Christian Arsov

T cells specific for HPV16 E7 epitopes in patients with squamous cell carcinoma of the oropharynx

Squamous cell carcinomas of the oropharynx (SCCO) are often infected with oncogenic human papilloma virus (HPV) subtype 16. To determine the frequency of T cells specific for human leukocyte antigen (HLA)‐A2.1 restricted HPV16 E7 protein‐derived epitopes, tetramer analysis was performed using peripheral blood lymphocytes of 20 HLA‐A2.1+ patients and 20 HLA‐A2.1+ healthy individuals. Tetramers specific for 3 HPV16 peptides (E711–20, E782–90 and E786–93), an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were used in multicolor flow analysis. The HPV‐specific T‐cell frequencies were correlated with the HPV16 E7 and p16 status in tumor sections. In vitro stimulation (IVS) with autologous dendritic cells (DC) pulsed with HPV16 E7 epitopes was performed to demonstrate proliferation and antitumor activity of the HPV‐responsive T cells. Frequencies of CD8+ T cells specific for HPV16 E7 peptides were not significantly different in patients with SCCO relative to normal donors. However, patients with tumors expressing HPV16 E7 (60%) and p16 (50%) had an increased frequency (p < 0.05) of T cells specific for the E711–20 epitope compared to those with tumors negative for both markers. HPV16 E711–20 and HPV16 E786–93 specific T cells were expandable upon IVS with cognate peptide‐pulsed DC and were reactive against peptide‐pulsed targets or, in case of the E711–20 epitope‐specific T cells, against HPV16 E7 expressing CaSki cell line. Thus, in patients with HPV16+ SCCO, precursor T cells specific for E711–20 epitope are present (1/3,947) in the circulation, are responsive to stimulation with the cognate viral peptide and recognize in vitro HPV16 E7+ tumor cells. Further studies have to elucidate why those T cells are unable to eliminate the tumor in vivo and this might also allow for finding potential strategies that will increase the chances of developing a future HPV‐based vaccine in patients with SCCO.

Prospective Randomized Trial Comparing Magnetic Resonance Imaging (MRI)-guided In-bore Biopsy to MRI-ultrasound Fusion and Transrectal Ultrasound-guided Prostate Biopsy in Patients with Prior Negative Biopsies

BACKGROUND A significant proportion of prostate cancers (PCas) are missed by conventional transrectal ultrasound-guided biopsy (TRUS-GB). It remains unclear whether the combined approach using targeted magnetic resonance imaging (MRI)-ultrasound fusion-guided biopsy (FUS-GB) and systematic TRUS-GB is superior to targeted MRI-guided in-bore biopsy (IB-GB) for PCa detection. OBJECTIVE To compare PCa detection between IB-GB alone and FUS-GB + TRUS-GB in patients with at least one negative TRUS-GB and prostate-specific antigen ≥4 ng/ml. DESIGN, SETTING, AND PARTICIPANTS Patients were prospectively randomized after multiparametric prostate MRI to IB-GB (arm A) or FUS-GB + TRUS-GB (arm B) from November 2011 to July 2014. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The study was powered at 80% to demonstrate an overall PCa detection rate of ≥60% in arm B compared to 40% in arm A. Secondary endpoints were the distribution of highest Gleason scores, the rate of detection of significant PCa (Gleason ≥7), the number of biopsy cores to detect one (significant) PCa, the positivity rate for biopsy cores, and tumor involvement per biopsy core. RESULTS AND LIMITATIONS The study was halted after interim analysis because the primary endpoint was not met. The trial enrolled 267 patients, of whom 210 were analyzed (106 randomized to arm A and 104 to arm B). PCa detection was 37% in arm A and 39% in arm B (95% confidence interval for difference, -16% to 11%; p=0.7). Detection rates for significant PCa (29% vs 32%; p=0.7) and the highest percentage tumor involvement per biopsy core (48% vs 42%; p=0.4) were similar between the arms. The mean number of cores was 5.6 versus 17 (p<0.001). A limitation is the limited number of patients because of early cessation of accrual. CONCLUSIONS This trial failed to identify an important improvement in detection rate for the combined biopsy approach over MRI-targeted biopsy alone. A prospective comparison between MRI-targeted biopsy alone and systematic TRUS-GB is justified. PATIENT SUMMARY Our randomized study showed similar prostate cancer detection rates between targeted prostate biopsy guided by magnetic resonance imaging and the combination of targeted biopsy and systematic transrectal ultrasound-guided prostate biopsy. An important improvement in detection rates using the combined biopsy approach can be excluded.

