Abstract 5894: Detection of a novel GNA11 processed pseudogene from cfDNA and implications for liquid biopsy

Abstract Processed pseudogenes are messenger RNAs that have been reverse transcribed and inserted into the genome. While the functional implications of these processed pseudogenes remain unknown, their presence can lead to variants being mistakenly detected and classified as copy number amplifications and deletions in comprehensive genomic profiling (CGP) assays for cancer treatment selection. False positive amplifications run the risk of patients being prescribed ineffective therapies. Northstar Select is a liquid biopsy CGP assay which covers 84 genes. In this retrospective analysis of 1800 patients, amplification signals due to a pseudogene were observed in 4 patients on SMAD4 and 7 patients on GNA11 and confirmed via orthogonal methods. These samples showed abnormal exonic amplifications and intronic deletions in the SMAD4 and GNA11 genes. Splice site variants were present at low variant allele fraction (VAF) on either side of each exon. Notably, the copy number detected does not correlate with estimated tumor fractions or other ctDNA mutations detected in these samples. Whole genome sequencing (WGS) of selected patients harboring the SMAD4 amplifications revealed the presence of a poly-A tail and a breakpoint in SCAI on chromosome 9, consistent with a pseudogene previously described in the literature. WGS of GNA11 amplification-positive samples revealed the presence of a novel processed pseudogene in chromosome 20 that has not been previously described, which may have further implications on cancer-driving mutations as GNA11 is an oncogene. Northstar Select’s copy number calling algorithm measures a copy number of 3 for GNA11, indicating an additional copy from the pseudogene. qPCR of extracted buffy coat gDNA across the suspected breakpoint in chromosome 20 confirms the insertion and its germline nature. The presence of the same breakpoint in multiple patients suggests this is an ancestral integration event. Similar to the previously described SMAD4 pseudogene, false positive splice site variants are results of mismapping of reads. In conclusion, we have determined a framework to confirm pseudogene presence that is not tumor DNA derived. Bioinformatic analysis of CGP tests should carefully screen suspicious results for the possibility of as-yet undiscovered processed pseudogenes to avoid reporting such false positive amplifications and deletions. Citation Format: Jan Wignall, Wen Zhou. Detection of a novel GNA11 processed pseudogene from cfDNA and implications for liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5894.