Barriers to initiation of hepatitis C virus therapy in Germany: A retrospective, case-controlled study.

Despite the availability of highly effective and well-tolerated direct-acting antivirals, not all patients with chronic hepatitis C virus infection receive treatment. This retrospective, multi-centre, noninterventional, case-control study identified patients with chronic hepatitis C virus infection initiating (control) or not initiating (case) treatment at 43 sites in Germany from September 2017 to June 2018. It aimed to compare characteristics of the two patient populations and to identify factors involved in patient/physician decision to initiate/not initiate chronic hepatitis C virus treatment, with a particular focus on historical barriers. Overall, 793 patients were identified: 573 (72%) who received treatment and 220 (28%) who did not. In 42% of patients, the reason for not initiating treatment was patient wish, particularly due to fear of treatment (17%) or adverse events (13%). Other frequently observed reasons for not initiating treatment were in accordance with known historical barriers for physicians to initiate therapy, including perceived or expected lack of compliance (14.5%), high patient age (10.9%), comorbidities (15.0%), alcohol abuse (9.1%), hard drug use (7.7%), and opioid substitution therapy (4.5%). Patient wish against therapy was also a frequently reported reason for not initiating treatment in the postponed (35.2%) and not planned (47.0%) subgroups; of note, known historical factors were also common reasons for postponing treatment. Real-world and clinical trial evidence is accumulating, which suggests that such historical barriers do not negatively impact treatment effectiveness. Improved education is key to facilitate progress towards the World Health Organization target of eliminating viral hepatitis as a major public health threat by 2030.

Treatment effects of palliative care consultation and patient contentment: A monocentric observational study.

ABSTRACT: Palliative care is a central component of the therapy in terminally ill patients. During treatment in non-palliative departments this can be realized by consultation.To analyze the change in symptom burden during palliative care consultation.In this observational study, we enrolled all cancer cases (n = 163) receiving inpatient treatment for 2015 to 2018 at our institution. We used the MDASI-questionnaire (0 = 'not present' and 10 = "as bad as you can imagine") and the FAMCARE-6 (1 = very satisfied, 5 = very dissatisfied) to analyze the treatment effect and patient satisfaction, respectively.We examined the association of symptom burden and patient satisfaction using Spearman-correlation. Comparing mean values, we applied the Wilcoxon-test and one-way ANOVA.An improvement in MDASI-core-items after treatment completion was significant (P < .05) in 14/18 symptoms. The change in perception of pain showed the strongest improvement (median: 5 to 3). Initially the MDASI-items "activity" (median = 8) and emotional distress (median = 5 and 6) were viewed as especially incriminating. There was no evidence for a correlation between patients' age, the type of diagnosis and time since diagnosis.The analysis of FAMCARE-6 patient contentment was lower or equal to two in all of the six items. There was a weak negative association between the change in symptom burden of psycho-emotional items "distress/feeling upset" (P = .006, rSp = -0,226), "sadness" and patient satisfaction in FAMCARE-6.A considerable improvement of the extensive symptom burden particularly of pain relief was achieved by integrating palliative consultation in clinical practice.

Sofosbuvir, velpatasvir, and voxilaprevir for patients with failure of previous direct-acting antiviral therapy for chronic hepatitis C: Results from the German Hepatitis C-Registry (DHC-R).

Despite the high effectiveness of direct-acting antivirals for the treatment of hepatitis C, a small proportion of patients do not respond to approved regimens. The combination regimen of SOF/VEL/VOX was recently approved for patients with failure to prior NS5A-based treatment. In this German real-world cohort including patients with cirrhosis (27.3 %) and previous decompensation events, 12 weeks of SOF/VEL/VOX resulted in high virologic response rates irrespective of disease severity and prior DAA regimen. Adverse events were mostly mild or moderate and comparable to those seen in the approval studies.

Treatment Response Monitoring in Patients with Advanced Malignancies Using Cell-Free SHOX2 and SEPT9 DNA Methylation in Blood: An Observational Prospective Study.

