Prognostic Role of Survivin and Macrophage Infiltration Quantified on Protein and mRNA Level in Molecular Subtypes Determined by RT-qPCR of KRT5, KRT20, and ERBB2 in Muscle-Invasive Bladder Cancer Treated by Adjuvant Chemotherapy.

OBJECTIVES: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/BIRC5 and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS). MATERIALS AND METHODS: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. BIRC5, KRT5, KRT20, ERBB2, and CD68 mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence. RESULTS: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy (p = 0.0138 and p = 0.001, respectively). These results were validated by the quantitation of BIRC5 mRNA by PCR (p = 0.0004 and p = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with BIRC5 mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/BIRC5 levels based on molecular subtype being assessed by KRT20 expression improved the predictive value, with tumors having low survivin/BIRC5 and KRT20 mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, p = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis (p = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or ERBB2-positive tumors was associated with worse DSS. CONCLUSIONS: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor-biological axes of high prognostic relevance, which warrant further investigation and validation.

Evaluation of a New Survivin ELISA and UBC® Rapid for the Detection of Bladder Cancer in Urine.

Urine-based biomarkers for non-invasive diagnosis of bladder cancer are urgently needed. No single marker with sufficient sensitivity and specificity has been described so far. Thus, a combination of markers appears to be a promising approach. The aim of this case-control study was to evaluate the performance of an in-house developed enzyme-linked immunosorbent assay (ELISA) for survivin, the UBC®Rapid test, and the combination of both assays. A total of 290 patients were recruited. Due to prior bladder cancer, 46 patients were excluded. Urine samples were available from 111 patients with bladder cancer and 133 clinical controls without urologic diseases. Antibodies generated from recombinant survivin were utilized to develop a sandwich ELISA. The ELISA and the UBC®Rapid test were applied to all urine samples. Receiver operating characteristic (ROC) analysis was used to evaluate marker performance. The survivin ELISA exhibited a sensitivity of 35% with a specificity of 98%. The UBC®Rapid test showed a sensitivity of 56% and a specificity of 96%. Combination of both assays increased the sensitivity to 66% with a specificity of 95%. For high-grade tumors, the combination showed a sensitivity of 82% and a specificity of 95%. The new survivin ELISA and the UBC®Rapid test are both able to detect bladder cancer, especially high-grade tumors. However, the performance of each individual marker is moderate and efforts to improve the survivin assay should be pursued. A combination of both assays confirmed the benefit of using marker panels. The results need further testing in a prospective study and with a high-risk population.

A recombinant polypeptide of the megakaryocyte potentiating factor is a potential biomarker in plasma for the detection of mesothelioma.

Malignant mesothelioma (MM) is a fatal disease mostly associated with asbestos exposure and difficult to detect by non-invasive methods. This study aimed to use recombinant fragments of the megakaryocyte potentiating factor (MPF) for the development of cost-effective MPF ELISAs. Three polypeptides spanning the MPF region (MPF1-148, MPF 34-288, MPF/MSLN254-400) were produced in E.coli as maltose-binding protein hybrids. After isolation, Factor Xa digest, and purification, the polypeptides were used for the generation of rabbit antibodies and development of ELISAs. Forty-one MM patients with known histological subtype before tumor-specific treatment and 70 asbestos-exposed individuals free of any cancer were matched according to age, gender, and smoking. Plasma of all subjects was tested with the three newly developed polyclonal antibody-based ELISAs and a commercial mesothelin assay (MESOMARK™). The latter differentiated patients (median concentration 1.95 nM) from controls (median 1.07 nM, p < 0.0001) and showed an area under curve (AUC) of 0.77 in receiver operating characteristics (ROC) analysis. Of the MPF variants, exclusively the ELISA based on antibodies against the MPF34-288 fragment displayed significantly (p = 0.0002) higher values in patients than in controls (median 1.61 nM versus 0.88 nM; AUC = 0.72). The combination of the MPF34-288 and mesothelin displayed an improved ROC performance (AUC = 0.80). The MPF34-288 ELISA could be a cost-effective and minimal-invasive contribution to support a diagnosis of mesothelioma, especially in regions with a limited medical care.

Membrane-binding mechanism of a bacterial phospholipid N-methyltransferase.

