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Supplementary patient data provides a summary on the clinical course of patient 16.

This file contains supplementary methods and references.

Supplementary Figure S1 shows circos plots including 17 PDACs of the patient cohort and a PDCM.

Supplementary Table S1 lists potentially clinically relevant somatic alterations in the PDAC patient cohort.

This file contains supplementary figures S1-S10.

This excel file contains supplementary tables S1-S7.

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

SummaryZygosityPredictor provides functionality to evaluate how many copies of a gene are affected by mutations in next generation sequencing data. In cancer samples, both somatic and germline mutations are accurately processed. In particular, ZygosityPredictor computes the number of affected copies for single nucleotide variants and small insertions and deletions (Indels). In addition, the tool integrates information at gene level by performing haplotype phasing and subsequent logic to derive how strongly a gene is affected by mutations. This information is of particular interest in precision oncology, e.g. when assessing whether unmutated copies of tumor-suppressor genes remain.Availability and implementationZygosityPredictor was implemented as an R-package and is available via Bioconductor athttps://bioconductor.org/packages/ZygosityPredictor. Detailed documentation is provided in the vignette including application to an example genome.

Background Epithelioid haemangioma (EH) arising from the skin is a benign vascular tumour with marked inflammatory cell infiltration, which exhibits a high tendency to persist and frequently recurs after resection. So far, the underlying pathogenesis is largely elusive. Objectives To identify genetic alterations by next-generation sequencing and/or droplet digital polymerase chain reaction (ddPCR) in cutaneous EH. Methods DNA and RNA from an EH lesion of an index patient were subjected to whole-genome and RNA sequencing. Multiplex PCR-based panel sequencing of genomic DNA isolated from archival formalin-fixed paraffin-embedded tissue of 18 patients with cutaneous EH was performed. ddPCR was used to confirm mutations. Results We identified somatic mutations in genes of the mitogen-activated protein kinase (MAPK) pathway (MAP2K1 and KRAS) in cutaneous EH biopsies. By ddPCR we could confirm the recurrent presence of activating, low-frequency mutations affecting MAP2K1. In total, nine out of 18 patients analysed showed activating MAPK pathway mutations, which were mutually exclusive. Comparative analysis of tissue areas enriched for lymphatic infiltrate or aberrant endothelial cells, respectively, revealed an association of these mutations with the presence of endothelial cells. Conclusions Taken together, our data suggest that EH shows somatic mutations in genes of the MAPK pathway which might contribute to the formation of this benign tumour.

The identification of gene fusions from RNA sequencing data is a routine task in cancer research and precision oncology. However, despite the availability of many computational tools, fusion detection remains challenging. Existing methods suffer from poor prediction accuracy and are computationally demanding. We developed Arriba, a novel fusion detection algorithm with high sensitivity and short runtime. When applied to a large collection of published pancreatic cancer samples (n = 803), Arriba identified a variety of driver fusions, many of which affected druggable proteins, including ALK, BRAF, FGFR2, NRG1, NTRK1, NTRK3, RET, and ROS1. The fusions were significantly associated with KRAS wild-type tumors and involved proteins stimulating the MAPK signaling pathway, suggesting that they substitute for activating mutations in KRAS. In addition, we confirmed the transforming potential of two novel fusions, RRBP1-RAF1 and RASGRP1-ATP1A1, in cellular assays. These results show Arriba's utility in both basic cancer research and clinical translation.

Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract. Inactivating mutations or epigenetic deregulation of succinate dehydrogenase complex (SDH) genes are considered defining features of a subset of GIST occurring in the stomach. Based on comprehensive molecular profiling and biochemical analysis within a precision oncology program, we identified hallmarks of SDH deficiency (germline SDHB-inactivating mutation accompanied by somatic loss of heterozygosity, lack of SDHB expression, global DNA hypermethylation, and elevated succinate/fumarate ratio) in a 40-year-old woman with undifferentiated gastric spindle cell sarcoma that did not meet the diagnostic criteria for other mesenchymal tumors of the stomach, including GIST. These data reveal that the loss of SDH function can be involved in the pathogenesis of non-GIST sarcoma of the gastrointestinal tract.