Patients with incurable cancer usually receive palliative treatment with significant toxicity and limited efficacy. Methylation analysis of circulating cell-free DNA (ccfDNA) in blood from cancer patients represents a promising approach for minimally invasive, real-time monitoring of treatment response. Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) methylation was analyzed in N = 8865 malignant and N = 746 normal adjacent tissues across 33 different malignancies from The Cancer Genome Atlas. Furthermore, we performed quantitative SHOX2 and SEPT9 ccfDNA methylation analysis in plasma obtained before and consecutively during treatment from prospectively enrolled N = 115 patients with various advanced cancers. SHOX2 and/or SEPT9 hypermethylation in malignant tissues is present in various carcinomas, sarcoma, melanoma, brain tumors, mesothelioma, and hematopoietic malignancies. Among the prospectively enrolled cancer patients, 61% (70/115) of patients had a baseline-positive blood cumulative ccfDNA methylation score (CMS) and were eligible for response monitoring. Dynamic changes of CMS during treatment were strongly associated with treatment response. A CMS increase indicated response up to 80 days before conventional monitoring. SHOX2 and SEPT9 ccfDNA methylation represents a pan-cancer biomarker and has the potential to be a powerful tool for monitoring treatment response in patients with solid tumors and lymphomas. The early identification of nonresponders might allow for a timely change of treatment regimen.

Risk factors for resistance development against lamivudine during long-term treatment of chronic hepatitis B virus infections.

BACKGROUND/AIM: The use of lamivudine for the treatment of chronic hepatitis B (CHB) is limited by high rates of lamivudine resistance. However, it is still in use in many regions. Factors associated with lamivudine resistance development have been studied in only a few European cohorts. The aim of our study was to assess the rate and risk factors for lamivudine resistance in a large real-life European cohort. PATIENTS AND METHODS: We retrospectively analyzed patients with CHB treated in three German University centers over up to 12 years. Lamivudine resistance was defined as virologic breakthrough and presence of genotypic lamivudine resistance. The probability of resistance was estimated by Kaplan-Meier analysis and resistance predictors by Cox regression. RESULTS: A total of 227 patients were included into the analysis (hepatitis B envelope antigen positive or negative). Rates of lamivudine resistance by years 1-7 were 7, 26, 35, 41, 46, 53, and 55%, respectively. Interestingly, two hepatitis B envelope antigen-negative patients developed resistance during the year 12 of treatment. Independent risk factors for resistance development were hepatitis B virus DNA levels of at least 10 copies/ml before and detectable hepatitis B virus DNA by month 6 of treatment. CONCLUSION: Even after long-term response to lamivudine more than 10 years, resistance may still develop. Our findings further discourage the use of lamivudine for the treatment of CHB.

Cell-Free SHOX2 DNA Methylation in Blood as a Molecular Staging Parameter for Risk Stratification in Renal Cell Carcinoma Patients: A Prospective Observational Cohort Study.

BACKGROUND: Novel targeted treatments and immunotherapies have substantially changed therapeutic options for advanced and metastatic renal cell carcinomas (RCCs). However, accurate diagnostic tests for the identification of high-risk patients are urgently needed. Here, we analyzed SHOX2 mRNA expression in RCC tissues and SHOX2 gene body methylation quantitatively in circulating cell-free DNA (ccfDNA) and RCC tissues with regard to risk stratification. METHODS: The clinical performance of SHOX2 methylation was tested retrospectively and prospectively in a training and testing cohort of RCC tissue samples (n = 760 in total). SHOX2 mRNA expression analysis was included in the training cohort. In matched blood plasma samples from the testing cohort (n = 100), we prospectively examined the capability of pretherapeutic quantitative SHOX2 ccfDNA methylation to assess disease stage and identify patients at high risk of death. RESULTS: SHOX2 gene body methylation was positively correlated with mRNA expression in RCC tissues (training cohort: Spearman ρ = 0.23, P < 0.001). SHOX2 methylation in tissue and plasma strongly correlated with an advanced disease stage (training cohort: ρ = 0.28, P < 0.001; testing cohort/tissue: ρ = 0.40, P < 0.001; testing cohort/plasma: ρ = 0.34, P = 0.001) and risk of death after initial partial or radical nephrectomy [training cohort: hazard ratio (HR) = 1.40 (95% CI, 1.24-1.57), P < 0.001; testing cohort/tissue: HR = 1.16 (95% CI, 1.07-1.27), P = 0.001; testing cohort/plasma: HR = 1.50 (95% CI, 1.29-1.74), P < 0.001]. CONCLUSIONS: Pretherapeutic SHOX2 ccfDNA methylation testing allows for the identification of RCC patients at high risk of death after nephrectomy. These patients might benefit from an adjuvant treatment or early initiation of a palliative treatment.

