Investigating intratumour heterogeneity analysis methods and their application in GBM

Glioblastoma (GBM) is an incurable cancer with a median survival of 15 months. Despite debulking surgery, cancer cells are inevitably left behind in the surrounding brain, with a minority able to resist subsequent chemoradiotherapy and eventually form a recurrent tumour. This resistance is likely influenced by the cells’ genotypes, which show high variability (intratumour heterogeneity), as a result of tumour evolution. Characterising changes in the genetic architecture of tumours through therapy, may allow us to understand the effect that different mutations and pathways have on cell survival, and potentially identify novel targets for counteracting resistance in GBM. Such analyses involve detection of mutations from bulk tumour samples, and then delineating them into individual genetically distinct ‘subclones’, through subclonal deconvolution. This is a complex process, with no reliable guidelines for the best pipelines to use. I therefore developed methods to allow simulation and in silico sequencing of genomes from realistically complex, artificial tumour samples, so that I could benchmark such pipelines. This revealed that no tested pipelines, using single bulk samples, showed a high level of accuracy, though mutation calling with Mutect2 and FACETS, followed by subclonal deconvolution with Ccube, showed the best results. I then used alternative approaches with the largest longitudinal GBM dataset investigated to date. I found that evidence of strong subclonal selection is absent in many samples, and not associated with therapy. Nonetheless, this does not negate the possibility of smaller, or less frequent, pockets of altered fitness. Using pathway analysis combined with variants that are informative of tumour progression, I identified processes that may confer increased resistance, or sensitisation to therapy, and which warrant further investigation. Lastly, I apply subclonal deconvolution to investigate mouse-specific evolution in GBM patient-derived orthotopic xenografts and found no clear evidence to suggest these models are unsuitable for investigations relevant to humans

IDHwt glioblastomas can be stratified by their transcriptional response to standard treatment, with implications for targeted therapy

Background: Glioblastoma (GBM) brain tumors lacking IDH1 mutations (IDHwt) have the worst prognosis of all brain neoplasms. Patients receive surgery and chemoradiotherapy but tumors almost always fatally recur.Results: Using RNA sequencing data from 107 pairs of pre- and post-standard treatment locally recurrent IDHwt GBM tumors, we identify two responder subtypes based on longitudinal changes in gene expression. In two thirds of patients, a specific subset of genes is upregulated from primary to recurrence (Up responders), and in one third, the same genes are downregulated (Down responders), specifically in neoplastic cells. Characterization of the responder subtypes indicates subtype-specific adaptive treatment resistance mechanisms that are associated with distinct changes in the tumor microenvironment. In Up responders, recurrent tumors are enriched in quiescent proneural GBM stem cells and differentiated neoplastic cells, with increased interaction with the surrounding normal brain and neurotransmitter signaling, whereas Down responders commonly undergo mesenchymal transition. ChIP-sequencing data from longitudinal GBM tumors suggests that the observed transcriptional reprogramming could be driven by Polycomb-based chromatin remodeling rather than DNA methylation.Conclusions: We show that the responder subtype is cancer-cell intrinsic, recapitulated in in vitro GBM cell models, and influenced by the presence of the tumor microenvironment. Stratifying GBM tumors by responder subtype may lead to more effective treatment.</p

Primary and recurrent glioma patient-derived orthotopic xenografts (PDOX) represent relevant patient avatars for precision medicine

Patient-derived cancer models are essential tools for studying tumor biology and preclinical interventions. Here, we show that glioma patient-derived orthotopic xenografts (PDOXs) enable long-term propagation of patient tumors and represent clinically relevant patient avatars. We created a large collection of PDOXs from primary and recurrent gliomas with and without mutations in IDH1, which retained histopathological, genetic, epigenetic and transcriptomic features of patient tumors with no mouse-specific clonal evolution. Longitudinal PDOX models recapitulate the limited genetic evolution of gliomas observed in patient tumors following treatment. PDOX-derived standardized tumor organoid cultures enabled assessment of drug responses, which were validated in mice. PDOXs showed clinically relevant responses to Temozolomide and to targeted treatments such as EGFR and CDK4/6 inhibitors in (epi)genetically defined groups, according to MGMT promoter and EGFR/CDK status respectively. Dianhydrogalactitol, a bifunctional alkylating agent, showed promising potential against glioblastoma. Our study underlines the clinical relevance of glioma PDOX models for translational research and personalized treatment studies

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology

Longitudinal molecular trajectories of diffuse glioma in adults.

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

Glioma progression is shaped by genetic evolution and microenvironment interactions

© 2022 Elsevier Inc.The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and 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 were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic 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 myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.N