Non-Response of Epstein-Barr Virus-Associated Breast Cancer after Primary Chemotherapy: Report of Two Cases.

Based on epidemiological evidence and molecular findings, a possible association of Epstein-Barr virus (EBV) with the carcinogenesis of breast cancer has been described. However, the frequency of EBV in breast cancer and the role of EBV regarding tumor progression or therapeutic results is largely unexplored. Here, we report on two cases of advanced, lymph node-positive invasive breast cancer of no special type (NST), histologically showing no clinical or histological evidence of tumor regression as an equivalent of a lack of response to primary systemic therapy. Both tumors were considered to be EBV-associated due to their positivity in EBV-encoded RNA (EBER) in situ hybridization (ISH) and their immunoreactivity against EBV Epstein-Barr nuclear antigen 1 (EBNA1). We hypothesize that the unusual non-response to chemotherapy in these cases of breast cancer classified as triple-negative and HER2-positive may be linked to the EBV co-infection of tumor cells. Therefore, EBV tumor testing should be considered in patients with breast cancer presenting with resistance to chemotherapy. This hypothesis may provide a new aspect in the context of EBV-associated mechanisms of tumor progression.

Comprehensive genomic and epigenomic analysis in cancer of unknown primary guides molecularly-informed therapies despite heterogeneity.

The benefit of molecularly-informed therapies in cancer of unknown primary (CUP) is unclear. Here, we use comprehensive molecular characterization by whole genome/exome, transcriptome and methylome analysis in 70 CUP patients to reveal substantial mutational heterogeneity with TP53, MUC16, KRAS, LRP1B and CSMD3 being the most frequently mutated known cancer-related genes. The most common fusion partner is FGFR2, the most common focal homozygous deletion affects CDKN2A. 56/70 (80%) patients receive genomics-based treatment recommendations which are applied in 20/56 (36%) cases. Transcriptome and methylome data provide evidence for the underlying entity in 62/70 (89%) cases. Germline analysis reveals five (likely) pathogenic mutations in five patients. Recommended off-label therapies translate into a mean PFS ratio of 3.6 with a median PFS1 of 2.9 months (17 patients) and a median PFS2 of 7.8 months (20 patients). Our data emphasize the clinical value of molecular analysis and underline the need for innovative, mechanism-based clinical trials.

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.

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.

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.

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.

RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples.

Oncogenic gene fusions are important drivers in many cancer types, including carcinomas, with diagnostic and therapeutic implications. Hence, sensitive and rapid methods for parallel profiling in formalin-fixed and paraffin-embedded (FFPE) specimens are needed. In this study we analyzed gene fusions in a cohort of 517 cases where standard treatment options were exhausted. To this end the Archer® DX Solid tumor panel (AMP; 285 cases) and the Oncomine Comprehensive Assay v3 (OCA; 232 cases) were employed. Findings were validated by Sanger sequencing, fluorescence in situ hybridization (FISH) or immunohistochemistry. Both assays demonstrated minimal dropout rates (AMP: 2.4%; n = 7/292, OCA: 2.1%; n = 5/237) with turnaround times of 6-9 working days (median, OCA and AMP, respectively). Hands-on-time for library preparation was 6 h (AMP) and 2 h (OCA). We detected n = 40 fusion-positive cases (7.7%) with TMPRSS2::ERG in prostate cancer being most prevalent (n = 9/40; 22.5%), followed by other gene fusions identified in cancers of unknown primary (n = 6/40; 15.0%), adenoid cystic carcinoma (n = 7/40; 17.5%), and pancreatic cancer (n = 7/40; 17.5%). Our results demonstrate that targeted RNA-sequencing of FFPE samples is feasible, and a well-suited approach for the detection of gene fusions in a routine clinical setting.

Spatial and Temporal Heterogeneity of Panel-Based Tumor Mutational Burden in Pulmonary Adenocarcinoma: Separating Biology From Technical Artifacts.

