Viral Etiology of Human Cancer:
A historical perspective

Etienne de Harven

The hypothesis according to which some human cancers might be caused by filterable micro-organisms such as viruses is almost one hundred years old. It was indeed in 1903 that Borrel, in France, suggested such a possible relationship. To put this hypothesis in a historical perspective one should refer to the book The Riddle of Cancer (1) which Charles Oberling published in 1952 and in which the possible role of viruses in human cancer was presented with extensive references to contributions of initial pioneers such as Rous, Shope, and Bittner. Since our purpose is, to some extent, focused on the evolution of methodologies which cancer researchers utilized in attempts to verify the hypothesis, one should emphasize that the approach of Rous and his followers was essentially based on establishing the difference between transmission of tumors and leukemias by cell transplants, i.e. grafts, or by cell-free filtrates. Transmission of tumors in experimental animals by cell-free filtrates was always interpreted as demonstration of viral etiology.

During the past fifty years, viral oncology has been studied in almost all cancer research centers, world wide. Practical results in terms of effective therapy of human malignancies have been nil. But still, recent issues of all the main oncology publications contain numbers of studies related to viral oncology, clearly indicating that the hypothesis still has considerable momentum!

The past fifty years can be analyzed in two distinct periods. The first, between 1945-1970 was dominated by electron microscopy; the second, from 1970 until now, being dominated by molecular biology.

Electron microscopy (EM) contributed a considerable amount of data, which can briefly be summarized as follows.

1. EM can readily demonstrate associations between viruses and cancers in several laboratory animal species, such as chickens and mice; (2,3)
2. EM data by themselves do not, however, prove any role of these viruses in the etiology of the tumors. (4) The EM data did, however, trigger microbiological experiments, based on ultrafiltration, which frequently yielded scientific evidence for etiological relationships;
3. as pointed out by André Lwoff et al. (5) in 1962, electron microscopy is probably the most efficient approach to viral classification;
4. viruses shown to be associated with several cancers of laboratory animals belong to various families of viruses (herpes, vaccinia, papova, retroviruses, DNA, RNA,...) and are not restricted to any one family;
5. viruses associated with some cancers and those responsible for infectious diseases look identical. There is no such thing as a family of oncogenic viruses, a terminology which never appears in general classifications of viruses and should actually be regarded as a misnomer;
6. practically, EM is essential to monitor the level of success in the sequential steps leading to virus isolation and purification. Therefore, the success of biochemical characterization of viral markers depends on electron microscopy to ascertain the purity of viral isolates and the absence (or minimal amounts) of non-viral contaminants;
7. finding particles with typical viral morphology does not mean that these viruses are pathogenic. Actually, there are probably many more non-pathogenic than pathogenic viruses. This point was well illustrated in a special conference sponsored around 1960 by the New York Academy of Sciences under the title Viruses in Search of Disease;
8. viruses, infectious or cancer-associated, rarely satisfy all the Koch postulates which, incidentally, were presented before viruses were discovered;
9. while the association between viruses and numerous malignancies of laboratory animals has been readily demonstrated by electron microscopy, and in spite of considerable efforts, similar associations have never been observed in human cancers (4) (with very rare exceptions such as the common wart and molluscum contagiosum...)

Of mice and men...

Research in viral oncology changed drastically around 1970, when methods of molecular biology took the lead, while electron microscopy was relegated to a distant background.

Dedicated virus hunters, as Peter Duesberg (6) would call them, were obviously not discouraged by the negative results of twenty years of active search for viruses by electron microscopy in many types of human cancers. To the contrary, large research programs were initiated, based primarily on the identification of viral, molecular markers such as enzymes, nucleic acids or proteins identified most frequently in cell cultures derived from human malignancies, rarely directly from the tumor tissues or blood plasma. The fact that viruses had never been directly observed in human tumors by electron microscopy was conveniently explained in terms of virus latency, and/or by integration of a provirus in the genome of tumor cells.

