Molecular characterization of gliomas has uncovered genomic signatures with significant impact on tumor diagnosis and prognostication. CDKN2A is a tumor suppressor gene involved in cell cycle control. Homozygous deletion of the CDKN2A/B locus has been implicated in both gliomagenesis and tumor progression through dysregulated cell proliferation. In histologically lower grade gliomas, CDKN2A homozygous deletion is associated with more aggressive clinical course and is a molecular marker of grade 4 status in the 2021 WHO diagnostic system. Despite its prognostic utility, molecular analysis for CDKN2A deletion remains time consuming, expensive, and is not widely available. This study assessed whether semi-quantitative immunohistochemistry for expression of p16, the protein product of CDKN2A, can serve as a sensitive and a specific marker for CDKN2A homozygous deletion in gliomas. P16 expression was quantified by immunohistochemistry in 100 gliomas, representing both IDH-wildtype and IDH-mutant tumors of all grades, using two independent pathologists’ scores and QuPath digital pathology analysis. Molecular CDKN2A status was determined using next-generation DNA sequencing, with homozygous CDKN2A deletion detected in 48% of the tumor cohort. Classifying CDKN2A status based on p16 tumor cell expression (0–100%) demonstrated robust performance over a wide range of thresholds, with receiver operating characteristic curve area of 0.993 and 0.997 (blinded and unblinded pathologist p16 scores, respectively) and 0.969 (QuPath p16 score). Importantly, in tumors with pathologist-scored p16 equal to or less than 5%, the specificity for predicting CDKN2A homozygous deletion was 100%; and in tumors with p16 greater than 20%, specificity for excluding CDKN2A homozygous deletion was also 100%. Conversely, tumors with p16 scores of 6–20% represented gray zone with imperfect correlation to CDKN2A status. The findings indicate that p16 immunohistochemistry is a reliable surrogate marker of CDKN2A homozygous deletion in gliomas, with recommended p16 cutoff scores of ≤ 5% for confirming and > 20% for excluding biallelic CDKN2A loss.

Background Mutations in mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2) are associated with microsatellite instability and a hypermutator phenotype in numerous systemic cancers, and germline MMR mutations have been implicated in multi-organ tumor syndromes. In gliomas, MMR mutations can function as an adaptive response to alkylating chemotherapy, although there are well documented cases of germline and sporadic mutations, with detrimental effects on patient survival. Methods The clinical, pathologic, and molecular features of 18 IDH-mutant astrocytomas and 20 IDH-wildtype glioblastomas with MMR mutations in the primary tumor were analyzed in comparison to 361 IDH-mutant and 906 IDH-wildtype tumors without MMR mutations. In addition, 12 IDH-mutant astrocytomas and 18 IDH-wildtype glioblastomas which developed MMR mutations between initial presentation and tumor recurrence were analyzed in comparison to 50 IDH-mutant and 104 IDH-wildtype cases that remained MMR-wildtype at recurrence. Results In both IDH-mutant astrocytoma and IDH-wildtype glioblastoma cohorts, the presence of MMR mutation in primary tumors was associated with significantly higher tumor mutation burden (TMB) (p<0.0001), however MMR mutations only resulted in worse overall survival in the IDH-mutant astrocytomas (p=0.0069). In addition, gain of MMR mutation between the primary and recurrent surgical specimen occurred more frequently with temozolomide therapy (p=0.0073), and resulted in a substantial increase in TMB (p<0.0001), higher grade (p=0.0119), and worse post-recurrence survival (p=0.0022) in the IDH-mutant astrocytoma cohort. Conclusions These results suggest that whether present initially or in response to therapy, MMR mutations significantly affect TMB but appear to only influence the clinical outcome in IDH-mutant astrocytoma subsets.

Background IDH mutations are thought to represent an early oncogenic event in glioma evolution, found with high penetrance across tumor cells, however in rare cases IDH mutation may exist only in a small subset of the total tumor cells (subclonal IDH-mutation). Methods We present two institutional cases with subclonal IDH1 R132H mutation. In addition, two large publicly-available cohorts of IDH-mutant astrocytomas were mined for cases harboring subclonal IDH mutations (defined as tumor cell fraction (TCF) with IDH mutation ≤0.67) and the clinical and molecular features of these subclonal cases were compared to clonal IDH-mutant astrocytomas. Results Immunohistochemistry (IHC) performed on two institutional WHO grade 4 IDH-mutant astrocytomas revealed only a minority of tumor cells in each case with IDH1 R132H mutant protein, and next-generation sequencing (NGS) revealed remarkably low IDH1 variant allele frequencies compared to other pathogenic mutations, including TP53 and/or ATRX. DNA methylation classified the first tumor as high-grade IDH-mutant astrocytoma with high confidence (0.98 score). In the publicly-available datasets, subclonal IDH mutation was present in 3.9% of IDH-mutant astrocytomas (18/466 tumors). Compared to clonal IDH-mutant astrocytomas (n=156), subclonal cases demonstrated worse overall survival in grades 3 (p=0.0106) and 4 (p=0.0184). Conclusions While rare, subclonal IDH1 mutations are present in a subset of IDH-mutant astrocytomas of all grades, which may lead to a mismatch between IHC results and genetic/epigenetic classification. These findings suggest a possible prognostic role of IDH mutation subclonality, and highlight the potential clinical utility of quantitative IDH1 mutation evaluation by IHC and NGS.

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