Smoley, Stephanie A.

Abstract Introduction: Clinical staging of multifocal non-small cell lung cancer (NSCLC) is facilitated by comparative genomic analysis of separate tumor nodules, but the role of genome-wide copy number profiling for this purpose is uncertain. Methods: We analyzed single nucleotide variants (SNVs), gene fusions, small copy number variants (gene amplifications and homozygous deletions) (sCNVs), and large-scale copy number variants (lCNVs) in a retrospective cohort of 34 patients with >1 lung cancer nodule tested on a previously validated tumor-only 515-gene next generation sequencing (NGS) panel (MayoComplete Solid Tumor Panel). For patients with <2 shared SNVs, a Bayesian approach was formulated to calculate the probability of shared versus independent events occurring in different tumor nodules in the same patient. The prevalence of specific lCNVs in NSCLC was derived from an independent dataset of 100 NSCLC peers tested by the same panel, and the prevalence of SNVs, fusions, and sCNVs in NSCLC was derived from publicly available data. Alterations unique to different tumor nodules and suspected germline alterations were excluded from the Bayesian analysis. Results: There were 54 total comparisons for 34 patients. Eleven of the patients (32.4%) had >=2 shared SNVs along with one or more shared lCNVs also identified in each patient. Eight patients (23.5%) had no shared SNVs, gene fusions, sCNVs, or lCNVs. Using the Bayesian approach for the remaining fifteen (44.1%) patients with at least one shared alteration (SNVs, sCNVs, and lCNVs), the probability of clonal relatedness ranged from 73% for a shared whole arm or whole chromosome lCNV to >99% for tumors with two or more shared alterations including multiple or complex lCNVs and SNVs. Whole chromosome or whole arm lCNVs were sometimes shared between tumor nodules with different driver SNVs. Conclusions: Incorporating lCNVs into comparative genomic analysis provides additional evidence of clonal relatedness in a subset of multifocal NSCLCs. However, differences in bioinformatics pipelines for calling lCNVs, concerns over the imprecision of genome-wide copy number profiling, and uncertainty regarding the temporal relationship between lCNVs and carcinogenesis (including somatic events possibly related to “field cancerization”) create challenges for integrating lCNVs into comparative genomic analysis with standard clinical workflows for tumor-only NGS on paraffin embedded tissue sections. Our simplified approach mitigates some of the uncertainty introduced by incorporating lCNVs into clonality determination. Citation Format: Claire Teigen, Ying-Chun Lo, Sounak Gupta, Stephanie Smoley, Beth Pitel, Hussam Al Kateb, Gopi Sivasankaran, Benjamin Kipp, Ande Rumilla, Gang Zheng, Kevin Halling, Katherine Geiersbach, . A Bayesian framework for evaluating somatic copy number variants into comparative genomic analysis of multifocal non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5423.

Abstract Diffuse hemispheric glioma, H3 G34‐mutant (DHG‐H3 G34) has been primarily molecularly characterized by methylation profiling and sequencing studies. We describe an integrated histomolecular evaluation including high‐resolution copy number profiling of a series of 60 DHG‐H3 G34 to further our understanding of the spectrum of genetic changes associated with this tumor type. Cases were clinically tested using an 187‐gene mutation and fusion targeted neuro‐oncology next‐generation sequencing panel ( n = 60) and Oncoscan chromosomal microarray ( n = 26) by a single laboratory (2018–2022). A subset of cases had immunohistochemical results for OLIG2 ( n = 42), p53 ( n = 48), and ATRX ( n = 46), and methylation array data ( n = 8). Median age at testing was 21 years (range, 12–50). No significant difference was noted in clinical, histopathological, and mutational profile between pediatric and adult patients. H3‐3A G34 mutations included G34R ( n = 56; 94%), G34V ( n = 3), and a non‐canonical G34E ( n = 1). Concurrent mutations most often involved TP53 ( n = 55; 92%), ATRX ( n = 50; 83%), and PDGFRA ( n = 34; 57%). A reportedly primary tumor was confirmed to be hypermutant and had a PMS2 mutation. A single case also showed an FGFR3::FAM184B fusion. All cases with available chromosomal microarray data had unbalanced genomes, which were often complex (14/26; 54%). The most frequent recurrent copy number abnormalities were losses involving 3q, 4q, 10q, 13q, and 18q, and 17p copy‐neutral loss of heterozygosity (cnLOH) encompassing TP53 . This copy number profile was reminiscent of that seen in Grade 4 IDH‐mutant astrocytomas. Collectively, a TP53 abnormality at copy number (12/26, all cnLOH), sequence (55/60) and protein expression (46/48) level was detected in all 60 cases. In conclusion, integrated high‐resolution copy number and histomolecular analysis expanded the spectrum of genetic changes associated with DHG‐H3 G34, including the presence of universal TP53 abnormalities with frequent cnLOH—a copy number abnormality that has been largely unrecognized—for this new 2021 World Health Organization central nervous system tumor type.

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.