Notch signaling induces cell proliferation in the labrum in a regulatory network different from the thoracic legs

The insect head is composed of several segments and an anterior non-segmental region. While patterning of the segmental region relies - at least in part - on the known trunk patterning mechanisms, development of the anterior most region remains poorly understood. The labrum is an enigmatic structure of the anterior median region (AMR) of the insect head. Based on similar development and gene expression patterns it has been suggested to be a serial homolog of trunk appendages. However, its position in the non-segmental region indicated an independent origin. In order to learn more about development of the AMR including the labrum, we screened the results of the large scale RNAi screen iBeetle to identify novel genes. We found the Notch ligand Tc-Serrate and the ubiquitin ligase Tc-mind bomb1 to be required for labrum formation. Further studies showed that Notch signaling is acting upstream of the genetic hierarchy and is required for regulating cell proliferation. We combined our work with previous data to compare the regulatory gene networks of labrum and trunk appendage formation. This reveals that despite the involvement of a similar set of genes, the genetic interactions are quite different. (C) 2015 The Authors. Published by Elsevier Inc

Biomarkers for Homologous Recombination Deficiency in Cancer

DNA double-strand breaks foster tumorigenesis and cell death. Two distinct mechanisms can be activated by the cell for DNA repair: the accurate mechanism of homologous recombination repair or the error-prone non-homologous end joining. Homologous Recombination Deficiency (HRD) is associated with sensitivity towards PARP inhibitors (PARPi) and its determination is used as a biomarker for therapy decision making. Nevertheless, the biology of HRD is rather complex and the application, as well as the benefit of the different HRD biomarker assays, is controversial. Acquiring knowledge of the underlying molecular mechanisms is the main prerequisite for integration of new biomarker tests. This study presents an overview of the major DNA repair mechanisms and defines the concepts of HRR, HRD and BRCAness. Moreover, currently available biomarker assays are described and discussed with respect to their application for routine clinical diagnostics. Since patient stratification for efficient PARP inhibitor therapy requires determination of the BRCA mutation status and genomic instability, both should be established comprehensively. For this purpose, a broad spectrum of distinct assays to determine such combined HRD scores is already available. Nevertheless, all tests require careful validation using clinical samples to meet the criteria for their establishment in clinical testing

Enhanced genome assembly and a new official gene set for Tribolium castaneum

Abstract Background The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. Results Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. Conclusions The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis

Molecular Characteristics of Radon Associated Lung Cancer Highlights MET Alterations

Simple Summary Lung cancer (LC) is the leading cause of cancer death worldwide. After smoking, one of the most prominent risk factors for LC development is radon (Rn) exposure. In our study we analysed and compared the genetic landscape of LC patients from a Rn exposed village with local matched non-exposed patients. Within the concordant genetic landscape, an increase in genetic MET proto-oncogene, receptor tyrosine kinase (MET) alteration in the Rn-exposed cohort was monitored, underlining the importance of routine MET testing and potential to enable a more effective treatment for this specific subgroup. Effective targeted treatment strategies resulted from molecular profiling of lung cancer with distinct prevalent mutation profiles in smokers and non-smokers. Although Rn is the second most important risk factor, data for Rn-dependent driver events are limited. Therefore, a Rn-exposed cohort of lung cancer patients was screened for oncogenic drivers and their survival and genetic profiles were compared with data of the average regional population. Genetic alterations were analysed in 20 Rn-exposed and 22 histologically matched non-Rn exposed LC patients using targeted Next generation sequencing (NGS) and Fluorescence In Situ Hybridization (FISH). Sufficient material and sample quality could be obtained in 14/27 non-exposed versus 17/22 Rn-exposed LC samples. Survival was analysed in comparison to a histologically and stage-matched regional non-exposed lung cancer cohort (n = 51) for hypothesis generating. Median overall survivals were 83.02 months in the Rn-exposed and 38.7 months in the non-exposed lung cancer cohort (p = 0.22). Genetic alterations of both patient cohorts were in high concordance, except for an increase in MET alterations and a decrease in TP53 mutations in the Rn-exposed patients in this small hypothesis generating study

Managing Difficulties of Microsatellite Instability Testing in Endometrial Cancer-Limitations and Advantages of Four Different PCR-Based Approaches