The most significant example illustrating this drastic change in approach is given by the reverse transcriptase enzyme, discovered in 1970 by Temin in purified Rous sarcoma virus, (7) and by Baltimore in Rauscher mouse leukemia virus. (8) This discovery was regarded as historical. It resulted in two Nobel prizes and in the renaming of all RNA tumor viruses as retroviruses. DNA synthesis from an RNA template was indeed a very surprising observation in 1970. The enzyme was initially thought to represent a unique feature of RNA tumor viruses and was, therefore, regarded as a reliable marker of the presence of retroviruses, even when retrovirus particles were never convincingly observed by EM. We learned, later on, that reverse transcription is a common phenomenon, that the enzyme (RT) is present in many different cells,9 and that demonstration of RT activity is far from enough to substantiate any claim for the isolation of a retrovirus.

An exhaustive review of viral oncology literature over the past 25 years would be beyond the purpose of this editorial. Instead, it may be appropriate to mention publications which have appeared over the past few years, in an attempt to evaluate how much progress has been made toward the consolidation of a century old hypothesis.

Two main areas of viral oncology, namely: herpes viruses with emphasis on EBV and herpes-8 virus, and papova viruses with emphasis on human papilloma virus, HPV, will be considered. Retroviruses, although they have been the topic of my own research, will not be specifically emphasized here because the currently used anti-HIV antibody tests are non-specific (vide infra), making any attempt to analyze the current literature very hazardous.

Herpes

Abundant reports in oncology literature refer to the Epstein-Barr virus. This human herpes virus infects most people early in life. However, when primary infection is delayed until adolescence or adulthood it frequently causes infectious mononucleosis. It is a highly contagious agent (the kissing disease). Most EBV carriers are disease-free. However, in some cases EBV appears to be associated with several forms of cancers: lymphomas in immunosuppressed patients, Hodgkin's disease, Burkitt's lymphoma in central Africa, nasopharyngeal carcinoma, thymic lymphoepithelioma, primary nasal lymphomas and gastric carcinoma, of much more sizeable epidemiological importance. These malignancies, and their relatedness to EBV, have been recently reviewed in a 1998 Gann monograph edited by Toyoro Osato. (10) Several well characterized virus proteins (the EBNA proteins) are involved in the immortalization of B lymphocytes and, most interestingly, cyclin D2, driving B cells from the resting stage into G1, is induced within 24 hours of EBV infection.

Although B cells appear as the primary targets of EBV, T cells might also be involved as suggested by Kanegane et al. (11) in a study of patients with severe, chronic active EBV infection who came down with EBV-positive T-cell lymphoma.

Methods of in situ hybridization have been applied to the study of familial Hodgkin's disease by Lin et al., (12) suggesting that EBV does not play an important role in familial Hodgkin's disease.

Studies at the University of Padova, by Ometto et al., (13) support the notion that lymphomas arise from clonal expansion of EBV+ cells. That latent EBV infection can be reactivated by EBV-specific CD8+ T cells was demonstrated by Nazaruk et al. (14)

The danger of EBV-induced lymphoproliferative disease after allogeneic stem cell transplantation was studied, using a semiquantitative EBV-PCR technology, by Lucas et al. (15) Recently, Hale and Waldmann (16) analyzed the occurrence of EBV driven lymphoproliferative disorders in patients receiving T-cell depleted allogeneic bone marrow transplantation. They suggested that additional depletion of B cells is beneficial possibly because it reduces the virus load or the virus target which is hardly compatible with the limited success experienced with antiviral agents in such cases.

In America and in Europe, 50% of the cases of Hodgkin's disease are associated with EBV. According to Roskrow et al., (17) EBV-specific cytotoxic T lymphocytes generated from normal donors may persist long-term in vivo and reconstitute the immune response to EBV, this possibly being an effective prophylaxis and treatment of immunoblastic lymphoma. The approach could be useful for cases failing to respond to salvage chemotherapy.

