sign in sign up
<<
Home , Herpes labialis, Herpes simplex

, ..

Neutralizing to Type 1 in Patients with Oral

EDWARD J. SHILLITOE, BDS, PHD, DEBORAH GREENSPAN, BDS, JOHN'S. GREENSPAN, BDS, PHD, MRCPATH, LOUIS S. HANSEN, DDS, MS, AND SOL SILVERMAN JR, MA, DDS

Neutralizing antibody to type 1 (HSV-1), type 2, and virus was IQeasured in the serum of patients with , patients with oral , and in control subjects who were smokers and nonsmokers. Significantly higher titers to HSV-1 were found in contrQJs who smoked than in controls who did not smoke. Patients with untreated oral cancer had HSV-1 neutralizing titers siQI~Iar to those of the controls who smqked, but those with later stage tumors bad higher titers tha!l those with earlier stage tumors. In patients who were tumor free after treatment for oral cancer, higher antibody titers to HSV-1 were associated with longer survival times. No association was found between clinical status and antibody to measles virus. The data are consistent with a role for both HSV-1 and smoking in the pathogenesis of orid cancer. Cancer 49:2315-2320, 1982.

RAGMENTARY EVIDENCE has accumulated to sug­ neutralizing antib9dies in oral cancer patients, in pa­ F gest a connection between the H~rpes simplex vi­ tients with leukoplakia who are therefore at risk of de­ rus type 1 (HSV-1) and cancer of the mouth. 1 Lehner veloping oral cancer,9 in smokers, who also have an et al. 2 found an increased cell-medi~ted immune re­ incre&sed risk pf oral cancer, 10 and in control subjects sponse to HSV-1 in patients having oral leukoplakia who do not smoke. For comparison we also measured with epithelial atypia as compared with patients having antibody to measles virus, which has not been associated oral leukoplakia without atypia, or control subjects. with any human cancer. The results were comp~red Ma~ignant change was accompanied by a specific fall with the clinical and histologic status of each patient, in the response. 3 Oral cancer patients may have cir­ and a prospective study has been started. culating antibody to tumor-associated derived 4 5 from HSV-1, • but the tumor specificity of such anti­ Materials and Methods gens is in doubt because independent efforts to confirm 6 7 8 Patients their specificity have failed, • • sera from patients with acute or recurrent herpetic have not been The 102 patients in this study were drawn from pa­ tested, and other have not been used as controls. tients attending the Oral Medjcine clinic at the Uni­ In fact, it is not well established whether patients with versity of California, San Francisco. This clinic is part oral cancer have higher or lower antibody responses to of a regional center for the treatment of head and neck HSV when standard tests, such as virus neu­ cancer that serves all of Northern California. All pa­ tralization, are applied. tients with head and neck cancer who attend the med­ This communication describes HSV-1 and HSV-2 ical center are examined in this clinic before treatment is begun, and are recalled at least twice a year after From the Department of Oral Medicine and Hospital Dentistry, treatme~t is concluded. About 50 patients with un­ School of Dentistry, University of California, San Francisco, Cali- treated oral are seen per ye~r. fornia. · Supported by the Cancer Research Coordinating Committee of the Consecutive patients were entered into this study for University of California, by grant RR05305-18 from the Biomedical up to one year, until enough had been included: 1\bout Research Support Grant Program, and by NIH grant 1 ROl one-third of the patients in the series were excluded DE05330-0l. because of a history of malignant at another Edward J. Shillitoc is ~ecipient of Research Career Development Award No. 1 K04 DE 00088-01. site, treatment with immunosuppressive drugs for any Address for reprints: Edward J. Shillitoc, BDS, PhD, S-653, Uni­ reason, or a history of recurrent herpetic ; the versity of California, San Francisco CA 94143. The authors thank Belma A. Enriquez for technical assistance. patients were otherwise unselected. Subjects were di­ Accepted for publication March 23, 1981. vided into these five groups:

0008-543X/82/0601/2315 $0.80@ American Cancer Society 2315 ..

