Tumor Heterogeneity in Glioblastomas: From Light Microscopy to Molecular Pathology

Abstract

One of the main reasons for the aggressive behavior of glioblastoma (GBM) is its intrinsic intra-tumor heterogeneity, characterized by the presence of clonal and subclonal differentiated tumor cell populations, glioma stem cells, and components of the tumor microenvironment, which affect multiple hallmark cellular functions in cancer. "Tumor Heterogeneity" usually encompasses both inter-tumor heterogeneity (population-level differences); and intra-tumor heterogeneity (differences within individual tumors). Tumor heterogeneity may be assessed in a single time point (spatial heterogeneity) or along the clinical evolution of GBM (longitudinal heterogeneity). Molecular methods may detect clonal and subclonal alterations to describe tumor evolution, even when samples from multiple areas are collected in the same time point (spatial-temporal heterogeneity). In GBM, although the inter-tumor mutational landscape is relatively homogeneous, intra-tumor heterogeneity is a striking feature of this tumor. In this review, we will address briefly the inter-tumor heterogeneity of the CNS tumors that yielded the current glioma classification. Next, we will take a deeper dive in the intra-tumor heterogeneity of GBMs, which directly affects prognosis and response to treatment. Our approach aims to follow technological developments, allowing for characterization of intra-tumor heterogeneity, beginning with differences on histomorphology of GBM and ending with molecular alterations observed at single-cell level.

Keywords: glioblastoma; glioma; neoplastic stem cells; precision medicine; prognosis; review; tumor heterogeneity; tumor microenvironment.

Figure 1

Overall profile of the selected articles cited in the review. (a) Type of tumors included in the studies. “Others” includes meningiomas, brain metastases, and other CNS tumors. (b) Types of tumor heterogeneity. (c) Types of tumor heterogeneity according to the origin of samples and time point of collection. Tumor evolution studies are counted in the “Spatial and temporal” category.

Figure 2

Timeline of methods by year of publication based on the articles selected in this review. “Histopathology” = H&E and IHC stains (the 1st WHO classification is from 1979). “Multi-omics” = studies with independent results from two or more high-throughput methods; studies with multiple methods for determination of groups in the study were not included in this count.

Figure 3

Saint Anne-Mayo histopathological criteria for astrocytoma grading. (a) Nuclear atypia in grade II—diffuse astrocytoma; (b) High mitotic activity (arrowheads) in grade III—anaplastic astrocytoma; (c) microvascular proliferation and (d) necrosis in GBM “multiforme”. Grade I gliomas are not part of this classification. Representative images captured from The Ohio State University (OSU) Tissue Archives.

Figure 4

Additional features of glioblastoma. (a) Multiple histopathological grades can be seen in the same tumor—in this example, the upper fragments represent lower grade, while the bottom part depicts a fully developed GBM. (b) Extensive pleomorphism with giant cells may be seen, but the small cells are the most invasive component. (c) Inflammatory cells may invade GBM (same case as Figure 6a). Representative images captured from OSU Tissue Archives.

Figure 5

Molecular features of GBM assessed by immunohistochemistry. (a) IDH1 R132H, (b) ATRX loss (positive stain is seen in endothelial cells), and (c) p53 expression are diffuse in the tumor. Representative images captured from OSU Tissue Archives.

Figure 6

Characterization of TME with IHC markers. (a) CD45 expression: hematopoietic cells (both lymphocytes and macrophages). (b) CD163 expression: M2 macrophages. (c) PDL-1 expression: presence of positive tumor cells in the perivascular space. (d) CD34 expression: endothelial cells. Representative images captured from OSU Tissue Archives.