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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

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types by combining proteogenomics with phenotypic and functional analyses. By using an optimized computational approach, we discovered a large number of novel tumor-specific and tumor-associated antigens including shared common target candidates. To create a pipeline for the identification of neoantigens in our cohort, we combined deep DNA and RNA sequencing with MS- based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity. In fact, we could detect a broad variety of non-wild type HLA-binding peptides in the majority of patients and confirmed the immunogenicity of 24 neoantigens. Most interestingly, the majority of total and immunogenic neoantigens originated from variants identified in the RNA dataset, illustrating the importance of RNA as a still understudied source of cancer antigens. Moreover, the amount of these mainly RNA-based immunogenic neoantigens correlated positively with overall CD8+ tumor-infiltrating T cells. This study therefore underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.Statement of significanceThe significance of this study lies not only in the potential of our optimized proteogenomic workflow for the discovery of neoantigens (in particular RNA-derived neoantigens) for clinical application, but sheds light on the entity-agnostic prevalence of HLA class I peptide presentation of RNA processing events to be used for tumor targeting.

The impact of neoadjuvant chemotherapy on tumor cells is largely unknown with resistance to therapy remaining a significant challenge. We analyzed 171 chemo-naïve and post-chemotherapy resected patient samples (chemoradiotherapy excluded) using RNAseq and multiplexed immunofluorescence. Neoadjuvant chemotherapy enriched for distinct neoplastic persister phenotypes expressing Classical and Basal-like biomarkers GATA6 and KRT17. The enrichment of GATA6HiClassical-like and KRT17Hi Basal-like cells in post-chemotherapy patient samples was associated with poor survival after mFOLFIRINOX, but not gemcitabine. CYP3A subfamily enzymes (CYP3A4 and CYP3A5) were expressed in GATA6Hi and KRT17Hi persister cells and can degrade mFOLFIRINOX constituent irinotecan. Taken together these data support a model in which pre-existing subpopulations of GATA6Hi (Classical) and/or KRT17Hi (Basal) neoplastic cells emerge after mFOLFIRINOX treatment by expressing CYP3A. The identification of CYP3A-enabled persister cell phenotypes is an important step in the design of effective therapies to overcome drug-tolerance and prevent recurrence after neoadjuvant therapy. Citation Format: Xu Zhou, Roma Kurilov, John P. Neoptolemos, Benedikt Brors, Kai Hu, Teresa Peccerella, Jing-Yu An, Stephanie Roessler, Katrin Pfütze, Angela Schulz, Stephan Wolf, Nicolas Hohmann, Ulrike Heger, Oliver Strobel, Simon T Barry, Christoph Springfeld, Christine Tjaden, Frank Bergmann, Markus Büchler, Thilo Hackert, Franco Fortunato, Peter Bailey. Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A010.

Identification of cancer vulnerabilities is pivotal for the success of precision oncology. Comprehensive molecular profiling technologies improved clinical outcome of targeted drugs. However, the complexity of molecular interactions still challenges appropriate data interpretation. Hence, new strategies are needed to support current treatment decisions. One approach would be to consider additional molecular information, for instance microRNA (miRNA) expression patterns. By analyzing various cancer datasets, we observed that inhibition of miRNA expression occurs in a directed manner. Strong cancer driving mutations, such as in KRAS, result in a block of miRNAs inhibiting the cancer driver gene itself, or its associated downstream pathways. As many cancer drivers can activate diverse downstream pathways, we examined whether groups of strongly repressed miRNAs can uncover essential pathways. Therefore, we developed a miRNA depletion based cancer gene dependency model and related drug prediction workflow using miRNA sequencing data from cancer cell lines. We defined a pathway ranking score (PRS) and focused on druggable target genes. To validate our drug prediction workflow, we performed miRNA sequencing in various patient derived 3D cancer models (PDCMs). We predicted top hit pathway dependencies and individual druggable target genes for each culture. Subsequently, samples were used for in vitro drug response assays. Here, samples were significantly more sensitive to predicted targeted drugs, compared to standard of care (SOC) chemotherapy, or non-predicted targeted drugs. Next, we applied our miRNA based drug prediction workflow to four patients and compared three sources for RNA sequencing for each patient: PDCM, primary tumor, and microdissected purified tumor from FFPE tissue. Of note, FFPE purified miRNAs extraction led to comparable results to PDCMs and primary tumors. Finally, we were interested whether our prediction workflow matches with a comprehensive genomic based drug prediction workflow from the MASTER precision oncology trial. Therefore, we sequenced miRNAs from 95 patients and performed subsequent drug prediction. Interestingly, a complete overlap was overserved in only 20% of patients. In 35% of the patients, MASTER drug recommendation was biologically related to our drug prediction, and 40% revealed no match. In 75 out of 91 patients (82%) with available RNA Seq data, at least one of our predicted target genes was noticeably overexpressed on mRNA level. In total, 40% of all with our workflow predicted target genes, and 52% of druggable predicted target genes showed an increase of 1.5 fold or higher on RNA level compared to the entire cohort. In conclusion, unidirectional silenced miRNA expression signatures give further insights into cancer cell pathways and druggable gene dependencies and might be suitable tools for precision oncology. Citation Format: Alexander A. Wurm, Silke Brilloff, Zunamys I. Carrero, Stephan Drukewitz, Alexander Krüger, Sandra Oster, Sofia Kolovich, Melanie Hüther, Sebastian Uhrig, Dana Westphal, Friedegund Meier, Katrin Pfütze, Daniel Hübschmann, Peter Horak, Simon Kreutzfeldt, Christoph Heining, Daniela Richter, Stefan Fröhling, Claudia R. Ball, Hanno Glimm. Pathway activation driven miRNA depletion highlights cancer signaling vulnerabilities and targeted therapy sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4110.