BACKGROUND: Tumor mutational burden (TMB) is an emerging biomarker used to identify patients who are more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, biological variables such as tumor cell content and intratumor heterogeneity may play an important role in determining TMB. METHODS: TMB estimates were determined applying the TruSight Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multiregion sequencing (two to six samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in five patients. RESULTS: On average, a coding region of 1.28 Mbp was covered with a mean read depth of 609x. Manual validation of the mutation-calls confirmed a good performance, but revealed noticeable misclassification during germline filtering. Different regions within a tumor showed considerable spatial TMB variance in 30% (7 of 24) of the cases (maximum difference, 14.13 mut/Mbp). Lymph node-derived TMB was significantly lower (p = 0.016). In 13 cases, distinct mutational profiles were exclusive to different regions of a tumor, leading to higher values for simulated aggregated TMB. Combined, intratumor heterogeneity and the aggregated TMB could result in divergent TMB designation in 17% of the analyzed patients. TMB variation between primary tumor and distant metastases existed but was not profound. CONCLUSIONS: Our data show that, in addition to technical aspects such as germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of single-sample-based TMB estimations in a clinical context.

Variant classification in precision oncology.

Next-generation sequencing has become a cornerstone of therapy guidance in cancer precision medicine and an indispensable research tool in translational oncology. Its rapidly increasing use during the last decade has expanded the options for targeted tumor therapies, and molecular tumor boards have grown accordingly. However, with increasing detection of genetic alterations, their interpretation has become more complex and error-prone, potentially introducing biases and reducing benefits in clinical practice. To facilitate interdisciplinary discussions of genetic alterations for treatment stratification between pathologists, oncologists, bioinformaticians, genetic counselors and medical scientists in specialized molecular tumor boards, several systems for the classification of variants detected by large-scale sequencing have been proposed. We review three recent and commonly applied classifications and discuss their individual strengths and weaknesses. Comparison of the classifications underlines the need for a clinically useful and universally applicable variant reporting system, which will be instrumental for efficient decision making based on sequencing analysis in oncology. Integrating these data, we propose a generalizable classification concept featuring a conservative and a more progressive scheme, which can be readily applied in a clinical setting.

Defective homologous recombination DNA repair as therapeutic target in advanced chordoma.

Chordomas are rare bone tumors with few therapeutic options. Here we show, using whole-exome and genome sequencing within a precision oncology program, that advanced chordomas (n = 11) may be characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair and alterations affecting HR-related genes, including, for example, deletions and pathogenic germline variants of BRCA2, NBN, and CHEK2. A mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and co-occurred with genomic instability. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells, led to prolonged clinical benefit in a patient with refractory chordoma, and whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain. These findings uncover a therapeutic opportunity in chordoma that warrants further exploration, and provide insight into the mechanisms underlying PARP inhibitor resistance.

Response to olaparib in a PALB2 germline mutated prostate cancer and genetic events associated with resistance.

Prostate cancers harboring DNA repair gene alterations are particularly sensitive to PARP inhibitor treatment. We report a case of an advanced prostate cancer patient profiled within the NCT-MASTER (Molecularly Aided Stratification for Tumor Eradication Research) precision oncology program using next-generation sequencing. Comprehensive genomic and transcriptomic analysis identified a pathogenic germline PALB2 variant as well as a mutational signature associated with disturbed homologous recombination together with structural genomic rearrangements. A molecular tumor board identified a potential benefit of targeted therapy and recommended PARP inhibition and platinum-based chemotherapy. Single-agent treatment with the PARP inhibitor olaparib as well as subsequent combination with platinum-based chemotherapy resulted in disease stabilization and substantial improvement of clinical symptoms. Upon progression, we performed whole-exome and RNA sequencing of a liver metastasis, which demonstrated up-regulation of several genes characteristic for the neuroendocrine prostate cancer phenotype as well as a novel translocation resulting in an in-frame, loss-of-function fusion of RB1. We suggest that multidimensional genomic characterization of prostate cancer patients undergoing PARP inhibitor therapy will be necessary to capture and understand predictive biomarkers of PARP inhibitor sensitivity and resistance.

Combined targeted DNA and RNA sequencing of advanced NSCLC in routine molecular diagnostics: Analysis of the first 3,000 Heidelberg cases.