DNA Methylation Analysis of Free-Circulating DNA in Body Fluids.

Circulating cell-free DNA in body fluids is an analyte of great interest in basic and clinical research. The analyses of DNA methylation and hydroxymethylation patterns in body fluids might allow one to determine the certain state of a disease, in particular of cancer. DNA methylation biomarkers in liquid biopsies, i.e. blood plasma samples, may help optimizing personalized therapy for individual patients. DNA methylation analyses of specific loci usually require a bisulfite conversion of the DNA, which requires a sufficiently high amount of DNA at the appropriate concentration. However, free-circulating DNA is generally low concentrated. Therefore, high volumes of body fluids need to be analyzed. This high volume needs to be reduced in order to facilitate the bisulfite conversion. In addition, disease-related free-circulating DNA is even less abundant than normal DNA in the total amount of free-circulating DNA. Accordingly, analytical and pre-analytical methods are needed, which permit an accurate and sensitive quantification of single methylated DNA copies in the presence of unmethylated DNA in abundance.This protocol describes two methods for DNA enrichment from body fluids: DNA extraction by means of magnetic beads and polymer-mediated enrichment of DNA. Subsequent bisulfite conversion is achieved by means of a high-speed conversion protocol. Adaptions of the workflow required for the analysis of hydroxymethylation via oxidation 5-hydroxymethylcytosines to 5-formylcytosines prior to the bisulfite conversion are introduced. A quantitative real-time PCR based on the methylation-specific and HeavyMethyl PCR methodologies is introduced. This qPCR assay allows for an accurate and sensitive quantification of single copies of the DNA methylation biomarkers SHOX2 and SEPT9 in blood plasma. Specific issues regarding the analysis of body fluids and respective trouble shooting approaches are discussed.

Bisulfite Conversion of DNA from Tissues, Cell Lines, Buffy Coat, FFPE Tissues, Microdissected Cells, Swabs, Sputum, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine.

Locus-specific analyses of DNA methylation patterns usually require a bisulfite conversion of the DNA, where cytosines are deaminated to uracils, while methylated and hydroxymethylated cytosines remain unaffected. The specific discrimination of hydroxymethylation and methylation can be achieved by introducing an oxidation of 5-hydroxymethylcytosines to 5-formylcytosines and subsequent bisulfite-mediated deamination of 5-formylcytosines.DNA methylation analysis of cell-free circulating DNA in liquid biopsies, i.e., blood samples (serum and plasma), urine, aspirates, bronchial lavages, pleural effusions, and ascites, is of great interest in clinical research. However, due to the generally low concentration of circulating cell-free DNA in body fluids, high volumes need to be analyzed. A reduction of this volume, e.g., by means of a polymer-mediated enrichment, is required in order to facilitate the bisulfite conversion. Further, these sample types usually contain a cellular fraction which is of additional interest and requires specific protocols for the sample preparation.Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used source for tissue-based clinical research. Due to degradation and covalent modifications of DNA in FFPE tissue samples, optimized protocols for the DNA preparation and bisulfite conversion are required.This chapter describes methods and protocols for the sample preparation and subsequent high-speed bisulfite conversion and DNA clean-up for several types of relevant samples, i.e., serum, plasma, urine, buffy coat, aspirates, sputum, lavages, effusions, ascites, swabs, fresh tissues, cell lines, FFPE tissues, and laser microdissected cells.Additionally, two real-time PCR assays for DNA quantification and quality control are described. The cytosine-free fragment (CFF) assay allows for the simultaneous quantification of bisulfite converted and total DNA and thus the determination of bisulfite conversion efficiency. The Mer9 real-time PCR assay amplifies the bisulfite converted sequence of the repetitive element Mer9 and enables the accurate quantification of minute DNA amounts, as present in microdissected cells and body fluids.