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

DNA sequencing and RNA sequencing are increasingly applied in precision oncology, where molecular tumor boards evaluate the actionability of genetic events in individual tumors to guide targeted treatment. To work toward an additional level of patient characterization, we assessed the abundance and activity of 27 proteins in 134 patients whose tumors had previously undergone whole-exome and RNA sequencing within the Molecularly Aided Stratification for Tumor Eradication Research (MASTER) program of National Center for Tumor Diseases, Heidelberg. Proteomic and phosphoproteomic targets were selected to reflect the most relevant therapeutic baskets in MASTER. Among six different therapeutic baskets, the proteomic data supported treatment recommendations that were based on DNA and RNA analyses in 10% to 57% and frequently suggested alternative treatment options. In several cases, protein activities explained the patients' clinical course and provided potential explanations for treatment failure. Our study indicates that the integrative analysis of DNA, RNA and protein data may refine therapeutic stratification of individual patients and, thus, holds potential to increase the success rate of precision cancer therapy. Prospective validation studies are needed to advance the integration of proteomic analysis into precision oncology.

PURPOSE CATCH (Comprehensive Assessment of clinical feaTures and biomarkers to identify patients with advanced or metastatic breast Cancer for marker driven trials in Humans) is a prospective precision oncology program that uses genomics and transcriptomics to guide therapeutic decisions in the clinical management of metastatic breast cancer. Herein, we report our single-center experience and results on the basis of the first 200 enrolled patients of an ongoing trial. METHODS From June 2017 to March 2019, 200 patients who had either primary metastatic or progressive disease, with any number of previous treatment lines and at least one metastatic site accessible to biopsy, were enrolled. DNA and RNA from tumor tissue and corresponding blood-derived nontumor DNA were profiled using whole-genome and transcriptome sequencing. Identified actionable alterations were brought into clinical context in a multidisciplinary molecular tumor board (MTB) with the aim of prioritizing personalized treatment recommendations. RESULTS Among the first 200 enrolled patients, 128 (64%) were discussed in the MTB, of which 64 (50%) were subsequently treated according to MTB recommendation. Of 53 evaluable patients, 21 (40%) achieved either stable disease (n = 13, 25%) or partial response (n = 8, 15%). Furthermore, 16 (30%) of those patients showed improvement in progression-free survival of at least 30% while on MTB-recommended treatment compared with the progression-free survival of the previous treatment line. CONCLUSION The initial phase of this study demonstrates that precision oncology on the basis of whole-genome and RNA sequencing is feasible when applied in the clinical management of patients with metastatic breast cancer and provides clinical benefit to a substantial proportion of patients.

