Hypermutated Gliomas Research Articles

Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis

Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient's primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term "genome shredding." Importantly, in the patient's recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. "Cancer shredding" leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.

Pediatric Hemispheric Glioma

About one-third of pediatric low-grade glioma and a half of pediatric high-grade glioma occur in the cerebrum. Pediatric hemispheric glioma may harbor diagnostic and therapeutically targetable genetic abnormalities, including BRAF V600E, H3.3 G34R/V, FGFR1 alternation, MYB or MYBL1 alternation, and NTRK/ALK/ROS1/MET fusion. In addition, the efficacy of molecular-targeted agents, such as BRAF inhibitors, MEK inhibitors, and NTRK inhibitors, against pediatric glioma with the relevant mutations has been demonstrated in several clinical trials. Furthermore, checkpoint inhibitors are considered a choice of treatment for hypermutated glioma, which is typically observed in patients with constitutional mismatch repair deficiency syndrome. Cancer gene panel testing, approved for insurance coverage in Japan in 2019, has been beneficial to pediatric cancer patients. However, to promote the clinical application of the recent molecular understanding of pediatric neuro-oncology, some issues have to be addressed. Herein, we review the genetic profiles of pediatric hemispheric glioma and introduce the current medical situation of precision medicine for pediatric patients with glioma in Japan.

Abstract 5705: Mechanisms and therapeutic implications of hypermutation in gliomas

Abstract High tumor mutational burden (hypermutation) is observed in some gliomas; however, its mechanisms of development and whether it predicts immunotherapy response are poorly understood. Here, we comprehensively analyze the molecular determinants of mutational burden and signatures in 10,294 gliomas including AACR Project GENIE and institutional datasets. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after temozolomide treatment. Experimentally, the mutational signature of post-treatment hypermutated gliomas was only recapitulated by temozolomide-induced damage in cells harboring MMR deficiency. MMR-deficient gliomas exhibited unique features including the lack of prominent T-cell infiltrates, extensive intratumoral heterogeneity, poor survival and low response rate to PD-1 blockade. Moreover, while microsatellite instability in MMR-deficient gliomas was not detected by bulk analyses, single-cell whole-genome sequencing of post-treatment hypermutated glioma cells demonstrated microsatellite mutations. This study shows that chemotherapy can drive acquisition of hypermutated populations without promoting response to PD-1 blockade and supports diagnostic use of mutational burden and signatures in cancer. Citation Format: Mehdi Touat, Yvonne Y. Li, Adam N. Boynton, Liam F. Spurr, Bryan Iorgulescu, Craig L. Bohrson, Isidro Cortes-Ciriano, Jack E. Geduldig, Kristine Pelton, Mary J. Lim-Fat, Sangita Pal, Shakti H. Ramkissoon, Frank Dubois, Charlotte Bellamy, Naomi Currimjee, Kenin Qian, Seth Malinowski, Aniket Shetty, Kin-Hoe Chow, Maïté Verreault, Erell Guillerm, Samy Ammari, Frédéric Beuvon, Karima Mokhtari, Agusti Alentorn, Caroline Dehais, Caroline Houillier, Florence Laigle-Donadey, Dimitri Psimaras, Alexandre Carpentier, Philippe Cornu, Laurent Capelle, Bertrand Mathon, Jill S. Barnholtz-Sloan, Arnab Chakravarti, Wenya L. Bi, Garrett M. Frampton, Marc Sanson, Brian M. Alexander, Andrew Cherniack, Patrick Y. Wen, David A. Reardon, Aurelien Marabelle, Peter J. Park, Ahmed Idbaih, Rameen Beroukhim, Pratiti Bandopadhayay, Franck Bielle, Keith L. Ligon. Mechanisms and therapeutic implications of hypermutation in gliomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5705.

Hypermutated Gliomas Respond Poorly to Immunotherapy.

Unlike melanomas and colorectal cancers with a high tumor mutation burden, hypermutated gliomas generally do not respond to checkpoint blockade, a study concludes. Hypermutation shortens patient survival, and the results suggest that it occurs because of selective pressure from the chemotherapeutic temozolomide.

Mechanisms and therapeutic implications of hypermutation in gliomas.