DNA Methylation Signatures for Prediction of Biochemical Recurrence After Radical Prostatectomy of Clinically Localized Prostate Cancer

PURPOSE Diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, causing overtreatment of indolent PC and risk of delayed treatment of aggressive PC. Here, we identify six novel candidate DNA methylation markers for PC with promising diagnostic and prognostic potential. METHODS Microarray-based screening and bisulfite sequencing of 20 nonmalignant and 29 PC tissue specimens were used to identify new candidate DNA hypermethylation markers for PC. Diagnostic and prognostic potential was evaluated in 35 nonmalignant prostate tissue samples, 293 radical prostatectomy (RP) samples (cohort 1, training), and 114 malignant RP samples (cohort 2, validation) collected in Denmark, Switzerland, Germany, and Finland. Sensitivity and specificity for PC were evaluated by receiver operating characteristic analyses. Correlations between DNA methylation levels and biochemical recurrence were assessed using log-rank tests and univariate and multivariate Cox regression analyses. RESULTS Hypermethylation of AOX1, C1orf114, GAS6, HAPLN3, KLF8, and MOB3B was highly cancer specific (area under the curve, 0.89 to 0.98). Furthermore, high C1orf114 methylation was significantly (P < .05) associated with biochemical recurrence in multivariate analysis in cohort 1 (hazard ratio [HR], 3.10; 95% CI, 1.89 to 5.09) and was successfully validated in cohort 2 (HR, 3.27; 95% CI, 1.17 to 9.12). Moreover, a significant (P < .05) three-gene prognostic methylation signature (AOX1/C1orf114/HAPLN3), classifying patients into low- and high-methylation subgroups, was trained in cohort 1 (HR, 1.91; 95% CI, 1.26 to 2.90) and validated in cohort 2 (HR, 2.33; 95% CI, 1.31 to 4.13). CONCLUSION We identified six novel candidate DNA methylation markers for PC. C1orf114 hypermethylation and a three-gene methylation signature were independent predictors of time to biochemical recurrence after RP in two PC patient cohorts.

Cabazitaxel Plus Prednisone for Metastatic Castration-resistant Prostate Cancer Progressing After Docetaxel: Results from the German Compassionate-use Programme

BACKGROUND Cabazitaxel (Cbz) is an approved second-line treatment in metastatic castration-resistant prostate cancer (mCRPC) following docetaxel therapy with a significant survival benefit compared with mitoxantrone. However, grade 3/4 toxicities were reported in 82% of patients. OBJECTIVE To report on the safety results of mCRPC patients treated within a compassionate-use programme in Germany. DESIGN, SETTING, AND PARTICIPANTS A total of 111 patients with a mean age of 67.9 yr (range: 49-81 yr) and progressive mCRPC were included. Patients had received a mean number of 12.7 ± 10.8 cycles (range: 6-69 cycles) of docetaxel with a mean cumulative dose of 970.9 mg/m(2); mean time from last docetaxel application to progression was 6.95 mo (range: 2-54 mo). Of the patients, 31.5% progressed by prostate-specific antigen (PSA) increase only; the remainder had a combination of PSA increase and clinical progression. INTERVENTION Cbz at a dosage of 25mg/m(2) intravenously every 3 wk combined with 5mg of oral prednisone twice a day. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Treatment-associated toxicity was the primary study end point; progression-free and overall survival were secondary end points. A descriptive statistical analysis was performed. RESULTS AND LIMITATIONS Patients received a mean number of 6.5 ± 2.2 cycles of Cbz and a mean cumulative dose of 160.3 ± 51.5mg/m(2). Grade 3 and 4 treatment-emergent adverse events were recorded in 34 patients (30.6%) and 18 patients (16.2%), respectively. Grade 3/4 anaemia, neutropenia, and thrombocytopenia were reported in 4.5%, 7.2%, and 0.9% of the patients, respectively. Neutropenic fever was reported in 1.8% of the patients. Grade 3/4 gastrointestinal toxicity was identified in 4.5% of the patients. Three patients died because of Cbz-related toxicity. Granulocyte colony-stimulating growth factors were used in 17.1% of patients. The limitations are due to the nonrandomised nature of the trial. CONCLUSIONS Treatment with Cbz is tolerable and is associated with a low incidence of serious adverse events in a real-world patient population with CRPC. The outcome of serious adverse events can be minimised with proactive treatment management and conscientious monitoring.

