Immunologic Targeting and Biologic Underpinnings of Human Cytomegalovirus in Glioblastoma

Abstract

Glioblastoma (GBM) is a grade IV astrocytoma in which the median overall survival is approximately 15 months at time of diagnosis. Even with the current multi- modal therapeutic approach of surgery, chemotherapy with the DNA alkylating agent temozolomide, and radiation therapy, GBM remains uniformly lethal. Immunotherapeutic interventions are a burgeoning field in many different cancer treatments. They offer the exquisite specificity endowed by the immune system with minimal toxicities and new methods are being developed to enhance the endogenous immune responses.With the recent identification of human cytomegalovirus (CMV) present within glioblastoma tissue but void in the surrounding normal healthy parenchyma there have been significant efforts aimed at understanding the biologic implications of the presence of the virus within GBM tissues with preliminary work demonstrating several capabilities of the virus to enhance the oncogenic process. Likewise, a key area of importance in the development and design of effective immunotherapeutic platforms is the identification and targeting of tumor-specific antigens. The success of any immunotherapy platform relies heavily on the ability to selectively target antigens present within tumors but absent on healthy tissue, regardless of its role in tumorigenesis, as well as having robust immunogenic properties.CMV offers a plethora of possible targets, as it is the largest known DNA virus that infects humans, yet very little is known about its biological significance in glioblastoma pathogenesis as well as the most efficacious and immunogenic targets for immunotherapeutic development. We have been able to elucidate more thoroughly the feasibility and potency of an immunologic platform targeting CMV within glioblastoma utilizing a multi-antigen multi-component peptide based strategy that demonstrated significant immunogenicity and anti-tumor activity in pre-clinical models utilizing various assays. We have also developed several sensitive and specific detection methodologies including: 1) custom gene expression microarrays, 2) multiplex real time quantitative polymerase chain reaction (RT-qPCR) assays, 3) a massively parallel RNA deep sequencing platform, and 4) immunological assays. We have also successfully determined the capacity for endogenous CMV gene expression to be maintained in primary glioblastoma cell lines as well as examining the preponderance of CMV gene expression in a subpopulation of glioma stem cell-like cells, the slow cycling GBM cells established from primary tumor tissues, in an attempt to illuminate some of the biologic underpinnings of CMV with respect to GBM pathogenesis.Taken together, these data lay the groundwork for the development of a more efficacious vaccination strategy targeting CMV in GBM. The screening strategies employed throughout this work will allow for an accurate antigenic profile of CMV in GBM which will subsequently permit the design of a more robust peptide vaccine for the next generation of cancer vaccine. We have also begun to describe some of the interesting biologic phenomena associated with CMV in GBM, as our results demonstrate continued viral gene expression in glioma stem cell-like cell populations indicating viral tropism for certain cell types.