Introduction: Thymic epithelial tumors (TETs) are very rare. Thymoma A and AB have a better prognosis than more aggressive thymoma B, thymic carcinoma (TC) and neuroendocrine tumors of the thymus (NET). While previous efforts such as TCGA have mainly characterized thymomas (Radovich et al., Cancer Cell 2018), the molecular landscape of TCs and NETs is still elusive. Patients and Methods: Between 03/2014 and 07/2020, we enrolled 44 TET patients (27 TCs, 11 thymomas, 6 NETs) in a prospective observational study (MASTER) conducted by the National Center for Tumor Diseases (NCT) Heidelberg, NCT Dresden and the German Cancer Consortium (DKTK). MASTER applied whole genome/exome sequencing (WGS, n=22; WES, n=22), transcriptome (n=40) and germline analysis to inform therapy recommendations by a dedicated molecular tumor board (MTB). We systematically gathered follow-up data to evaluate outcome and compared progression-free survival (PFS) of the first treatment according to an MTB recommendation (PFS2) to the last prior systemic treatment (PFS1) in each patient (PFS ratio). Results: Tumor mutational burden (TMB) was low (median=0.99 mutations/Mb, range 0.08-3.48) but higher than in TCGA (p 6 months and a PFS ratio > 1.3. The best outcome was achieved using imatinib in a patient with a KIT mutation (p.W557R). After progression, the MTB recommended ponatinib based on a secondary KIT mutation (p.V654A). The patient was still on ponatinib when the observation period ended. Conclusion: We demonstrate that comprehensive molecular analysis provides clinically relevant information in a subgroup of TET patients. Thymoma, TCs, and NETs present with different molecular characteristics. Distinction between immunologically hot and cold TCs may have value for risk stratification and therapeutic strategies. PARP inhibition could be a potential new treatment option in a small subgroup of TETs. Molecular testing of KIT, germline analysis and genetic counseling should be recommended for all patients with advanced TETs. Citation Format: Lino Möhrmann, Lysann Rostock, Małgorzata Oleś, Arne Jahn, Marie Arlt, Nagarajan Paramasivam, Korinna Jöhrens, Luise Rupp, Marc Schmitz, Daniela Richter, Sebastian Uhrig, Martina Fröhlich, Barbara Hutter, Jennifer Hüllein, Elena E. Wolf, Dorothea Hanf, Laura Gieldon, Simon Kreutzfeldt, Christoph E. Heilig, Veronica Teleanu, Daniel B. Lipka, Andreas Mock, Ivan Jelas, Damian T. Rieke, Marcel Wiesweg, Melanie Boerries, Anna L. Illert, Alexander Desuki, Thomas Kindler, Angela M. Krackhardt, C. Benedikt Westphalen, Heidrun Grosch, Leonidas Apostolidis, Albrecht Stenzinger, Irina A. Kerle, Christoph Heining, Daniel Hübschmann, Evelin Schröck, Stefan Fröhling, Hanno Glimm. Genomics-based personalized oncology of advanced thymic epithelial tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 926.

Background Approvals of cancer therapeutics are primarily disease entity specific. Current molecular diagnostic approaches frequently identify actionable alterations in rare cancers or rare subtypes of common cancers for which the corresponding treatments are not approved and unavailable within clinical trials due to entity-related eligibility criteria. Access may be negotiated with health insurances. However, approval rates vary, and critical information required for a scientific evaluation of treatment-associated risks and benefits is not systematically collected. Thus clinical trials with optimized patient selection and comprehensive molecular characterization are essential for translating experimental treatments into standard care. Patients and methods Continuous ReAssessment with Flexible ExTension in Rare Malignancies (CRAFT) is an open-label phase II trial for adults with pretreated, locally advanced, or metastatic solid tumors. Based on the evaluation by a molecular tumor board, patients are assigned to combinations of six molecularly targeted agents and a programmed death-ligand 1 (PD-L1) antagonist within seven study arms focusing on (i) BRAF V600 mutations; (ii) ERBB2 amplification and/or overexpression, activating ERBB2 mutations; (iii) ALK rearrangements, activating ALK mutations; (iv and v) activating PIK3CA and AKT mutations, other aberrations predicting increased PI3K–AKT pathway activity; (vi) aberrations predicting increased RAF–MEK–ERK pathway activity; (vii) high tumor mutational burden and other alterations predicting sensitivity to PD-L1 inhibition. The primary endpoint is the disease control rate (DCR) at week 16; secondary and exploratory endpoints include the progression-free survival ratio, overall survival, and patient-reported outcomes. Using Simon’s optimal two-stage design, 14 patients are accrued for each study arm. If three or fewer patients achieve disease control, the study arm is stopped. Otherwise, 11 additional patients are accrued. If the DCR exceeds 7 of 25 patients, the null hypothesis is rejected for the respective study arm. Conclusions CRAFT was activated in October 2021 and will recruit at 10 centers in Germany. Trial registration numbers EudraCT: 2019-003192-18; ClinicalTrials.gov: NCT04551521., Highlights • Actionable genetic alterations are detected in many cancers with unknown efficacy of the corresponding targeted therapies. • CRAFT, an open-label multicenter phase II trial, uses six molecularly targeted agents and a PD-L1 antagonist in seven treatment arms. • Patient allocation to trial arms is based on evaluation of molecular tumor characteristics by the German Cancer Consortium. • The in-depth molecular characterization helps to understand mechanisms underlying primary and acquired therapy resistance. • The CRAFT trial has been active in Germany since October 2021 and will open at 10 sites during 2022.

Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. The oncogenic potential of BRAF fusions has been attributed to the loss of critical N-terminal domains that mediate BRAF autoinhibition. We used whole-exome and RNA sequencing in a patient with glioblastoma multiforme to identify a rearrangement between TTYH3, encoding a membrane-resident, calcium-activated chloride channel, and BRAF intron 1, resulting in a TTYH3–BRAF fusion protein that retained all features essential for BRAF autoinhibition. Accordingly, the BRAF moiety of the fusion protein alone, which represents full-length BRAF without the amino acids encoded by exon 1 (BRAFΔE1), did not induce MEK/ERK phosphorylation or transformation. Likewise, neither the TTYH3 moiety of the fusion protein nor full-length TTYH3 provoked ERK pathway activity or transformation. In contrast, TTYH3–BRAF displayed increased MEK phosphorylation potential and transforming activity, which were caused by TTYH3-mediated tethering of near-full-length BRAF to the (endo)membrane system. Consistent with this mechanism, a synthetic approach, in which BRAFΔE1 was tethered to the membrane by fusing it to the cytoplasmic tail of CD8 also induced transformation. Furthermore, we demonstrate that TTYH3–BRAF signals largely independent of a functional RAS binding domain, but requires an intact BRAF dimer interface and activation loop phosphorylation sites. Cells expressing TTYH3–BRAF exhibited increased MEK/ERK signaling, which was blocked by clinically achievable concentrations of sorafenib, trametinib, and the paradox breaker PLX8394. These data provide the first example of a fully autoinhibited BRAF protein whose oncogenic potential is dictated by a distinct fusion partner and not by a structural change in BRAF itself.