2316 CANCER June 1 1982 • Vol. 49

512 (1) Oral cancer, untreated. Twenty-one patients had untreated primary squamo~s cell carcinoma of the tongue, , floor of mouth, or gingiva. 256 ...... -- Their ages ranged from 34-76, with a mean of 59.5 years. There were 14 q~.en and seven women, 128 ~ ... •• -·· ... . •• and 20 (95%) were cigarette smokers. The stag€l of each tumor was a~sessed according to the method of the Americ;an Joint for li4 ...... Com~ittee + 11 1/HS¥~1 Cancer Staging and Epd Results reporting. Tltor (2) Oral cancer, treated. Nineteen patients had be~n 32 t • • t f - treated in the past for oral squamous cell car- - cinoma at sites-comparable to those of the un- 16 treated patients, and had remained tumor-free since treatment was completed. Treatment had + consisted of surgery, radiatiop therapy, or both. These patients were in good health at the time • of testing and were ranged ip age from 26-72, Dr1l C1ncer Dr1l C1ncer Loukopllktl Non·SIIIIIktng Smaktng Untr ..tod Trutod Controls Controls with a mean of 59.6. Twelve were men and seven

Z51i were women. Sixteen (84%) had been cigarette smokers when the tumor was diagnosed, at w~ich time six had then quit. 128 .. • •• (3) Leukoplakia. The WHO definition of leuko- plakia was adopted-namely, a white plaque of 64 • • •• the oral mucos!l that cannpt be characterized 9 1/HSV-2 -- - clinically or histologically as any other disease. Titer A group of 21 patients with this condition was 32 .. -·· ·- - -· studied; their ages ranged from 36-84 years with a mean of 63 years. Niqe were men and 12 were 16 !'"- women. Eight were cigarette smokers, one was l -· -· =F a pipe smoker, and four had quit smoking several + years previously. At the time of diagnosis, there- • t ..... - + fore, 62% were smokers. (4) Dr1l Concer Oral C1ncer Leukopli~i• llan-...,king Smoking Controls whopid not smoke. A group of 21 non- Untrutod Tl'!lited Controls Controls smokers were assembled from patients attending the same clinic as the cancer patients. None had ever'"'been smokers. They were ultimately diag- i I nosed as having nonmalignant conditions unre- 204 .. - -- .. - lated to virus infections, such as aphthous , ( lichen pla,nus, or . 1024 ...... • Ages ranged from 30-94, with a mean of 54.8 \ - ·-· years; nine were men and 12 were women. I I (5) Controls who ·were smokers. A group of 20 cig- 512 .... I -:1: I 1/Heaslos arette smokers was assembled in the same way Titer f ·+ T as were the nonsmokers. Ages ranged from 28- 256 - •• - •• 66, with a mean of 45.4 years. There were 12 :r men and eight women. ~ 128 • ••• The majority of patients in each group were social ·-· - - drinkers. One patient with untreated oral cancer was l an alc;oholic, but there were no other differences in 64 ••• drinking habits among the various groups. Serum was l <64 .. i Ural Concer Dr1l Concer Leukophkh Non-sa~~ktng Sllaking obtained from each patient and was stored at -20 C ' Untreated . Treoted Control,. Controls I before testing. I FIG. 1. Neutralization titers to HSV-1 (top), HSV-2 (center), and measles virus (bottom) of serum from patients with untreated oral Virus Neutralization Tests can~;er, treated oral cancer, and leukoplakia; controls who were non- smokers; and control subjects who smoked. Bars represent the geo- HSV-1 (14-012 strain), HSV-2 (333 strain), and l metric mean titer of each group ± 1 standard error of the mean. measles virus (Edmunston strain) were obtained from No. II HERPES IN ORAL CANCER • Shil/itoe et al. 2317

