Search

Diffuse glioma is characterized by a poor prognosis and a universal resistance to therapy, though the evolutionary processes behind this resistance remain unclear. The Glioma Longitudinal Analysis (GLASS) Consortium has previously demonstrated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single-cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the microenvironment and the administration of standard-of-care therapy. To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA- and/or DNA-sequencing data from temporally separated tumor pairs of over 300 adult patients with IDH-wild-type or IDH-mutant glioma. In a subset of these tumor pairs, we complemented these data with single-nucleus RNAseq and multiplex imaging mass cytometry at each time point. Recurrent tumors exhibited diverse changes that were attributable to changes in histological features, somatic alterations, and microenvironment interactions. IDH-wild-type tumors overall were more invasive at recurrence and exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. In contrast, recurrent IDH-mutant tumors exhibited a significant increase in proliferative expression programs that correlated with discrete genetic changes. Hypermutation and acquired CDKN2A homozygous deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor expansion. A transition to the mesenchymal phenotype was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells, providing opportunities to target this transition through therapy. Collectively, our results uncover recurrence-associated changes in genetics and the microenvironment that can be targeted to shape disease progression following initial diagnosis.

SummaryTumor adaptation or selection is thought to underlie therapy resistance of gliomas. To investigate the longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 143 matched initial and recurrent patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) gliomas. IDHwt gliomas showed a longitudinally stable epigenome with relatively low levels of global methylation, whereas the epigenome of IDHmut gliomas showed initial high levels genome-wide of DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. By integrating DNA methylation and gene expression data, adaptive changes of putative master regulators of the cell cycle and of differentiation were seen in IDHmut recurrent tumors. Furthermore, relapses of IDHmut tumors were accompanied by histological progression which in turn influenced survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment differed between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment for IDHmut gliomas. Our study provides one of the largest cohorts of paired glioma samples profiled with epigenomics, transcriptomics and genomics; and our results demonstrate that the treatment of IDHmut gliomas reshapes the epigenome towards an IDHwt-like phenotype. Accordingly, the prevalent practice of early genotoxic treatment in this patient population may need to be revisited.

Ionizing radiation causes DNA damage and is a mainstay for cancer treatment, but understanding of its genomic impact is limited. We analyzed mutational spectra following radiotherapy in 190 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium and 3,693 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified radiotherapy-associated significant increases in the burden of small deletions (5–15 bp) and large deletions (20+ bp to chromosome-arm length). Small deletions were characterized by a larger span size, lacking breakpoint microhomology and were genomically more dispersed when compared to pre-existing deletions and deletions in non-irradiated tumors. Mutational signature analysis implicated classical non-homologous end-joining-mediated DNA damage repair and APOBEC mutagenesis following radiotherapy. A high radiation-associated deletion burden was associated with worse clinical outcomes, suggesting that effective repair of radiation-induced DNA damage is detrimental to patient survival. These results may be leveraged to predict sensitivity to radiation therapy in recurrent cancer. Radiotherapy induces small and large deletions as well as inversions across the genome in multiple cancer types. The genomic changes associated with radiotherapy correlate with poorer clinical outcomes.

SummaryTo interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA and/or DNA sequencing data from temporally separated tumor pairs of 292 adult patients with IDH-wild-type or IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their malignant cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells. Collectively, our results uncover recurrence-associated changes that could be targetable to shape disease progression following initial diagnosis.

Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain unknown. We analyzed the mutational spectra following treatment with ionizing radiation in sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 2249 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified a significant increase in the frequency of small deletions following radiation therapy that was independent of other factors. These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and metastatic cohort, P = 1.2e-02 and P = 8e-11, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P < 2.2e-16, Kolmogorov-Smirnov test) and a reduction in microhomology at breakpoints (P = 3.2e-02, paired Wilcoxon signed-rank test). These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, radiotherapy resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions. Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and metastatic cohort, P = 4.7e-02, HR = 2.59 [95% CI: 1.01, 6.60] and P = 2.5e-02, HR = 1.43 [95% CI: 1.05, 1.94], respectively; multivariable Cox regression), suggesting that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Taken together, the identified genomic scars as a result of radiation therapy reflect a more aggressive tumor with increased levels of resistance to follow up treatments.

The evolutionary processes that drive universal therapeutic resistance in adult patients with diffuse glioma remain unclear1,2. Here we analysed temporally separated DNA-sequencing data and matched clinical annotation from 222 adult patients with glioma. By analysing mutations and copy numbers across the three major subtypes of diffuse glioma, we found that driver genes detected at the initial stage of disease were retained at recurrence, whereas there was little evidence of recurrence-specific gene alterations. Treatment with alkylating agents resulted in a hypermutator phenotype at different rates across the glioma subtypes, and hypermutation was not associated with differences in overall survival. Acquired aneuploidy was frequently detected in recurrent gliomas and was characterized by IDH mutation but without co-deletion of chromosome arms 1p/19q, and further converged with acquired alterations in the cell cycle and poor outcomes. The clonal architecture of each tumour remained similar over time, but the presence of subclonal selection was associated with decreased survival. Finally, there were no differences in the levels of immunoediting between initial and recurrent gliomas. Collectively, our results suggest that the strongest selective pressures occur during early glioma development and that current therapies shape this evolution in a largely stochastic manner.

