Real-World Implementation of genomic Tumor Boards: Experiences from the German National Network Genomic Medicine (nNGM)

Andreas Till¹, Tatjana Huebner¹, Britta Haenisch¹, Rudolf Klatt¹, Luca Pötschke¹ & Catharina Scholl¹, Roman A. Siddiqui²,Friedrich von Kessel²,Sebastian C. Semler² & michael Krawczak², Christian altbürger³,⁴, Peter Schirmacher³,⁴ & Albrecht stenzinger³,⁴, Ronald Black⁵, Jürgen Wolf⁶,⁷, Anna Kron⁶,⁷, Uwe Lührig⁶,⁷ & Anna Rasokat⁶,⁷, dorothee Andres⁸, Anika Anker⁸, Sophia Schade⁸ & Jana straßburger⁸, Stefan Aretz⁹,¹⁰,¹¹ & Markus M. Nöthen⁹, Tobias B. Haack¹²,Stephan Ossowski¹² & Olaf Riess¹²,Daniela Hauke¹³,Katharina Jäger¹³,Michael Knecht¹³ & Thomas Thum¹³,Frank Lehmann¹⁴,¹⁵,Katharina M. Schueler¹⁴ & Jochen Wölfel¹⁴,¹⁵, Martin Schwab¹⁶ & Claudia Müller-Tidau¹⁶, Heiko Wittig¹⁷,¹⁸ & Johannes Schmutzler¹⁷,¹⁸, Christoph Wölfel¹⁹,²⁰ & Michael Hummel¹⁹,²⁰

Affiliations:

¹Federal institute for Drugs and Medical Devices (BfArM), Bonn, Germany
²Technology, Methods, and Infrastructure for Networked Medical Research (TMF), Berlin, Germany
³Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany
⁴Centre for Personalized Medicine (ZPM) Heidelberg, Heidelberg, Germany
⁵National Association of Statutory Health Insurance Funds, GKV-Spitzenverband, Berlin, Germany
⁶Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Düsseldorf (CIO ABCD), University Hospital of Cologne, Cologne, Germany
⁷National Network Genomic Medicine (nNGM) Lung Cancer, University Hospital of Cologne, Cologne, Germany
⁸Federal ministry of Health, Bonn, Germany
⁹Institute of Human Genetics, University Hospital Bonn, Bonn, Germany
¹⁰Center for Hereditary Tumor Syndromes, University Hospital Bonn, Bonn, Germany
¹¹European Reference Network on Genetic Tumour Risk Syndromes (ERN GENTURIS), Bonn, Germany
¹²Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tubingen, Germany
¹³German Center for Cardiovascular Research (DZHK), Berlin, Germany
¹⁴Institute of Medical Informatics, University of Münster, Münster, Germany
¹⁵University Hospital Münster, Münster, Germany
¹⁶University Cancer Center Hamburg – UCC Hamburg, Hamburg, Germany
¹⁷University Hospital Freiburg, Freiburg, Germany
¹⁸SignGene Expert Center for Personalized Cancer therapy, Freiburg, Germany
¹⁹Institute of Pathology, Klinikum rechts der isar, technical University of munich, Munich, Germany
²⁰German cancer Consortium (DKFZ), Heidelberg, Germany

Germline Variants in RAD51C are Associated with Increased risk of Childhood-Onset Neuroblastoma

Abstract

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, characterized by high heterogeneity and aggressive clinical behavior. While MYCN amplification remains the strongest genetic risk factor, a substantial proportion of NB cases lack this amplification, suggesting the involvement of other genetic factors. Here, we report a thorough analysis of germline variants in a large cohort of NB patients and controls, identifying rare, damaging germline variants in RAD51C as considerably associated with increased NB risk, notably in non-amplified cases. Functional studies demonstrate that these variants impair RAD51C protein function, leading to impaired DNA repair and increased genomic instability. Our findings highlight RAD51C as a novel susceptibility gene for NB and provide insights into the genetic mechanisms underlying tumor development.

Introduction

Neuroblastoma (NB) is a neuroectodermal tumor arising from immature neural crest cells. despite advances in treatment, high-risk NB remains a significant cause of cancer-related mortality in children.The genetic basis of NB is complex, with both inherited and somatic factors contributing to tumorigenesis.MYCN amplification is a well-established oncogenic driver, present in approximately 30% of NB cases. However, the majority of NB cases do not harbor MYCN amplification, indicating the involvement of other genetic factors.

Genome-wide association studies (GWAS) have identified several common genetic variants associated with NB risk, but these typically explain only a small fraction of the heritability. Rare, highly penetrant germline variants are increasingly recognized as significant contributors to cancer susceptibility. Here, we aimed to identify rare germline variants associated with NB risk through whole-exome sequencing (WES) of a large cohort of NB patients and controls.

Materials and Methods

Study Cohort: We analyzed germline DNA from 632 NB patients and 648 healthy controls. Patient samples were collected from multiple centers across Germany. Clinical data,including age at diagnosis,stage,and MYCN amplification status,were obtained from medical records.

Whole-Exome Sequencing: Exome sequencing was performed on Illumina NovaSeq 6000 platforms. Sequencing data were aligned to the human reference genome (GRCh38) using BWA-MEM. Variant calling was performed using GATK HaplotypeCaller. Variants were annotated using ANNOVAR.

Variant filtering and prioritization: We focused on rare, damaging variants with a minor allele frequency (MAF) < 0.01 in the gnomAD database. Damaging variants were predicted using multiple algorithms, including SIFT, PolyPhen-2, and CADD.

Functional Studies: RAD51C variants were introduced into a human cell line using CRISPR-Cas9 gene editing.RAD51C protein expression and DNA repair capacity were assessed using Western blotting and a comet assay, respectively.

statistical Analysis: Association between RAD51C variants and NB risk was assessed using logistic regression, adjusting for age and sex. Stratified analyses were performed based on MYCN amplification status.

Results

Identification of RAD51C as a Susceptibility Gene: WES analysis revealed a significant association between rare, damaging germline variants in RAD51C and increased NB risk (OR = 2.8, 95% CI: 1.5-5.2, p = 0.001). This association was particularly strong in NB cases without MYCN amplification (OR = 4.1, 95% CI: 2.0-8.4, p = 0.0002). We identified 12 distinct RAD51C variants in 15 NB patients, all of which were predicted to be damaging.

Functional Impact of RAD51C Variants: Functional studies demonstrated that the identified RAD51C variants significantly reduced RAD51C protein expression and impaired DNA repair capacity in cells. Comet assays showed increased DNA damage in cells expressing mutant RAD51C compared to wild-type RAD51C.

Clinical Characteristics of RAD51C mutation Carriers: NB patients carrying RAD51C variants tended to be diagnosed at a younger age compared to non-carriers (median age: 18 months vs. 36 months, p = 0.03). There was no significant difference in stage or overall survival between RAD51C mutation carriers and non-carriers.

discussion

Our findings establish RAD51C as a novel susceptibility gene for childhood neuroblastoma. RAD51C encodes a key protein involved in homologous recombination,a critical DNA repair pathway.