A Search for Herpes Simplex Virus Type 2 Markers in Cervical Carcinoma

[CANCER RESEARCH 36, 795-799, February 1976]

Robert J. Goldberg 2 and Maneth Gravell

Viral Oncology, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland 20014

Summary tually facilitated the identification of several cultural and sexual factors associated with increased risk (35, 61). Thus, Carcinoma of the uterine cervix is now clearly recognized it became clear that the pivotal demographic and epidemio- as the second most common malignant disease of women logical characteristics that distinguished women at greater in the United States. Epidemiological studies provided the risk to developing cervical cancer were low socioeconomic first suggestive evidence that an infectious, venereally trans- status, early age of 1st coitus, and number of sex partners. mitted agent was involved in this disease in either a causal The suggested etiological involvement of a venereally trans- or a casual fashion. Later cytohistopathological, virologi- mitted agent in cervical carcinoma was a natural extension cal, and seroepidemiological studies confirmed this obser- of these studies and it was not surprising that such specula- vation and identified the suspect agent as herpes simplex tion included the possible involvement of a DNA virus (15, virus type 2. Recently, several laboratories have directed 60). During the last decade, a virtual plethora of evidence their energies towards establishing lines of direct evidence has emerged which unequivocally associates HSV-23 with linking herpes simplex virus type 2 etiologically with human human cervical carcinoma. These studies have utilized the cervical carcinoma. Some of these approaches have in- disciplines of cytohistopathology, virology, seroepidemiol- volved attempts to detect infectious virus, viral compo- ogy, immunology, and molecular biology. The seroepidemi- nents, or virus-specific modifications in neoplastic cervical ological studies have been extensively reviewed (35, 43, 55) tissues. Results obtained utilizing human tissues will be and will be discussed elsewhere in this Symposium. This reviewed and discussed. discussion will focus on those studies which have dealt with the detection of infectious HSV, subviral components, or virus-specific modifications in anaplastic human cervical The 2nd most common malignant disease of women in tissues. the United States is now clearly recognized as carcinoma of The isolation of HSV from the primary lesions of 3 cases the uterine cervix. Aithoughthere are approximately 35,000 of acute vulvovaginitis by Slavin and Gavett (64) in 1946 new cases of cervical carcinoma and 10,000 deaths re- provided the 1st evidence for herpesvirus infection of the ported annually, the incidence of anaplastic cervical pathol- human female genitalia. Neutralizing antibody was detected ogy is surely much higher when one considers the myriad in the convalescent, but not acute, sera of the 3 patients, cases of cervical dysplasia that may or may not progress to and evidence suggesting a venereal mode of transmission frank carcinoma. The mortality statistics from this disease was detected in 1 case. This report had signal importance in would be far more depressing were it not for the test devel- explaining cervicovaginal cytological findings reported oped in 1943 by Papanicolaou and Traut (53) correlating the later, since it clearly described the ability of HSV to initiate cytopathology of exfoliated cervical cells with the histologi- infection and induce pathological lesions on the female genitalia. The genital cytological findings were heralded cal diagnosis of cancer. In a historical sense, the Papanico- by the demonstration of characteristic HSV-induced se- laou (Pap) test was the sine qua non for much of our present understanding of cervical carcinoma. In addition to provid- quential cytomorphic changes in cells of primary or recur- ing an invaluable tool for early diagnosis and posttreatment rent nongenital lesions (10). Although similar changes were monitoring, this test furnished the 1st practical means of reported in cervicovaginal smears (36, 69), convincing evi- studying the pathogenesis, epidemiology, and etiology of dence relevant to the involvement of HSV in their etiology cervical carcinoma on a large scale. was not presented until 1963. It was at that time that Stern Fortunately, the epidemiology of cervical carcinoma has and Longo (65) described characteristic HSV cytological received considerable attention for over 30 years. An excel- features in vaginal and cervical Pap smears from a case of lent perspective on the key findings in this area has been acute vulvovaginitis. More importantly, the cytological diag- presented by aotkin (61). In spite of the lack of total agree- nosis was supported by virus isolation and by the demon- ment among various investigators regarding individual as- stration of an elevated titer of neutralizing antibody in the pects of these studies, certain trends emerged that even- patients' convalescent phase serum. The efficacy of cytolog-