A high tumour mutational burden (hypermutation) is observed in some gliomas 1–5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.

The efficacy of DNA mismatch repair enzyme immunohistochemistry as a screening test for hypermutated gliomas

A subset of gliomas has DNA repair defects that lead to hypermutated genomes. While such tumors are resistant to alkylating chemotherapies, they may also express more mutant neoantigens on their cell surfaces, and thus be more responsive to immunotherapies. A fast, inexpensive method of screening for hypermutated gliomas would therefore be of great clinical value. Since immunohistochemistry (IHC) for the DNA mismatch repair (MMR) proteins Msh2, Msh6, Mlh1, and Pms2 is already used to screen for hypermutated colorectal cancers, we sought to determine whether that panel might have similar utility in gliomas. MMR IHC was scored in 100 WHO grade I-IV gliomas (from 96 patients) with known tumor mutation burden (TMB), while blinded to TMB data. Cases included 70 grade IV GBMs, 13 grade III astrocytomas, 4 grade II astrocytomas (3 diffuse astrocytomas and 1 pleomorphic xanthoastrocytoma), 1 grade I pilocytic astrocytoma, 2 grade III oligodendrogliomas, 7 grade II oligodendrogliomas, and 3 grade I glioneuronal tumors. Eight of 100 tumors showed loss of one or more MMR proteins by IHC, and all 8 were hypermutated. Among the remaining 92 gliomas with intact MMR IHC, only one was hypermutated; that tumor had an inactivating mutation in another DNA repair gene, ATM. Overall accuracy, sensitivity, and specificity for DNA MMR IHC compared to the gold standard of TMB were 99, 89, and 100%, respectively. The strongest correlates with hypermutation were prior TMZ treatment, MGMT promoter methylation, and IDH1 mutation. Among the 8 MMR-deficient hypermutated gliomas, 4 (50%) contained both MMR-lost and MMR-retained tumor cells. Together, these data suggest that MMR IHC could be a viable front-line screening test for gliomas in which immunotherapy is being considered. They also suggest that not all cells in a hypermutated glioma may actually be MMR-deficient, a finding that might need to be considered when treating such tumors with immunotherapies.

PATH-39. DNA MISMATCH REPAIR ENZYME IMMUNOHISTOCHEMISTRY IS A RAPID, EFFECTIVE SCREENING TEST FOR HYPERMUTATED GLIOMAS

Abstract BACKGROUND While gliomas with hypermutated DNA are resistant to alkylating agents like temozolomide, they may be especially responsive to immunotherapy, since they express abundant neoantigens on their cell surfaces. Screening for hypermutated gliomas is currently being done through next-generation sequencing (NGS) panels that cover large portions of tumor DNA, although this is costly and access to such testing is not universal. Since hypermutated gliomas typically contain inactivating mutations in one of the main DNA mismatch repair (MMR) proteins, and cancers with an MMR mutation usually show loss of normal MMR protein, we sought to determine the feasibility of rapidly screening for hypermutated gliomas with an MMR immunohistochemistry (IHC) panel that is already in widespread use for colorectal adenocarcinomas. METHODS Tumor mutation burden (TMB) was determined via NGS for 101 gliomas, including 64 GBMs, 24 grade II-III astrocytomas, 9 grade II-III oligodendrogliomas, and 4 grade I gliomas. IHC for MSH2, MSH6, MLH1, and PMS2 was performed and analyzed on all gliomas while blinded to mutation profile and TMB. RESULTS Seven of 101 gliomas (7%) showed loss of an MMR protein by IHC. All 7 had matching MMR gene mutations and were hypermutated (100%), defined as TMB >20 per megabase of DNA. Of the remaining 94 with intact MMR IHC, only one was hypermutated. That case had an inactivating splice region mutation in another gene involved in DNA repair, ATM, although ATM is not part of the IHC panel originally developed for colorectal cancer. Overall sensitivity and specificity of the current MMR IHC panel for hypermutated gliomas was therefore 88% and 100%, respectively. CONCLUSION The colorectal MMR IHC panel, available in virtually all clinical IHC labs, is also a good screening test for hypermutated gliomas. Expansion of the panel to include even more DNA repair proteins, like ATM, would enhance its utility.

