Kollmeyer, Thomas M.

ABSTRACT IDH-mutant astrocytomas maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, producing extreme inter-arm telomere length heterogeneity, yet how this heterogeneity shapes structural genome evolution remains unknown. Using Oxford Nanopore long-read sequencing of 20 IDH-mutant astrocytomas, we profiled structural variants (SVs), copy number variants, extrachromosomal DNA (ecDNA) and measured allele-specific telomere lengths from individual long reads. We identified pervasive complex rearrangements, including chromothripsis and foldback events consistent with breakage-fusion-bridge cycles, and widespread ecDNAs. SV breakpoints were enriched at telomeric and centromeric regions regardless of local telomere length, revealing constitutive structural fragility. Arm-level telomere length analysis uncovered a dual-mode model: arms with short telomeres preferentially harbored breakage-associated events, while arms with long ALT-maintained telomeres were enriched for ecDNA and amplification-associated events. These findings identify chromosome-arm-specific telomere length as a determinant of structural genome evolution in ALT-driven tumors.

Abstract Background: Alternative lengthening of telomeres (ALT) is common in isocitrate dehydrogenase (IDH) mutant astrocytomas, but how telomere length variability contributes to genome instability remains unclear. We hypothesized that chromosome arm–specific telomere heterogeneity underlies distinct patterns of somatic structural variation (SV), copy-number alteration (CNA), and extrachromosomal DNA (ecDNA) formation. Methods: Oxford Nanopore long-read whole-genome sequencing (∼20–40x coverage) was performed in a clinical cohort of n = 20 IDH-mutant astrocytomas. Somatic SVs were called with Severus and filtered to a high-confidence tumor-specific set. CNAs were inferred by read-depth segmentation (DNAcopy) and further refined using a long-read CNA framework (Wakhan). Chromosome arm–specific telomere length was estimated with Telogator, and SV breakpoints were annotated for enrichment near subtelomeric and pericentromeric regions. Associations between arm-level telomere length and SV/CNA features (rate and proportion) were tested using linear mixed-effects models with patient as a random effect. High-level amplification regions were defined directly from CNA segments with high copy number, and ecDNA-like circular amplicons were subsequently reconstructed from amplified intervals using Decoil. Results: IDH-mutant astrocytomas displayed pronounced SV heterogeneity, with complex breakends (BNDs) dominating the somatic SV landscape. SVs were spatially patterned, as deletions and insertions preferentially accumulated near subtelomeric and pericentromeric domains, implicating chromosome ends and repeat-rich regions as recurrent substrates for rearrangement. At the tumor level, markedly shortened telomeres were associated with increased SV burden and CNA burden, consistent with telomere attrition as a correlate of genome-wide structural instability in highly rearranged genomes. At higher resolution, telomere length varied substantially across chromosome arms and classified distinct structural outcomes: arms with shorter telomeres were enriched for complex insertional SVs, whereas arms with relatively elongated telomeres more frequently carried focal high-level amplifications and ecDNA-like circular amplicons. Conclusions: These findings reveal a link between arm-specific telomere heterogeneity and the emergence of structural and amplification architectures in IDH-mutant astrocytomas. Telomere remodeling may influence where rearrangements and ecDNA structures arise, offering potential biomarkers of genome instability and tumor evolution. Generative AI was used to improve language clarity; all content was verified by the authors. Citation Format: Maryam Jehangir, Kristen Drucker, T. Rhyker Ranallo-Benavidez, Thomas M. Kollmeyer, Ayse Keskus, Daniel Lachance, Jeanette E. Eckel-Passow, Ofer Shoshani, Mikhail Kolmogorov, Robert B. Jenkins, Floris P. Barthel. Telomere length heterogeneity shapes structural and amplification landscapes in IDH-mutant astrocytoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr PR009.

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 In adult-type diffuse gliomas CDKN2A and/or CDKN2B (CDKN2A/B) deletions often co-occur with deletion of MTAP, suggesting that MTAP immunohistochemistry (IHC) may be a surrogate marker of CDKN2A/B status. However, the association between CDKN2A/B and MTAP deletion at the genomic level remains unknown. We assessed CDKN2A/B and MTAP deletions by chromosomal microarray in 333 adult-type diffuse gliomas and performed MTAP IHC on a subset (n = 63). CDKN2A/B and MTAP deletions were detected in 216 and 215 cases, respectively, and were concurrent in 99.5% (215/216). While most tumors with CDKN2A/B homozygous deletion (n = 148) showed concurrent MTAP homozygous deletion (108/148; 73.0%), a subset harbored MTAP heterozygous deletion (39/148; 26.4%). By analyzing the size of the chromosomal alterations, we demonstrate that initial large chromosomal 9p losses result in concurrent heterozygous deletion of CDKN2A/B and MTAP whereas smaller “second hit” deletions leading to homozygous CDKN2A/B deletion do not always encompass the MTAP locus. Discordant CDKN2A/B and MTAP tumors affect the association between MTAP IHC and copy number status of MTAP and CDKN2A/B. These findings suggest that adult-type diffuse gliomas, regardless of IDH status, follow a stereotypic pathway involving concurrent CDKN2A/B and MTAP heterozygous deletion but may diverge for CDKN2A/B and MTAP homozygous deletion.