Folate‐mediated one‐carbon metabolism (FOCM) is a key pathway essential for nucleotide synthesis, DNA methylation, and repair. This pathway is a critical target for 5‐fluorouracil (5‐FU), which is predominantly used for colorectal cancer (CRC) treatment. A comprehensive assessment of polymorphisms in FOCM‐related genes and their association with prognosis has not yet been performed. Within 1,739 CRC cases aged ≥30 years diagnosed from 2003 to 2007 (DACHS study), we investigated 397 single nucleotide polymorphisms (SNPs) and 50 candidates in 48 FOCM‐related genes for associations with overall‐ (OS) and disease‐free survival (DFS) using multiple Cox regression (adjusted for age, sex, stage, grade, BMI, and alcohol). We investigated effect modification by 5‐FU‐based chemotherapy and assessed pathway‐specific effects. Correction for multiple testing was performed using false discovery rates (FDR). After a median follow‐up time of 5.0 years, 585 patients were deceased. For one candidate SNP in MTHFR and two in TYMS, we observed significant inverse associations with OS (MTHFR: rs1801133, C677T: HR het = 0.81, 95% CI: 0.67–0.97; TYMS: rs1001761: HR het = 0.82, 95% CI: 0.68–0.99 and rs2847149: HR het = 0.82, 95% CI: 0.68–0.99). After FDR correction, one polymorphism in paraoxonase 1 (PON1; rs3917538) was significantly associated with OS (HR het = 1.28, 95% CI: 1.07–1.53; HR hzv = 2.02, 95% CI:1.46–2.80; HR logAdd = 1.31, pFDR = 0.01). Adjusted pathway analyses showed significant associations for pyrimidine biosynthesis (P = 0.04) and fluorouracil drug metabolism (P

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.

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.

DNA methylation variations in gene promoter regions are well documented tumor-specific alterations in human malignancies including colon cancer, which may influence tumor behavior and clinical outcome. As a subset of colon cancer patients does not benefit from adjuvant chemotherapy, predictive biomarkers are desirable. Here, we describe that DNA methylation levels at CpG loci of hyaluronoglucosaminidase 2 (HYLA2) could be used to identify stage II and III colon cancer patients who are most likely to benefit from 5-flourouracil (5-FU) chemotherapy with respect to overall survival and progression-free survival.

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.

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.

Breast cancer (BC) is the leading cause of cancer-related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC-associated DNA methylation signatures in peripheral blood were largely unknown. Here, we performed a genome-wide methylation screening and identified a BC-associated differentially methylated CpG site cg27091787 in the hyaluronoglucosaminidase 2 gene (HYAL2) (discovery round with 72 BC case and 24 controls: p = 2.61 × 10(-9) adjusted for cell-type proportions). The substantially decreased methylation of cg27091787 in BC cases was confirmed in two validation rounds (first validation round with 338 BC case and 507 controls: p < 0.0001; second validation round with 189 BC case and 189 controls: p < 0.0001). In addition to cg27091787, the decreased methylation of a 650-bp CpG island shore of HYAL2 was also associated with increased risk of BC. Moreover, the expression and methylation of HYAL2 were inversely correlated with a p-value of 0.006. To note, the BC-associated decreased HYAL2 methylation was replicated in the T-cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early-stage BC cases (stages 0 and I) from healthy controls [area under curve (AUC) = 0.89], and was robust for the detection of BC in younger women as well (age < 50, AUC = 0.87). Our study reveals a strong association between decreased HYAL2 methylation in peripheral blood and BC, and provides a promising blood-based marker for the detection of early BC.

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.

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.

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.

Colorectal cancer (CRC) is one of the most common cancer worldwide. However, a large number of genetic risk factors involved in CRC have not been understood. Copy number variations (CNVs) might partly contribute to the 'missing heritability' of CRC. An increased overall burden of CNV has been identified in several complex diseases, whereas the association between the overall CNV burden and CRC risk is largely unknown. We performed a genome-wide investigation of CNVs on genomic DNA from 384 familial CRC cases and 1285 healthy controls by the Affymetrix 6.0 array. An increase of overall CNV burden was observed in familial CRC patients compared with healthy controls, especially for CNVs larger than 50kb (case/control ratio = 1.66, P = 0.025). In addition, we discovered for the first time a novel structural variation at 12p12.3 and determined the breakpoints by strategic PCR and sequencing. This 12p12.3 structural variation was found in four of 2862 CRC cases but not in 6243 healthy controls (P = 0.0098). RERGL gene (RERG/RAS-like), the only gene influenced by the 12p12.3 structural variation, sharing most of the conserved regions with its close family member RERG tumor suppressor gene (RAS-like, estrogen-regulated, growth inhibitor), might be a novel CRC-related gene. In conclusion, this is the first study to reveal the contribution of the overall burden of CNVs to familial CRC risk and identify a novel rare structural variation at 12p12.3 containing RERGL gene to be associated with CRC.