The potential usefulness of therapeutic protocols based on EBV has been demonstrated recently by Neyts et al. (18) who studied xenografts of EBV-associated nasopharyngeal carcinomas in athymic nude mice. Administration of the antiviral agent Cidofovir had a pronounced antitumor effect in these tumor-bearing mice, apparently as a result of rapid cell death, through apoptosis, of EBV-transformed epithelial cells.

Primary effusion lymphomas have a more complex association, not with one but with two distinct viruses of the herpes group, namely EBV and human herpesvirus-8 (HHV-8), the Kaposi's sarcoma associated agent. This has recently been described by Horenstein et al. (19) The various patterns of EBV latency expression and the interaction with HHV-8 may contribute to a better understanding of the pathobiology of this form of lymphoproliferative disease.

The association between HHV-8 infection and multiple myeloma was initially reported by Said et al. (20) in 1997 and confirmed by Broussais et al. (21) However, Cathomas et al., (22) in Switzerland, had difficulties in confirming the PCR results and stressed the absence of anti-HHV-8 antibodies in 17/18 multiple myeloma patients. In sharp contrast, anti-HHV8 antibodies were readily identified in the majority of a group of patients with Kaposi's sarcoma.

The fact, initially reported in 1994, that a large majority of cases of Kaposi's sarcoma are associated with a virus of the herpes group, HHV-8, seems generally accepted. This observation may have interesting therapeutic implications as indicated recently by Low et al., (23) working in Fleckenstein's laboratory in Erlangen. Low observed a transient disappearance of HHV-8 DNA in the PBMC of a patient with disseminated Kaposi's sarcoma. The positive PCR results in PBMC were interpreted as reflecting viremia, although viremia classically means the presence of free virus particles in blood plasma.

Considerable progress has been made in the understanding of virus-host cell interactions. Thirty years ago, viruses were subclassified into cytolytic and non-cytolytic. The Epstein-Barr virus was regarded as cytolytic because it was, most of the time, observed by electron microscopy in cells apparently in a state of degeneration, with pyknotic nuclei. Important advances have been made in the understanding of apoptosis, which is modulated by many positive and negative controls. For example, the overexpression of the anti-apoptotic BCL-2 protein contributes to some forms of cancer, while the loss of p53 function reduces sensitivity of cells to the apoptosis inducing activity of genotoxic drugs or irradiation. These fundamental aspects of cell growth control are currently studied in many laboratories, including the St. Mary's Branch of the Ludwig Institute in London. (24) That these aspects of cell growth control are somehow related to viral infection, and in particular to EBV infection is clearly indicated by recent observations made in Erlangen by Fleckstein et al., (25) where the main research emphasis is placed on the anti-apoptotic strategies of lymphotropic viruses, evasion of cytotoxic T-cell effects being part of these strategies.

Papova viruses

Human papillomaviruses (HPV), mainly types 16 and 18, are believed to be responsible for the development of invasive cancer of the uterine cervix. It is still not clear, however, whether HPV is a passenger, a driver, or both, as recently discussed by Leopold Koss (26) in New York. In an early PCR study, 46% of a group of healthy female university students were shown to carry the virus. It is unlikely that such a number of young women are candidates for cervical cancer; most likely HPV is only a passenger in most cases. For many oncologists there seems to be little doubt that HPV plays some role in human cancer. However, what transforms a passenger virus into a driver is still an open question. One interesting line of research is related to possible interactions between a protein product of the open reading frames E6 and E7 with protein products of cancer inhibitory genes Rb and p53. Type 16 HPV E7 is a viral oncoprotein, which plays a major role in cervical neoplasia according to Wang-Johanning et al. (27) who prepared antibodies against this oncoprotein with possible therapeutic applications.

