Christoph E. Heilig

Desmoplastic small round cell tumor (DSRCT) is an ultra-rare sarcoma with limited treatment options. Here, we show that comprehensive molecular profiling informs diagnosis and individualized therapy in this disease. We report the results of whole-genome/exome, transcriptome, and DNA methylome analyses performed in 30 refractory DSRCT patients, complemented by (phospho)proteomic profiling in nine, within a nationwide precision oncology program. In eight patients (27%), DSRCT was diagnosed only after molecular profiling. Although DSRCTs have “quiet” genomes, 28 patients (93%) received 107 molecular-based management recommendations, including assessment of clinical trial eligibility in 17 (57%). Most recommendations are informed by overexpression of tyrosine kinases, SSTR3/5, and CLDN6, detected in 45%, 33%, and 20% of cases, respectively. Thirteen patients (46%) received recommended therapies, yielding disease control in eight (62%), including three long-lasting responses to pazopanib and trastuzumab deruxtecan, the latter administered based on ERBB2 overexpression in the absence of aberrant ERBB2 kinase activation. These findings demonstrate that multi-omics profiling provides clinically actionable insights for DSRCT management.

Precision cancer medicine aims to improve patient outcomes by providing individually tailored recommendations for clinical management based on the evaluation of biological disease profiles in multidisciplinary molecular tumor boards (MTBs). The quality of MTB decisions depends on the comprehensive, reliable, and reproducible interpretation of increasingly complex molecular data. We developed and implemented, as part of a multicenter precision oncology program, the Knowledge Connector (KC), a decision support system that integrates individual patients’ molecular and clinical data with world knowledge to generate and document MTB recommendations. The KC supports data curation, database integration, and discussion based on multiomics data and provides an interface for creating a cross-institutional knowledge base. Furthermore, it extracts relevant biomarker-drug associations and increases the efficacy of data interpretation in a clinically relevant manner by reducing reliance on external sources and optimizing inter-curator concordance. Our results demonstrate that the KC is a versatile tool that supports medical decision-making in MTBs, thus enabling the scalability of precision cancer medicine.

Precision oncology is becoming a mainstay in the standard of care for cancer patients. Recent technological advancements have significantly lowered the cost of various tumor profiling approaches, broadening the reach of molecular diagnostics and improving patient access to precision oncology. In parallel, drug development and discovery pipelines continue to evolve, driving targeted therapeutic options forward. Yet, not all patients harboring actionable molecular alterations respond to these interventions, and existing therapies do not cover the entire spectrum of potential molecular targets. In this review, we examine the current suite of omics technologies employed in clinical settings and underscore their roles in deepening our understanding of tumor biology and optimizing patient stratification for targeted treatments. We also highlight relevant precision oncology trials and share our own experiences using multi-omics data within a molecular tumor board framework. Finally, we discuss areas for future exploration aimed at propelling precision oncology to new heights.

Desmoplastic small round cell tumor (DSRCT) is an ultra-rare sarcoma with limited treatment options. We performed whole-genome/exome, transcriptome, and DNA methylome analysis in 30 refractory DSRCT patients, complemented by (phospho)proteomic profiling in nine, within a nationwide precision oncology program. In eight patients (27%), DSRCT was diagnosed based on molecular profiling. Although all patients had “quiet” genomes, 28 (93%) received 107 molecular-based management recommendations, including assessment of clinical trial eligibility in 17 (57%). Nearly half of recommendations (45%) were based on overexpression of tyrosine kinases, as well as SSTR3/5 and CLDN6, detected in 33% and 20% of cases, respectively. Thirteen patients (46%) received recommended therapies, yielding disease control in eight (62%; partial response, n = 5; stable disease, n = 3), including three long-lasting responses (≥ 12 months) to pazopanib and trastuzumab deruxtecan, triggered by ERBB2 overexpression in the absence of constitutive ERBB2 signaling. Thus, multi-omics profiling enables individualized DSRCT treatment.

Precision oncology offers new cancer treatment options, yet sequencing methods vary in type and scope. In this study, we compared whole-exome/whole-genome (WES/WGS) and transcriptome sequencing (TS) with broad panel sequencing by resequencing the same tumor DNA and RNA as well as normal tissue DNA for germline assessment, from 20 patients with rare or advanced tumors, who were originally sequenced by WES/WGS ± TS within the DKFZ/NCT/DKTK MASTER program from 2015 to 2020. Molecular analyses resulted in a median number of 2.5 (gene panel) to 3.5 (WES/WGS ± TS) treatment recommendations per patient. Our results showed that approximately half of the therapy recommendations (TRs) of both sequencing programs were identical, while approximately one-third of the TRs in WES/WGS ± TS relied on biomarkers not covered by the panel. Eight of 10 molecularly informed therapy implementations were supported by the panel, the remaining two were based on biomarkers absent from the panel, highlighting the potential additional clinical benefit of WGS and TS.

Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.

Analysis of selected cancer genes has become an important tool in precision oncology but cannot fully capture the molecular features and, most importantly, vulnerabilities of individual tumors. Observational and interventional studies have shown that decision-making based on comprehensive molecular characterization adds significant clinical value. However, the complexity and heterogeneity of the resulting data are major challenges for disciplines involved in interpretation and recommendations for individualized care, and limited information exists on how to approach multilayered tumor profiles in clinical routine. We report our experience with the practical use of data from whole-genome or exome and RNA sequencing and DNA methylation profiling within the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) program of the National Center for Tumor Diseases (NCT) Heidelberg and Dresden and the German Cancer Research Center (DKFZ). We cover all relevant steps of an end-to-end precision oncology workflow, from sample collection, molecular analysis, and variant prioritization to assigning treatment recommendations and discussion in the molecular tumor board. To provide insight into our approach to multidimensional tumor profiles and guidance on interpreting their biological impact and diagnostic and therapeutic implications, we present case studies from the NCT/DKFZ molecular tumor board that illustrate our daily practice. This manual is intended to be useful for physicians, biologists, and bioinformaticians involved in the clinical interpretation of genome-wide molecular information.

In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAF ^Δβ3-αC oncoproteins usually lack five amino acids in the β3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAF ^Δβ3-αC oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAF ^ΔLNVTAP>F and two novel mutants, BRAF ^delinsFS and BRAF ^ΔLNVT>F , and compare them with other BRAF ^Δβ3-αC oncoproteins. We show that BRAF ^Δβ3-αC oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAF ^Δβ3-αC oncoproteins, e.g., BRAF ^ΔLNVTAP>F , increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAF ^Δβ3-αC mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.