Accurate tumor purity determination is critical for the analysis of homologous recombination deficiency (HRD).

Homologous recombination deficiency (HRD) is a predictive marker for response to poly (ADP-ribose) polymerase inhibitors (PARPi) in ovarian carcinoma. HRD scores have entered routine diagnostics, but the influence of algorithms, parameters and confounders has not been analyzed comprehensively. A series of 100 poorly differentiated ovarian carcinoma samples was analyzed using whole exome sequencing (WES) and genotyping. Tumor purity was determined using conventional pathology, digital pathology, and two bioinformatic methods. HRD scores were calculated from copy number profiles determined by Sequenza and by Sclust either with or without fixed tumor purity. Tumor purity determination by digital pathology combined with a tumory purity informed variant of Sequenza served as reference method for HRD scoring. Seven tumors had deleterious mutations in BRCA1/2, 12 tumors had deleterious mutations in other homologous recombination repair (HRR) genes, 18 tumors had variants of unknown significance (VUS) in BRCA1/2 or other HRR genes, while the remaining 63 tumors had no relevant alterations. Using the reference method for HRD scoring, 68 tumors were HRD-positive. HRDsum determined by WES correlated strongly with HRDsum determined by single nucleotide polymorphism (SNP) arrays (R = 0.85). Conventional pathology systematically overestimated tumor purity by 8% compared to digital pathology. All investigated methods agreed on classifying the deleterious BRCA1/2-mutated tumors as HRD-positive, but discrepancies were observed for some of the remaining tumors. Discordant HRD classification of 11% of the tumors was observed comparing the tumor purity uninformed default of Sequenza and the reference method. In conclusion, tumor purity is a critical factor for the determination of HRD scores. Assistance by digital pathology helps to improve accuracy and imprecision of its estimation.

BAP1 and PTEN mutations shape the immunological landscape of clear cell renal cell carcinoma and reveal the intertumoral heterogeneity of T cell suppression: a proof-of-concept study.

Clear cell renal cell carcinoma (ccRCC) is an immunologically vulnerable tumor entity, and immune checkpoint inhibitors are now widely used to treat patients with advanced disease. Whether and to what extent immune responses in ccRCC are shaped by genetic alterations, however, is only beginning to emerge. In this proof-of-concept study, we performed a detailed correlative analysis of the mutational and immunological landscapes in a series of 23 consecutive kidney cancer patients. We discovered that a high infiltration with CD8 + T cells was not dependent on the number of driver mutations but rather on the presence of specific mutational events, namely pathogenic mutations in PTEN or BAP1. This observation encouraged us to compare mechanisms of T cell suppression in the context of four different genetic patterns, i.e., the presence of multiple drivers, a PTEN or BAP1 mutation, or the absence of detectable driver mutations. We found that ccRCCs harboring a PTEN or BAP1 mutation showed the lowest level of Granzyme B positive tumor-infiltrating lymphocytes (TILs). A multiplex immunofluorescence analysis revealed a significant number of CD8 + TILs in the vicinity of CD68 + macrophages/monocytes in the context of a BAP1 mutation but not in the context of a PTEN mutation. In line with this finding, direct interactions between CD8 + TILs and CD163 + M2-polarized macrophages were found in BAP1-mutated ccRCC but not in tumors with other mutational patterns. While an absence of driver mutations was associated with more CD8 + TILs in the vicinity of FOXP3 + Tregs and CD68 + monocytes/macrophages, the presence of multiple driver mutations was, to our surprise, not found to be strongly associated with immunosuppressive mechanisms. Our results highlight the role of genetic alterations in shaping the immunological landscape of ccRCC. We discovered a remarkable heterogeneity of mechanisms that can lead to T cell suppression, which supports the need for personalized immune oncological approaches.

Genomic architecture of FGFR2 fusions in cholangiocarcinoma and its implication for molecular testing.