Abstract Background Current literature suggestsisocitrate dehydrogenase (IDH)-mutant astrocytoma contains several molecular subgroups. In this study, we are interested in determining the connection between different molecular subgroups with grade and/or survival. Methods A cohort of 470 Mayo Clinic adult patients (≥18 years, 56.2% male) with primary IDH-mutant astrocytoma diagnosed by World Health Organization (WHO) 2021 criteria were examined. Results were validated in an independent cohort of 614 Mayo Clinic Neuropathology consult patients and 235 The Cancer Genome Atlas (TCGA) patients. Results The Mayo Clinic Practice cohort confirmed the association of CDKN2A/B deletion with overall survival (OS, homozygous vs hemizygous vs intact, 2.7 vs 9.6 vs 17.2 years, P < .001). Phosphatase and tensin homolog (PTEN) deletion was also associated with poor OS (7.3 vs 17.4 years, P < .001). Increased number of copy number alterations was associated with OS (continuous variable, HR = 1.027, P < .001). Carrying one or more copies of the germline risk allele at rs55705857 was associated with earlier age of onset (median age 33 vs 35 years, P = .01), and a shorter OS after adjusting for age, grade, sex and treatment (HR = 1.81, P = .007). The Mayo Clinic Neuropathology Consult cohort and TCGA were utilized to validate age of onset and survival, respectively. Unsupervised clustering of the copy number alterations identified several clinically significant groups that may define pathways to disease progression. Losses of chromosomes 11p, 13q, 1p, and 10q were all associated with reduced overall survival in the Mayo Clinic cohort. Conclusions Patients with hemizygous loss of CDKN2A/B, loss of PTEN, increased number of copy number alterations, specific chromosomal arm losses or rs55705857 germline risk allele have reduced overall survival.

Abstract Background Glioblastoma IDH-wildtype (GBM) may be diagnosed histologically (hGBM) or based on molecular features when microvascular proliferation and histologic necrosis are absent (mGBM). Clinical and radiographic distinction between hGBM and mGBM remains unclear. Methods 101 hGBM cases were matched to 109 mGBM cases by age, sex, year of diagnosis, and TERT promoter mutation (TERTp) status. Clinical outcome, radiographic, and molecular features were compared across mGBM and hGBM. Results Median time from first MRI to diagnosis was longer for mGBM versus hGBM (34 vs 7 days, p < .001). mGBM more often presented with seizures (51% vs 26%, p < .001), gliomatosis pattern (12% vs 2%, p = .005), basal ganglia involvement (17% vs 0%, p < .001). Contrast enhancement appeared on MRI for 98% (96/98) of hGBM and 50% (54/107) of mGBM (p < .001). mGBM with non-enhancing MRI were rarely multifocal (2% vs 30%, p < .001). mGBM harbored fewer total copy number alterations (median 20 vs 16.5, p = .029), less often had EGFRamp (34% vs 48%, p = .051), isolated chromosome 10 loss (84% vs 96%, p = .013), and CDKN2A/B homozygous deletion (56% vs 77%, p = .005). Median overall survival was longer for mGBM (2.16 vs 1.44 years, p < .001), and for mGBM without MRI contrast enhancement versus hGBM with enhancement (3.11 vs 1.62 years, adjusted p = .003). mGBM without +7/−10 or without CDKN2A/B homozygous deletion had longer survival and time-to-progression versus hGBM with these alterations. Conclusion mGBM with MRI enhancement may represent undersampled or early/evolving hGBM. mGBM that are non-enhancing, present with seizures, prolonged time to diagnosis, absent CDKN2A/B alteration or +7/−10 may represent other IDH-wildtype entities.

Abstract Glioblastoma (GBM) diagnosis integrates morphology and tumor molecular alterations. Patients with classic morphology are designated as histologic GBM (hGBM). Patients without classic morphology harboring TERT promoter mutation (TERTp), or epidermal growth factor receptor amplification (EGFRamp), or combined gain of chromosome 7 and loss of 10 (+7/-10) designate molecular GBM (mGBM). We compared radiographic features, genomic alterations, and clinical outcome between hGBM and mGBM. 109 mGBM Mayo Clinic cases diagnosed between 1993-2021 were matched to 101 cGBM by age, sex, year of diagnosis, and TERT promoter (TERTp) status. Clinical and radiographic data abstraction was blinded. Progression-free and overall survival were compared across groups in the subset of patients who received standard of care. Contrast enhancement was present on index MRI (MRI at diagnosis) for 98% (96/98) hGBM and 50% (54/107) mGBM (p<0.0001) patients. mGBM tumors harbored fewer total copy number alterations (median of 16.5 vs 20, p=0.029) and were less likely to have CDKN2A/B homozygous deletion (56% vs 77%, p=0.005). Among the 87 mGBM and 82 hGBM receiving standard of care, median overall survival was longer for mGBM versus hGBM (2.24 vs 1.62 years, p=0.016). mGBM patients with +7/-10 (HR=2.60, p=0.035) or CDKN2A/B (HR=3.61, p=0.002) had poorer overall survival compared to mGBM patients without the corresponding alteration. On index MRI mGBM patients without contrast enhancement had better overall survival compared to hGBM (HR=0.50; p=0.003) whereas there was no survival difference between mGBM with enhancement and hGBM. mGBM with enhancement on index MRI likely represent undersampled, or early / evolving hGBM. mGBM patients with non-enhancing MRI, younger than 60, with long symptom profile including seizures, and some combination of non-anaplastic morphology, TERTp only, no homozygous deletion of CDKN2A/B, and low copy number complexity may represent other IDHwt glioma entities. Caution is advised in counseling, management, discussing prognosis, and inclusion in clinical trials.