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

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on increasingly accurate, high-resolution molecular diagnostics as well as the functional and mechanistic understanding of individual tumors. While molecular stratification of patients can be achieved through different means, a promising approach is next-generation sequencing of tumor DNA and RNA, which can reveal genomic alterations that have immediate clinical implications. Furthermore, certain genetic alterations are shared across multiple histologic entities, raising the fundamental question of whether tumors should be treated by molecular profile and not tissue of origin. We here describe MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a clinically applicable platform for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors. We illustrate how a standardized workflow for selection and consenting of patients, sample processing, whole-exome/genome and RNA sequencing, bioinformatic analysis, rigorous validation of potentially actionable findings, and data evaluation by a dedicated molecular tumor board enables categorization of patients into different intervention baskets and formulation of evidence-based recommendations for clinical management. Critical next steps will be to increase the number of patients that can be offered comprehensive molecular analysis through collaborations and partnering, to explore ways in which additional technologies can aid in patient stratification and individualization of treatment, to stimulate clinically guided exploratory research projects, and to gradually move away from assessing the therapeutic activity of targeted interventions on a case-by-case basis toward controlled clinical trials of genomics-guided treatments.

Onimomotarō (Der Dämonenpfirsichjunge, 1891) ist eine ursprünglich für Kinder verfasste Kurzgeschichte des Schriftstellers Ozaki Kōyō, einer der bedeutendsten Schriftsteller der Meiji-Zeit. Kōyō, der vor allem durch seinen Roman Konjiki yasha (Goldfarbener Teufel, 1897–1902) Berühmtheit erlangte, engagierte sich in der so genannten genbun ichi Bewegung, eine Reformbewegung, die sich dafür stark machte, die Schriftsprache an die Umgangssprache anzupassen. Die hier erstmals in deutscher Übersetzung vorgelegte Erzählung Onimomotarō, eine Parodie des berühmten Volksmärchens Momotarō (Der Pfirsichjunge), ist hingegen noch ganz der klassischen japanischen Schriftsprache verpflichtet.

Pancreatic acinar cell carcinoma (PAC) is a rare disease with a poor prognosis. Treatment options for metastatic PAC are limited and often follow chemotherapeutic regimens for pancreatic ductal adenocarcinoma. Although recurrent genomic alterations, such as BRAF fusions and defects in genes involved in homologous recombination DNA repair, have been described in PAC, data on the clinical efficacy of molecularly guided, targeted treatment are scarce. Here we describe the case of a 27-yr-old patient with BRAFV600E-mutated PAC who was successfully treated with a combination of BRAF and MEK inhibitors. The patient presented to our clinic with abdominal pain and weight loss. Imaging showed extensive retroperitoneal disease as well as mediastinal lymphadenopathy. Because of elevated α-fetoprotein (AFP) levels and inconclusive histologic findings, a germ cell tumor was suspected; however, PEI chemotherapy was unsuccessful. A repeat biopsy yielded the diagnosis of PAC and treatment with FOLFIRINOX was initiated. Comprehensive molecular profiling within the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) precision oncology program revealed a somatic BRAFV600E mutation and a germline PALB2 stop-gain mutation. Therapy was therefore switched to BRAF/MEK inhibition, resulting in almost complete remission and disease control for 12 mo and a remarkable improvement in the patient's general condition. These results indicate that BRAF alterations are a valid therapeutic target in PAC that should be routinely assessed in this patient population.