DRES-08. CLINICAL SIGNIFICANCE OF HYPERMUTATION IN GLIOMAS

Abstract BACKGROUND Hypermutation is an emerging biomarker for predicting response to immunotherapy in cancer patients, however its clinical value in gliomas is not established. We sought to assess the determinants of hypermutation in gliomas, and its value for predicting response to standard of care and immune checkpoint blockade (ICB). METHODS We performed comprehensive genomic characterization of 2,420 clinically annotated gliomas. We assessed the clinical and molecular characteristics associated with hypermutation and relationships between hypermutation and response to cancer treatments. RESULTS Hypermutation occurred predominantly as an adaptive resistance mechanism to temozolomide in gliomas and was most prevalent in recurrent gliomas with MGMTpromoter methylation (33.8%), IDH1/2mutation (41.0%) or 1p/19q co-deletion (59.5%). Hypermutation was almost always associated with molecular defects in DNA mismatch repair (MMR), and was associated with shorter survival after its appearance based on multivariate analysis (hazard ratio 1.91; 95% CI 1.24–2.94; P=0.004). The molecular mechanisms whereby gliomas undergo hypermutation during therapy with alkylating agents were dissected using patient-derived glioma models in vitro and in vivo. Outcomes of hypermutated gliomas treated with immune checkpoint blockade or with standard of care agents will be presented at the conference. CONCLUSIONS Using the largest set of hypermutated gliomas described to date, this study establishes that mutational burden and mutation signatures are clinically and biologically significant biomarkers that can be used to predict therapy response and guide treatment decisions in gliomas

OS9.1 Clinical significance of hypermutation in gliomas

Abstract BACKGROUND Among patients with glioma, little is known about the clinical significance of hypermutation. We sought to define the determinants of hypermutation in gliomas, and to assess the value of this biomarker for predicting response to standard of care and immune checkpoint blockade. MATERIAL AND METHODS We performed comprehensive molecular characterization of 2420 pediatric and adult gliomas with clinical annotation. We determined the clinical and molecular characteristics associated with hypermutation, and assessed the relationship between hypermutation and clinical response to cancer therapies. RESULTS Overall, 114 hypermutated gliomas were identified. Hypermutation occurred predominantly in therapy-responsive subtypes characterized by methylated MGMT promoter, IDH1/2mutation or 1p/19q co-deletion. Hypermutation was almost always associated with prior treatment with temozolomide and molecular defects in DNA mismatch repair (MMR), which were identified in one-third of IDH1/2-mutated recurrent gliomas and was associated with shorter survival in multivariate analyses (hazard ratio 2.11 [95% CI 1.24–3.6], P=0.006). The molecular mechanisms whereby gliomas undergo hypermutation during therapy with alkylating agents were dissected using patient-derived glioma models in vitro and in vivo. Outcomes of hypermutated gliomas treated with immune checkpoint blockade or with standard of care agents will be presented at the conference. CONCLUSION This study establishes that mutation burden and mutation signatures are clinically and biologically significant biomarkers that can be used to predict therapy response and guide treatment decisions in gliomas.

Hypermutagenesis in untreated adult gliomas due to inherited mismatch mutations.

Hypermutagenesis refers to marked increase in the number of mutations due to continuous mutagenic process. Hypermutated tumors, have being found in several tumor types, are associated with inherited or acquired alterations in the DNA repair pathways. Hypermutation has been observed in a subset of adult glioma patients as a direct result of temozolomide(TMZ)‐induced mutagenesis. In our study, we have identified a rare subset of treatment‐naïve adult gliomas with de novo hypermutator phenotype and explored the evolution of spontaneous and treatment‐induced hypermutagenesis. We conducted Whole‐Exome Sequencing (WES), Whole‐Transcriptome Sequencing (WTS), and Single‐Cell Sequencing (SCS) of TMZ‐naïve and post‐TMZ‐treated hypermutated tumors to identify distinct clinical or genomic manifestations that contribute to the development of hypermutation in untreated adult gliomas. TMZ‐naïve hypermutated tumors were marked by absence of IDH1 somatic mutation and MGMT promoter (pMGMT) methylation, two genomic traits that were significantly associated with the TMZ‐induced hypermutagenic event in glioblastoma, and harbored inherited alterations in the mismatch repair (MMR) machinery. The immediate family members of the TMZ‐naive hypermutated glioma patients were also previous diagnosed with cancer development history, suggesting that germline dysfunction of the MMR pathway could potentially pose hereditary risk to genetic predisposition of carcinogenesis in gliomas. Lastly, both TMZ‐naïve and post‐TMZ‐treated hypermutated tumors exhibited a significant accumulation of neoantigen loads, suggesting immunotherapeutic alternatives. Our results present new and unique understanding of hypermutagenic process in adult gliomas and an important step towards clinical implication of immunotherapy in glioma treatment.

