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.
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.
Therapeutic options for neuroendocrine neoplasms (NEN) are limited. Within the MASTER program, a multi-institutional registry trial for prospective 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, we apply prospective whole-exome/genome sequencing (WES/WGS) and RNA sequencing (RNA-seq) to determine therapeutic choices for individual patients who have exhausted standard therapy options. We here report the clinical and molecular characteristics of the NEN cohort within this study. Between 2013 and 2018, 108 patients (male, n=65; female, n=43) were enrolled. Histologies according to the 2017 WHO Classification were neuroendocrine tumor grade 1 (NET G1), n=8; NET G2, n=31; NET G3, n=9; neuroendocrine carcinoma, n=49; and mixed neuroendocrine/non-neuroendocrine neoplasm, n=11. Primary tumor sites were gastrointestinal tract, n=30; pancreas, n=31; thorax, n=21; genitourinary system, n=11; head and neck, n=5; and other regions, n=10. All patients had advanced-stage disease and had received a median of 2 prior lines of systemic therapy. WES, WGS, and RNA-seq were performed in 69, 40, and 87 patients, respectively. Clinical evaluation of germline and somatic molecular data (single-nucleotide variants, small insertions and deletions, copy number variations, mutational burden, mutational signatures, homologous recombination deficiency scores, gene expression patterns, etc.) from 105 patients by a dedicated molecular tumor board yielded evidence-based recommendations for clinical management in 91 cases (87%). Treatment recommendations were grouped as follows: PARP inhibition, n=35; immunotherapy, n=27; mTOR inhibition, n=22; CDK4/6 inhibition, n=15; tyrosine or serine/threonine kinase inhibition, n=57 (ALK, n=1; ERBB, n=6; FGFR, n=10; MET, n=5; RET, n=12; VEGFR, n=4, MEK, n=9; other, n=10), DNA-crosslinking chemotherapy, n=16; anti-claudin18.2 antibody, n=5; BET inhibition, n=5; and DLL3 antibody, n=5. As of November 2018, at least 18 patients had received molecularly guided treatment (PARP inhibition, immunotherapy, mTOR inhibition, tyrosine or serine/threonine kinase inhibition) of which 11 were evaluable for response (partial response, n=4; stable disease, n=2; progressive diseases, n=5). Twenty-seven patients died before therapy could be started, 16 are currently receiving other regimens, and for the remaining patients no follow-up data are available yet. In conclusion, comprehensive molecular profiling offers valuable insight into to the genomic and transcriptomic landscape of NEN and creates additional therapeutic opportunities in a subset of patients.Citation Format: Simon Kreutzfeldt, Leonidas Apostolidis, Malgorzata Oles, Peter Horak, Christoph E. Heilig, Christoph Heining, Barbara Hutter, Laura Gieldon, Barbara Klink, Mario Lamping, Damian T. Rieke, Sebastian Uhrig, Henning Jann, Ulrich F. Pape, Albrecht Stenzinger, Eva C. Winkler, Bertram Wiedenmann, Dirk Jäger, Benedikt Brors, Evelin Schröck, Ulrich Keilholz, Marianne Pavel, Hanno Glimm, Stefan Fröhling. Clinical relevance of comprehensive genomic analysis in patients with advanced-stage neuroendocrine neoplasms: 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 919.
ABSTRACT Background Salivary gland cancers (SGC) are rare and heterogeneous malignant tumors. Advanced SGC lack established treatment options and show poor response to immunotherapy. Here, an integrative multi-omics analysis in a large cohort of advanced SGC revealed insights into the tumor immune microenvironment (TIM) and distinct mechanisms of immune evasion. Methods A total of 104 patients with recurrent/metastatic SGC from the DKTK MASTER program were included in this study. Whole-exome or whole-genome sequencing and RNA-sequencing was performed on fresh frozen tumor tissue. The tumor immune microenvironment was analyzed using CIBERSORT deconvolution analysis and immune gene expression scores in bulk RNA-sequencing data. Single-nuclei sequencing and immunohistochemistry analyses were performed in selected samples. Results were validated in bulk RNA-sequencing data of a previously published independent dataset. Results Bulk transcriptome analysis revealed an immune-deserted TIM in the majority of advanced SGC samples. Immune exclusion was most prominent in adenoid cystic carcinoma (ACC) subgroup 1 exhibiting a downregulation of the antigen processing machinery. Only a small subset of advanced SGC, including few adenoid cystic carcinoma, exhibited T-cell inflammation, which was correlated with tumor mutational burden in Non-ACC samples. Subtype specific expression of immune checkpoints as well as cancer testis antigens were identified with prominent expression of VTCN1 in luminal cells within ACC. Single-cell RNA-sequencing and bulk RNA-seq deconvolution analysis validated immune cell exclusion and revealed a TIM that was dominated by M2 macrophages across SGC subtypes. Among evaluable patients treated with immune checkpoint inhibitors, a high T-cell to macrophage ratio was associated with clinical benefit. Conclusions These data support biomarker-based development of immune-checkpoint inhibition and the development of novel immune-checkpoint inhibitors and cellular therapies in SGC. Trial Registration Retrospectively registered, NCT05852522
Abstract 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.
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.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.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.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.
