Letter to the Editor

Re: Investigation of Human Brain Tumors for the Presence of Polyomavirus Genome Sequences by Two

Independent Laboratories by Rollison et al. (Published Online 21 October 2004)

Jennifer Gordon, Luis Del Valle, Sidney Croul, Krzysztof Reiss, Martyn White and Kamel Khalili*

Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, PA, USA

Dear Sir,In the 21 October 2004 issue of the International Journal of

Cancer, Rollison and colleagues from 2 laboratories, the Labo-ratory of Molecular Medicine and Neuroscience, NINDS(LMMN) and the Johns Hopkins Bloomberg School of PublicHealth (JHU), published a study on the presence of human pol-yomaviruses BKV, JCV and simian virus 40 (SV40) in a seriesof brain tumors.1 In their effort, a total of 9 of 225 samples ofbrain tumor tissue were found positive for one of the polyoma-viruses by one laboratory (LMMN), but the other laboratory(JHU) reported all 9 of these samples as negative. JHUdetected polyomavirus in only 1 of 165 samples, a finding thatwas not reproduced by their collaborators at LMMN. The lackof reproducibility between 2 laboratories evaluating the sameset of clinical samples raises several important issues that areboth technical as well as conceptual in nature, particularly interms of sensitivity as well as specificity of the methods thatwere employed.

Several aspects of the methodologies used by the authors shouldbe considered when interpreting their results. In LMMN, the DNAextraction step yielded highly variable results and only 5 ml oftemplate was used regardless of concentration, even if sampleswere negative. Furthermore, no information was provided on thesensitivity of either the PCR or the Southern blot. Of note, theSouthern blot was performed with random primed full-lengthprobes, rather than oligonucleotide probes specific for the targetamplicon. The authors indicate that 1.0 ng of JCV and BKV and0.1 ng of SV40 plasmid were used on the blots as controls, whichwould be equivalent to 10,000 and 1,000 copies, respectively.Under these conditions, it is likely that the sensitivity of the South-ern blot remained off by several orders of magnitude.

The JHU laboratory did not seem to have any difficulty inextracting the DNA, and 150 ng of template was used in eachreaction. Using real-time quantitative PCR, JCV and BKV wereco-amplified using the same primer pair in one reaction, and the 2were distinguished by melting temperatures of 82�C and 83�Cusing SYBR Green. It should be noted that quantitative PCR ofparaffin-embedded tumor specimens is not well established andassays at present may not be as sensitive as conventional PCR.2

Nevertheless, positive PCR samples were verified by hybridizationusing biotinylated oligonucleotide probes, which, as acknowl-edged by the authors, may not be as sensitive a method as radio-labeling. The authors reported a sensitivity of 10 copies for theirassay, although how this number was reached is unclear. Thedetection of positive controls is also a concern. Whereas LMMNdid not report the sensitivity or specificity of their assay, JHU indi-cated that the positive JCV mouse tumor controls only contained1.7 and 495.5 copies, which are quite low, especially given thatthe sensitivity of the assay is reported as 10 copies. In fact, theonly human tumor found positive by this laboratory was a medul-loblastoma where 0.12 was reported as the copy number for SV40.Copy numbers for PML tissue and the hamster SV40 model wereexponentially higher, as one would expect in samples with repli-cating 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 sim-ian 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 reprodu-cible methods are utilized and that all appropriate controls are per-formed to rule out false positives. It is particularly important thatnegative data are obtained using robust and reproducible methodsbecause such findings can be misleading. Variability in laboratorytechniques can lead to differences in sensitivity and/or specificity,which could account for the discord in published results. It shouldbe noted that preservation of paraffin-embedded archived tissuemay be one significant source of variability that is difficult to over-come.

We wish to extend an open invitation to work with any indi-viduals who may experience difficulty in amplifying human poly-omaviruses from their samples and are willing to share reagents,protocols, and expertise to establish the most appropriate method-ologies for such studies. We encourage multicenter studies thatshare blinded and coded samples similar to the format of thisstudy, but such studies should attempt to standardize methods orutilize assays with comparable levels of sensitivity and specific-ity. It is unfortunate that the present study using large setsof well-characterized tissues did not include laboratories whohave routinely published results where polyomaviruses have beensuccessfully and reproducibly detected by complementarytechniques.

