CMV and glioma–are we there yet?

Findings describing the potential role of human cytomegalovirus (HCMV) infection in glioblastoma (GBM) have been controversial since their initial description in 2002.1 We hope to provide some perspective on their evolution and, hopefully, offer some suggestions about how to move forward in a constructive fashion. With regard to HCMV in glioma, difficulties in arriving at definitive conclusions are due in part to the technical intricacies of detecting viral epitopes by immunohistochemistry (IHC) as well as the lack of success of ancillary corroborating methodologies to demonstrate the virus in brain tumors. These factors have generated a healthy degree of skepticism and caution, which, in our opinion, is warranted until findings can be reasonably proven and accepted on the basis of the totality of evidence. In this regard, we can borrow a phrase often used in the legal system, that of reasonable doubt. As applied in this case, it is probably wisest to retain doubt about the role of HCMV in glioma until its relevance can be widely reproduced. Continuing this analogy, the burden of proof is best placed on those making the claim, and caution is warranted in the absence of clear and convincing data. A paper in this issue of Neuro-Oncology by Baumgarten et al.2 reports an inability to detect HCMV in GBM, casting further doubt on its relevance in glioma. Robust scientific conclusions should be based on the totality of evidence from complementary sources and provide mutual support for a consensus conclusion. Accordingly, the absent HCMV detection by complementary and ancillary methods (mRNA, DNA sequencing, etc) invites skepticism about the pathogenic role the virus may play in GBM. Reliance on one method, like IHC, under a specific set of iron-clad conditions raises red flags in terms of drawing definitive conclusions. Even when read as convincingly positive, IHC can be misleading, as illustrated by the well-recognized cross-reactivity of some antikeratin antibodies in GBM.3 Directly on point is a recent paper that failed to identify HCMV DNA using high-coverage unbiased/whole genome sequencing in 34 samples of GBM.4 While this does not rule out low-level HCMV in GBM, it is important to note that evidence of HHV4 (Epstein-Barr) and other viral DNA sequences were identified, suggesting that the abundance of HCMV would not exceed 1 virus per 240 000 tumor cells even if present. Similarly, additional studies using RNA sequencing have not yielded evidence of HCMV in GBM.5–7 The absolute absence of HCMV in glioma is extremely difficult to prove definitively and, in an important way, that is not the point of this paper by Baumgarten et al. Instead, the report details a carefully performed study that failed to detect HCMV signal by IHC in a setting in which known positive controls were in order. As its title indicates, the virus was not detected in 123 brain samples using known standard techniques that detect HCMV in other settings. One can always argue that the technical limitations of a specific assay preclude positive detection, and the antigen would have been detected if only the assay had been done differently or the samples processed appropriately, etc. In our opinion, the most judicious approach is one in which conclusions are reached based on the totality of evidence, ideally from diverse methods and sources. In this setting, we believe that the totality of the evidence to date does not warrant the conclusion that HCMV is present in GBM. In fairness to proponents of the HCMV hypothesis, it is important to note that this paper only reports an inability to detect HCMV. However, as the saying goes, absence of proof should not be construed as proof of absence. A fair conclusion of this paper might be that HCMV was undetectable in a given set of samples, using a specific technical approach. In this light, the report represents an important contribution to the literature as a whole. Moving forward, we must remain open to the possibility of improved methods in the future that would allow widespread and reliable detection of HCMV across laboratories, However, until its detection can be robustly reproduced and independently confirmed in a range of assays and with appropriate analytical and clinical validity, one can only conclude that “the jury is still out” with respect to the relevance of HCMV in glioma.