a study to discover novel tumor-specific mutations

Background Splenic marginal zone lymphoma (SMZL) is an indolent B-cell non- Hodgkin lymphoma and represents the most common primary malignancy of the spleen. Its precise molecular pathogenesis is still unknown and specific molecular markers for diagnosis or possible targets for causal therapies are lacking. Methods We performed whole exome sequencing (WES) and copy number analysis from laser-microdissected tumor cells of two primary SMZL discovery cases. Selected somatic single nucleotide variants (SNVs) were analyzed using pyrosequencing and Sanger sequencing in an independent validation cohort. Results Overall, 25 nonsynonymous somatic SNVs were identified, including known mutations in the NOTCH2 and MYD88 genes. Twenty-three of the mutations have not been associated with SMZL before. Many of these seem to be subclonal. Screening of 24 additional SMZL for mutations at the same positions found mutated in the WES approach revealed no recurrence of mutations for ZNF608 and PDE10A, whereas the MYD88 L265P missense mutation was identified in 15 % of cases. An analysis of the NOTCH2 PEST domain and the whole coding region of the transcription factor SMYD1 in eight cases identified no additional case with a NOTCH2 mutation, but two additional cases with SMYD1 alterations. Conclusions In this first WES approach from microdissected SMZL tissue we confirmed known mutations and discovered new somatic variants. Recurrence of MYD88 mutations in SMZL was validated, but NOTCH2 PEST domain mutations were relatively rare (10 % of cases). Recurrent mutations in the transcription factor SMYD1 have not been described in SMZL before and warrant further investigation

Whole exome sequencing of microdissected splenic marginal zone lymphoma: a study to discover novel tumor-specific mutations

BACKGROUND: Splenic marginal zone lymphoma (SMZL) is an indolent B-cell non-Hodgkin lymphoma and represents the most common primary malignancy of the spleen. Its precise molecular pathogenesis is still unknown and specific molecular markers for diagnosis or possible targets for causal therapies are lacking. METHODS: We performed whole exome sequencing (WES) and copy number analysis from laser-microdissected tumor cells of two primary SMZL discovery cases. Selected somatic single nucleotide variants (SNVs) were analyzed using pyrosequencing and Sanger sequencing in an independent validation cohort. RESULTS: Overall, 25 nonsynonymous somatic SNVs were identified, including known mutations in the NOTCH2 and MYD88 genes. Twenty-three of the mutations have not been associated with SMZL before. Many of these seem to be subclonal. Screening of 24 additional SMZL for mutations at the same positions found mutated in the WES approach revealed no recurrence of mutations for ZNF608 and PDE10A, whereas the MYD88 L265P missense mutation was identified in 15 % of cases. An analysis of the NOTCH2 PEST domain and the whole coding region of the transcription factor SMYD1 in eight cases identified no additional case with a NOTCH2 mutation, but two additional cases with SMYD1 alterations. CONCLUSIONS: In this first WES approach from microdissected SMZL tissue we confirmed known mutations and discovered new somatic variants. Recurrence of MYD88 mutations in SMZL was validated, but NOTCH2 PEST domain mutations were relatively rare (10 % of cases). Recurrent mutations in the transcription factor SMYD1 have not been described in SMZL before and warrant further investigatio

Hepatitis C-associated B-cell non-Hodgkin lymphomas. Epidemiology, molecular signature and clinical management

n/

Serum MicroRNA-21 as Marker for Necroinflammation in Hepatitis C Patients with and without Hepatocellular Carcinoma

Background: MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC. Methodology/Principal Findings: 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%. Conclusions/Significance: The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC

Promoter methylation of MLH1, PMS2, MSH2 and p16 is a phenomenon of advanced-stage HCCs

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future

Evaluation of the Integrated Pulmonary Index® during non-anesthesiologist sedation for percutaneous endoscopic gastrostomy

