Evidence supporting <i>RPS20</i> as a colorectal cancer susceptibility gene from a case-control MGPT study.

10617 Background: Ribosomal protein S20 ( RPS20 ) is increasingly included in colorectal cancer (CRC) germline multigene panel testing (MGPT) and clinical management guidelines, despite limited evidence of gene-disease association. Germline loss-of-function (LOF) variants in RPS20 have been observed in a small number of individuals with CRC, without tumor loss-of-heterozygosity, and have typically been classified as variants of uncertain significance (VUS). We conducted a case/control study of individuals heterozygous for RPS20 LOF variants to evaluate the CRC and colorectal polyp risk compared to controls and cases of established CRC susceptibility genes. Methods: Cases and controls underwent MGPT at Invitae (now part of Labcorp) from May 2018-May 2025. Cases included individuals with a single LOF variant in RPS20 who had no identified pathogenic variants (PV) in other hereditary cancer genes and no VUS in established CRC risk genes; controls consisted of individuals with no PV or VUS identified. To reduce ascertainment bias, cases and controls were identified from requisitioned (clinician-ordered, reported) and unrequisitioned (run on MGPT backbone but unreported) data, under WCG protocol 1167406. Ninety controls were randomly sampled and age/sex matched to cases. CRC/polyp diagnoses were determined by clinician-reported ICD10 codes and keywords and compared between cases and controls utilizing Fisher’s exact tests. To compare risk estimates with a well-defined hereditary CRC syndrome, odds of CRC/polyps in cases with RPS20 LOF variants and in cases with mismatch repair gene PVs ( MLH1, MSH2, MSH6, PMS2 ), both compared to controls, were estimated with multivariable logistic regression that included sex, age, requisition status, family history of cancer, and race/ethnicity. Results: Twenty-seven heterozygous RPS20 LOF cases were identified (median age at testing 48 years [IQR 35-37]). CRC prevalence was significantly higher in RPS20 LOF cases (63.0%) compared with controls (11.1%, p= &lt;0.001), while polyp prevalence did not differ (7.4% vs. 10.0%, p=1). In multivariable analysis, RPS20 LOF variants were associated with increased odds of CRC compared with controls (odds ratio [OR] 18.0; 95% confidence interval [CI], 5.2–62.1), comparable to odds observed for MMR gene PGV cases (OR 9.5; 95% CI, 6.6–13.7). Conclusions: Heterozygous RPS20 LOF variants identified through MGPT are associated with substantially increased CRC risk. By incorporating age- and sex-matched controls and unrequisitioned testing data, this study reduces ascertainment bias and supports RPS20 as a CRC susceptibility gene. Larger studies are needed to inform clinical management. Multivariable logistic regression, RPS20 LOF cases compared to controls. Cancer/polyps OR CI p-value CRC 18.0 5.2 - 62.1 &lt;0.001 Colon polyps 0.8 0.2 - 3.0 0.719 CRC/polyps 10.2 3.5 - 29.5 &lt;0.001 Other Cancer 2.5 0.9 - 6.6 0.114