' Presented at the symposium "Immunological Control of Virus-associated 3 The abbreviations used are: HSV-2, herpes simplex virus type 2; HSV, Tumors in Man: Prospects and Problems, April 7 to 9, 1975, Bethesda, Md. herpes simplex virus; HSV-1, herpes simplex virus type 1; cRNA, complimen- = Presenter. tary RNA; EBV, Epstein-Barr virus.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1976 American Association for Cancer Research. R. J. Goldberg and M. Gravell ical techniques in detecting genital herpes infections was changes, and to distinguishing between primary and recur- later corroborated in similar, although more extensive, hu- rent infections (45, 49). In this respect, it is pertinent that man (49) and murine (44) studies. Successful viral isolations approximately 5 to 14% of genital HSV isolates were typed were nearly always (>90%) paralleled by positive cytological as HSV-1 (34, 39, 40). A comparison of the data from previ- findings, while there was a 65 to 75% correlation by the ously discussed studies indicates that proced u res for detect- reverse comparison (44, 47-49). However, infectious virus ing genital HSV infections decrease in sensitivity according (3) and HSV structural antigens (2, 7) have not been identi- to the order serological > virological > cytological > clini- fied in cervical specimens taken between recrudescences of cal examination. Histological diagnosis of cervical HSV in- cytologically detectable herpetic cervicitis. Thus, genital fection has been reported to be more difficult than cytologi- HSV mimics the classic picture of viral latency displayed by cal diagnosis since it appears to depend on the time at HSV-1 in nongenital tissues (17, 66). which specimens are obtained, the origin and size of the It is now known that herpes genitalis is a common vene- specimens, and the thoroughness with which the tissues real disease (41,42, 49, 56) that shares several epidemiologi- are examined (49). However, differentiation of herpetic from cal characteristics with cervical carcinoma (30, 31). The anaptastic cytomorphic modifications was sometimes eas- clinical features and epidemiological patterns of the infec- ier to resolve by histological rather than cytological exami- tion have been reviewed recently (34, 43). The observation nation when both types of changes appeared in the same that HSV isolates of facial-oral (HSV-1) and genital (HSV-2) specimen (47, 49). Review of biopsy or hysterectomy speci- origin possessed different serological, biological, and physi- mens, as opposed to cervicovaginal smears, also permitted cal properties was particularly significant (20, 40, 54, 63). localization of the respective changes relative to particular An overwhelming majority of the cases of herpes genitalis, cell types and associations within tissues. including herpes cervicitis, have been shown to be caused Taken together, these facts point to a rather restricted use by HSV-2 (20, 32, 40).. of cytohistopathological techniques for detecting genital The 1st evidence associating HSV with genital cancer HSV-2 and for studying its relationship to cervical carci- came from simultaneous screens of Pap smears for anaplas- noma. Cytohistopathological techniques, however, are ex- tic and herpetic cytological markers (47). In this and later tremely useful as diagnostic tools to identify individuals studies (1, 9, 29, 48-50, 52, 70), an increased incidence of with an increased risk of developing cervical cancer. Pap cervical anaplasia was noted in women with cytologically smears offer the simplest, least expensive method currently detectable genital herpes virus infections. These observa- available to screen large numbers of women for evidence of tions stimulated extensive seroepidemiological studies that cervical neoplasia. It would seem a pity to exclude simulta- largely confirmed the associative relationship between HSV neous examination of smears or tissue sections for HSV and cervical anaplasia (35, 43, 55). In spite of inherent markers since HSV-2 infection has been highly associated difficulties related to the serological specificity and cytohis- with cervical cancer. topathological interpretation of the tests, women with inva- The search for HSV markers in cervical tumor cells has sive cervical carcinoma, carcinoma in situ, or cervical dys- been extensive. Infectious virus, viral structural antigens, plasia tended to have higher titers of HSV-2-neutralizing and HSV-specific cytoplasmic changes have not been de- antibodies than controls. In addition, women with cervical tected directly in cervical cancer biopsies (2, 5, 7). However, anaplasia showed an increased frequency of genital herpes- virion structural antigens were observed in exfoliated tumor virus infection as determined by serological, rather than cells and tumor cells on the periphery of neoplastic lesions cytological, examination. This finding is consistent with the (2, 7, 62). The observed serological reactivity seemed to be ability of serological analyses to detect evidence of both directed against virion proteins made relatively late in the past and recurrent infections and with the often latent na- HSV-2 growth cycle. Fluorescence-positive anaplastic cells ture of the infection when the patient is clinically and cyto- contained no virus particles but did exhibit cytological logically asymptomatic (30, 32). changes similar to those induced by HSV. There was no Utilization of cytopathological techniques to obtain evi- correlation between the stage of the disease and the num- dence for an etiological relationship between genital HSV ber of HSV antigen-positive cells. Extensive control studies and cervical anaplasia has led to an awareness of the advan- pointed to the specificity of the immunofluorescent tech- tages and limitations of this approach. Detection of cytologi- niques used for both HSV-2 and cervical carcinoma cells. cal changes induced by genital HSV is entirely dependent Further evidence for the persistence of the HSV genome on the ability of the observer to recognize and differentiate in cervical tumor cells was garnered from in vitro experi- specific HSV-induced changes from nonspecific cyto- ments in which infectious virus, viral antigens, and charac- morphic alterations. Nonspecific changes include multinu- teristic HSV nuclear alteration were detected in sponta- cleated endocervical repair, trophoblastic, anaplastic, and neously degenerating cell cultures derived from a carci- foreign-body giant cells (49) and genital cells infected with noma in situ (3, 8). Changes similar to those appearing lymphogranuloma venereum (37), condyloma acuminata spontaneously could be induced by exposure of the cells to (24, 46), and cytomegalovirus (45). Although HSV and vari- medium of high pH, but they were undetected in cultures cella-zoster virus induce practically identical cytological showing no signs of degeneration. A culture derived from changes (10), the latter virus has not been isolated from the an invasive cervical carcinoma behaved quite differently (3). cervix. Other limitations relate to the difficulty of detecting Virus expression was limited to 2% of the cells and ap- infection between periods of recrudescence, to differentiat- peared as a membrane fluorescence that was detected with ing between HSV-1- and HSV-2-induced cytological human sera containing antibody to either HSV-2 or HSV-I.