Cancers of unknown primary site (CUPs) represent a heterogeneous group of metastatic tumors, which accounts for 3-5% of malignancies. Due to the poor prognosis and limited local and systemic treatment options, there is an urgent need for improvement of molecularly driven treatment strategies. We investigated the molecular profile and clinical course of 70 patients enrolled in a prospective precision oncology registry trial conducted by the National Center for Tumor Diseases (NCT) Heidelberg/Dresden and the German Cancer Consortium (DKTK) that addresses younger adults with advanced-stage cancer across histologies as well as patients with rare tumors (NCT/DKTK MASTER). Molecular analyses included whole genome sequencing (WGS, n=29), whole exome sequencing (WES, n=41) and transcriptome analysis (n=55). All patients were diagnosed with CUP-syndrome, 61/70 (87.1%) of diagnoses fulfilled the ESMO Clinical Practice Guidelines. Progression free survival (PFS) of the first treatment based on MASTER (PFS2) was compared to the PFS of the last prior systemic treatment (PFS1) in each individual patient. Within the coding sequence, we identified 0 to 1386 nonsynonymous point mutations (SNVs, median=41) and 0 to 38 insertions/deletions (indels, median=3) per sample. Hypermutation (≥100 SNVs and indels) was observed in 14 samples. Mutations of TP53 and KRAS were significantly enriched. Analysis of copy-number changes (CNVs) was performed in 51 samples (27 WGS and 24 WES) and revealed complex CNV profiles in most cases. Gains and losses involved single arms or whole chromosomes. Gains in chromosome 8q, 1q and 7 and losses in chromosome 6q and 17p occurred in more than 40% of the patients. Fusions of EML4-ALK and FGFR2 were found in three and six cases, respectively. In one case, pathological reevaluation for NUT midline carcinoma was recommended based on a NUTM1-MXI1 fusion. In total, pathological reevaluation based on characteristic genetic events was recommended in five cases. Germline analysis of 70 cases revealed five pathogenic variants (ACMG Class 5) in CHEK2, BRCA1, CDKN2A, NBN and ERCC3. In addition, one likely pathogenic variant (ACMG Class 4) was found in FH. The molecular tumor board recommended targeted therapy in 56/70 (80.0%) patients which could be applied in 20/56 (35.7%) cases. The molecularly driven treatment approaches translated into a median PFS2/1 ratio of 2.25 (n=17). Median PFS1 was 89 days (range 31-304, n=17) compared to a median PFS2 of 180 days (range 50-805, n=17). For three patients in which PFS1 could not be determined median PFS2 was 305 days (range 182-336). We demonstrate that a comprehensive molecular analysis of CUPs provides clinically relevant information and additional, molecularly stratified treatment approaches in many cases. These targeted therapies can be highly beneficial even in heavily pretreated patients. Citation Format: Maximilian Werner, Lino Möhrmann, Małgorzata Oleś, Andreas Mock, Arne Jahn, Simon Kreutzfeldt, Sebastian Uhrig, Martina Fröhlich, Barbara Hutter, Daniela Richter, Gina Rüter, Ivan Jelas, Rainer Hamacher, Johanna Falkenhorst, Sebastian Wagner, Christian Brandts, Melanie Börries, Anna Illert, Klaus Metzeler, Benedikt Westphalen, Alexander Desuki, Thomas Kindler, Albrecht Stenzinger, Evelin Schröck, Benedikt Brors, Peter Horak, Christoph Heining, Stefan Fröhling, Hanno Glimm. Genomics based personalized oncology of cancer of unknown primary [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 820.

Systems biology has experienced dramatic growth in the number, size, and complexity of computational models. To reproduce simulation results and reuse models, researchers must exchange unambiguous model descriptions. We review the latest edition of the Systems Biology Markup Language (SBML), a format designed for this purpose. A community of modelers and software authors developed SBML Level 3 over the past decade. Its modular form consists of a core suited to representing reaction‐based models and packages that extend the core with features suited to other model types including constraint‐based models, reaction‐diffusion models, logical network models, and rule‐based models. The format leverages two decades of SBML and a rich software ecosystem that transformed how systems biologists build and interact with models. More recently, the rise of multiscale models of whole cells and organs, and new data sources such as single‐cell measurements and live imaging, has precipitated new ways of integrating data with models. We provide our perspectives on the challenges presented by these developments and how SBML Level 3 provides the foundation needed to support this evolution., Over the past two decades, scientists from different fields have been developing SBML, a standard format for encoding computational models in biology and medicine. This article summarizes recent progress and gives perspectives on emerging challenges.

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., Chordomas are rare bone tumors with limited therapeutic options. Here, the authors identify molecular alterations associated with defective homologous recombination DNA repair in advanced chordomas and report prolonged response in a patient treated with a PARP inhibitor, which later acquired resistance due to a newly gained PARP1 mutation.

Comprehensive molecular profiling can be successfully applied to guide targeted treatment in cancer patients, an approach commonly referred to as precision oncology. Over the past years, several clinical trials that employed subgenomic molecular profiling have demonstrated that molecularly informed decision-making across tumor entities is associated with improved clinical outcome in approximately one third of patients. To investigate the feasibility and clinical relevance of comprehensive genomic analysis, i.e. whole-exome/genome sequencing (WES/WGS) and RNA sequencing (RNA-seq), in younger adults with advanced-stage cancer across all histologies and patients with rare tumors, we established MASTER (Molecularly Aided Stratification for Tumor Eradication Research) - a prospective, multicenter precision oncology platform - at NCT Heidelberg/Dresden in 2013, which was extended to the German Cancer Consortium (DKTK) in 2016. Based on a standardized workflow, we have analyzed more than 1,700 poor-prognosis (median overall survival, 12 months) patients with advanced, heavily pretreated (median number of prior therapies, n=2) malignancies representing a broad spectrum of rare histopathologic entities. We here report the actionable findings and clinical outcomes for the first 1,311 patients discussed in cross-institutional molecular tumor board (MTB) conferences. Each MTB recommendation was based on the individual molecular profile and specific predictive molecular biomarkers identified by WES/WGS and RNA-seq. In addition to DNA alterations (single-nucleotide variants, small insertions/deletions, copy number alterations), we also used alterations identified by RNA-seq (gene fusions, aberrant gene expression) to support clinical decision-making. We categorized therapy recommendations into seven different intervention baskets and assigned evidence levels to each recommendation according to a dedicated NCT/DKTK classification system, which addresses the complexity of evaluating predictive molecular biomarkers in clinical routine. MTB recommendations were implemented in one third of cases, and overall response and disease control rates on molecularly guided treatment were improved compared to prior systemic therapies, which translated into a progression-free survival ratio of greater than 1.3 in a significant proportion of patients. Furthermore, comprehensive genomic profiling in combination with histopathologic reevaluation allowed reclassification of approximately 4% of cases, in particular soft-tissue sarcomas not otherwise specified and carcinomas of unknown primary site. This prospective study demonstrates that comprehensive molecular profiling based on WES/WGS and RNA-seq in a multiinstitutional clinical setting creates meaningful therapeutic opportunities for patients with rare cancers. Our data demonstrate the added benefit of germline and RNA analysis, providing a rationale for their routine clinical implementation. Current and future activities of the MASTER network are focused on the standardization of variant classification and evidence levels in MTB conferences, the implementation of molecularly stratified basket trials, and the integration of additional layers of patient characterization. Citation Format: Peter Horak, Christoph Heining, Andreas Mock, Simon Kreutzfeldt, Andreas Lassmann, Lino Möhrmann, Jennifer Hüllein, Dorothea Hanf, Arne Jahn, Leo Ruhnke, Laura Gieldon, Christoph E. Heilig, Veronica Teleanu, Martina Fröhlich, Sebastian Uhrig, Katja Beck, Daniela Richter, Stephan Wolf, Katrin Pfütze, Christina Geörg, Bettina Meissburger, Frederick Klauschen, Ulrich Keilholz, Sebastian Ochsenreither, Gunnar Folprecht, Jens Siveke, Sebastian Bauer, Thomas Kindler, Christian Brandts, Melanie Boerries, Anna L. Illert, Nikolas von Bubnoff, Karsten Spiekermann, Philipp J. Jost, Klaus Schulze-Osthoff, Michael Bitzer, Peter Schirmacher, Christof von Kalle, Richard F. Schlenk, Barbara Klink, Barbara Hutter, Daniel Hübschmann, Albrecht Stenzinger, Wilko Weichert, Evelin Schröck, Benedikt Brors, Hanno Glimm, Stefan Fröhling, German Cancer Consortium (DKTK). Comprehensive genomic analysis of rare cancers: Results of the MASTER precision oncology trial of the German Cancer Consortium [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 821.