BACKGROUND Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain largely unknown. MATERIAL AND METHODS We analyzed the mutational spectra following treatment with RT in whole genome or exome sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 3693 post-treatment metastatic tumors from the Hartwig Medical Foundation (HMF). RESULTS We identified a significant increase in the burden of small deletions following radiation therapy that was independent of other factors (P = 3e-03, multivariable log-linear regression). These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and HMF, P = 1.5e-04 and P = 6e-16, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P < 2.2e-16, Kolmogorov-Smirnov test) and a lack of microhomology at breakpoints (P = 6.6e-05, paired Wilcoxon signed-rank test). Furthermore, mutational signature analysis confirmed the distinct genomic characteristics of RT-associated deletions when compared to deletions arising via homologous recombination deficiency or microsatellite instability. These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, RT resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions in IDHmut glioma (P= 1.9e-05, Fisher’s exact test). Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and HMF, P = 3.4e-02 and P < 1e-04, respectively; log-rank test). CONCLUSION Our results collectively suggest that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Furthermore, CDKN2A homozygous deletion at recurrence may be leveraged as a promising clinical biomarker of RT-resistance in IDHmut glioma. Taken together, the identified genomic scars as a result of RT reflect a more aggressive tumor with increased levels of resistance to follow up treatments.

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.

Treatment options for adult patients with glioma has remained largely unchanged over the past three decades. Targeted inhibitors and immunotherapies have improved outcomes for many cancer types but their relevance in glioma is unclear. The inevitability of glioma disease recurrence demands an understanding of mechanisms driving therapy resistance. The Glioma Longitudinal Analysis (GLASS) Consortium was initiated to establish a definitive portrait of the recurrence process and to discover vulnerabilities that render the tumor sensitive to therapeutic intervention. GLASS is a community-driven effort that seeks to overcome the logistical challenges in constructing adequately powered longitudinal genomic glioma datasets by pooling data from patients treated at institutions worldwide. Currently, the GLASS Data Resource comprises DNA sequencing data (exome and/or whole-genome) from 288 patients of whom high-quality data in at least two time points are present from 222 patients (n = 134 IDHwt, n = 63 IDHmutant-noncodel, n = 25 IDHmutant-codel). We inferred longitudinal mutation, copy number, clonal frequency, and neoantigen profiles and demonstrated that driver genes found at initial disease persisted into recurrence. Treatment with alkylating-agents resulted in a hypermutator phenotype at different rates across glioma subtypes, most frequently among IDHmutant-noncodels, and hypermutation was not associated with differences in overall survival. Acquired aneuploidy was frequently detected in recurrent IDHmutant-noncodel gliomas and further converged with acquired cell cycle pathway alterations and poor outcomes. We showed that the clonal architecture of each tumor remains largely intact over time and that genetic drift was associated with increased survival. Finally, we found that neoantigens were exposed to stable selective pressures throughout a tumor’s progression. Our results collectively suggest that the strongest selective pressures occur early during glioma development and that current therapies shape this evolution in a largely stochastic manner. The GLASS Data Resource provides a genomic reference to study the patterns of glioma evolution.

SummarySporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole genome-, exome-, transcriptome-and methylation-sequencing of 81 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, p53 and cell cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.SignificanceDiffuse gliomas are the most common malignant brain tumors, with high-grade tumors carrying a dismal prognosis. Preclinical models have proven themselves as poor predictors of clinical efficacy. Spontaneous glioma in dogs provides an attractive alternative model, because of their comparable tumor microenvironment and tumor life history. We determined the similarities and differences between human and canine gliomas through genomic profiling, and leveraged our datasets to identify conserved somatic drivers, mutational processes and temporal ordering of somatic glioma events across species. We show that canine gliomas resemble human gliomas at (epi-)genetic levels and are more reminiscent of pediatric than adult disease, thus rationalizing sporadic canine glioma as a preclinical model tailored to measuring treatment efficacies in patients with canine or human glioma.

Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain unknown. We analyzed the mutational spectra following treatment with ionizing radiation in sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 2116 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified a significant increase in the burden of small deletions following radiation therapy that was independent of other factors and was significantly associated with the clinically applied RT-dosage in Gy (P = 1e-02, multivariable log-linear regression). These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and metastatic cohort, P = 1.2e-02 and P = 8e-11, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P < 2.2e-16, Kolmogorov-Smirnov test) and a reduction in microhomology at breakpoints (P = 3.2e-02, paired Wilcoxon signed-rank test). These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, radiotherapy resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions. Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and metastatic cohort, P < 1e-04 and P = 2.6e-02, respectively; Wald test). Our results collectively suggest that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Taken together, the identified genomic scars as a result of radiation therapy reflect a more aggressive tumor with increased levels of resistance to follow up treatments. Citation Format: Emre Kocakavuk, Kevin J. Anderson, Kevin C. Johnson, Frederick S. Varn, Samirkumar B. Amin, Erik P. Sulman, Floris P. Barthel, Roel G. Verhaak. Radiotherapy is associated with a deletion signature that contributes to poor cancer patient outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2068.