Hypermethylation of the GABRE∼miR-452∼miR-224 Promoter in Prostate Cancer Predicts Biochemical Recurrence after Radical Prostatectomy

Purpose: Available tools for prostate cancer diagnosis and prognosis are suboptimal and novel biomarkers are urgently needed. Here, we investigated the regulation and biomarker potential of the GABRE∼miR-452∼miR-224 genomic locus. Experimental Design: GABRE/miR-452/miR-224 transcriptional expression was quantified in 80 nonmalignant and 281 prostate cancer tissue samples. GABRE∼miR-452∼miR-224 promoter methylation was determined by methylation-specific qPCR (MethyLight) in 35 nonmalignant, 293 prostate cancer [radical prostatectomy (RP) cohort 1] and 198 prostate cancer tissue samples (RP cohort 2). Diagnostic/prognostic biomarker potential of GABRE∼miR-452∼miR-224 methylation was evaluated by ROC, Kaplan–Meier, uni- and multivariate Cox regression analyses. Functional roles of miR-224 and miR-452 were investigated in PC3 and DU145 cells by viability, migration, and invasion assays and gene-set enrichment analysis (GSEA) of posttransfection transcriptional profiling data. Results: GABRE∼miR-452∼miR-224 was significantly downregulated in prostate cancer compared with nonmalignant prostate tissue and had highly cancer-specific aberrant promoter hypermethylation (AUC = 0.98). Functional studies and GSEA suggested that miR-224 and miR-452 inhibit proliferation, migration, and invasion of PC3 and DU145 cells by direct/indirect regulation of pathways related to the cell cycle and cellular adhesion and motility. Finally, in uni- and multivariate analyses, high GABRE∼miR-452∼miR-224 promoter methylation was significantly associated with biochemical recurrence in RP cohort 1, which was successfully validated in RP cohort 2. Conclusion: The GABRE∼miR-452∼miR-224 locus is downregulated and hypermethylated in prostate cancer and is a new promising epigenetic candidate biomarker for prostate cancer diagnosis and prognosis. Tumor-suppressive functions of the intronic miR-224 and miR-452 were demonstrated in two prostate cancer cell lines, suggesting that epigenetic silencing of GABRE∼miR-452∼miR-224 may be selected for in prostate cancer. Clin Cancer Res; 20(8); 2169–81. ©2014 AACR.

Comparison of different mathematical models of diffusion-weighted prostate MR imaging

PURPOSE To evaluate which mathematical model (monoexponential, biexponential, statistical, kurtosis) fits best to the diffusion-weighted signal in prostate magnetic resonance imaging (MRI). MATERIALS AND METHODS 24 prostate 3-T MRI examinations of young volunteers (YV, n=8), patients with biopsy proven prostate cancer (PC, n=8) and an aged matched control group (AC, n=8) were included. Diffusion-weighted imaging was performed using 11 b-values ranging from 0 to 800 s/mm(2). RESULTS Monoexponential apparent diffusion coefficient (ADC) values were significantly (P<.001) lower in the peripheral (PZ) zone (1.18±0.16 mm(2)/s) and the central (CZ) zone (0.73±0.13 mm(2)/s) of YV compared to AC (PZ 1.92±0.17 mm(2)/s; CZ 1.35±0.21 mm(2)/s). In PC ADC(mono) values (0.61±0.06 mm(2)/s) were significantly (P<.001) lower than in the peripheral of central zone of AC. Using the statistical analysis (Akaike information criteria) in YV most pixels were best described by the biexponential model (82%), the statistical model, respectively kurtosis (93%) each compared to the monoexponential model. In PC the majority of pixels was best described by the monoexponential model (57%) compared to the biexponential model. CONCLUSION Although a more complex model might provide a better fitting when multiple b-values are used, the monoexponential analyses for ADC calculation in prostate MRI is sufficient to discriminate prostate cancer from normal tissue using b-values ranging from 0 to 800 s/mm(2).