The identification of HPV subtypes seems to contribute little to the clinical management of patients, as already indicated by Koss and recently confirmed by Herrington et al. (28)

Pathogenicity of human papillomavirus is not restricted to the uterine cervix. As well documented by Steinberg et al., (29) in 1996, HPVs cause benign tumors in the respiratory tract, and probably play a role in the etiology of a subset of head and neck cancers. Here again, HPV-16 and 18 are associated with a higher probability of malignant conversion (high risk viruses). Clues to the mechanism of action of E7 are discussed by these authors in terms of possible interaction with several cell-cycle regulator proteins which may further contribute to abnormal cell cycle progression. However, approximately 1/3 of all women with cervical cancer have never been infected with HPV. Therefore, HPV could possibly be a co-factor in some cases. In HPV positive patients, however, one wonders whether possible anti-viral therapy might be considered in view of the fact, reported by Neyts et al., (18) that the anti-DNA virus Cidofovir produces complete regression of Shope papilloma virus-induced lesions in rabbits.

General considerations on studies related to the hypothetical viral etiology of some human cancers

The two examples just considered - EBV and HPV - clearly indicate that contemporary viral oncology research is primarily based on the identification of viral markers such as proteins or nucleic acids.

However, the specificity of viral markers depends on the success of virus isolation and purification. Without fully demonstrated success in virus isolation and purification, identification of viral markers is extremely hazardous and can lead to severe misinterpretation of clinical data. A dramatic illustration of this is to be found in current HIV research. In this case, the virus (HIV) has never been properly isolated, since sedimentation in sucrose gradient at the density of 1.16 g/mL was erroneously considered to yield pure virus, systematically ignoring that material sedimenting at that density contains large amounts of cell debris and microvesicles. (30,31) Therefore, proteins and nucleic acids found in such 1.16 bands are very likely to be of cellular origin and cannot be used as viral markers. Such a faulty methodology has had extremely serious consequences, i.e. the world-wide use of HIV-antibody tests, Elisa and Western Blot, which dangerously lack specificity, as demonstrated in 1993 by Papadopulos et al., (32) in Australia.

Admitting, however, that some viral markers are specific, their presence within tumor cells will probably never show more than an association. Etiological relationships are unlikely to be demonstrated by the presence of markers, even if these markers are related to the viral genome. One has difficulties in following Levin and Levine (33) when they state that the identification of the viral genome in tumor cells is the strongest evidence for its activity as an oncogenic agent. This is reminiscent of an old problem when electron microscopy was only showing association with viruses, but never their etiological significance.

In microbiology, most viral diseases are highly contagious. If some forms of cancer had viral etiology, how is it that we don't see more cancer clusters? Clusters have been occasionally observed, but their number is very small and is certainly not compatible with the concept of primary infections. We know that EBV is a ubiquitous virus. And, as T. Osato (10) points out, ubiquity and oncogenicity are seemingly incompatible. But we are not aware of the ubiquity of HHV-8, and we don't see any evidence for clusters of HHV-8 associated malignancies.

An area in which progresses have been highly significant is unquestionably that of apoptosis. Thirty years ago, viruses were regarded as either cytolytic or non-cytolytic. This property was considered as an intrinsic characteristic of the virus itself. Today, factors controlling cell cycling are much better understood, and the cell cycle appears as a fragile balance between apoptotic cell death and cell immortalization. Suppression of apoptosis may contribute to cancer. As studied at the Ludwig Institute in London, (24) it appears, for example, that over-expression of the anti-apoptotic BCL-2 protein is a key event in follicular lymphoma. Factors interfering with the progression through the cell cycle are many; some are endogenous, some are exogenous; some are chemical in nature, others are physical; some could probably be added by the activation of latent viruses, such as EBV. All experiments supporting this view are, however, in vitro experiments, and it will take considerable clinical skill to demonstrate that these in vitro observations are of any significance in the sudden development of tumors in latently EBV infected individuals.