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.

Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. The oncogenic potential of BRAF fusions has been attributed to the loss of critical N-terminal domains that mediate BRAF autoinhibition. We used whole-exome and RNA sequencing in a patient with glioblastoma multiforme to identify a rearrangement between TTYH3, encoding a membrane-resident, calcium-activated chloride channel, and BRAF intron 1, resulting in a TTYH3-BRAF fusion protein that retained all features essential for BRAF autoinhibition. Accordingly, the BRAF moiety of the fusion protein alone, which represents full-length BRAF without the amino acids encoded by exon 1 (BRAF

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

Purpose Rapidly evolving genomics technologies, in particular comprehensive next-generation sequencing (NGS), have led to exponential growth in the understanding of cancer biology, shifting oncology toward personalized treatment strategies. However, comprehensive NGS approaches, such as whole-exome sequencing, have limitations that are related to the technology itself as well as to the input source. Hence, clinical implementation of comprehensive NGS in a quality-controlled diagnostic workflow requires both the standardization of sequencing procedures and continuous validation of sequencing results by orthogonal methods in an ongoing program to enable the determination of key test parameters and continuous improvement of NGS and bioinformatics pipelines. Patients and Methods We present validation data on 220 patients who were enrolled between 2013 and 2016 in a multi-institutional, genomics-guided precision oncology program (Molecularly Aided Stratification for Tumor Eradication Research) of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. Results More than 90% of clinically actionable genomic alterations identified by combined whole-exome sequencing and transcriptome sequencing were successfully validated, with varying frequencies of discordant results across different types of alterations (fusions, 3.7%; single-nucleotide variants, 2.6%; amplifications, 1.1%; overexpression, 0.9%; deletions, 0.6%). The implementation of new computational methods for NGS data analysis led to a substantial improvement of gene fusion calling over time. Conclusion Collectively, these data demonstrate the value of a rigorous validation program that partners with comprehensive NGS to successfully implement and continuously improve cancer precision medicine in a clinical setting.

Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. Oncogenic BRAF fusion proteins usually consist of the BRAF kinase domain and an N-terminal fusion partner that replaces the critical domains required for BRAF autoinhibition. We applied whole-exome and RNA sequencing in a patient with glioblastoma multiforme (GBM) to identify a rearrangement between TTYH3, encoding a membrane-resident, calcium-activated chloride channel, and BRAF intron 1, resulting in a TTYH3-BRAF fusion protein that retained all structural prerequisites for BRAF autoinhibition. Indeed, the BRAF moiety of the fusion protein alone, which represented near-full-length BRAF without the amino acids encoded by exon 1 (BRAFΔE1), did not induce MEK/ERK phosphorylation or cellular transformation. Similarly, neither the TTYH3 moiety of the fusion protein nor full-length TTYH3 provoked ERK pathway activity or transformation. In contrast, TTYH3-BRAF displayed increased MEK phosphorylation potential and transforming activity, which were caused by TTYH3-mediated tethering of BRAFΔE1 to the (endo)membrane system. Consistent with this mechanism, a synthetic approach in which BRAFΔE1 was localized to the membrane by fusing it to the cytoplasmic tail of CD8 also induced transformation. Furthermore, we demonstrate that TTYH3-BRAF signals largely independent of a functional RAS-binding domain, but relies on an intact BRAF dimer interface and forms homo- and heterodimers with RAF1 or ARAF. Moreover, the MEK phosphorylation and transformation potential of TTYH3-BRAF requires the activation loop phosphorylation sites T599 and S602. Various cell line models, including primary human astrocytes and a GBM stem cell line, expressing TTYH3-BRAF exhibited increased MEK/ERK signaling. TTYH3-BRAF-induced MEK/ERK phosphorylation was blocked by clinically achievable concentrations of sorafenib, trametinib, and the paradox breaker PLX8394. These data provide the first example of a fully autoinhibited BRAF protein whose oncogenic potential is dictated by a distinct fusion partner and not by a structural change in BRAF itself. Citation Format: Florian Weinberg, Ricarda Herr, Martina Fröhlich, Christoph Heining, Sandra Braun, Corinna Spohr, Mary Iconomou, Viola Hollek, Michael Röring, Peter Horak, Simon Kreutzfeldt, Gregor Warsow, Barbara Hutter, Sebastian Uhrig, Olaf Neumann, David Reuss, Dieter Hendrik Heiland, Christof von Kalle, Wilko Weichert, Albrecht Stenzinger, Benedikt Brors, Hanno Glimm, Stefan Fröhling, Tilman Brummer. Identification and characterization of an unusual BRAF fusion oncoprotein with retained autoinhibitory domains [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B08. doi:10.1158/1535-7163.TARG-19-LB-B08