Standard monitoring of heart rate, blood pressure and arterial oxygen saturation during endoscopy is recommended by current guidelines on procedural sedation. A number of studies indicated a reduction of hypoxic (art. oxygenation  15 s) and severe hypoxic events (art. oxygenation  10 s, partial pressure of end-tidal carbon dioxide [PetCO2]). The aim of this paper was to analyze the value of IPI as parameter to monitor the respiratory status in patients receiving propofol sedation during PEG-procedure. Patients reporting for PEG-placement under sedation were randomized 1:1 in either standard monitoring group (SM) or capnography monitoring group including IPI (IM). Heart rate, blood pressure and arterial oxygen saturation were monitored in SM. In IM additional monitoring was performed measuring PetCO2, respiratory rate and IPI. Capnography and IPI values were recorded for all patients but were only visible to the endoscopic team for the IM-group. IPI values range between 1 and 10 (10 = normal; 8–9 = within normal range; 7 = close to normal range, requires attention; 5–6 = requires attention and may require intervention; 3–4 = requires intervention; 1–2 requires immediate intervention). Results on capnography versus standard monitoring of the same study population was published previously. A total of 147 patients (74 in SM and 73 in IM) were included in the present study. Hypoxic events occurred in 62 patients (42%) and severe hypoxic events in 44 patients (29%), respectively. Baseline characteristics were equally distributed in both groups. IPI = 1, IPI  10 s had a high sensitivity for hypoxic and severe hypoxic events, respectively (IPI = 1: 81%/81% [hypoxic/severe hypoxic event], IPI  10 s: 84%/84%). All four parameters had a low specificity for both hypoxic and severe hypoxic events (IPI = 1: 13%/12%, IPI  10 s: 7%/7%). In multivariate analysis, only SM and PetCO2 = 0 mmHg were independent risk factors for hypoxia. IPI (IPI = 1 and IPI  10 s allow a fast and convenient conclusion on patients’ respiratory status in a morbid patient population. Sensitivity is good for most parameters, but specificity is poor. In conclusion, IPI can be a useful metric to assess respiratory status during propofol-sedation in PEG-placement. However, IPI was not superior to PetCO2 and apnea > 10 s

MicroRNA Profiling of Laser-Microdissected Hepatocellular Carcinoma Reveals an Oncogenic Phenotype of the Tumor Capsule

Several microRNAs (miRNAs) are associated with the molecular pathogenesis of hepatocellular carcinoma (HCC). However, previous studies analyzing the dysregulation of miRNAs in HCC show heterogeneous results. We hypothesized that part of this heterogeneity might be attributable to variations of miRNA expression deriving from the HCC capsule or the fibrotic septa within the peritumoral tissue used as controls. Tissue from surgically resected hepatitis C–associated HCC from six well-matched patients was microdissected using laser microdissection and pressure catapulting technique. Four distinct histologic compartments were isolated: tumor parenchyma (TP), fibrous capsule of the tumor (TC), tumor-adjacent liver parenchyma (LP), and cirrhotic septa of the tumor-adjacent liver (LC). MiRNA expression profiling analysis of 1105 mature miRNAs and precursors was performed using miRNA microarray. Principal component analysis and consecutive pairwise supervised comparisons demonstrated distinct patterns of expressed miRNAs not only for TP versus LP (e.g., intratumoral down-regulation of miR-214, miR-199a, miR-146a, and miR-125a; P< .05) but also for TC versus LC (including down-regulation within TC of miR-126, miR-99a/100, miR-26a, and miR-125b; P< .05). The tumor capsule therefore demonstrates a tumor-like phenotype with down-regulation of well-known tumor-suppressive miRNAs. Variations of co-analyzed fibrotic tissue within the tumor or in controls may have profound influence on miRNA expression analyses in HCC. Several miRNAs, which are proposed to be HCC specific, may indeed be rather associated to the tumor capsule. As miRNAs evolve to be important biomarkers in liver tumors, the presented data have important translational implications on diagnostics and treatment in patients with HCC

Microsatellite instability occurs rarely in patients with Cholangiocarcinoma : a retrospective study from a German tertiary care hospital

Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA

MSP analysis.

<p>Representative examples of MSP analysis for MLH1, MSH2, PMS2 and p16 methylation in HCC and non-tumour adjacent tissues were shown. Bisulfite-modified DNA was amplified using MSP primers specific to a CpG-rich region of each gene promoter. PCR-amplified products were resolved by 2% agarose gel electrophoresis. (U) Lanes represent amplification of unmethylated alleles, and (M) lanes contain only methylated alleles.</p