Simple Summary Due to the approval of immune checkpoint inhibitor therapy for microsatellite instability-high or mismatch repair-deficient advanced solid tumors, testing of both biomarkers has gained interest in recent years. Available testing systems were established in the context of Lynch Syndrome for colorectal cancer, thus differences between microsatellite profiles across cancer types may lead to false data interpretation using validated tests for another tumor entity. The present study deals with challenges during microsatellite instability testing in endometrial cancer (EC) and provides a comprehensive comparative study of four different PCR-based approaches which could help to improve microsatellite instability (MSI) testing in future screenings. A validation strategy has been developed for the Idylla system, which can guide the method transfer to other tumor entities, and a screening procedure for EC has been proposed. By direct comparison, this study was able to highlight advantages and limitations of each system in an extensive manner. Microsatellite instability (MSI), a common alteration in endometrial cancers (EC) is known as a biomarker for immune checkpoint therapy response alongside screening for Lynch Syndrome (LS). However, former studies described challenging MSI profiles in EC hindering analysis by using MSI testing methods intensively validated for colorectal cancer (CRC) only. In order to reduce false negatives, this study examined four different PCR-based approaches for MSI testing using 25 EC samples already tested for mismatch repair deficiency (dMMR). In a follow up validation set of 75 EC samples previously tested both for MMR and MSI, the efficiency of a seven-marker system corresponding to the Idylla system was further analyzed. Both Bethesda and Promega marker panels require trained operators to overcome interpretation complexities caused by either hardly visible additional peaks of one and two nucleotides, or small shifts in microsatellite repeat length. Using parallel sequencing adjustment of bioinformatics is needed. Applying the Idylla MSI assay, an evaluation of input material is more crucial for reliable results and is indispensable. Following MMR deficiency testing as a first-line screening procedure, additional testing with a PCR-based method is necessary if inconclusive staining of immunohistochemistry (IHC) must be clarified

FGFR2-amplified tumor clones are markedly heterogeneously distributed in carcinomas of the upper gastrointestinal tract

Background FGFR2 is a therapy-relevant target in tumors of the upper gastrointestinal tract (GIT), and clinical trials are currently underway to test the efficacy of FGFR2 inhibitors. Tumor heterogeneity is one of the relevant causes of treatment failure. Almost nothing is known about the heterogeneous distribution of FGFR2-amplified clones in adenocarcinomas of the upper GIT. Patients and methods To assess FGFR2 gene copy number alteration and intratumoral heterogeneity of upper GIT adenocarcinomas, we analyzed 893 patient-derived formalin-fixed paraffin-embedded tumor specimens, including primary operated and neoadjuvant-treated tumors (462 gastric carcinomas and 429 esophageal adenocarcinomas) as well as complementary lymph node and distant metastasis by fluorescence in situ hybridization. Results Twenty-six gastric tumors (5.6%) and 21 esophageal adenocarcinomas (4.9%) showed FGFR2 amplification. Overall, 93% of gastric carcinomas and 83% of esophageal carcinomas showed heterogeneous amplification. FGFR2 amplification was found in different histological growth patterns, including intestinal and diffuse type according to the Lauren classification. In the primary gastric carcinoma group, FGFR2 amplification was associated with poor prognosis (p = 0.005). Conclusion Homogeneous FGFR2 amplification in tumors of the upper GIT is the exception. This has highly relevant implications in the nature of FGFR2 diagnostics (sufficient tumor cell number, determination of amplification at metastasis versus primary tumor, etc.) and on the response probability of appropriate inhibitors. It is relevant that the often poorly treatable and aggressive subtype of diffuse carcinomas (poorly cohesive carcinomas) also shows FGFR2 amplification and that an individualized therapy option with FGFR2 inhibitors could be an option in this group

Screens in fly and beetle reveal vastly divergent gene sets required for developmental processes

Background: Most of the known genes required for developmental processes have been identified by genetic screens in a few well-studied model organisms, which have been considered representative of related species, and informative-to some degree-for human biology. The fruit fly Drosophila melanogaster is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. A quantification of conservation versus divergence of gene functions has been missing, without which it is unclear how representative data from model systems actually are. Results: Here, we systematically compare the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes. Then we compared the gene sets required for four different developmental processes between beetle and fly. We found that around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (similar to 10%) or beetle (similar to 40%) reflecting both technical and biological differences. Accordingly, we were able to annotate novel developmental GO terms for 96 genes studied in this work. With this work, we publish the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). Conclusions: We conclude that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor

Occurrence of High Microsatellite-Instability/Mismatch Repair Deficiency in Nearly 2,000 Human Adenocarcinomas of the Gastrointestinal Tract, Pancreas, and Bile Ducts: A Study From a Large German Comprehensive Cancer Center

Introduction: Knowledge of the high microsatellite-instability (MSI-H)/mismatch repair deficiency (MMRd) status is of increasing interest for personalized neoadjuvant or adjuvant therapy planning. Only a few studies are available on MSI-H distribution in the Northern European Caucasian patient population. In this study, we focused on a large cohort of tumors of the upper gastrointestinal tract. Materials and Methods: Surgical material from a total of 1,965 patients was analyzed for MSI-H/MMRd status (including 1,267 carcinomas of the esophagus or stomach). All tumors were analyzed with an internationally recommended immunohistochemical panel consisting of four antibodies (MLH1, MSH2, PMS2, and MSH6). The results were molecularly objectified. Results: Adenocarcinomas with MSI-H/MMRd were detected with the following distribution: esophagus (1.4%), stomach (8.3%), small intestine (18.2%), large intestine (8.5%), intrahepatic bile ducts (1.9%), and pancreas (0%). In case of gastric tumors with MSI-H/MMRd, neoadjuvant therapy did not influence the prognosis of patients (p = 0.94). Within all tumor entities with MSI-H/MMRd, patients with a UICC stage 4 were also represented. In this advanced stage, 11.7% of patients with MSS tumors were diagnosed compared to 0.5% of patients with MSI-H tumors relative to the entire tumor collective. Discussion: In this study, the proportion of MSI-H/MMRd tumors in the stomach is smaller than would have been expected in knowledge of the data published by TCGA or AGRC. Negative prognostic effects regarding MSI-H status and neoadjuvant therapy as described by the MAGIC study group were not seen in our cohort. The extent to which the MSI-H/MMRd status should be known for neoadjuvant therapy planning must be clarified in prospective studies in the future. At present, there is no convincing data to dispense the neoadjuvant therapy for gastric carcinoma. Due to the very convincing, positive data regarding the response rates of MSI-H tumors to treatment with PD1/PD-L1 inhibitors, every metastatic carcinoma of the gastrointestinal tract should be tested for its MSI-H status

Modern day screening for Lynch syndrome in endometrial cancer: the KEM experience

Purpose Current guidelines for Lynch syndrome detection in endometrial cancer (EC) patients rely either on risk evaluation, based on personal/family history, or detection of mismatch repair (MMR) deficiency on tumor tissue. We present a combined screening algorithm for Lynch syndrome. Methods In this study, 213 consecutive patients treated for EC at Kliniken Essen-Mitte between 2014 and 2018 were included. Personal/family history was evaluated by the Amsterdam II, revised Bethesda/German-DKG criteria and prediction model PREMM5. MMR testing was performed by immunohistochemistry (IHC) and/or polymerase chain reaction (PCR) based microsatellite analysis on tumor tissue. MLH1 promoter methylation analysis was performed in case of MLH1 loss or microsatellite instability. Results Based on personal/family history 2/213 (Amsterdam II), 31/213 (revised Bethesda/German-DKG) and 149/213 (PREMM5) patients were identified as at risk for Lynch syndrome. MMR analysis was performed by IHC in 51.2%, by PCR in 32.4%, and in 16.4% of patients both methods were used. MMR deficiency was detected in 20.6% (44/213). Methylation analysis was performed in 27 patients of whom, 22 (81.4%) showed MLH1 promoter hypermethylation. Only 9% of MMR deficient patients were identified as at risk for Lynch syndrome by the revised Bethesda/German-DKG criteria. A pathogenic germline mutation was discovered in 3 out of 20 patients that underwent genetic testing. None of these patients were younger than 50 years or had a family history of Lynch syndrome-associated malignancies. Conclusion General MMR assessment is a feasible strategy to improve the detection of Lynch Syndrome in patients with EC