Introduction: Treatment strategies incorporating proteasome inhibitors, immunomodulators, and autologous transplantation induce durable remissions in most newly diagnosed multiple myeloma (NDMM) patients. However, for 20% of patients even the most intensive therapies have not resulted in satisfactory outcomes. Currently available risk scores do not fully appreciate the complex biology of MM and have limited sensitivity and/or specificity for identification of high risk (HR) disease. We therefore aimed to characterize the mutational landscape of transplant-eligible NDMM patients who relapsed within 2 years after treatment initiation, thereby defining true clinical HRMM. To elucidate the clonal structure and evolution in these patients, we performed deep whole genome sequencing (WGS, ~80x) and RNAseq of samples collected at baseline and first relapse. Methods: We included 34 transplanted NDMM patients who experienced early relapse during maintenance within 2 years after treatment initiation. Tumor samples were collected from 20 and 31 patients at baseline and first relapse, respectively. Paired samples taken at both time points were available from 17 patients. WGS and RNAseq data were pre-processed using in-house pipelines. Single nucleotide variants (SNVs), indels, translocations, and copy number variants (CNVs) were called using Platypus, SOPHIA and ACESeq. Subclones were identified using SciClone. RNAseq data was aligned using STAR. Fusion genes were called by Arriba. Differential gene expression was assessed using DESeq2. Results: At baseline, only 12/20 patients would have been classified as HR according to conventional markers, including presence of t(4;14), t(14;16), amp(1q), clonal del(17p) or ISS3. In 5 patients del(17p) was solely observed in a minor sublone, which was selected during treatment and became dominant at relapse in 3 of them. Selection of amp(1q)-positive subclones was seen in 2 patients, illustrating that subclonal amp(1q) or del(17p) are frequent events in HR patients, and - in contrast to recent results - could contribute to early relapse. Translocations involving MYC have also been reported to be of prognostic impact. At baseline 9 of 20 patients were positive for this event, with BMP6 and the lambda locus being the translocation partner in 2 patients each. At relapse we found an additional MYC-lambda, and two MYC-kappa translocations, supporting recent observations that MYC-light chain translocations are associated with aggressive disease. We identified a median of 40 (range: 17-233) nonsilent somatic SNVs per patient at baseline and 61 (range: 14-322) at relapse. Yet, comparing paired samples there was no significant increase in SNVs. In our HRMM set, 21 of 64 recently identified driver genes were mutated at baseline with KRAS (n = 6), TP53 (n = 6), NRAS (n = 2), and DIS3 (n = 2) being the most frequently affected genes. 6 of them - ACTG1, DIS3, FAM46C, NFKB2, RB1, and TRAF3 - were involved in fusion genes. At relapse the number of mutated driver genes increased to 29, and 10 of 31 patients presented with a clonal TP53 mutation. All patients with a TP53 mutation also showed deletion of the second allele or LOH. Including other tumor suppressor genes, such as RB1, CDKN2C, or TRAF3, 12/20 NDMM and 20/31 relapsed patients had at least one bi-allelic aberration, doubling the number of HR patients with such events compared to considering TP53 alone. Longitudinally, we observed all patterns of clonal evolution that were recently described for unselected patients. Stable evolution was primarily seen in patients achieving partial remission, supporting a model where some tumor cells survive in a protective microenvironment (ME). In deep responders, however, branching evolution was the dominant patterns. This observation rather supports strong cell-intrinsic mechanisms and rapid selection of aggressive minor subclones in clinically defined HRMM. Conclusions: Understanding the mutational landscape in HRMM and drivers of early relapse is crucial in order to improve treatment options. Our study highlights the importance of bi-allelic events in HR and suggests that focusing on TP53 is not sufficient, if all HR cases are to be identified. Of note, 3 patients in the entire cohort would have been classified as low risk by conventional risk scores and even at relapse did not carry any bi-allelic event, indicating the existence of unknown somatic HR aberrations or a protective ME. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Molecular Partners: Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding.