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Spontaneous or high pH-induced cell degeneration failed to in this tumor was determined to be covalently linked to host augment virus expression in this culture. cell DNA. In yet another approach, soluble membrane antigens In contrast to the above results, other investigators have were extracted from cervical, vaginal, and vulvar carcino- failed to demonstrate HSV-2 DNA in cervical carcinoma mas and were shown to react in complement fixation tests biopsies, zur Hausen et al. (73) found no HSV DNA in 10 with an antiserum prepared against partially purified HSV-2 cervical carcinomas examined with cRNA (probes synthe- (27). Interpretation of these results is difficult since data sized from HSV-1 or HSV-2 DNA with E. coli RNA polym- relevant to the recovery of infectious HSV from the speci- erase). It was also reported (73) that both Schulte- mens prior to extraction or to the genital HSV status of the Holthausen and Petersen, also using HSV-2 cRNA as probe, patients at the time of tumor removal was not reported. In failed to find HSV-2 DNA in approximately 30 cervical tumor later studies, the nature of this serological reactivity was biopsies. As acknowledged by zur Hausen et al. (73), re- defined by showing that soluble membrane antigens from sults obtained with cRNA do not negate results obtained by cervical carcinomas fixed complement in the presence of the more sensitive assays used by Frenkel et al. (23) since antibody directed against HSV nonvirion antigens (28). The E. coli RNA polymerase may not synthesize RNA transcripts nonvirion antigens used in these tests were prepared from of the complete HSV-2 genome. Thus, if only fractional HSV-infected cultured mammalian cells and were shown to HSV-2 genomes are present in tumor cells, they will go un- contain no immunologically demonstrable virus structural detected unless RNA transcripts complementary to these components. In addition, sera from cervical cancer patients sequences are present in the probe, cRNA probes have with no anti-HSV structural antigen activity were reported to been used successfully to detect EBV DNA in lympho- react in complement fixation tests with partially purified blastoid cell lines, in Burkitt's and nasopharyngeal tumors, HSV nonvirion antigens. Antibodies to HSV nonvirion anti- and in cells from infectious mononucleosis patients (51, gens were also detected in a large percentage of patients 71-73). The sensitivity of the cRNA hybridization assay with squamous cell carcinomas of the head and neck (26). for detecting EBV DNA has been estimated to be between 2 In similar, although more extensive studies, human sera and 3 genome equivalents/cell (71). However, it should be from cervical cancer cases and matched controls were kept in mind that the complete EBV genome is present in screened for reactivity with antigens that appeared in cul- cells of EBV-associated diseases (Burkitt's lymphoma, naso- tured human cells 4 hr after infection with HSV-2 (4, 6). This pharyngeal carcinoma, and infectious mononucleosis) or in antigen, called AG-4, appeared to be distinct from those cells transformed in vitro by EBV, a situation not found for viral structural proteins involved in neutralization. Antibody HSV in cervical tumors and in vitro-transformed cells. Using to AG-4 was detected in women with cervical neoplasia and the more sensitive DNA-DNA renaturation kinetics tech- displayed a pattern of increased prevalence in patients with nique, it has been reported (73) that Pagano was also una- more advanced stages of the disease. Antibody to AG-4 was ble to detect evidence for HSV DNA in biopsies of 5 cervical not detected in the majority of matched controls, in patients tumors. Hence, to date, only 1 cervical carcinoma biopsy who had undergone successful therapy for cervical carci- has been reported to contain nucleic acids homologous to noma, and in women with carcinomas at other sites. AG-4 those of HSV-2. reactivity was also shown to be present in extracts of cervical cancer biopsies and in cell cultures derived from 2 Discussion different cervical carcinomas. In this communication a large body of information has By use of molecular hybridization techniques, Frenkel et been reviewed concerning the relationship of HSV-2 to squa- al. (23), and Ro~zman and Frenkel (59) have obtained evi- mous cell carcinoma of the human uterine cervix. A definite dence for the presence of HSV nucleic acids in 1 cervical pattern of association between HSV-2 and cervical carci- carcinoma biopsy. Analysis of the kinetics of hybridization noma has emerged from these studies, but etiological in- in liquid of purified radiolabeled HSV DNA with excess volvement has not been proven. Seroepidemiological stud- unlabeled RNA from the tumor yielded evidence that the ies in which higher titers of neutralizing antibody to HSV-2 tumor contained RNA transcripts complementary to only 5% have been found in sera of cancer patients compared to of HSV-2 DNA. Preliminary results further indicated that matched controls have provided the strongest associative both early and late HSV-2 functions were expressed by the evidence. More direct approaches, however, have met with 5% of the genome transcribed and that about one-half of the only qualified success. sequences transcribed were shared in common by HSV-1 A major question that needs clarification is whether HSV- and HSV-2 DNA. 2 genetic information is present in all cervical tumor cells The same cervical tumor biopsy was also examined for and, if so, in what state. As discussed previously, infectious DNA sequences homologous to those found in HSV-1 and HSV-2 was isolated from a cell culture derived from a carci- HSV-2 DNA (23, 59). Evidence obtained from the rate of noma in situ (3, 8) and HSV-2 membrane antigens were reassociation of labeled HSV-2 DNA in the presence of detected in a small percentage of cells from a culture, not unlabeled DNA from the tumor indicated that only a frag- yielding infectious virus, originating from an invasive cervi- ment consisting of about 39% of HSV-2 DNA was present in cal carcinoma (3). The observed variations in virus expres- tumor cells. Because of the uncertainty of the exact ploidy sion in these cultures may simply reflect the selection in of cells from this tumor, the DNA fragment was calculated to vitro of cell populations not representative of the respective be present at from 1 copy/heteroploid cell to 3.5 cop- tumors. Nevertheless, the isolation of infectious HSV-2 from ies/diploid cell. At least a portion of the HSV-2 DNA present cultured cells of a carcinoma in situ certainly suggests that

