The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.

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.

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.

Outcome of immune checkpoint inhibition in cancer can be predicted by measuring PDL1 expression of tumor cells. Search for additional biomarkers led to tumor mutational burden (TMB) as surrogate marker for neoantigens presented. While TMB was previously determined via whole exome sequencing (WES), there have been approaches with comprehensive gene panels as well. We sequenced samples derived from formalin-fixed tumors, aPOLEmutated cell line and standard DNA by WES and five different panels. If available, normal tissue was also exome sequenced. Sequencing data was analyzed by commercial software solutions and an in-house pipeline. A robust Pearson correlation (R = 0.9801 ± 0.0167; mean ± sd; N = 7) was determined for the different panels in a tumor paired normal setting for WES. Expanded analysis on tumor only exome sequenced samples yielded similar correlation (R = 0.9439 ± 0.0632; mean ± sd; N = 14). Remaining germline variants increased TMB in WES by 5.761 ± 1.953 (mean ± sd.; N = 7) variants per megabase (v/mb) for samples including synonymous variants and 3.883 ± 1.38 v/mb for samples without synonymous variants compared to tumor-normal paired calling results. Due to limited sample numbers in this study, additional replication is suggested for a clinical setting. Remaining germline variants in a tumor-only setting and artifacts caused by different library chemistries construction might affect the results.

Gene fusions involving the three neurotrophic tyrosine receptor kinase genesNTRK1,NTRK2, orNTRK3were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of aNTRKfusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next‐generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA‐/DNA‐based tNGS for detection ofNTRKfusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH‐based and 18 in the NGS‐based round robin test, the latter additionally subdivided into low‐input and high‐input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin‐fixed and paraffin‐embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS‐based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH‐ and tNGS‐basedNTRKtesting can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150NTRKfusion variants by several commercially available RNA‐based tNGS panels.