Chordomas are rare tumors of the axial skeleton and skull base with few therapeutic options and no clinically validated molecular drug targets. The value of comprehensive genomic analyses for guiding medical therapy of patients with advanced-stage chordoma is unknown. We performed whole-exome and genome sequencing of tumor and matched germline control samples from 11 patients with locally advanced or metastatic chordoma within the MASTER program, a prospective clinical sequencing program of the German Cancer Consortium. All patients were pretreated and had progressive disease prior to molecular analysis. Genomic profiling showed that advanced chordomas are frequently characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair. First, DNA copy number profiles showed high numbers of structural variants greater than 10 million base pairs in size in the majority of cases. Second, all patients harbored somatic aberrations of at least 2 genes known to be involved in HR, and 10/11 cases harbored somatic alterations in 3 or more HR pathway genes. For example, 8 patients showed heterozygous BRCA2 deletions, which were associated with heterozygous deletions of ERCC6 in 6 patients and RAD54L in 7 patients, as well as PTEN alterations (heterozygous deletion, heterozygous mutation and deletion of the wildtype allele or loss of heterozygosity). Other recurrently altered HR genes included ATR, CHEK2, FANCC, FANCD2, FANCG, RAD18, RAD51B, and XRCC3. Third, pathogenic germline alterations were detected in 3 patients. A heterozygous BRCA2 frameshift mutation (p.T3085fs*26; ACMG Class 5), a heterozygous NBN frameshift mutation (p.K219Nfs*16; ACMG Class 5), and a heterozygous CHEK2 missense mutation (p.R145W; ACMG Class 4) were accompanied by somatic deletion of the respective wildtype alleles. Fourth, a mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and coincided with genomic instability. The high prevalence of an HR deficiency “footprint” in chordoma patients prompted us to explore the clinical efficacy of the poly(ADP-ribose) polymerase(PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells. Olaparib treatment of a patient whose tumor showed a prominent exposure to an HR deficiency-associated mutational signature, a high degree of genomic instability, and 13 heterozygous HR gene alterations halted tumor growth for 10 months. Whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain, providing novel insight into the mechanisms of PARP inhibitor resistance. In summary, our study has uncovered a key biological feature of advanced chordoma that represents an immediately actionable therapeutic target and provides a rationale for genomics-guided clinical trials of PARP inhibition in this intractable tumor entity. Citation Format: Stefan Gröschel, Daniel Hübschmann, Francesco Raimondi, Peter Horak, Gregor Warsow, Martina Fröhlich, Barbara Klink, Laura Gieldon, Barbara Hutter, Kortine Kleinhenz, David Bonekamp, Oliver Marschal, Priya Chudasama, Jagoda Mika, Marie Groth, Sebastian Uhrig, Stephen Krämer, Christoph Heining, Christoph Heilig, Daniela Richter, Eva Reisinger, Katrin Pfütze, Roland Eils, Stephan Wolf, Christof von Kalle, Christian Brandts, Claudia Scholl, Wilko Weichert, Stephan Richter, Sebastian Bauer, Roland Penzel, Evelin Schröck, Albrecht Stenzinger, Richard Schlenk, Benedikt Brors, Robert Russell, Hanno Glimm, Matthias Schlesner, Stefan Fröhling. Defective homologous recombination DNA repair as therapeutic target in advanced chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2723.

Most adult gastrointestinal stromal tumors (GIST) are driven by activating KIT or PDGFRA mutations. The remaining 10-15% of cases, often referred to as wildtype (WT) GIST, display either alterations of the succinate dehydrogenase complex (SDH) or RAS pathway mutations. To gain additional insight into the biology of GIST, we performed whole-exome or genome and RNA sequencing in 38 GIST patients (WT, n=15; KIT-mutant, n=21; PDGFRA-mutant, n=2) enrolled in a prospective molecular stratification trial of NCT Heidelberg/Dresden and the German Cancer Consortium (DKTK) designed for younger adults with advanced-stage cancer across histologies and patients with rare tumors (NCT/DKTK MASTER). Of the 15 patients with WT GIST, 3 had pathogenic germline mutations in NF1 and 9 harbored SDH alterations (germline, n=5; somatic, n=4). In the 3 patients with quadruple-negative GIST - defined by the absence of KIT, PDGFRA, SDH, or RAS pathway alterations - we detected novel gene fusions affecting RET, FGFR2, and FGF4, respectively. To delineate biologically relevant subgroups of GIST based on RNA sequencing data from the entire cohort (n=34), we used 3 different clustering methods and 4 different measures of stability and consistency. Despite the underlying clinical and molecular heterogeneity, we identified 3 distinct transcriptional subgroups that were characterized by (i) SDH deficiency, (ii) recurrent somatic RB1 alterations and mutational signatures associated with defective homologous recombination DNA repair, and (iii) elevated PDGFRA expression, respectively. Furthermore, we used random forest analysis to identifiy genes that are significantly (p Citation Format: Peter Horak, Matea Hajnic, Laura Gieldon, Mario Hlevnjak, Susan Richter, Barbara Hutter, Johanna Falkenhorst, Sebastian Uhrig, Gregor Warsow, Nagarajan Paramasivam, Stefan Gröschel, Barbara Klink, Simon Kreutzfeldt, Christoph Heining, Christoph E. Heilig, Martina Fröhlich, Stephan Richter, Christian Brandts, Wilko Weichert, Philipp Jost, Olaf Neumann, Marc Zapatka, Albrecht Stenzinger, Alexander Marx, Benedikt Brors, Evelin Schröck, Sebastian Bauer, Peter Hohenberger, Hanno Glimm, Claudia Scholl, Stefan Fröhling. Comprehensive genomic and transcriptomic profiling of gastrointestinal stromal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1686.