Abstract The 2021 WHO classification redefined glioblastoma IDH-wildtype (GBM), integrating histopathological and tumor molecular features. Diffuse astrocytic glioma lacking histologic features of necrosis and microvascular proliferation are classified as (molecular) GBM if the tumor is IDH- and H3-wildtype and has one or more additional tumor molecular alterations: TERT promoter mutation, EGFR amplification, or +7/-10. Due to the absence of histopathological features of classic GBM and presentation without enhancement on MRI in approximately 50% of cases, molecular GBM frequently resemble low-grade IDH-mutant gliomas. We hypothesized that MRI-based deep learning models could distinguish IDH-mutant glioma from molecular GBM. We retrospectively identified 97 individuals with molecular GBM, who were matched to 97 individuals with IDH-mutant glioma by age, sex, year of diagnosis, and enhancement on MRI. Of the 97 IDH-mutant gliomas, 51% were 1p/19q codeleted, 42% grade 2, 47% grade 3, and 10% grade 4. Preoperative postcontrast T1-weighted (T1C) and T2-weighted (T2) MRI sequences were used to train a 3D-DenseNet121 model. The model was validated on an independent prospective set of 150 IDH-mutant gliomas and 71 molecular GBM. The MRI-based deep learning model achieved an area under the receiver operator curve (AUC) of 0.75 (95% CI: 0.68-0.82) in the prospective validation set. Sex-stratified AUCs were 0.72 (95% CI: 0.59-0.85) for females and 0.76 (95% CI: 0.68-0.85) for males. Age stratified AUCs were 0.71 (95% CI: 0.60-0.83) for patients under 55 years of age and 0.64 (95% CI: 0.50-0.79) for patients older than 55. These results suggest that discriminative MRI features of IDH-mutant glioma and molecular GBM exist and can be identified from deep learning models. However, radiographic ambiguity exists and underscores the need for further optimization of the deep learning architecture or by integrating additional non-invasive modalities. For example, future work entails integrating germline variants into the model, including rs55705857, which is a causal variant for IDH-mutant glioma. With further validation and integration of multi-modal data, a noninvasive tool may support more personalized decision-making.

Abstract IDH-wildtype diffuse astrocytic gliomas with lower-grade histological features alongside specific genomic alterations (e.g., TERT promoter mutation, EGFR amplification, gain Chr7/loss Chr10) are presently diagnosed as glioblastoma, IDH-wildtype (GBM) under WHO 2021 criteria. Previous analyses categorized these into “early/evolving” or “surgically undersampled” GBMs. However, it remains unclear whether these tumors share identical clinical and prognostic features with histologic GBM. Data from IDH-wt molecular GBMs across six institutions were aggregated. Astrocytic IDH-wt tumors of any grade that lacked necrosis or microvascular proliferation on initial histopathology yet exhibited molecular alterations consistent with WHO 2021 GBM diagnosis, were included. Recursive partitioning analysis (RPA) was used to categorize patients into risk groups based on overall survival (OS). Among the 264 patients included, the median age was 60 years (IQR: 51-67), with 162 (61%) being male. Additionally, 210 (80%) were diagnosed since 2016, and 216 (82%) received chemoradiation plus adjuvant temozolomide (GBM-SOC). The median follow-up period was 3.9 years (95% CI: 3.4, 4.6). Identified univariate risk factors for worse survival included older age, biopsy-only status, EGFR amplification, absence of initial adjuvant treatment, low post-operative KPS, and tumor histologic grade of 3. RPA divided patients into four distinct survival groups (p<0.0001). The “best” risk group comprised lower grade (<=2) tumors (n=58, median OS (mOS): 40 months (95% CI: 33, NA)). The “second best” group included Grade 3 patients receiving GBM-SOC without steroids during initial RT (n=139, mOS: 25 months (95% CI: 22, 28)). The “second worst” group encompassed Grade 3 patients who were on steroids during initial RT while receiving GBM-SOC (n=38, mOS: 15 months (95% CI: 13, 20)). The “worst” group contained Grade 3 tumors that had not undergone SOC (n=29, mOS: 9 months (95% CI: 4, 26)). These data show that histologic features and treatment are strong indicators of patient outcomes.