Tyrosine kinase inhibitors currently confer the greatest survival gain for nonsmall cell lung cancer (NSCLC) patients with actionable genetic alterations. Simultaneously, the increasing number of targets and compounds poses the challenge of reliable, broad and timely molecular assays for the identification of patients likely to benefit from novel treatments. Here, we demonstrate the feasibility and clinical utility of comprehensive, NGS-based genetic profiling for routine workup of advanced NSCLC based on the first 3,000 patients analyzed in our department. Following automated extraction of DNA and RNA from formalin-fixed, paraffin-embedded tissue samples, parallel sequencing of DNA and RNA for detection of mutations and gene fusions, respectively, was performed using PCR-based enrichment with an ion semiconductor sequencing platform. Overall, 807 patients (27%) were eligible for currently approved, EGFR-/BRAF-/ALK- and ROS1-directed therapies, while 218 additional cases (7%) with MET, ERBB2 (HER2) and RET alterations could potentially benefit from experimental targeted compounds. In addition, routine capturing of comutations, e.g. TP53 (55%), KEAP1 (11%) and STK11 (11%), as well as the precise typing of fusion partners and involved exons in case of actionable translocations including ALK and ROS1, are prognostic and predictive tools currently gaining importance for further refinement of therapeutic and surveillance strategies. The reliability, low dropout rates (<5%), minimal tissue requirements, fast turnaround times (6 days on average) and lower costs of the diagnostic approach presented here compared to sequential single-gene testing, highlight its practicability in order to support individualized decisions in routine patient care, enrollment in molecularly stratified clinical trials, as well as translational research.

Size matters: Dissecting key parameters for panel-based tumor mutational burden analysis.

Tumor mutational burden (TMB) represents a new determinant of clinical benefit from immune checkpoint blockade that identifies responders independent of PD-L1 expression levels and is currently being explored in clinical trials. Although TMB can be measured directly by comprehensive genomic approaches such as whole-genome and exome sequencing, broad availability, short turnaround times, costs and amenability to formalin-fixed and paraffin-embedded tissue support the use of gene panel sequencing for approximating TMB in routine diagnostics. However, data on the parameters influencing panel-based TMB estimation are limited. Here, we report an extensive in silico analysis of the TCGA data set that simulates various panel sizes and compositions. We demonstrate that panel size is a critical parameter that influences confidence intervals (CIs) and cutoff values as well as important test parameters including sensitivity, specificity, and positive predictive value. Moreover, we evaluate the Illumina TSO500 panel, which will be made available for TMB estimation, and propose dynamic, entity-specific cutoff values based on current clinical trial data. Optimizing the cost-benefit ratio, our data suggest that panels between 1.5 and 3 Mbp are ideally suited to estimate TMB with small CIs, whereas smaller panels tend to deliver imprecise TMB estimates for low to moderate TMB (0-30 muts/Mbp), connected with insufficient separation of hypermutated tumors from non-hypermutated tumors.

Next generation sequencing of the cellular and liquid fraction of pancreatic cyst fluid supports discrimination of IPMN from pseudocysts and reveals cases with multiple mutated driver clones: First findings from the prospective ZYSTEUS biomarker study.