Breast cancer (BC) is the leading cause of cancer-related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC-associated DNA methylation signatures in peripheral blood were largely unknown. Here, we performed a genome-wide methylation screening and identified a BC-associated differentially methylated CpG site cg27091787 in the hyaluronoglucosaminidase 2 gene (HYAL2) (discovery round with 72 BC case and 24 controls: p = 2.61 × 10(-9) adjusted for cell-type proportions). The substantially decreased methylation of cg27091787 in BC cases was confirmed in two validation rounds (first validation round with 338 BC case and 507 controls: p0.0001; second validation round with 189 BC case and 189 controls: p0.0001). In addition to cg27091787, the decreased methylation of a 650-bp CpG island shore of HYAL2 was also associated with increased risk of BC. Moreover, the expression and methylation of HYAL2 were inversely correlated with a p-value of 0.006. To note, the BC-associated decreased HYAL2 methylation was replicated in the T-cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early-stage BC cases (stages 0 and I) from healthy controls [area under curve (AUC) = 0.89], and was robust for the detection of BC in younger women as well (age50, AUC = 0.87). Our study reveals a strong association between decreased HYAL2 methylation in peripheral blood and BC, and provides a promising blood-based marker for the detection of early BC.

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.

Introduction Angiogenesis, the generation of new blood vessels, is crucial in tumor growth, progression, and metastasis. The process involves a variety of factors including signaling, adhesion and chemotactic molecules, ECM (extracellular matrix) proteins, proteinases, transcription factors, growth factors and receptors. Variation in genes encoding these factors potentially influence angiogenic processes. We thus evaluated the association between variants in angiogenesis-related genes and risk as well as prognosis of colorectal cancer (CRC). Approach In a candidate pathway approach, we investigated 437 variants in 36 angiogenesis-related genes for association with CRC risk and prognosis in ∼1800 patients and ∼1800 controls of the German DACHS/IMPACT study. Patients were aged 30 years or older and diagnosed between 2003 and 2007. CRC risk was estimated using conditional logistic regression based on the co-dominant inheritance model. In addition, we investigated the association between polymorphisms and overall survival using multivariable Cox regression. Correction for multiple testing was performed using false discovery rates (FDR). Results Risk Several of the investigated variants in ANGTP1, ETS1, FLT4, MMP2, NOTCH4, PDGFRB, TGFB2 were associated with risk of CRC, and one variant in DLL1 (delta-like 1) remained significant after accounting for multiple testing. DLL1 rs9348307 was associated with decreased risk of CRC (ORGC 0.81, 95% CI 0.68-0.96; ORCC 0.62, 95% CI 0.36-1.06, p=0.02). Survival After a median follow up time of 5 years, variants in twelve genes (ANGPT1, EFNB2, ETS1, FLT4, JAG1, KDR, MMP2, MMP9, NRP1, NRP2, PDGFRB, TGFB2) were associated with overall survival in CRC patients. When accounting for multiple testing, variants in EFNB2, JAG1 and MMP2 remained significant. Most of the EFNB2 variants were associated with poorer survival, while one variant in EFNB2 was associated with better survival (rs2391333: HRCT 0.88, 95% CI 0.74-1.05; HRTT 0.60, 95% CI 0.45-0.78, p Conclusion Variants in angiogenesis-related genes were associated with CRC risk and overall survival in CRC patients. Two of the identified genes (DLL1 and JAG1) are involved in NOTCH signaling, which is crucial to cell differentiation, proliferation, apoptosis and angiogenesis, underlining the importance of this oncogenic pathway in colorectal carcinogenesis. Citation Format: Dominique Scherer, Yesilda Balavarca, Nina Habermann, Katharina Buck, Petra Seibold, Lisanne Kap, Katja Butterbach, Katrin Pfütze, Axel Benner, Michael Hoffmeister, Hermann Brenner, Barbara Burwinkel, Jenny Chang-Claude, Cornelia M. Ulrich. Genetic variation in angiogenesis-related genes is associated with colorectal cancer risk and prognosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2188. doi:10.1158/1538-7445.AM2014-2188