PITX2 DNA Methylation as Biomarker for Individualized Risk Assessment of Prostate Cancer in Core Biopsies.

Hypermethylation of the paired-like homeodomain transcription factor 2 (PITX2) gene is a strong predictor of the risk of biochemical recurrence in patients with prostate cancer (PCa) after radical prostatectomy. We investigate whether PITX2 methylation is feasible for individualized risk assessment in prostate core biopsies before surgery. A quantitative, methylation-specific real-time PCR was used to measure PITX2 in three cohorts: i) matched samples of neoplastic and nonneoplastic tissue from 24 patients with PCa, ii) a well-characterized cohort of 300 patients with PCa after radical prostatectomy, and iii) core biopsy specimens from 32 patients with PCa and 31 patients with benign prostatic disease. PITX2 methylation discriminated between neoplastic and nonneoplastic tissue in patients with PCa (P < 0.001). In the second cohort, PITX2 methylation significantly correlated with clinicopathologic parameters, and PITX2 hypermethylation predicted an increased risk of biochemical recurrence in univariate Cox proportional hazards regression analysis (hazard ratio, 1.77; P = 0.046) and Kaplan-Meier analysis (P = 0.043). In 753 prostate biopsies, 720 (95.6%) were applicable for analysis, rendering the assay feasible for diagnostic biopsies. PITX2 methylation was furthermore significantly increased in tumor-positive biopsies and strongly correlated with International Society of Urological Pathology (ISUP) grade groups. This study indicates that the PITX2 methylation assay is feasible in prostate biopsies and might add valuable prognostic information for risk assessment in a presurgical diagnostic setting.

CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients.

Molecular biomarkers may facilitate the distinction between aggressive and clinically insignificant prostate cancer (PCa), thereby potentially aiding individualized treatment. We analyzed cysteine dioxygenase 1 (CDO1) promoter methylation and mRNA expression in order to evaluate its potential as prognostic biomarker. CDO1 methylation and mRNA expression were determined in cell lines and formalin-fixed paraffin-embedded prostatectomy specimens from a first cohort of 300 PCa patients using methylation-specific qPCR and qRT-PCR. Univariate and multivariate Cox proportional hazards and Kaplan-Meier analyses were performed to evaluate biochemical recurrence (BCR)-free survival. Results were confirmed in an independent second cohort comprising 498 PCa cases. Methylation and mRNA expression data from the second cohort were generated by The Cancer Genome Atlas (TCGA) Research Network by means of Infinium HumanMethylation450 BeadChip and RNASeq. CDO1 was hypermethylated in PCa compared to normal adjacent tissues and benign prostatic hyperplasia (P < 0.001) and was associated with reduced gene expression (ρ = -0.91, P = 0.005). Using two different methodologies for methylation quantification, high CDO1 methylation as continuous variable was associated with BCR in univariate analysis (first cohort: HR = 1.02, P = 0.002, 95% CI [1.01-1.03]; second cohort: HR = 1.02, P = 0.032, 95% CI [1.00-1.03]) but failed to reach statistical significance in multivariate analysis. CDO1 promoter methylation is involved in gene regulation and is a potential prognostic biomarker for BCR-free survival in PCa patients following radical prostatectomy. Further studies are needed to validate CDO1 methylation assays and to evaluate the clinical utility of CDO1 methylation for the management of PCa.

PITX3 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients after radical prostatectomy.