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1976 American Association for Cancer Research. R. J. Goldberg and M. Gravell the complete viral genome persisted in these cells. Two ence of HSV-2 RNA transcripts in cervical tumors and in possibilities could explain the failure to isolate infectious vitro transformed cells also appear warranted. virus from the invasive cancer: (a) a more stringent control The soluble nonvirion antigens which have been ex- of virus expression at the transcriptional or translational tracted from HSV-2-infected cell cultures and cervical tu- level; or (b) a defective viral genome. It is unfortunate that mors must be characterized more fully. It is unknown cell cloning and molecular hybridization analyses were not whether these antigens are viral-coded products or cellular used to quantitate and identify the HSV-2 genetic informa- products produced as a consequence of HSV-2 infection in tion in these cell cultures. a manner analogous to fetal antigen and fetal thymidine The relative ease with which HSV-2 structural and nonvi- kinase induction in SV40-infected cells (11-13). It would be rion antigens have been detected in exfoliated tumor cells interesting to know whether HSV nonvirion antigens appear and in tumor specimens, respectively, stands in sharp con- in cultured cells infected with other oncogenic and nonon- trast to the limited success enjoyed in detecting HSV-2 cogenic viruses or in chemically transformed cells. Regard- genetic information in cervical tumors. It is presently unless of whether herpes nonvirion antigens are virus or cell known whether the appearance of these antigens reflects coded, continued study appears warranted based on their the release from repression of endogenous HSV-2 genetic potential diagnostic and prognostic significance in cervical information or exogenous infection of susceptible tumor carcinoma. cells by genital HSV. The failure to detect signs of active genital herpetic infection in cervical cancer patients ex- pressing HSV-2 antigens in tumor cells would not unequivo- References cally rule out the latter possibility since infectious HSV-2 has been shown to produce an abortive infection in certain 1. An, S. H. Herpes Simplex Virus Infection Detected on Routine Gynecolog- ical Cell Specimens. Acta Cytol., 13: 354-358, 1969. classes of transformed cells (12, 18, 19, 68). 2. Aurelian, L. Possible Role of Herpesvirus Hominus Type 2 in Human There is a good rationale for proposing that defective, Cervical Cancer. 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FEBRUARY 1976 799

Robert J. Goldberg and Maneth Gravell

Cancer Res 1976;36:795-799.

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