The membrane lipid phosphatidylcholine (PC) is crucial for stress adaptation and virulence of the plant pathogen Agrobacterium tumefaciens. The phospholipid N-methyltransferase PmtA catalyzes three successive methylations of phosphatidylethanolamine to yield PC. Here, we asked how PmtA is recruited to its site of action, the inner leaflet of the membrane. We found that the enzyme attaches to the membrane via electrostatic interactions with anionic lipids, which do not serve as substrate for PmtA. Increasing PC concentrations trigger membrane dissociation suggesting that membrane binding of PmtA is negatively regulated by its end product PC. Two predicted alpha-helical regions (αA and αF) contribute to membrane binding of PmtA. The N-terminal helix αA binds anionic lipids in vitro with higher affinity than the central helix αF. The latter undergoes a structural transition from disordered to α-helical conformation in the presence of anionic lipids. The basic amino acids R8 and K12 and the hydrophobic amino acid F19 are critical for membrane binding by αA as well as for activity of full-length PmtA. We conclude that a combination of electrostatic and hydrophobic forces is responsible for membrane association of the phospholipid-modifying enzyme.

Combination of calretinin, MALAT1, and GAS5 as a potential prognostic biomarker to predict disease progression in surgically treated mesothelioma patients.

BACKGROUND: The role of cytoreductive surgery for epithelioid pleural mesothelioma within a multimodal treatment approach remains controversial. Carefully selected patients benefit from cytoreductive surgery and adjuvant chemotherapy, but there is no established biomarker to predict tumor recurrence or progression during the course of the disease. The aim of this study was to identify potential biomarkers to predict therapeutic response in terms of progression-free survival. METHODS: Between 03/2014 and 08/2022, preoperative blood samples were collected from 76 patients with epithelioid pleural mesothelioma who underwent cytoreductive surgery as part of a multimodal treatment approach. Identification of potential biomarkers was performed by determination of mesothelin and calretinin, as well as specific long non-coding RNAs and microRNAs. Receiver operating characteristic analysis, Kaplan-Meier survival analysis, and Cox regression were used to assess the association between biomarker concentrations and patient recurrence status and survival. RESULTS: MALAT1, GAS5, and calretinin showed statistically significant increased biomarker levels in patients with recurrence in contrast to recurrence-free patients after surgical treatment (p < 0.0001, p = 0.0190, and p = 0.0068, respectively). The combination of the three biomarkers resulted in a sensitivity of 68 % and a specificity of 89 %. CONCLUSION: MALAT1, GAS5, and calretinin could be potential biomarkers for the prediction of tumor recurrence, improving the benefit from multimodal treatment including cytoreductive surgery.

Exploring solid-phase proximity ligation assay for survivin detection in urine.

Urine-based biomarkers are a rational and promising approach for the detection of bladder cancer due to the proximity of urine to the location of the tumor site and the non-invasive nature of its sampling. A well-known and highly investigated biomarker for bladder cancer is survivin. For detection of very small amounts of urinary survivin protein a highly sensitive assay was developed. The assay is based on the immuno-PCR technology, more precisely a solid-phase proximity ligation assay (spPLA). The limit of detection for the survivin spPLA was 1.45 pg/mL, resulting in an improvement of the limit of detection by a factor of approximately 23 compared to the previously in-house developed survivin ELISA. A key step in development was the initial isolation of survivin by a molecular fishing rod based on magnetic beads. Interfering matrix compounds pose a special challenge for further analytical application, but can be overcome by this isolation step. The assay is designed to work with only 500 µL of voided urine. The survivin spPLA showed a sensitivity of 30% and specificity of 89% for bladder cancer detection in this study of 110 bladder cancer cases and 133 clinical controls. Moreover, the results demonstrated again that survivin is a useful complementary marker in combination with UBC® Rapid by increasing the overall sensitivity to 70% with a specificity of 86%. Although the performance for detection of bladder cancer was rather low, the herein developed assay might serve as a new tool for survivin biomarker research in diverse human fluids, even if the biological matrix is complex or survivin is only present in small amounts.

S-adenosylmethionine-binding properties of a bacterial phospholipid N-methyltransferase.

The presence of the membrane lipid phosphatidylcholine (PC) in the bacterial membrane is critically important for many host-microbe interactions. The phospholipid N-methyltransferase PmtA from the plant pathogen Agrobacterium tumefaciens catalyzes the formation of PC by a three-step methylation of phosphatidylethanolamine via monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine. The methyl group is provided by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) during transmethylation. Despite the biological importance of bacterial phospholipid N-methyltransferases, little is known about amino acids critical for binding to SAM or phospholipids and catalysis. Alanine substitutions in the predicted SAM-binding residues E58, G60, G62, and E84 in A. tumefaciens PmtA dramatically reduced SAM-binding and enzyme activity. Homology modeling of PmtA satisfactorily explained the mutational results. The enzyme is predicted to exhibit a consensus topology of the SAM-binding fold consistent with cofactor interaction as seen with most structurally characterized SAM-methyltransferases. Nuclear magnetic resonance (NMR) titration experiments and (14)C-SAM-binding studies revealed binding constants for SAM and SAH in the low micromolar range. Our study provides first insights into structural features and SAM binding of a bacterial phospholipid N-methyltransferase.