BACKGROUND: Molecular biomarkers that might help to distinguish between more aggressive and clinically insignificant prostate cancers (PCa) are still urgently needed. Aberrant DNA methylation as a common molecular alteration in PCa seems to be a promising source for such biomarkers. In this study, PITX3 DNA methylation (mPITX3) and its potential role as a prognostic biomarker were investigated. Furthermore, mPITX3 was analyzed in combination with the established PCa methylation biomarker PITX2 (mPITX2). METHODS: mPITX3 and mPITX2 were assessed by a quantitative real-time PCR and by means of the Infinium HumanMethylation450 BeadChip. BeadChip data were obtained from The Cancer Genome Atlas (TCGA) Research Network. DNA methylation differences between normal adjacent, benign hyperplastic, and carcinomatous prostate tissues were examined in the TCGA dataset as well as in prostatectomy specimens from the University Hospital Bonn. Retrospective analyses of biochemical recurrence (BCR) were conducted in a training cohort (n = 498) from the TCGA and an independent validation cohort (n = 300) from the University Hospital Bonn. All patients received radical prostatectomy. RESULTS: In PCa tissue, mPITX3 was increased significantly compared to normal and benign hyperplastic tissue. In univariate Cox proportional hazards analyses, mPITX3 showed a significant prognostic value for BCR (training cohort: hazard ratio (HR) = 1.83 (95 % CI 1.07-3.11), p = 0.027; validation cohort: HR = 2.56 (95 % CI 1.44-4.54), p = 0.001). A combined evaluation with PITX2 methylation further revealed that hypermethylation of a single PITX gene member (either PITX2 or PITX3) identifies an intermediate risk group. CONCLUSIONS: PITX3 DNA methylation alone and in combination with PITX2 is a promising biomarker for the risk stratification of PCa patients and adds relevant prognostic information to common clinically implemented parameters. Further studies are required to determine whether the results are transferable to a biopsy-based patient cohort. Trial registration: Patients for this unregistered study were enrolled retrospectively.

CXCL12 promoter methylation and PD-L1 expression as prognostic biomarkers in prostate cancer patients.

BACKGROUND: The CXCR4/CXCL12 axis plays a central role in systemic metastasis of prostate carcinoma (PCa), thereby representing a promising target for future therapies. Recent data suggest that the CXCR4/CXCL12 axis is functionally linked to the PD-1/PD-L1 immune checkpoint. We evaluated the prognostic value of aberrant CXCL12 DNA methylation with respect to PD-L1 expression in primary PCa. RESULTS: CXCL12 methylation showed a consistent significant correlation with Gleason grading groups in both cohorts (p < 0.001 for training and p = 0.034 for testing cohort). Short BCR-free survival was significantly associated with aberrant CXCL12 methylation in both cohorts and served as an independent prognostic factor in the testing cohort (hazard ratio = 1.92 [95%CI: 1.12-3.27], p = 0.049). Concomitant aberrant CXCL12 methylation and high PD-L1 expression was significantly associated with shorter BCR-free survival (p = 0.005). In comparative analysis, the CXCL12 methylation assay was able to provide approximately equivalent results in biopsy and prostatectomy specimens. MATERIALS AND METHODS: CXCL12 methylation was determined by means of a methylation specific quantitative PCR analysis in a radical prostatectomy patient cohort (n = 247, training cohort). Data published by The Cancer Genome Atlas served as a testing cohort (n = 498). CXCL12 methylation results were correlated to clinicopathological parameters including biochemical recurrence (BCR)-free survival. CONCLUSIONS: CXCL12 methylation is a powerful prognostic biomarker for BCR in PCa patients after radical prostatectomy. Further studies need to ascertain if CXCL12 methylation may aid in planning active surveillance strategies.

Diagnostic and prognostic value of SHOX2 and SEPT9 DNA methylation and cytology in benign, paramalignant, and malignant ascites.