Despite the inconsistencies in the data from LMMN and JHU,which the authors acknowledge may be due to differences inassay sensitivity, they conclude that polyomaviruses may bepresent at low frequencies and at low copy numbers. Althoughbroad surveys of tumor tissue are important, we suggest that thediscussion should move beyond PCR studies, which do notappear to reach consensus but merely add more discordantresults to the debate. The authors should acknowledge othermethodologies that have detected polyomaviruses in humanCNS and non-CNS tumors. For example, to rule out latentlyinfected B cells as the source of polyomavirus, the use of lasercapture microdissection (LCM) combined with PCR techniqueshas demonstrated the presence of polyomavirus sequences spe-cifically within tumor cells. Most notably, a number of groupshave reported the unambiguous detection of JCV and SV40 T-antigen in the nuclei of neoplastic cells, as well as the JCV lateauxiliary agnoprotein in perinuclear regions, findings that couldnot result from any type of laboratory contamination.3–8 Consis-tent with these findings, viral capsid proteins have not beendetected, suggesting that the virus is nonreplicating. Techniquessuch as LCM and immunohistochemistry should be used in par-allel for validation of PCR amplification.

The detection of viral proteins with known transforming capa-bilities should not be overlooked. T-antigen and agnoprotein arecapable of altering a number of cellular signaling pathways

*Correspondence to: Center for Neurovirology and Cancer Biology,1900 North 12th St., 015-96, Room 203, Temple University, Philadelphia,PA, 19122. Fax:1215-204-0679. E-mail: kamel.khalili@temple.eduReceived 13 December 2004; Accepted after revision 2 February 2005DOI 10.1002/ijc.21161Published online 23 May 2005 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 117, 693–694 (2005)' 2005 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

involved in transcriptional regulation, cell cycle progression, andDNA repair. Alterations in these pathways have been detected inmany clinical samples. Indeed, in a recent overview of cancer, itwas stated, ‘‘(it) may be that it is impossible for a tumor of epithe-lial origin to form unless the p53 and Rb tumor-suppressor genepathways have been inactivated.’’9 The polyomaviral proteins dis-

rupt 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 and Kamel KHAILILI *

References

1. Rollison DE, Utaipat Y, Ryschkewitsch C, Hou J, Godlthwaite P,Daniel R, Helzlsouer KJ, Burger PC, Shah KV, Major EO. Investiga-tion of human brain tumors for the presence of polyomavirus genomesequences by two independent laboratories. Int J Cancer 2005;113:769–74.

2. Biedermann K, Dandachi N, Trattner M, Vogl G, Doppelmayr H,More E, Staudach A, Dietze O, Hauser-Kronberger C. Comparison ofreal-time PCR signal-amplified in situ hybridization and conventionalPCR for detection and quantification of human papillomavirus inarchival cervical cancer tissue. J Clin Microbiol 2004;42:3758–65.

3. Corallini A, Tognon M, Negrini M, Barbanti-Brodano G. Evidencefor BK virus as a human tumor virus. In: Khalili K, Stoner GL, eds.Human polyomaviruses: molecular and clinical perspectives. NewYork: Wiley-Liss, 2001. p. 431–60.

4. Del Valle L, Enam S, Lara C, Miklossy J, Khalili K, Gordon J. Pri-mary central nervous system lymphoma expressing the human neuro-tropic polyomavirus, JC virus, genome. J Virol 2004;78:3462–9.

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6. Flaegstad T, Andresen PA, Johnsen JI, Asomani SK, Jorgensen G-E,Vignarajan S, Kjuul A, Kogner P, Traavik T. A possible contributoryrole of BK virus infection in neuroblastoma development. Cancer Res1999;59:1160–3.

7. Khalili K, White MK, Sawa H, Nagashima K, Safak M. The agnopro-tein of polyomaviruses: a multifunctional auxiliary protein. J CellPhysiol 2004;204:1–7.

8. Testa JR, Carbone M, Hirvonen A, Khalili K, Krynska B, LinnainmaaK, Pooley FD, Rizzo P, Rusch V, Xiao G-H. A multi-institutionalstudy confirms the presence and expression of Simian Virus 40 inhuman malignant mesothelioma. Cancer Res 1998;58:4505–9.

9. Vogelstein B, Kinzler KW. Cancer genes and the pathways they con-trol. Nature Med 2004;10:789–99.

694 HUMAN BRAIN TUMORS AND POLYOMAVIRUS SEQUENCES