Circulating long non-coding RNA GAS5 (growth arrest-specific transcript 5) as a complement marker for the detection of malignant mesothelioma using liquid biopsies.

BACKGROUND: For the detection of malignant mesothelioma additional markers are needed besides the established panel consisting of calretinin and mesothelin. The aim of this study was the identification and verification of long non-coding RNAs (lncRNAs) as complementing circulating markers. METHODS: Candidate lncRNAs were identified in silico using previously published RNA expression profiles and verified using quantitative PCR (qPCR) in mesothelioma cell lines as well as human plasma samples from mesothelioma patients and asbestos-exposed controls. RESULTS: GAS5 (growth arrest-specific transcript 5) as a single marker is marked by a low sensitivity of 14%, but the combination of GAS5 with calretinin and mesothelin increased the panel's sensitivity from 64 to 73% at a predefined specificity of 97%. Circulating GAS5 is not affected by pleurectomy before blood collection, age, or smoking status. CONCLUSIONS: GAS5 is verified as an appropriate circulating marker for the supplement of calretinin and mesothelin to detect malignant mesothelioma. Although the sensitivity of GAS5 is too low for the use as a single marker, the addition of GAS5 as a third marker improves the performance of the established marker panel. The benefit of GAS5 for the detection of malignant mesothelioma at early stages needs to be validated in a prospective study.

Toward noninvasive follow-up of low-risk bladder cancer - Rationale and concept of the UroFollow trial.

BACKGROUND: Follow-up recommendations for patients with nonmuscle invasive bladder cancer (NMIBC) are largely based upon expert opinion. A growing body of evidence suggests that current follow-up strategies for bladder cancer patients with low and intermediate risk represent overdiagnosis and may lead to overtreatment. The goal of this study is to explore the options of a noninvasive follow-up in patients with pTa G1-2/low-grade NMIBC. METHODS: The risks and options for a urine marker-guided, noninvasive follow-up of patients with pTa G1-2/low-grade NMIBC were defined and the study design for a prospective randomized trial (UroFollow) was developed based upon the current literature. RESULTS: The investigators postulated that follow-up of patients with pTa G1-2/low-grade NMIBC requires a high sensitivity of urinary tumor markers. However, data from prospective studies with prediagnostic urine samples are scarce, even for approved markers, and cross-sectional studies with symptomatic patients overestimate the sensitivity. So far, cell-based markers (e.g., uCyt+ and UroVysion) in urine appeared to have higher sensitivities and specificities in low-grade NMIBC than urine cytology and markers analyzing soluble tumor-associated antigens. Marker panels are more sensitive than single-marker approaches at the expense of a lower specificity. Given a prospective randomized comparison with a marker sensitivity of 80% compared to usual care with cystoscopy, the sample size calculation yielded that 62 to 185 patients under study per arm are needed depending on different recurrence rates. CONCLUSIONS: Based upon these findings the UroFollow trial has been designed as a prospective randomized study comparing a noninvasive marker-based (UroVysion, NMP22, urine cytology, and ultrasound) follow-up with the current standard of care over a period of 3 years.

Phosphatidylcholine biosynthesis and its significance in bacteria interacting with eukaryotic cells.

Phosphatidylcholine (PC), a typical eukaryotic membrane phospholipid, is present in only about 10% of all bacterial species, in particular in bacteria interacting with eukaryotes. A number of studies revealed that PC plays a fundamental role in symbiotic and pathogenic microbe-host interactions. Agrobacterium tumefaciens mutants lacking PC are unable to elicit plant tumors. The human pathogens Brucella abortus and Legionella pneumophila require PC for full virulence. The plant symbionts Bradyrhizobium japonicum and Sinorhizobium meliloti depend on wild-type levels of PC to establish an efficient root nodule symbiosis. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (Pcs) pathway. The methylation pathway involves a three-step methylation of phosphatidylethanolamine by at least one phospholipid N-methyltransferase to yield phosphatidylcholine. In the Pcs pathway, choline is condensed directly with CDP-diacylglycerol to form PC. This review focuses on the biosynthetic pathways and the significance of PC in bacteria with an emphasis on plant-microbe interactions.