Re: Investigation of human brain tumors for the presence of polyomavirus genome sequences by two independent laboratories by Rollisonet al. (published online 21 October 2004) (2005)

In the 21 October 2004 issue of the International Journal of Cancer, Rollison and colleagues from 2 laboratories, the Laboratory of Molecular Medicine and Neuroscience, NINDS (LMMN) and the Johns Hopkins Bloomberg School of Public Health (JHU), published a study on the presence of human polyomaviruses BKV, JCV and simian virus 40 (SV40) in a series of brain tumors.1 In their effort, a total of 9 of 225 samples of brain tumor tissue were found positive for one of the polyomaviruses by one laboratory (LMMN), but the other laboratory (JHU) reported all 9 of these samples as negative. JHU detected polyomavirus in only 1 of 165 samples, a finding that was not reproduced by their collaborators at LMMN. The lack of reproducibility between 2 laboratories evaluating the same set of clinical samples raises several important issues that are both technical as well as conceptual in nature, particularly in terms of sensitivity as well as specificity of the methods that were employed. Several aspects of the methodologies used by the authors should be considered when interpreting their results. In LMMN, the DNA extraction step yielded highly variable results and only 5 μl of template was used regardless of concentration, even if samples were negative. Furthermore, no information was provided on the sensitivity of either the PCR or the Southern blot. Of note, the Southern blot was performed with random primed full-length probes, rather than oligonucleotide probes specific for the target amplicon. The authors indicate that 1.0 ng of JCV and BKV and 0.1 ng of SV40 plasmid were used on the blots as controls, which would be equivalent to 10,000 and 1,000 copies, respectively. Under these conditions, it is likely that the sensitivity of the Southern blot remained off by several orders of magnitude. The JHU laboratory did not seem to have any difficulty in extracting the DNA, and 150 ng of template was used in each reaction. Using real-time quantitative PCR, JCV and BKV were co-amplified using the same primer pair in one reaction, and the 2 were distinguished by melting temperatures of 82°C and 83°C using SYBR Green. It should be noted that quantitative PCR of paraffin-embedded tumor specimens is not well established and assays at present may not be as sensitive as conventional PCR.2 Nevertheless, positive PCR samples were verified by hybridization using biotinylated oligonucleotide probes, which, as acknowledged by the authors, may not be as sensitive a method as radiolabeling. The authors reported a sensitivity of 10 copies for their assay, although how this number was reached is unclear. The detection of positive controls is also a concern. Whereas LMMN did not report the sensitivity or specificity of their assay, JHU indicated that the positive JCV mouse tumor controls only contained 1.7 and 495.5 copies, which are quite low, especially given that the sensitivity of the assay is reported as 10 copies. In fact, the only human tumor found positive by this laboratory was a medulloblastoma where 0.12 was reported as the copy number for SV40. Copy numbers for PML tissue and the hamster SV40 model were exponentially higher, as one would expect in samples with replicating virus (PML) or injection of large viral inoculum (hamster). No positive controls for BKV were used in this study. The presence of the human polyomaviruses JCV, BKV and simian virus 40 (SV40) in human tumors has been investigated by many groups by PCR analysis with a wide range of conclusions. Care must be taken to ensure that the most sensitive and reproducible methods are utilized and that all appropriate controls are performed to rule out false positives. It is particularly important that negative data are obtained using robust and reproducible methods because such findings can be misleading. Variability in laboratory techniques can lead to differences in sensitivity and/or specificity, which could account for the discord in published results. It should be noted that preservation of paraffin-embedded archived tissue may be one significant source of variability that is difficult to overcome. We wish to extend an open invitation to work with any individuals who may experience difficulty in amplifying human polyomaviruses from their samples and are willing to share reagents, protocols, and expertise to establish the most appropriate methodologies for such studies. We encourage multicenter studies that share blinded and coded samples similar to the format of this study, but such studies should attempt to standardize methods or utilize assays with comparable levels of sensitivity and specificity. It is unfortunate that the present study using large sets of well-characterized tissues did not include laboratories who have routinely published results where polyomaviruses have been successfully and reproducibly detected by complementary techniques. Despite the inconsistencies in the data from LMMN and JHU, which the authors acknowledge may be due to differences in assay sensitivity, they conclude that polyomaviruses may be present at low frequencies and at low copy numbers. Although broad surveys of tumor tissue are important, we suggest that the discussion should move beyond PCR studies, which do not appear to reach consensus but merely add more discordant results to the debate. The authors should acknowledge other methodologies that have detected polyomaviruses in human CNS and non-CNS tumors. For example, to rule out latently infected B cells as the source of polyomavirus, the use of laser capture microdissection (LCM) combined with PCR techniques has demonstrated the presence of polyomavirus sequences specifically within tumor cells. Most notably, a number of groups have reported the unambiguous detection of JCV and SV40 T-antigen in the nuclei of neoplastic cells, as well as the JCV late auxiliary agnoprotein in perinuclear regions, findings that could not result from any type of laboratory contamination.3, 4, 5, 6, 7, 8 Consistent with these findings, viral capsid proteins have not been detected, suggesting that the virus is nonreplicating. Techniques such as LCM and immunohistochemistry should be used in parallel for validation of PCR amplification. The detection of viral proteins with known transforming capabilities should not be overlooked. T-antigen and agnoprotein are capable of altering a number of cellular signaling pathways involved in transcriptional regulation, cell cycle progression, and DNA repair. Alterations in these pathways have been detected in many clinical samples. Indeed, in a recent overview of cancer, it was stated, “(it) may be that it is impossible for a tumor of epithelial origin to form unless the p53 and Rb tumor-suppressor gene pathways have been inactivated.”9 The polyomaviral proteins disrupt these and other pathways, thus it is reasonable to suggest that, if expressed, they can contribute to tumor development. Yours sincerely, Jennifer Gordon, Luis Del Valle, Sidney Croul, Krzysztof Reiss, Martyn White, Kamel Khailili.