BACKGROUND: Cholangiocarcinoma (CCA) is a primary malignancy of the biliary tract with a dismal prognosis. Recently, several actionable genetic aberrations were identified with significant enrichment in intrahepatic CCA, including FGFR2 gene fusions with a prevalence of 10-15%. Recent clinical data demonstrate that these fusions are druggable in a second-line setting in advanced/metastatic disease and the efficacy in earlier lines of therapy is being evaluated in ongoing clinical trials. This scenario warrants standardised molecular profiling of these tumours. METHODS: A detailed analysis of the original genetic data from the FIGHT-202 trial, on which the approval of Pemigatinib was based, was conducted. RESULTS: Comparing different detection approaches and displaying representative cases, we described the genetic landscape and architecture of FGFR2 fusions in iCCA and show biological and technical aspects to be considered for their detection. We elaborated parameters, including a suggestion for annotation, that should be stated in a molecular diagnostic FGFR2 report to allow a complete understanding of the analysis performed and the information provided. CONCLUSION: This study provides a detailed presentation and dissection of the technical and biological aspects regarding FGFR2 fusion detection, which aims to support molecular pathologists, pathologists and clinicians in diagnostics, reporting of the results and decision-making.

Prognostic impact of copy number alterations and tumor mutational burden in carcinoma of unknown primary.

INTRODUCTION: Chromosomal aberrations are known to drive metastatic spread, but their profile is still elusive in carcinoma of unknown primary (CUP). Therefore, the aim of this study was to characterize the chromosomal aberration pattern in CUP depending on histological and clinical features and to assess its prognostic impact together with chromothripsis, tumor mutational burden (TMB), microsatellite instability (MSI), and mutational profiles as potential prognostic biomarkers. METHODS: Chromosomal aberrations and chromothripsis were detected by methylation-based copy number variation (CNV) analysis, whereas TMB and MSI were calculated based on large next-generation sequencing (NGS) panels. Putative primaries were assigned by consensus between two independent oncologists. RESULTS: CNV losses varied depending on putative primaries and were more abundant in patients harboring TP53 mutations and/or deletions 17p. CNV loss was prognostically adverse in localized CUP treated with surgery and/or radiotherapy, but not in disseminated poor-risk CUP treated with palliative chemotherapy. CNV loss also worsened the prognosis in squamous cell CUP. Chromothripsis was detected in 18/59 (30.5%) patients without prognostic effect. TMB was highest in cases with MSI, squamous cell histology, and with lung, anal or cervical putative primaries. CONCLUSION: Overall, CNV, chromothripsis, TMB, and MSI profiles in CUP are reminiscent of biological characteristics known from other cancer entities without a unifying CUP-specific signature. Markedly, high-level CNV loss is an adverse predictive biomarker in localized but not disseminated chemotherapy-treated CUP. This implies that chromosomal losses drive CUP progression, but also increase susceptibility to chemotherapy, with both effects apparently leveling out in disseminated CUP.

Assigning evidence to actionability: An introduction to variant interpretation in precision cancer medicine.

Modern concepts in precision cancer medicine are based on increasingly complex genomic analyses and require standardized criteria for the functional evaluation and reporting of detected genomic alterations in order to assess their clinical relevance. In this article, we propose and address the necessary steps in systematic variant evaluation consisting of bioinformatic analysis, functional annotation and clinical interpretation, focusing on the latter two aspects. We discuss the role and clinical application of current variant classification systems and point out their scope and limitations. Finally, we highlight the significance of the molecular tumor board as a platform for clinical decision-making based on genomic analyses.

Mutations in TP53 or DNA damage repair genes define poor prognostic subgroups in primary prostate cancer.

