI2 expression levels. Since NRP2 was shown to bind TGFß1, associate with TGFß receptors, and enhance TGFß1 signaling, we evaluated downstream signaling pathways using an epithelial-to-mesenchymal transition (EMT)-assay in combination with a PCR profiling array containing 84 genes related to EMT. Subsequent target validation in NRP2 knockout and knockdown models revealed secreted phosphoprotein 1 (SPP1/OPN/Osteopontin) as a downstream target positively regulated by NRP2.

Prognostic Value of RANKL/OPG Serum Levels and Disseminated Tumor Cells in Nonmetastatic Breast Cancer.

PURPOSE: We assessed serum concentrations of the receptor activator of NFκB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), two proteins implicated in the development and progression of breast cancer, in 509 patients with primary, nonmetastatic breast cancer. Then the results were evaluated with regards to the occurrence of bone metastases, the presence of disseminated tumor cells (DTC) in the bone marrow, survival, and risk of developing metastatic disease. EXPERIMENTAL DESIGN: Before surgery, two bone marrow aspirates were analyzed for DTC using density centrifugation followed by immunocytochemistry (pan-cytokeratin antibody A45-B/B3). RANKL and OPG levels in the serum were measured by ELISA. RESULTS: RANKL levels were significantly lower in women >60 years (P < 0.0001) and RANKL/OPG ratios higher in lymph node-positive patients (P < 0.05). High OPG serum levels were associated with a higher risk of death from breast cancer [HR 1.94; 95% confidence interval (CI) 1.23-3.07; P = 0.005] and OPG was an independent prognostic marker for breast cancer-specific survival (BCSS; multivariate analyses, P = 0.035). RANKL levels were 33% higher (P < 0.0001) in DTCpos patients (41%), whereas high levels were associated with a significantly better BCSS in DTCneg patients as compared with low levels (HR 0.524; 95% CI 0.30-0.95; P = 0.04). RANKL serum levels were significantly increased in patients who developed bone metastases (P = 0.01) and patients within the highest quartile of RANKL had a significantly increased risk of developing bone metastases compared with those in the lowest (HR 4.62; 95% CI 1.49-14.34; P = 0.03). CONCLUSIONS: These findings warrant further investigation as they provide a rationale for novel diagnostic or therapeutic approaches.

Quantification of MicroRNAs in Urine-Derived Specimens.

MicroRNAs are small noncoding RNAs which regulate the expression of genes involved in a multitude of cellular processes. Dysregulation of microRNAs and-in consequence-of the affected pathways is frequently observed in numerous pathologies including cancers. Therefore, tumor-related alterations in microRNA expression and function can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers.In particular, microRNAs with differential expression in bladder cancer (BCa) might represent promising tools for noninvasive tumor detection in urine. This would be helpful not only for diagnostic and monitoring purposes but also for therapeutic decisions. Detection and quantification of BCa-associated microRNAs in urine can be performed using the cellular sediment, which also contains BCa cells, or in exosomes originating from those cells. Methods for isolation of exosomes from urine, extraction of total RNA from cells and exosomes as well as techniques for RNA quantification, reverse transcription, and qPCR-based quantification of microRNA expression levels are described herein.