If viral markers show only association, without implying etiology, this does not mean that the presence of such markers within cancer cells is not of possible therapeutic usefulness. Targeting is an interesting approach to chemotherapy, or to CTLs lymphocytes. A significant example for this can be found in the paper by Roskrow et al. (17) on EBV-specific cytotoxic T lymphocytes for the possible treatment of patients with EBV-positive relapsed Hodgkin's disease.

But what about antiviral therapy? Could it possibly be that its eventual success would produce the evidence for the oncogenicity of some viruses which we are so eagerly trying to establish? When we learned that Cidofovir produces complete regression of Shope papilloma virus-induced lesions in rabbits and that its nephrotoxicity can be kept under control in humans, (18) it became a most attractive approach to therapy, as well as to understanding of the still hypothetical oncogenicity of the associated virus. Can the effects of Foscarnet on Kaposi's sarcoma and HHV-8 associated hemophagocytic syndrome (23) possibly be placed in the same perspective?

For DNA viruses associated malignancies, we have effective antiviral agents of manageable toxicity at hand. This is not the case for RNA virus associated diseases, and in particular for syndromes such as AIDS, hypothetically associated (6,32) with infection by the HIV retrovirus. In these cases, the currently used combined antivirals are unacceptably toxic, making the so-called therapy worse than the disease itself! Moreover, the effects of anti-retroviral therapy are currently measured by quantitative PCR technology. Unfortunately, Karry Mullis PCR technology is not reliable to measure what has been erroneously labeled viral load in AIDS patients. (34,35)

As a concluding remark, I wish to say that the cases for both herpes and papova viruses are worth considerable attention. This is in sharp contrast with retroviruses, which, to the best of my knowledge, have never been satisfactorily demonstrated to be associated with any human disease.

Etienne de Harven, MD Professor Emeritus of Pathology, University of Toronto, Ontario, Canada

Correspondence: "Le Mas Pitou", 2879 Route de Grasse, 06530 Saint Cézaire-sur-Siagne, France. - Tel & Fax: international +33-4-93602839 - E-mail: pitou.deharven@wanadoo.fr