BACKGROUND: Mutations in DNA damage repair genes, in particular genes involved in homology-directed repair, define a subgroup of men with prostate cancer with a more unfavorable prognosis but a therapeutic vulnerability to PARP inhibition. In current practice, mutational testing of prostate cancer patients is commonly done late i.e., when the tumor is castration resistant. In addition, most sequencing panels do not include TP53, one of the most crucial tumor suppressor genes in human cancer. In this proof-of-concept study, we sought to extend the clinical use of these molecular markers by exploring the early prognostic impact of mutations in TP53 and DNA damage repair genes in men with primary, nonmetastatic prostate cancer undergoing radical prostatectomy (RPX). METHODS: Tumor specimens from a cohort of 68 RPX patients with intermediate (n = 11, 16.2%) or high-risk (n = 57, 83.8%) disease were analyzed by targeted next generation sequencing using a 37 DNA damage repair and checkpoint gene panel including TP53. Sequencing results were correlated to clinicopathologic variables as well as PSA persistence or time to PSA failure. In addition, the distribution of TP53 and DNA damage repair gene mutations was analyzed in three large publicly available datasets (TCGA, MSKCC and SU2C). RESULTS: Of 68 primary prostate cancers analyzed, 23 (33.8%) were found to harbor a mutation in either TP53 (n = 12, 17.6%) or a DNA damage repair gene (n = 11, 16.2%). The vast majority of these mutations (22 of 23, 95.7%) were detected in primary tumors from patients with high-risk features. These mutations were mutually exclusive in our cohort and additional data mining suggests an enrichment of DNA damage repair gene mutations in TP53 wild-type tumors. Mutations in either TP53 or a DNA damage repair gene were associated with a significantly worse prognosis after RPX. Importantly, the presence of TP53/DNA damage repair gene mutations was an independent risk factor for PSA failure or PSA persistence in multivariate Cox regression models. CONCLUSION: TP53 or DNA damage repair gene mutations are frequently detected in primary prostate cancer with high-risk features and define a subgroup of patients with an increased risk for PSA failure or persistence after RPX. The significant adverse impact of these alterations on patient prognosis may be exploited to identify men with prostate cancer who may benefit from a more intensified treatment.

Local ablative treatment with surgery and/or radiotherapy in single-site and oligometastatic carcinoma of unknown primary.

BACKGROUND: Single-site carcinoma of unknown primary (CUP) is recognised as a distinct favourable subtype in the European Society of Medical Oncology (ESMO) classification. There is broad consensus that these patients are candidates for local ablative treatment strategies with surgery and/or radiotherapy, but data on their outcomes are scarce. PATIENTS AND METHODS: In this study, we have addressed the prospects of cure and prognostic factors in a retrospective cohort of 63 patients who were eligible for local treatment at our centre. RESULTS: Median event-free (EFS) and overall survival (OS) were 15.6 months and 52.5 months, respectively. Of 61 patients who received local treatment, 20 (32.8%) remained event-free over a median follow-up of 28 months. Baseline clinical parameters including affected organ, number, volume and histology of metastases had no significant impact on prognosis, whereas deleterious TP53 mutations and DNA copy number loss emerged as independent adverse risk factors with respect to EFS. Surgical treatment was associated with improved OS as compared to radiation-based therapy. CONCLUSION: Our study advocates to pursue localised treatment with surgery and/or radiotherapy whenever feasible and implies that genetic parameters might additionally determine the clinical course of single-site CUP patients.

Distinct Mutational Profile of Lynch Syndrome Colorectal Cancers Diagnosed under Regular Colonoscopy Surveillance.

Regular colonoscopy even with short intervals does not prevent all colorectal cancers (CRC) in Lynch syndrome (LS). In the present study, we asked whether cancers detected under regular colonoscopy surveillance (incident cancers) are phenotypically different from cancers detected at first colonoscopy (prevalent cancers). We analyzed clinical, histological, immunological and mutational characteristics, including panel sequencing and high-throughput coding microsatellite (cMS) analysis, in 28 incident and 67 prevalent LS CRCs (n total = 95). Incident cancers presented with lower UICC and T stage compared to prevalent cancers (p < 0.0005). The majority of incident cancers (21/28) were detected after previous colonoscopy without any pathological findings. On the molecular level, incident cancers presented with a significantly lower KRAS codon 12/13 (1/23, 4.3% vs. 11/21, 52%; p = 0.0005) and pathogenic TP53 mutation frequency (0/17, 0% vs. 7/21, 33.3%; p = 0.0108,) compared to prevalent cancers; 10/17 (58.8%) incident cancers harbored one or more truncating APC mutations, all showing mutational signatures of mismatch repair (MMR) deficiency. The proportion of MMR deficiency-related mutational events was significantly higher in incident compared to prevalent CRC (p = 0.018). In conclusion, our study identifies a set of features indicative of biological differences between incident and prevalent cancers in LS, which should further be monitored in prospective LS screening studies to guide towards optimized prevention protocols.