Feasibility of diffusional kurtosis tensor imaging in prostate MRI for the assessment of prostate cancer: preliminary results.

PURPOSE To assess the feasibility of full diffusional kurtosis tensor imaging (DKI) in prostate MRI in clinical routine. Histopathological correlation was achieved by targeted biopsy. MATERIALS AND METHODS Thirty-one men were prospectively included in the study. Twenty-one were referred to our hospital with increased prostate specific antigen (PSA) values (>4ng/ml) and suspicion of prostate cancer. The other 10 men were volunteers without any history of prostate disease. DKI applying diffusion gradients in 20 different spatial directions with four b-values (0, 300, 600, 1000s/mm(2)) was performed additionally to standard functional prostate MRI. Region of interest (ROI)-based measurements were performed in all histopathologically verified lesions of every patient, as well as in the peripheral zone, and the central gland of each volunteer. RESULTS DKI showed a substantially better fit to the diffusion-weighted signal than the monoexponential apparent diffusion coefficient (ADC). Altogether, 29 lesions were biopsied in 14 different patients with the following results: Gleason score 3+3=6 (n=1), 3+4=7 (n=7), 4+3=7 (n=6), 4+4=8 (n=1), and 4+5=9 (n=2), and prostatitis (n=12). Values of axial (Kax) and mean kurtosis (Kmean) were significantly different in the tumor (Kax 1.78±0.39, Kmean 1.84±0.43) compared with the normal peripheral zone (Kax 1.09±0.12, Kmean 1.16±0.13; p<0.001) or the central gland (Kax 1.40±0.12, Kmean 1.44±0.17; p=0.01 respectively). There was a minor correlation between axial kurtosis (r=0.19) and the Gleason score. CONCLUSION Full DKI is feasible to utilize in a routine clinical setting. Although there is some overlap some DKI parameters can significantly distinguish prostate cancer from the central gland or the normal peripheral zone. Nevertheless, the additional value of DKI compared with conventional monoexponential ADC calculation remains questionable and requires further research.

Prospective Randomized Evaluation of Risk-adapted Prostate-specific Antigen Screening in Young Men: The PROBASE Trial

Department of Urology, University Hospital, Heinrich-Heine-University, Dusseldorf, Germany; Division of Cancer Epidemiology, German Cancer Research Center Heidelberg, Heidelberg, Germany; Department of Urology, University Hospital, Ruprecht-Karls-University, Heidelberg, Germany; Department of Urology, University Hospital, Technical University of Munich, Munich, Germany; Department of Urology, University Hospital, Hannover Medical School, Hannover, Germany; University Dusseldorf, Medical Faculty, Department of Diagnostic and Interventional Radiology, Dusseldorf, Germany; Department of Pathology, University Hospital, Rheinische Friedrich-Wilhelms-University, Bonn, Germany; Department of Urology, University Hospital, Rheinische Friedrich-Wilhelms-University, Bonn, Germany; Department of Urology, University Hospital, Westfalische Wilhelms-University, Munster, Germany; Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom; Department of Laboratory Medicine, Surgery and Medicine, Memorial Sloan-Kettering Cancer Center, New York, USA; Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, USA; Department of Urology, Erasmus Medical Center Rotterdam, The Netherlands

Association of PITX2 mRNA down-regulation in prostate cancer with promoter hypermethylation and poor prognosis

