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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.

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.

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.