Earlier extracranial progression and shorter survival in ALK-rearranged lung cancer with positive liquid rebiopsies.

BACKGROUND: Liquid rebiopsies can detect resistance mutations to guide therapy of anaplastic lymphoma kinase-rearranged (ALK+) non-small-cell lung cancer (NSCLC) failing tyrosine kinase inhibitors (TKI). Here, we analyze how their results relate to the anatomical pattern of disease progression and patient outcome. METHODS: Clinical, molecular, and radiologic characteristics of consecutive TKI-treated ALK+ NSCLC patients were analyzed using prospectively collected plasma samples and the 17-gene targeted AVENIO kit, which covers oncogenic drivers and all TP53 exons. RESULTS: In 56 patients, 139 instances of radiologic changes were analyzed, of which 133 corresponded to disease progression. Circulating tumor DNA (ctDNA) alterations were identified in most instances of extracranial progression (58/94 or 62%), especially if concomitant intracranial progression was also present (89%, P<0.001), but rarely in case of isolated central nervous system (CNS) progression (8/39 or 21%, P<0.001). ctDNA detectability correlated with presence of "short" echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion variants (mainly V3, E6:A20) and/or TP53 mutations (P<0.05), and presented therapeutic opportunities in <50% of cases. Patients with extracranial progression and positive liquid biopsies had shorter survival from the start of palliative treatment (mean 52 vs. 69 months, P=0.002), regardless of previous and subsequent therapy and initial ECOG performance status. Furthermore, for patients with extracranial progression, ctDNA detectability was associated with shorter next-line progression-free survival (PFS) (3 vs. 13 months, P=0.003) if they were switched to another systemic therapy (49/86 samples), and with shorter time-to-next-treatment (TNT) (3 vs. 8 months, P=0.004) if they were continued on the same treatment due to oligoprogression (37/86). In contrast, ctDNA detectability was not associated with the outcome of patients showing CNS-only progression. In 6/6 cases with suspicion of non-neoplastic radiologic lung changes (mainly infection or pneumonitis), ctDNA results remained negative. CONCLUSIONS: Positive blood-based liquid rebiopsies in ALK+ NSCLC characterize biologically more aggressive disease and are common with extracranial, but rare with CNS-only progression or benign radiologic changes. These results reconcile the increased detection of ALK resistance mutations with other features of the high-risk EML4-ALK V3-associated phenotype. Conversely, most oligoprogressive patients with negative liquid biopsies have a more indolent course without need for early change of systemic treatment.

Status quo of ALK testing in lung cancer: results of an EQA scheme based on in-situ hybridization, immunohistochemistry, and RNA/DNA sequencing.

With this external quality assessment (EQA) scheme, we aim to investigate the diagnostic performance of the currently available methods for the detection of ALK alterations in non-small cell lung cancer on a national scale, namely, in situ hybridization (ISH), immunohistochemistry (IHC), and RNA/DNA sequencing (NGS). The EQA scheme cohort consisted of ten specimens, including four ALK positive and six ALK negative samples, which were thoroughly pretested using IHC, ISH, and RNA/DNA NGS. Unstained tumor sections were provided to the 57 participants, and the results were retrieved via an online questionnaire. ISH was used by 29, IHC by 38, and RNA/DNA sequencing by 19 participants. Twenty-eight institutions (97%) passed the ring trial using ISH, 33 (87%) by using IHC, and 18 (95%) by using NGS. The highest sensitivity and interrater agreement (Fleiss ' kappa) was observed for RNA/DNA sequencing (99%, 0.975), followed by ISH (94%, 0.898) and IHC (92%, 0.888). However, the proportion of samples that were not evaluable due to bad tissue quality was also higher for RNA/DNA sequencing (4%) compared with ISH (0.7%) and IHC (0.5%). While all three methods produced reliable results between the different institutions, the highest sensitivity and concordance were observed for RNA/DNA sequencing. These findings encourage the broad implementation of this method in routine diagnostic, although the application might be limited by technical capacity, economical restrictions, and tissue quality of formalin-fixed samples.