BACKGROUND Hypermethylation of the PITX2 (paired-like homeodomain transcription factor 2) gene promoter is strongly associated with recurrence after radical prostatectomy. We hypothesized that PITX2 hypermethylation leads to PITX2 silencing and that decreased PITX2 expression is likewise associated with poor prognosis in prostate cancers. Moreover, it is unknown so far how PITX2 hypermethylation relates to other molecular changes in prostate cancer, such as ERG oncogenic activation in about half of all cases. OBJECTIVE To investigate how PITX2 expression and methylation are related, whether biochemical recurrence after radical prostatectomy can be predicted by PITX2 mRNA levels, and how changes in PITX2 relate to ERG overexpression. MATERIAL AND METHODS We measured PITX2 and ERG expression in 45 cancerous and 13 benign tissues from patients undergoing radical prostatectomy (age range: 59-74 years). Methylation of the PITX2 gene was analyzed in an extended series of 93 cancers. Follow-up was performed for all patients for a 98-month median period. Additionally, expression and methylation changes of PITX2 were investigated in prostate carcinoma cell lines. Gene expression and methylation were determined by quantitative RT-PCR and methylation-specific PCR, respectively. Biochemical recurrence defined as a total PSA of >0.2 ng/ml on 2 consecutive tests was considered as the surrogate endpoint for survival analysis. RESULTS PITX2 expression was significantly and strongly decreased in prostate cancer compared to benign tissues. Cases with decreased PITX2 experienced significantly earlier biochemical recurrences. PITX2 down-regulation was associated with PITX2 promoter hypermethylation in tumor samples and cell lines. PITX2 hypermethylation was more pronounced in cases with ERG overexpression. CONCLUSIONS PITX2 down-regulation is associated with promoter hypermethylation and is a good predictor of clinical outcomes after radical prostatectomy. PITX2 methylation might be influenced by oncogenic ERG.

MRI-Guided In-Bore Biopsy: Differences Between Prostate Cancer Detection and Localization in Primary and Secondary Biopsy Settings

OBJECTIVE The objective of our study was to evaluate transrectal MRI-guided in-bore biopsy in patients who either were biopsy-naive (primary biopsy) or had undergone at least one previous negative transrectal ultrasound-guided biopsy (secondary biopsy) with regard to cancer detection rate, tumor localization, and lesion size. MATERIALS AND METHODS In total, 1602 biopsy cores from 297 consecutive patients (mean ± SD, 66.1 ± 7.8 years; median prostate-specific antigen value, 8.2 ng/mL) in primary (n = 160) and secondary (n = 137) prostate biopsy settings were evaluated in this retrospective study. All patients previously underwent prostate MRI (T2-weighted imaging, DWI, dynamic contrast-enhanced imaging) at 3 T. All described lesions were biopsied with MRI-guided in-bore biopsy and were examined histologically. RESULTS In 148 patients, overall 511 cores were positive for prostate cancer. Clinically significant prostate cancer (any Gleason pattern ≥ 4) was found in 82.4% of patients. The prostate cancer detection rate for patients who underwent primary biopsies was 55.6% and was 43.1% for patients who underwent secondary biopsies. In patients with primary versus secondary biopsies, prostate cancer was located peripherally in 62.9% versus 49.5% (p = 0.04), in the transition zone in 27.4% versus 27.5% (p = 1.0), and in the anterior stroma in 10.3% versus 22.9% (p < 0.01), respectively. The prostate cancer detection rates for patients with smaller prostate volumes (< 30 vs 30-50 vs > 50 mL; p < 0.01) or for patients with larger lesions (> 0.5 vs 0.25-0.5 vs < 0.25 cm(3); p < 0.01) were significantly higher. CONCLUSION MRI-guided in-bore biopsy led to high detection rates in primary and secondary prostate biopsies. Prostate cancer detection rates were significantly higher for patients with larger lesions and smaller prostate glands. In patients who underwent secondary biopsies, prostate cancer was located in the anterior stroma at a significantly more frequent rate.