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Diffuse glioma is an aggressive brain cancer that is characterized by a poor prognosis and a universal resistance to therapy. The evolutionary processes behind this resistance remain unclear. Previous studies by the Glioma Longitudinal Analysis (GLASS) Consortium have indicated 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. Interactions between these factors remain poorly understood, making it difficult to predict how a patient’s tumor will evolve from diagnosis to recurrence. 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 292 adult patients with IDH-wild-type or IDH-mutant glioma. Recurrent tumors exhibited diverse changes that were attributable to changes in anatomic composition, somatic alterations, and microenvironment interactions. 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. 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, 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.

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 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 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.

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Diffuse glioma is an aggressive brain cancer that is characterized by a poor prognosis and a universal resistance to therapy. The evolutionary processes behind this resistance remain unclear. Previous studies by the Glioma Longitudinal Analysis (GLASS) Consortium have indicated 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 capable of changing their cell state in response to diverse challenges in their microenvironment. The tumor immune response has been implicated as a major driver of these malignant cell state transitions, and is known to be affected by the administration of therapy, but the extent to which tumor genetics, therapy, and the different components of the immune response interact to influence a glioma’s evolutionary trajectory are poorly understood. To further investigate these factors, we collected DNA and RNA sequencing data on pre- and post-treatment tumor pairs from over 150 glioma patients that have received chemotherapy, radiotherapy, and/or immune checkpoint blockade agents. By integrating mutation, copy number, and in silico deconvolution analyses of bulk transcriptome data across the three molecular subtypes of diffuse glioma, we show that longitudinal increases in chromosomal instability and gene fusions associate with decreased immune infiltrate and altered cell states at recurrence. We additionally find that specific molecular alterations and malignant cell states associate with unique inflammatory and immunosuppressive programs in tumor-associated macrophages and microglia. Lastly, we show that the abundance of T cells in the tumor microenvironment does not associate with changes in neoantigen depletion and the acquisition of antigen presentation machinery defects, suggesting minimal immunoediting activity over time. Collectively, our results indicate that the administration of therapy can alter the dynamics of tumor-immune interactions in glioma, resulting in new steady-states at recurrence that can be subsequently targeted.

Diffuse glioma is an aggressive brain cancer that is characterized by a poor prognosis and a universal resistance to therapy. The evolutionary processes behind this resistance remain unclear. Previous studies by the Glioma Longitudinal Analysis (GLASS) Consortium have indicated 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 immune response and the administration of standard-of-care therapy. Interactions between these factors remain poorly understood, making it difficult to predict how a patient's tumor will evolve from diagnosis to recurrence. To investigate these factors, we collected RNAseq data from 151 pre- and post-treatment tumor pairs, 101 of which also had matched whole exome or whole genome sequencing. Together, this dataset represents the largest collection of longitudinal multiomic glioma data yet assembled. We integrated this dataset with representative glioma single cell RNAseq data to implement digital cytometry approaches that quantified the microenvironmental composition of each tumor and reconstructed their tumor and immune cell state-specific gene expression profiles. In both IDHwt and IDHmut glioma, the tumor microenvironment was dominated by myeloid cells. The myeloid compartment of IDHwt tumors more closely resembled blood-derived macrophages, while myeloid cells in IDHmut tumors were more similar to microglia. While therapy did not alter the balance between macrophages and microglia in IDHwt tumors, myeloid cells in IDHmut tumors that increased grade following therapy had fewer microglia characteristics. Myeloid cells in mesenchymal glioma exhibited a distinct gene expression signature compared to those in non-mesenchymal tumors, and this signature was upregulated in the myeloid cells of tumors that acquired this phenotype following therapy. Receptor-ligand analyses revealed that this mesenchymal phenotype was associated with interactions between differentiated-like tumor cells and myeloid cells, suggesting a potential mechanism of mesenchymal transformation. Radiation therapy did not alter the composition or transcriptional activity of the glioma microenvironment but did associate with increased synaptic plasticity in stem cell-like and differentiated-like tumor cells. Collectively, our results suggest that tumor-myeloid cell interactions in glioma are dynamic and capable of shaping tumor evolution following the administration of therapy. Clinically targeting these interactions may allow for better control of malignant cell state shifts over time, creating new opportunities for disease management. Citation Format: Frederick S. Varn, Kevin C. Johnson, Floris P. Barthel, Hoon Kim, Taylor Wade, Disha Lodha, Shoaib Ajaib, Nazia Ahmed, Luciano Garofano, Fulvio D'Angelo, Lucy Stead, Houtan Noushmehr, Antonio Iavarone, Roel Verhaak, The GLASS Consortium. Tumor-myeloid cell interactions are dynamic and influence the evolutionary trajectory of adult diffuse glioma [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 2169.