BACKGROUND: Cytology remains the gold standard for the detection of malignant cells in ascites. However, its sensitivity is limited. The aim of this study was to evaluate DNA methylation biomarkers for the differential diagnosis of benign (ascites in patients without malignancy), malignant (ascites in cancer patients directly caused by malignancy), and paramalignant (ascites in cancer patients caused by comorbidities but not by malignancy) ascites. METHODS: A cohort of 283 patients (134 cancer patients, 149 patients with benign diseases) presenting with ascites was prospectively enrolled. Ascites was evaluated by means of cytopathological investigation and DNA methylation of SHOX2 and SEPT9 in the cell-free and cellular fraction. DNA methylation in bisulfite-converted DNA was determined using quantitative methylation specific real-time PCR. Cytopathological and DNA methylation results were evaluated with regard to diagnosis and overall survival (OS). RESULTS: Patients with positive DNA methylation had a poor overall survival compared to methylation-negative patients (hazard ratio: HR = 1.97, p = 0.001). In multivariate survival analysis, DNA methylation was an independent prognostic parameter (p = 0.003) together with age (HR = 1.03, p < 0.001) and the presence of malignant disease (HR = 1.87, p < 0.001). The combination of methylation with cytopathological analyses led to a 42 % increase in the detection rate of malignant ascites, resulting in 37 % positively diagnosed cancer patients and a specificity of 97 %. Among cancer patients, patients with DNA methylation-positive ascites showed an adverse clinical course (HR = 1.63, p = 0.039). CONCLUSIONS: DNA methylation testing adds diagnostic and prognostic information and might constitute an effective ancillary method for the differential diagnosis of malignant, paramalignant, and benign ascites.

Symptoms of anxiety and depression are frequent in patients with acute hepatitis C and are not associated with disease severity.

OBJECTIVE: Neuropsychiatric symptoms of hepatitis C virus (HCV) infection and during peginterferon α therapy have been investigated in the chronic stage of the infection, but have not been described during the acute phase of the disease so far. We therefore evaluated anxiety and depression in patients with acute hepatitis C by the Hospital Anxiety and Depression Scale (HADS) within a clinical trial. METHODS: Data were analysed from the German Hep-Net Acute HCV-III study. Anxiety and depression were characterized by an anxiety (HADS-A) and a depression subscale (HADS-D). More than eight points in each subscale were considered clinically relevant. Data were prospectively collected at baseline, end of treatment and at the end of the study. RESULTS: At baseline, a HADS-A above eight points was observed significantly more frequently than a HADS-D above eight points [n=23/103 (22%) vs. n=12/103 (12%); P=0.041].A pathological HADS-A or HADS-D score did not correlate with age, sex, IL28B genotype, the probable mode of infection, HCV genotype or severity of disease as investigated by alanine aminotransferase and bilirubin levels.Antiviral therapy did not influence anxiety as 12/50 (24%) of patients had HADS-A above 8 at the end of therapy. The proportion of patients with HADS-D above eight points increased from 12% at baseline to 24% (n=12/50) at the end of therapy (P=0.06). HADS results were not associated with lost to follow-up or sustained virological response rates. CONCLUSION: HADS data in acute HCV infection indicate that anxiety and depression do not correlate with severity of the disease, mode of acquisition, lost to follow-up and sustained virological response rates.

The Immune Checkpoint Regulator PD-L1 Is Highly Expressed in Aggressive Primary Prostate Cancer.

PURPOSE: Therapies targeting the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway promote anti-tumor immunity and have shown promising results in various tumors. Preliminary data further indicate that immunohistochemically detected PD-L1 may be predictive for anti-PD-1 therapy. So far, no data are available on PD-L1 expression in primary prostate cancer. EXPERIMENTAL DESIGN: Following validation of a monoclonal antibody, immunohistochemical analysis of PD-L1 expression was performed in two independent, well-characterized cohorts of primary prostate cancer patients following radical prostatectomy (RP), and resulting data were correlated to clinicopathological parameters and outcome. RESULTS: In the training cohort (n= 209), 52.2% of cases expressed moderate to high PD-L1 levels, which positively correlated with proliferation (Ki-67,P< 0.001), Gleason score (P= 0.004), and androgen receptor (AR) expression (P< 0.001). Furthermore, PD-L1 positivity was prognostic for biochemical recurrence [BCR;P= 0.004; HR, 2.37; 95% confidence interval (CI), 1.32-4.25]. In the test cohort (n= 611), moderate to high PD-L1 expression was detected in 61.7% and remained prognostic for BCR in univariate Cox analysis (P= 0.011; HR, 1.49; 95% CI, 1.10-2.02). The correlation of Ki-67 and AR with PD-L1 expression was confirmed in the test cohort (P< 0.001). In multivariate Cox analysis of all patients, PD-L1 was corroborated as independently prognostic for BCR (P= 0.007; HR, 1.46; 95% CI, 1.11-1.92). CONCLUSIONS: We provide first evidence that expression of the therapy target PD-L1 is not only highly prevalent in primary prostate cancer cells but is also an independent indicator of BCR, suggesting a biologic relevance in primary tumors. Further studies need to ascertain if PD-1/PD-L1-targeted therapy might be a treatment option for hormone-naïve prostate cancers.

