Glioblastomas, (grade 4 astrocytomas), are aggressive primary brain tumors characterized by histopathological heterogeneity. High-resolution sequencing technologies have shown that these tumors also feature significant inter-tumoral molecular heterogeneity. Molecular subtyping of these tumors has revealed several predictive and prognostic biomarkers. However, intra-tumoral heterogeneity may undermine the use of single biopsy analysis for determining tumor genotype and has implications for potential targeted therapies. The clinical relevance and theories of tumoral molecular heterogeneity in glioblastoma are discussed.

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 (p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression (GPNMB, CCL5, and KLRC1) and polarization toward an M2 phenotype (CD163 and MSR1) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.

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Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. DNA repair mechanisms are key elements involved in the response to temozolomide with epigenetic silencing of the O6-methylguanine methyltransferase (MGMT) promoter being a predictive biomarker for temozolomide response. However, response to temozolomide is highly variable and not always predicted by MGMT promoter methylation status. The mismatch repair (MMR) and base excision repair (BER) pathways have also been shown to be involved in treatment response with aberrations in these pathways leading to chemo-resistance and poor response to therapy. Thus changes in these pathways may confer resistance to temozolomide which is independent of MGMT methylation. Further, intratumoral heterogeneity in these pathways may also exacerbate resistance leading to worse outcomes. This study investigated intratumoral heterogeneity in glioblastoma with a particular focus on the DNA repair pathways. The cohort comprised 14 cases of glioblastoma with 2 - 6 tumor tissue biopsies (5-10mm3) per case resected from regions at least 1cm apart. Classification of transcriptional subtype was performed by gene expression profiling (Fluidigm, Taqman assays). Pyrosequencing was used to identify MGMT promoter methylation. Expression of MMR and BER genes was determined using qRT-PCR and Taqman assays and deep sequencing of these genes was performed using the MiSeq Illumina platform and Avadis NGS software. Gene expression profiling using two different limited gene-sets classified tumor specimens into the 3 major transcriptional subtypes, proneural, classical and mesenchymal, with strong concordance. These clustering techniques were then applied to tumor biopsies from the same individual. Transcriptional intratumoral heterogeneity defined as biopsies from the same individual being classified into different subtypes was identified in 40% of the patients. Intratumoral heterogeneity was also identified in the DNA repair pathways. Variability in MGMT methylation status was found in 14% of cases. In each case the percentage methylation varied up to 4-fold and the methylation status was independent of transcriptional classification. Intratumoral variation in the expression of the MMR genes MSH2 and PMS2 was identified in 15% and 20% of cases respectively and in 50% and 30% of cases for the BER genes PARP1 and APEX1. Significant heterogeneity within specimens was not identified for MMR genes MSH6 or MLH1. Targeted next generation sequencing of these 6 genes confirmed the presence of intratumoral heterogeneity at the mutation level. Up to 80 sequence variants were identified in each specimen, with 35 – 56 variants common across all specimens from a case, up to 20 shared between at least 2 specimens in a case and between 1 and 37 unique to each specimen within a case. This study identified intratumoral heterogeneity of DNA repair pathways in glioblastomas, at the genomic, transcriptional and mutational levels. These pathways have roles in the responsiveness of glioblastomas to temozolomide. As such, intratumoral heterogeneity may confound patient management. Therefore, these results highlight the importance of assessing results from multiple tumor biopsies in order to correctly manage glioblastoma patients and their treatment. Citation Format: Nicole R. Parker, Amanda L. Hudson, Peter Khong, Jonathon F. Parkinson, Rowan Ikin, Zhangkai Jason Cheng, Fatemeh Vafaee, Helen R. Wheeler, Viive M. Howell. Intratumoral heterogeneity of DNA repair pathways in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B39.