Approximately half of all pancreatic cysts are neoplastic, mainly comprising intraductal papillary mucinous neoplasms (IPMN), which can progress to invasive carcinoma. Current Fukuoka guidelines have limited sensitivity and specificity in predicting progression of asymptomatic pancreatic cysts. We present first results of the prospective ZYSTEUS biomarker study investigating (i) whether detection of driver mutations in IPMN by liquid biopsy is technically feasible, (ii) which compartment of IPMN is most suitable for analysis, and (iii) implications for clinical diagnostics. Twenty-two patients with clinical inclusion criteria were enrolled in ZYSTEUS. Fifteen cases underwent endoscopic ultrasound (EUS)-guided fine-needle aspiration and cytological diagnostics. Cellular and liquid fraction of the cysts of each case were separated and subjected to deep targeted next generation sequencing (NGS). Clinical parameters, imaging findings (EUS and MRI), and follow-up data were collected continuously. All IPMN cases (n = 12) showed at least one mutation in either KRAS (n = 11) or GNAS (n = 4). Three cases showed both KRAS and GNAS mutations. Six cases harbored multiple KRAS/GNAS mutations. In the three cases with pseudocysts, no KRAS or GNAS mutations were detected. DNA yields were higher and showed higher mutation diversity in the cellular fraction. In conclusion, mutation detection in pancreatic cyst fluid is technically feasible with more robust results in the cellular than in the liquid fraction. Current results suggest that, together with imaging, targeted sequencing supports discrimination of IPMN from pseudocysts. The prospective design of ZYSTEUS will provide insight into diagnostic value of NGS in preoperative risk stratification. Our data provide evidence for an oligoclonal nature of IPMN.

Measurement of tumor mutational burden (TMB) in routine molecular diagnostics: in silico and real-life analysis of three larger gene panels.

Assessment of Tumor Mutational Burden (TMB) for response stratification of cancer patients treated with immune checkpoint inhibitors is emerging as a new biomarker. Commonly defined as the total number of exonic somatic mutations, TMB approximates the amount of neoantigens that potentially are recognized by the immune system. While whole exome sequencing (WES) is an unbiased approach to quantify TMB, implementation in diagnostics is hampered by tissue availability as well as time and cost constrains. Conversely, panel-based targeted sequencing is nowadays widely used in routine molecular diagnostics, but only very limited data are available on its performance for TMB estimation. Here, we evaluated three commercially available larger gene panels with covered genomic regions of 0.39 Megabase pairs (Mbp), 0.53 Mbp and 1.7 Mbp using i) in silico analysis of TCGA (The Cancer Genome Atlas) data and ii) wet-lab sequencing of a total of 92 formalin-fixed and paraffin-embedded (FFPE) cancer samples grouped in three independent cohorts (non-small cell lung cancer, NSCLC; colorectal cancer, CRC; and mixed cancer types) for which matching WES data were available. We observed a strong correlation of the panel data with WES mutation counts especially for the gene panel >1Mbp. Sensitivity and specificity related to TMB cutpoints for checkpoint inhibitor response in NSCLC determined by wet-lab experiments well reflected the in silico data. Additionally, we highlight potential pitfalls in bioinformatics pipelines and provide recommendations for variant filtering. In summary, our study is a valuable data source for researchers working in the field of immuno-oncology as well as for diagnostic laboratories planning TMB testing.

Implementing tumor mutational burden (TMB) analysis in routine diagnostics-a primer for molecular pathologists and clinicians.

Tumor mutational burden (TMB) is a new biomarker for prediction of response to PD-(L)1 treatment. Comprehensive sequencing approaches (i.e., whole exome and whole genome sequencing) are ideally suited to measure TMB directly. However, as their applicability in routine diagnostics is currently limited by high costs, long turnaround times and poor availability of fresh tissue, targeted next-generation sequencing (NGS) of formalin-fixed and paraffin-embedded (FFPE) samples appears to be a more feasible and straight-forward approach for TMB approximation, which can be seamlessly integrated in already existing diagnostic workflows and pipelines. In this work, we provide an overview of the clinical implications of TMB testing and highlight key parameters including pre-analysis, analysis and post-analytical steps that influence and shape TMB approximation by panel sequencing. Collectively, the data will not only serve as a field guide and state of the art knowledge source for molecular pathologists who consider implementation of TMB measurement in their lab, but also enable clinicians in understanding the specific parameters influencing TMB test results and reporting.

Subclonal evolution of pulmonary adenocarcinomas delineated by spatially distributed somatic mitochondrial mutations.