Comprehensive Genomic and Transcriptomic Analysis for Guiding Therapeutic Decisions in Patients with Rare Cancers.

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659.

Validation of a Targeted Next-Generation Sequencing Panel for Tumor Mutation Burden Analysis: Results from the Onconetwork Immuno-Oncology Consortium.

Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r2 = 0.998) and phase 2 (r2 = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research.

[Variant interpretation in molecular pathology and oncology : An introduction]. / Varianteninterpretation in der molekularen Pathologie und Onkologie : Eine Einführung.

Increasingly extensive genomic diagnostics in cancer precision medicine require uniform evaluation criteria for the classification of variants with regard to their functional and therapeutic implications. In this review we present the most important guidelines and classification systems currently used in daily clinical practice, explain their advantages and disadvantages as well as differences and similarities, and present the step-by-step, systematic process that enables successful variant interpretation.

KRAS/GNAS-testing by highly sensitive deep targeted next generation sequencing improves the endoscopic ultrasound-guided workup of suspected mucinous neoplasms of the pancreas.

Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.

Targeting rare and non-canonical driver variants in NSCLC - An uncharted clinical field.

OBJECTIVES: Implementation of tyrosine kinase inhibitors (TKI) and other targeted therapies was a main advance in thoracic oncology with survival gains ranging from several months to years for non-small-cell lung cancer (NSCLC) patients. High-throughput comprehensive molecular profiling is of key importance to identify patients that can potentially benefit from these novel treatments. MATERIAL AND METHODS: Next-generation sequencing (NGS) was performed on 4500 consecutive formalin-fixed, paraffin-embedded specimens of advanced NSCLC (n = 4172 patients) after automated extraction of DNA and RNA for parallel detection of mutations and gene fusions, respectively. RESULTS AND CONCLUSION: Besides the 24.9 % (n = 1040) of cases eligible for approved targeted therapies based on the presence of canonical alterations in EGFR exons 18-21, BRAF, ROS1, ALK, NTRK, and RET, an additional n = 1260 patients (30.2 %) displayed rare or non-canonical mutations in EGFR (n = 748), BRAF (n = 135), ERBB2 (n = 30), KIT (n = 32), PIK3CA (n = 221), and CTNNB1 (n = 94), for which targeted therapies could also be potentially effective. A systematic literature search in conjunction with in silico evaluation identified n = 232 (5.5 %) patients, for which a trial of targeted treatment would be warranted according to available evidence (NCT level 1, i.e. published data showing efficacy in the same tumor entity). In conclusion, a sizeable fraction of NSCLC patients harbors rare or non-canonical alterations that may be associated with clinical benefit from currently available targeted drugs. Systematic identification and individualized management of these cases can expand applicability of precision oncology in NSCLC and extend clinical gain from established molecular targets. These results can also inform clinical trials.

Longitudinal therapy monitoring of ALK-positive lung cancer by combined copy number and targeted mutation profiling of cell-free DNA.