Abstract 2955: A radiomic-based MRI phenotype is uniquely associated with hypermutated genotype in gliomas

Abstract INTRODUCTION Hypermutation is the excessive accumulation of DNA mutation in cancer cells. This specific hypermutated genotype has been reported in low and high grade gliomas, specifically post-temozolomide treatment and is associated with treatment-resistance. Herein, we sought to identify an imaging-based signature for hypermutated gliomas using a radiomics-based approach. MATERIALS AND METHODS In this IRB-approved retrospective study, we analyzed a total of 101 patients with primary gliomas from the University of Texas MD Anderson Cancer Center. Next generation sequencing (NGS) platforms (T200 and Foundation 1) were used to determine the Mutation burden status in post-biopsy (stereotactic/excisional). Patients were dichotomized based on their mutation burden; 77 hypomutated (<30 mutations) and 24 hypermutated (>=30 mutations or <30 with MMR gene or POLE/POLD gene mutations). Radiomic analysis was performed on the conventional MR images (FLAIR and T1 post-contrast) obtained prior to tumor tissue surgical sampling; and a total of 2480 rotation-invariant radiomic features were extracted using: (i) the first-order histogram and (ii) grey level co-occurrence matrix. The Maximum Relevance Minimum Redundancy technique was used to select the most relevant radiomic features. ROC analysis and leave-one-out cross-validation (LOOCV) were used to assess the performance of the Support Vector Machine (SVM) classifier as and AUC, Sensitivity, Specificity, and p-value were obtained. RESULTS We found 100 radiomic features that can discriminate between hypermutated versus hypomutated gliomas, AUC 96.3% (CI: 90.2%-98.9%), Sensitivity 100%, Specificity 95%, p-value=3.769e-6. CONCLUSION Hypermutated gliomas has a unique radiomic quantitative signature that can be used to predict mutation burden regardless of tumor grade or histopathology. Citation Format: Islam Hassan, Aikaterini Kotrotsou, Carlos Kamiya Matsuoka, Kristin D. Alfaro-Munoz, Nabil Elshafeey, Nancy El Shafei, Pascal O. Zinn, John F. de Groot, Rivka R. Colen. A radiomic-based MRI phenotype is uniquely associated with hypermutated genotype in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2955.

A unique MRI-based radiomic signature predicts hypermutated glioma genotype.

2022Background: Hypermutation is defined as the excessive accumulation of DNA mutation in cancer cells and is reported in several forms of cancer including low and high grade gliomas. Incidence of ...

Temozolomide-associated hypermutation in gliomas.

Low-grade gliomas cause considerable morbidity and most will recur after initial therapy. At recurrence, low-grade gliomas can undergo transformation to high-grade gliomas (grade III or grade IV), which are associated with worse prognosis. Temozolomide (TMZ) provides survival benefit in patients with glioblastomas, but its value in patients with low-grade gliomas is less clear. A subset of TMZ-treated, isocitrate dehydrogenase‒mutant, low-grade astrocytomas recur as more malignant tumors with thousands of de novo, coding mutations bearing a signature of TMZ-induced hypermutation. Preliminary studies raise the hypothesis that TMZ-induced hypermutation may contribute to malignant transformation, although with highly variable latency. On the other hand, hypermutated gliomas have radically altered genomes that present new opportunities for therapeutic intervention. In light of these findings and the immunotherapy clinical trials they inspired, how do patients and providers approach the risks and benefits of TMZ therapy? This review discusses what is known about the mechanisms and consequences of TMZ-induced hypermutation and outstanding questions regarding its clinical significance.