Saha, Neiladri

Abstract Introduction Accurate detection of gene fusions is critical for the diagnosis and management of sarcomas. However, occasional false-negative results may occur due to factors such as low tumor cellularity, limited assay coverage of relevant genomic regions, or mis-annotation by bioinformatic pipelines. We recently encountered a case in which our clinical SARCP fusion detection assay failed to identify an HMGA2::NCOR2 (exon 3–exon 16) fusion. Method The SARCP assay is a targeted, amplicon-based RNA-seq next-generation sequencing (NGS) assay designed to detect gene fusions across 138 genes, including HMGA2. Although NCOR2 is not among the targeted genes, the assay is capable of detecting fusions when only one gene partner is represented. Results Suspicion of a false-negative result arose when the pathologist noted that the tumor’s histologic features—keratin-positive, giant cell-rich soft tissue—were consistent with a subtype previously associated with the HMGA2::NCOR2 fusion. This prompted a review of the bioinformatic output, which had erroneously reported an HMGA2::MAML2 fusion. The misannotation was traced to sequence homology involving a CAA/CAG repeat at the 5′ end of NCOR2 exon 16, likely resulting from DNA polymerase slippage and misalignment in low-complexity, partially complementary repetitive regions. This erroneously lead to the target genomic sequence getting mapped to MAML2 exon 2 as opposed to the NCOR2 exon 16. Discussion This case highlights how bioinformatic mis-annotation can lead to occasional false-negative fusion calls. It underscores the importance of manual review in cases with strong clinical or pathologic suspicion and calls attention to the need for fusion-calling algorithms that better handle repetitive and homopolymeric sequences. Citation Format: Ganesh P. Pujari, Mitch L. Klebig, Kurt S. Willkom, Montha Chhunn, Stefan W. Nelson, Neiladri Saha, Kevin C. Halling. Mis-Annotation of an HMGA2::NCOR2 Fusion in FFPE Sarcoma Samples: A Case Illustrating Bioinformatic Limitations in Fusion Detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A012.

Abstract Most TERT promoter (TERTp) clinically relevant sequence variants in CNS tumors are the canonical C228T and C250T mutations. Non-canonical TERTp mutations have been reported mainly in non-CNS tumors. We describe 12 CNS tumors with non-canonical TERTp variants interpreted as clinically relevant. Using clinical targeted NGS panels (2020-2024), we detected c.-100_-79dup (n=2), c.-92_-91insGGCGGCCCCGCCCCTTCCTTTC (n=1), c.-118_-117insTCCCCGGCCCAGCCCCTTCCGGG (n=1), c.-123_-76dup (n=1), c.-110_-89dup (n=2), and c.-57A>C (n=5). Most cases had chromosomal microarray (n=11) and/or methylation array data analyzed using the NCI/Bethesda v.2 classifier (n=9). All non-canonical TERTp variants occurred at a variant allele frequency suggestive of somatic clonal origin (mean, 34%; range, 18-46), and in adults (mean age, 60 years; range, 36-88). Except for the c.-57A>C variant, all non-canonical TERTp variants occurred in tumors with histological and/or molecular diagnostic features of glioblastoma, IDH-wildtype (GBM), often epigenetically aligning to this tumor type. All five tested cases matched to superfamily glioblastoma (score ≥ 0.9), with three to class GBM_RTK_I (>0.9 in 2, 0.86 in 1) and two to GBM_RTK_II (>0.9). The size and sequence of such variants suggest that these may be functionally equivalent to canonical mutations or generate favorable TERTp activation motif conformations like previously reported activating duplications. The c.-57A>C variant is a well-established oncogenic mutation observed in other cancer types but has not been reported in CNS tumors. We describe five cases with the c.-57A>C variant: one GBM matching to methylation class GBM_MES_TYP (0.99); two oligodendroglioma, IDH-mutant and 1p/19q-codeleted, one of which had methylation data matching to class O_IDH (0.99); one diffuse glioma, IDH-mutant, which was partially 1p/19q-codeleted and matched to class O_IDH (0.99); and one anaplastic meningioma, aligning to superfamily meningioma (0.99)/MNG_MAL (0.79). Our findings show that non-canonical TERTp variants typically occur in tumor types that often harbor TERTp canonical mutations, expanding the spectrum of TERTp clinically relevant variants that are molecular biomarkers in CNS tumors.