BACKGROUND: Targeted therapies (TKI) have improved the prognosis of ALK-rearranged lung cancer (ALK+ NSCLC), but clinical courses vary widely. Early identification and molecular characterisation of treatment failure have key importance for subsequent therapies. We performed copy number variation (CNV) profiling and targeted panel sequencing from cell-free DNA (cfDNA) to monitor ALK+ NSCLC. METHODS: 271 longitudinal plasma DNA samples from 73 patients with TKI-treated metastatic ALK+ NSCLC were analysed by capture-based targeted (average coverage 4,100x), and shallow whole genome sequencing (sWGS, 0.5x). Mutations were called using standard algorithms. CNVs were quantified using the trimmed median absolute deviation from copy number neutrality (t-MAD). FINDINGS: cfDNA mutations were identified in 58% of patients. They included several potentially actionable alterations, e.g. in the genes BRAF, ERBB2, and KIT. sWGS detected CNVs in 18% of samples, compared to 6% using targeted sequencing. Several of the CNVs included potentially druggable targets, such as regions harboring EGFR, ERBB2, and MET. Circulating tumour DNA (ctDNA) mutations and t-MAD scores increased during treatment, correlated with markers of higher molecular risk, such as the EML4-ALK variant 3 and/or TP53 mutations, and were associated with shorter patient survival. Importantly, t-MAD scores reflected the tumour remission status in serial samples similar to mutant ctDNA allele frequencies, and increased with disease progression in 79% (34/43) of cases, including those without detectable single nucleotide variant (SNV). INTERPRETATION: Combined copy number and targeted mutation profiling could improve monitoring of ALK+ NSCLC. Potential advantages include the identification of treatment failure, in particular for patients without detectable mutations, and broader detection of genomic changes acquired during therapy, especially in later treatment lines and in high-risk patients. FUNDING: This work was supported by the German Center for Lung Research (DZL), by the German Cancer Consortium (DKTK), by the Heidelberg Center for Personalized Oncology at the German Cancer Research Center (DKFZ-HIPO), and by Roche Sequencing Solutions (Pleasanton, CA, USA).

Correction to: The majority of ß-catenin mutations in colorectal cancer is homozygous.

An amendment to this paper has been published and can be accessed via the original article.

The majority of ß-catenin mutations in colorectal cancer is homozygous.

BACKGROUND: ß-catenin activation plays a crucial role for tumourigenesis in the large intestine but except for Lynch syndrome (LS) associated cancers stabilizing mutations of ß-catenin gene (CTNNB1) are rare in colorectal cancer (CRC). Previous animal studies provide an explanation for this observation. They showed that CTNNB1 mutations induced transformation in the colon only when CTNNB1 was homozygously mutated or when membranous ß-catenin binding was hampered by E-cadherin haploinsufficiency. We were interested, if these mechanisms are also found in human CTNNB1 mutated CRCs. RESULTS: Among 869 CRCs stabilizing CTNNB1 mutations were found in 27 cases. Homo- or hemizygous CTNNB1 mutations were detected in 74% of CTNNB1 mutated CRCs (13 microsatellite instabile (MSI-H), 7 microsatellite stabile (MSS)) but only in 3% (1/33) of extracolonic CTNNB1 mutated cancers. In contrast to MSS CRC, CTNNB1 mutations at codon 41 or 45 were highly selected in MSI-H CRC. Of the examined three CRC cell lines, ß-catenin and E-cadherin expression was similar in cell lines without or with hetereozygous CTNNB1 mutations (DLD1 and HCT116), while a reduced E-cadherin expression combined with cytoplasmic accumulation of ß-catenin was found in a cell line with homozygous CTNNB1 mutation (LS180). Reduced expression of E-cadherin in human MSI-H CRC tissue was identified in 60% of investigated cancers, but no association with the CTNNB1 mutational status was found. CONCLUSIONS: In conclusion, this study shows that in contrast to extracolonic cancers stabilizing CTNNB1 mutations in CRC are commonly homo- or hemizygous indicating a higher threshold of ß-catenin stabilization to be required for transformation in the colon as compared to extracolonic sites. Moreover, we found different mutational hotspots in CTNNB1 for MSI-H and MSS CRCs suggesting a selection of different effects on ß-catenin stabilization according to the molecular pathway of tumourigenesis. Reduced E-cadherin expression in CRC may further contribute to higher levels of transcriptionally active ß-catenin, but it is not directly linked to the CTNNB1 mutational status.

NTRK testing: First results of the QuiP-EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA-based NGS assays.

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion 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 of NTRK fusions 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-based NTRK testing 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 150 NTRK fusion variants by several commercially available RNA-based tNGS panels.