References

1. Oberling C. The riddle of cancer. 2nd ed. New Haven: Yale University Press, 1952.
2. Bernhard W. The detection and study of tumor viruses with the electron microscope. Cancer Res 1960; 20: 712-27.
3. de Harven E. Remarks on the ultrastructure of type A, B, and C virus particles. Advances in virus research, Academic Press, 1974; 16:223-64.
4. de Harven E. Remarks on viruses, leukemia and electron microscopy. In: V. Defendi, ed. Methodological approaches to the study of leukemias. The Wistar Institute Symposium Monograph No. 4, Philadelphia: The Wistar Institute Press, 1965. p. 147-56.
5. Lwoff A, Horne R, Tournier P. Cold Spring Harbor Symposium on Quantitative Biology, New York; 1962. p. 27-51.
6. Duesberg P. Inventing the Aids Virus. Washington: Regnery, 1966.
7. Temin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 1970; 226:1211-3.
8. Baltimore D. Viral RNA-dependent DNA polymerase. Nature 1970; 226:1209-11.
9. Varmus H. Reverse transcription. Science Am 1987; 257:48-54.
10. Osato T. Epstein-Barr virus and human cancer. Gann Monograph on Cancer Research No. 45. Basel: S. Karger AG, 1998.
11. Kanegane H, Bathia K, Gutierrez M, et al. A syndrome of peripheral blood T-cell infection with Epstein-Barr virus (EBV) followed by EBV-positive T-cell lymphoma. Blood 1998; 91:2085-91.
12. Lin D, Kingma DW, Lennette ET, et al. Epstein-Barr virus and familial Hodgkin's disease. Blood 1996; 88: 3160-5.
13. Ometto L, Menin C, Masiero S, et al. Molecular profile of Epstein-Barr virus in human, immunodeficiency virus type-1 related lymphoadenopathies and lymphomas. Blood 1997; 90:313-22.
14. Nazaruk RA, Rochford R, Hobbs MV, et al. Functional diversity of the CD8+ T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders. Blood 1998; 91:3875-83.
15. Lucas KG, Burton RL, Zimmerman SE, et al. Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation. Blood 1998; 91:3654-61.
16. Hale G, Waldmann H. Risks of developing Epstein-Barr virus-related lymphoproliferative disorders after T-cell-depleted marrow transplants. Blood 1998; 91: 3079-83.
17. Roskrow MA, Suzuki N, Gan YJ, et al. Epstein-Barr-virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin's disease. Blood 1998; 91:2925-34.
18. Neyts J, Sadler R, De Clercq E, et al. The antiviral agent Cidofovir (S-1-3-hydroxy-2-phosphonyl-methoxypropylcytosine) has pronounced activity against nasopharyngeal carcinoma grown in nude mice. Cancer Res 1998; 58:384-8.
19. Horenstein MG, Nador RG, Chadburn A, et al. Epstein-Barr Virus latent gene expression in primary effusion lymphomas containing Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8. Blood 1997; 90:1186-91.
20. Said JW, Tazaka T, Takeuchi S, et al. Primary effusion lymphoma in women: report of two cases of Kaposi's sarcoma virus-associated effusion-based lymphoma in human immunodeficiency virus-negative women. Blood 1996; 88:3124-8.
21. Broussais P, Meggetto F, Attal M, et al. Kaposi's sarcoma-associated herpesvirus infection and multiple myeloma. Science 1997; 278:1972.
22. Cathomas G, Stalder A, Kurner MO. Multiple myeloma and HHV8 infection. Blood 1998; 91:4391-2.
23. Low P, Neipel F, Rascu A, et al. Suppression of HHV-8 viremia by Foscarnet in an HIV-infected patient with Kaposi's sarcoma and HHV-8 associated hemophagocytic syndrome. Eur J Med Res 1998;14:461-4.
24. Saville M, Watson RJ. B-Myb: a key regulator of the cell cycle. Adv Cancer Res 1998; 72:109-40.
25. Meinl E, Fickenscher H, Thome M, et al. Anti-apoptotic strategies of lymphotropic viruses. Immunol Today 1998; 19:474-9.
26. Koss L. Human papillomavirus - Passenger, driver, or both? Hum Pathol 1998; 29:309-10.
27. Wang-Johanning F, Yancey G, Grim J, et al. Intracellular expression of a single-chain antibody directed against human Papillomavirus type 16 E7 oncoprotein achieves targeted antineoplastic effects. Cancer Res 1998; 58:1893-900.
28. Herrington CS, Wells M. Can HPV typing predict the behaviour of cervical epithelial neoplasia? Histopathology 1997; 31:301-3.
29. Steinberg BM, DiLorenzo TP. A possible role for human papillomaviruses in head and neck cancer. Cancer Metast Rev 1996; 15:91-112.
30. Gluschankof P, Mondor I, Gelderblom HR, et al. Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations. Virology 1997; 230:125-33.
31. Bess JW Jr, Gorelick RJ, Bosche WJ, et al. Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations. Virology 1997; 230: 134-44.
32. Papadopulos-Eleopulos E, Turner VF, Papadimitriou JM. Is a positive Western Blot proof of HIV infection? Bio-Technol 1993; 11:696-707.
33. Levin LI, Levine PH. The epidemiology of Epstein-Barr virus-associated human cancers. In: Osato T, ed. Epstein-Barr virus and human cancer, Gann Monograph on Cancer Research No. 45. Basel: S. Karger AG, 1998.
34. Busch MP, Henrad DR, Hewlett IK, et al. Poor sensitivity, specificity, and reproducibility of detection of HIV-1 DNA in serum by polymerase chain reaction. J AIDS 1992; 5:872.
35. Eleopulos E, Turner VF, Papadimitriou J. Factor VIII, HIV, and AIDS in hemophiliacs: An analysis of their relationship. Genetica 1995; 95:25-50.