Integrating proteomics into precision oncology.

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 based on omics data: The NCT Heidelberg experience.

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.

Methylation status at HYAL2 predicts overall and progression-free survival of colon cancer patients under 5-FU chemotherapy.

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.

DNA methylation array analyses identified breast cancer-associated HYAL2 methylation in peripheral blood.

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.

Genome-wide analysis associates familial colorectal cancer with increases in copy number variations and a rare structural variation at 12p12.3.

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.

Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC.

Neoadjuvant chemotherapy can improve the survival of individuals with borderline and unresectable pancreatic ductal adenocarcinoma; however, heterogeneous responses to chemotherapy remain a significant clinical challenge. Here, we performed RNA sequencing (n = 97) and multiplexed immunofluorescence (n = 122) on chemo-naive and postchemotherapy (post-CTX) resected patient samples (chemoradiotherapy excluded) to define the impact of neoadjuvant chemotherapy. Transcriptome analysis combined with high-resolution mapping of whole-tissue sections identified GATA6 (classical), KRT17 (basal-like) and cytochrome P450 3A (CYP3A) coexpressing cells that were preferentially enriched in post-CTX resected samples. The persistence of GATA6hi and KRT17hi cells post-CTX was significantly associated with poor survival after mFOLFIRINOX (mFFX), but not gemcitabine (GEM), treatment. Analysis of organoid models derived from chemo-naive and post-CTX samples demonstrated that CYP3A expression is a predictor of chemotherapy response and that CYP3A-expressing drug detoxification pathways can metabolize the prodrug irinotecan, a constituent of mFFX. These findings identify CYP3A-expressing drug-tolerant cell phenotypes in residual disease that may ultimately inform adjuvant treatment selection.

Signaling-induced systematic repression of miRNAs uncovers cancer vulnerabilities and targeted therapy sensitivity.

Targeted therapies are effective in treating cancer, but success depends on identifying cancer vulnerabilities. In our study, we utilize small RNA sequencing to examine the impact of pathway activation on microRNA (miRNA) expression patterns. Interestingly, we discover that miRNAs capable of inhibiting key members of activated pathways are frequently diminished. Building on this observation, we develop an approach that integrates a low-miRNA-expression signature to identify druggable target genes in cancer. We train and validate our approach in colorectal cancer cells and extend it to diverse cancer models using patient-derived in vitro and in vivo systems. Finally, we demonstrate its additional value to support genomic and transcriptomic-based drug prediction strategies in a pan-cancer patient cohort from the National Center for Tumor Diseases (NCT)/German Cancer Consortium (DKTK) Molecularly Aided Stratification for Tumor Eradication (MASTER) precision oncology trial. In conclusion, our strategy can predict cancer vulnerabilities with high sensitivity and accuracy and might be suitable for future therapy recommendations in a variety of cancer subtypes.

Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification.

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 for proteogenomic-based discovery of neoantigens. By using an optimized computational approach, we discover a large number of tumor-specific and tumor-associated antigens. To create a pipeline for the identification of neoantigens in our cohort, we combine DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity and an in-depth validation process. We detect a broad variety of non-canonical HLA-binding peptides in the majority of patients demonstrating partially immunogenicity. Our validation process allows for the selection of 32 potential neoantigen candidates. The majority of neoantigen candidates originates from variants identified in the RNA data set, illustrating the relevance of RNA as a still understudied source of cancer antigens. This study underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.

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

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

CATCH: A Prospective Precision Oncology Trial in Metastatic Breast Cancer.

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.

Comprehensive Genomic and Transcriptomic Analysis for Guiding Therapeutic Decisions in Patients with Rare Cancers.

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659.

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

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

Pathway analysis of genetic variants in folate-mediated one-carbon metabolism-related genes and survival in a prospectively followed cohort of colorectal cancer 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: HRhet  = 0.81, 95% CI: 0.67-0.97; TYMS: rs1001761: HRhet  = 0.82, 95% CI: 0.68-0.99 and rs2847149: HRhet  = 0.82, 95% CI: 0.68-0.99). After FDR correction, one polymorphism in paraoxonase 1 (PON1; rs3917538) was significantly associated with OS (HRhet  = 1.28, 95% CI: 1.07-1.53; HRhzv  = 2.02, 95% CI:1.46-2.80; HRlogAdd  = 1.31, pFDR  = 0.01). Adjusted pathway analyses showed significant associations for pyrimidine biosynthesis (P = 0.04) and fluorouracil drug metabolism (P < 0.01) with significant gene-chemotherapy interactions, including PON1 rs3917538. This study supports the concept that FOCM-related genes could be associated with CRC survival and may modify effects of 5-FU-based chemotherapy in genes in pyrimidine and fluorouracil metabolism, which are relevant targets for therapeutic response and prognosis in CRC. These results require confirmation in additional clinical studies.

Validating Comprehensive Next-Generation Sequencing Results for Precision Oncology: The NCT/DKTK Molecularly Aided Stratification for Tumor Eradication Research Experience.

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.

Integration of genomics and histology revises diagnosis and enables effective therapy of refractory cancer of unknown primary with PDL1 amplification.

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.