OBJECTIVES: The potential role of cancer associated somatic mutations of the mitochondrial genome (mtDNA) is controversial and still poorly understood. Our group and others recently challenged a direct tumorigenic impact and suggested a passenger-like character. In combination with the known increased mutation rate, somatic mtDNA mutations account for an interesting tool to delineate tumor evolution. Here, we comprehensively analyzed the spatial distribution of somatic mtDNA mutations throughout whole tumor sections of pulmonary adenocarcinoma (ADC). MATERIALS AND METHODS: Central sections of 19 ADC were analyzed in a segmented manner (11-34 segments/tumor) together with non-neoplastic tissue samples and lymph node metastasis, if present. We performed whole mtDNA sequencing and real-time PCR based quantification of mtDNA copy numbers for all samples. Further, histological growth patterns were determined on H&E sections and the tumor cell content was quantified by digital pathology analyses. RESULTS: Somatic mtDNA mutations were present in 96% (18/19) of the analyzed tumors, either ubiquitously or restricted to specific tumor regions. Spatial and histological mapping of the mutations enabled the identification of subclonal structures and phylogenetic relations within a tumor section indicating different progression levels. In this regard, lymph node metastases seem to be related to early events in ADC development. There was no concurrence between histological and mtDNA mutation based clusters. However, micropapillary patterns occurred only in tumors with ubiquitous mutations. ADC with more than two ubiquitous mutations were associated with shorter disease-free survival (p < 0.01). CONCLUSION: Cancer related mtDNA mutations are interesting candidates for the understanding of subclonal ADC evolution and perspectively for monitoring tumor progression. Our data reveal a potential prognostic relevance of somatic mtDNA mutations.

Genetic profiling of melanoma in routine diagnostics: assay performance and molecular characteristics in a consecutive series of 274 cases.

A deeper understanding of melanoma biology has opened up new avenues for mechanistically informed therapies. However, data on the prevalence of druggable genetic lesions in melanoma are still conflicting and real-world performance data on high-throughput genetic profiling of melanoma cases using formalin fixed, paraffin embedded (FFPE) tissue with variable tumour cellularity and quality are lacking. We retrospectively analysed targeted next-generation sequencing data of 274 consecutive melanoma samples obtained for routine diagnostics between December 2013 and July 2017. Actionable mutations were detected in 197 cases (71.9%), of which activating BRAF (mostly p.V600E/K) and RAS (mostly p.Q61R/K) mutations occurred in 40.5% (n = 111) and 30.3% (n = 83) of cases, respectively. We identified driver mutations of the Triple-WT subgroup in 10.6% of cases (n = 29; 10 with activating KIT mutations). Median turnaround time was 5 working days with no dropouts. Tumour cellularity ranged from 5% to 95% and successful sequencing was possible at DNA concentrations as low as 0.03 ng/µL (median 10.58 ng/µL; range 0.03-209.05 ng/µL). Fast, quality-controlled high-throughput genetic profiling of FFPE melanoma samples is feasible and provides a landscape of genetic aberrations in melanoma that is currently relevant in clinical practice and approximates TCGA subtypes.

NRG1 Fusions in KRAS Wild-Type Pancreatic Cancer.

We used whole-genome and transcriptome sequencing to identify clinically actionable genomic alterations in young adults with pancreatic ductal adenocarcinoma (PDAC). Molecular characterization of 17 patients with PDAC enrolled in a precision oncology program revealed gene fusions amenable to pharmacologic inhibition by small-molecule tyrosine kinase inhibitors in all patients with KRAS wild-type (KRASWT) tumors (4 of 17). These alterations included recurrent NRG1 rearrangements predicted to drive PDAC development through aberrant ERBB receptor-mediated signaling, and pharmacologic ERBB inhibition resulted in clinical improvement and remission of liver metastases in 2 patients with NRG1-rearranged tumors that had proved resistant to standard treatment. Our findings demonstrate that systematic screening of KRASWT tumors for oncogenic fusion genes will substantially improve the therapeutic prospects for a sizeable fraction of patients with PDAC.Significance: Advanced PDAC is a malignancy with few treatment options that lacks molecular mechanism-based therapies. Our study uncovers recurrent gene rearrangements such as NRG1 fusions as disease-driving events in KRASwt tumors, thereby providing novel insights into oncogenic signaling and new therapeutic options in this entity. Cancer Discov; 8(9); 1087-95. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.