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on high-resolution molecular diagnostics. The value of comprehensive molecular profiling based on whole-exome/genome sequencing (WES/WGS) and global RNA sequencing to guide therapeutic decisions in individual patients remains to be established. We report the results of MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a multicenter registry trial for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors conducted under the auspices of NCT Heidelberg/Dresden and the German Cancer Consortium (DKTK). Based on a standardized workflow for selection and consenting of patients, sample processing, WES/WGS and RNA sequencing, bioinformatic analysis, and technical validation of potentially actionable findings, we have analyzed more than 1300 poor-prognosis (median overall survival, 12 months) patients representing a broad spectrum of entities. Evaluation of the data by a cross-institutional molecular tumor board has allowed categorization into 7 different intervention baskets and formulation of evidence-based recommendations for clinical management in more than 80% of patients, which were implemented in approximately one third of cases. Overall response and disease control rates on molecularly guided treatment were significantly improved compared to prior systemic therapies, which translated into a progression-free survival (PFS) ratio of greater than 1.3 in more than 40% of cases. In 5% of patients, comprehensive genomic profiling allowed to refine the clinical diagnosis, as exemplified by several soft-tissue sarcomas not otherwise specified and carcinomas of unknown primary site that could be categorized based on their genotypes and subsequent histopathologic re-evaluation. Finally, systematic analysis of germline alterations revealed that 11% of patients had pathogenic (ACMG Class 4 or 5) variants in known tumor predisposition genes, and that 4% were carriers for autosomal recessive disorders. This prospective trial demonstrates that molecular profiling based on WES/WGS and RNA sequencing in a multi-institutional clinical setting is feasible, complements and advances routine molecular diagnostics, and creates clinically meaningful therapeutic opportunities in a significant proportion of patients. To improve clinical translation, the MASTER platform is now linked to a growing portfolio of cross-institutional basket trials. In the intermediate term, genomic profiling within MASTER will be integrated with additional layers of patient characterization and extended to additional treatment modalities (e.g. radiotherapy and surgical interventions). Citation Format: Peter Horak, Christoph Heining, Simon Kreutzfeldt, Christoph E. Heilig, Lino Möhrmann, Laura Gieldon, Martina Fröhlich, Sebastian Uhrig, Daniel Hübschmann, Katja Beck, Daniela Richter, Stephan Wolf, Katrin Pfütze, Christina Geörg, Bettina Meißburger, Frederick Klauschen, Sebastian Ochsenreither, Gunnar Folprecht, Jens Siveke, Sebastian Bauer, Thomas Kindler, Christian Brandts, Melanie Börries, Nikolas von Bubnoff, Karsten Spiekermann, Philipp J. Jost, Klaus Schulze-Osthoff, Michael Bitzer, Peter Schirmacher, Christof von Kalle, Richard Schlenk, Barbara Klink, Barbara Hutter, Wilko Weichert, Albrecht Stenzinger, Evelin Schröck, Benedikt Brors, Hanno Glimm, Stefan Fröhling. Clinical relevance of comprehensive genomic analysis in advanced-stage cancers and rare malignancies: Results from the MASTER trial of the German Cancer Consortium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 468.

Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. We here aimed to identify actionable genetic alterations in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy.We used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733_1735del, p.D579del) as potentially druggable target in this patient and carried out cancer hotspot panel sequencing to detect secondary resistance-conferring mutations in KIT. Furthermore, as a step towards clinical exploitation of the recently described signatures of mutational processes in cancer genomes, we established and applied a novel bioinformatics algorithm that enables supervised analysis of the mutational catalogs of individual tumors.Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. KIT dependency was further validated by a secondary KIT exon 17 mutation (c.2459_2462delATTCinsG, p.D820_S821delinsG) that was detected upon tumor progression after 10 months of imatinib treatment and provided a rationale for salvage therapy with regorafenib, which has activity against KIT exon 11/17 mutant GIST.These observations highlight the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscore that KIT exon 11 mutations represent tractable therapeutic targets across different histologies.

Identification of the tissue of origin in cancer of unknown primary (CUP) poses a diagnostic challenge and is critical for directing site-specific therapy. Currently, clinical decision-making in patients with CUP primarily relies on histopathology and clinical features. Comprehensive molecular profiling has the potential to contribute to diagnostic categorization and, most importantly, guide CUP therapy through identification of actionable lesions. We here report the case of an advanced-stage malignancy initially mimicking poorly differentiated soft-tissue sarcoma that did not respond to multiagent chemotherapy. Molecular profiling within a clinical whole-exome and transcriptome sequencing program revealed a heterozygous, highly amplified KRAS G12S mutation, compound-heterozygous TP53 mutation/deletion, high mutational load, and focal high-level amplification of Chromosomes 9p (including PDL1 [CD274] and JAK2) and 10p (including GATA3). Integrated analysis of molecular data and histopathology provided a rationale for immune checkpoint inhibitor (ICI) therapy with pembrolizumab, which resulted in rapid clinical improvement and a lasting partial remission. Histopathological analyses ruled out sarcoma and established the diagnosis of a poorly differentiated adenocarcinoma. Although neither histopathology nor molecular data were able to pinpoint the tissue of origin, our analyses established several differential diagnoses including triple-negative breast cancer (TNBC). We analyzed 157 TNBC samples from The Cancer Genome Atlas, revealing PDL1 copy number gains coinciding with excessive PDL1 mRNA expression in 24% of cases. Collectively, these results illustrate the impact of multidimensional tumor profiling in cases with nondescript histology and immunophenotype, show the predictive potential of PDL1 amplification for immune checkpoint inhibitors (ICIs), and suggest a targeted therapeutic strategy in Chromosome 9p24.1/PDL1-amplified cancers.

Kato and colleagues ([1][1]) reported 4 cancer patients with metastatic disease, hyperprogression under treatment with anti-PD-1/PD-L1 monotherapy, and a cooccurring MDM2/4 amplification in the cancer cells. They defined hyperprogression as time-to-treatment failure 50% increase in tumor