Germline sequencing in children with cancer in Quebec: an integrated investigative approach

ABSTRACT Background The province of Quebec has progressively implemented paired tumour-germline sequencing in paediatric oncology through two coordinated precision research programs, preceding a province-wide mainstream clinical genomics initiative. We report the prevalence, spectrum, and clinical relevance of germline findings (GFs) in children with primary extracranial cancers, integrating molecular, phenotypic, and pathological data. Methods Patients enrolled between 2014 and 2022 underwent germline whole-exome sequencing (WES) using a virtual 352-cancer gene panel. Sequencing, bioinformatics and variant interpretation followed best practices standards based on GATK, ACMG/AMP and ClinGen recommendations. Somatic WES and transcriptomic data were integrated when available. GFs were categorised as diagnostic findings (DFs; established or suspicious association with the cancer phenotype) or as other findings further subcategorised according to actionability and age of disease onset. Findings Among 484 children, 130 (26.9%) carried 149 GFs, including 49 (10.1%) with a DF (42 with well-established associations with cancer phenotypes). DFs involved 21 genes related to childhood cancer predisposition, trisomy 21 and one clinical Beckwith-Wiedemann syndrome. Six DFs were initially missed by standard exome pipelines, and mosaic constitutional cancer predisposition syndrome (CPS) was confirmed in 4/49 children, underscoring the value of integrative analyses. A CPS was known at the time of primary cancer in 10/49 children. Among those diagnosed with a CPS after cancer onset, suggestive phenotypic features were present in 36/39. Other non-diagnostic findings were identified in 92 children; 21 (4.3% of the cohort) with actionable implications in childhood (n=7) or adulthood (n=14). Somatic sequencing was informative for refining causality, as somatic second hit alterations were identified in 29/33 (87.9%) DFs involving monoallelic tumour suppressor genes, whereas no such alterations were observed in non-DFs counterparts (0/57; p<0.0001). Interpretation: This provincial research experience highlights the analytical and practical challenges of germline evaluation in paediatric oncology and supports a shift toward integrative interpretation frameworks combining complementary germline, somatic, pathology, and phenotypic data. Flexibility in investigative strategies and nuanced categorisation of findings are warranted, guided by a child-centred interpretative framework. This approach underpins Quebec’s paediatric oncology genomics mainstreaming initiative.