TRPM4 protein expression in prostate cancer: a novel tissue biomarker associated with risk of biochemical recurrence following radical prostatectomy.

BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level with biochemical recurrence (BR) following radical prostatectomy (RP). MATERIAL AND METHODS: The study included 614 patients who had undergone RP. TRPM4 immunohistochemical staining was performed on samples of benign tissue, tissue containing PIN glands and PCa tissue using a commercially available polyclonal antibody. Staining intensity was recorded by two independent observers using a four-tired semi-quantitative grading system (0, 1+, 2+, 3+) converted into H-scores. Interobserver agreement was calculated by linear weighted kappa statistics. The association between staining intensity and BR was analysed using the Kaplan-Meier estimator and uni- and multiple Cox proportional hazard regression models. RESULTS: Significantly higher staining intensity was found in PCa glands compared to benign glands (p < 0.001). The concordance rate in TRPM4 staining intensities for benign, PIN and PCa tissue ranged from 86.0 to 91.5 %, corresponding to linear weighted kappa values of 0.566-0.789. After adjusting for patient and tumour characteristics, patients with a higher staining intensity in PCa glands compared to matched benign glands and an H-score equal to or above the median had an increased risk of BR (HR 1.79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high staining intensity and a high H-score) is associated with increased risk of BR after RP.

Outwitting EF-Tu and the ribosome: translation with d-amino acids.

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides. The rare appearance of d-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases. The system consists of wild-type components and d-aa are introduced via artificially charged, unmodified tRNA(Gly) that was selected according to the rules of 'thermodynamic compensation'. The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.

Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction.

The transcription factor T-bet regulates the production of interferon-γ and cytotoxic molecules in effector CD8 T cells, and its expression correlates with improved control of chronic viral infections. However, the role of T-bet in infections with differential outcome remains poorly defined. Here, we report that high expression of T-bet in virus-specific CD8 T cells during acute hepatitis B virus (HBV) and hepatitis C virus (HCV) infection was associated with spontaneous resolution, whereas T-bet deficiency was more characteristic of chronic evolving infection. T-bet strongly correlated with interferon-γ production and proliferation of virus-specific CD8 T cells, and its induction by antigen and IL-2 stimulation partially restored functionality in previously dysfunctional T-bet-deficient CD8 T cells. However, restoration of a strong interferon-γ response required additional stimulation with IL-12, which selectively induced the phosphorylation of STAT4 in T-bet(+) CD8 T cells. The observation that T-bet expression rendered CD8 T cells responsive to IL-12 suggests a stepwise mechanism of T cell activation in which T-bet facilitates the recruitment of additional transcription factors in the presence of key cytokines. These findings support a critical role of T-bet for viral clearance and suggest T-bet deficiency as an important mechanism behind chronic infection.

Inhibitory phenotype of HBV-specific CD4+ T-cells is characterized by high PD-1 expression but absent coregulation of multiple inhibitory molecules.

BACKGROUND: T-cell exhaustion seems to play a critical role in CD8+ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4+ T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4+ T-cell failure. METHODS: The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4+ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4+ T-cell proliferation and cytokine production. RESULTS: CD4+ T-cell responses during chronic HBV infection was characterized by reduced Tetramer+CD4+ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4+ T-cell expansion almost in treated patients with viral control. CONCLUSION: HBV-specific CD4+ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4+ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4+ T-cell functionality with heterogeneous patterns of CD4+ T-cell rejunivation.