Dr. F. Rapp (Hershey, PA). Each virus was propagated 256 .... ••• in Vero cells until the cytopathic effect involved 80% of the monolayer. The cells were scraped into the me­ 12 dium and disrupted by ultrasonic vibration, and debris • was pelleted by centrifugation at 1000g for 15 min­ utes. The supernatant was stored in 0.5 ml volumes at 64 ••• + -70 c. 1/HSV-1 Serum from each patient was inactivated at 56 C for Tfter 1 hour and serially diluted in Hank's Balanced Salt 32 •• Solution,_starting at a dilution of 1:8. Equal volumes 3 t of diluted serum and virus at i X 10 plaque-forming unitsfml were mixed and allowed to stand at room tem­ 16 perature for 1 hour. The mixture was plated in 0.2-ml volumes onto monolayers of Vero cells in 60..:mm Petri • dishes and left to adsorb for 1 hour. The dish was then <8 overlaid_with Hank's Balanced Sait Solution containing 1-11 'III-IV 2% fetal calf serum, 0.5% methylcellulose, and 2.25 gf Tumor staye liter of sodium bicarbonate, and incubated for four days at 37 C. The cells were then fixed, stained with crystal 256 • violet, and plaques were counted. The antibody neu­ tralization titer was taken to be the highest serum di­ lution that reduced plaques by over 50%, compared with 128 •• plaques in dishes with mediu~ in place of the patients' serum. The ratio of HSV-2 antibodies to HSV-1 anti­ 64 •• • bodies (the II/I ratio) was caiculated by the method 1/HSV-2 of Rawls et al. l'2 The neutralization end point was de­ T1ter termined by the method of proportional differences, 13 32 •• and the ratio of the reciprocals of the end points was calculated. 16 Statistical Analysis +· t • Data were analyzed by the two-tailed t test for un­ ~

Results 1024 •• • Neutralization titers to HSV-1 were essentially the same In untreated cancer patients, treated cancer pa­ 512 tients, and leukoplakia patients, although control sub­ 1/Heosles jects who smoked had slightly higher antibody titers T1ter t= (Fig. 1). Controls who did not smoke had titers that 256 •• were significantly lower than those of the controls who f. were smoker.s (P < 0.001 ), the untreated cancer patients (P < 0.02), and the leukoplakia patients (P < 0.02, Fig. 12H •• ••• 1). The difference between nonsmokers and smokers could be mainly attributed to antibodies to HSV-1 64 rather than to HSV-2 because the II/I ratio was 0.84 • (±S:E. 0.047) in the ffonsniokers and 0.67 (±0.054) in <64 1-11 III-IV the smokers (P < 0.05). Neutralization titers to measles Turuor staye virus were generally higher than titers to HSV-1 (Fig. FIG. 2. Neutralization titers to HSV-1 (top), HSV-2 (center), anil ., i ). Controls who smoked had the lowest titers to measles measles virus (bottom) of serum from patients with untreated oral \virus, but the difference from the controls who did not cancer, arranged according to the stage of the tumor. - --·-·---

t..

',•

2318 CANCER June 1 1982 Vol. 49

~5 • • smoke was not statistically significant (t = 1.5), and no \ other differences among the various groups were seen. ' In the untreated oral cancer patients, higher HSV- 4 • 1 titers were found in patien~s with Stage III and IV tumors than in patients with Stage I or II tumors (Fig. 2). However, the difference did not reach the 5% level 3 • of significance. A similar relationship was seen with YEARS HSV -2 but was much weaker, suggesting that the effect • was due to HSV -1 antibodies. No such relationship was 2 • • seen when measles virus antibodies were examined. The • mean .II/I ratio in the unt~eat~d oral cancer patients • • was 0. 79 ± 0.05, and no difference in the ratio was de- 1 • • • • tected among patients with tumors of different stages . ,0.5 • • In patients who had been treated for oral cancer, higher HSV -1 neutralization titers were associated with 1/HSV-1 Titer longer tumor-free survival times (r = 0.61, P < 0.01, Fig. 3 ). A very similar relationship was seen with HSV- )5 • • 2 (r = 0.61, P < 0.01), but no such relationship was found with measles virus antibody (r = 0.12, Fig. 3 ). Histologic examination of biopsy specimens from the 4 • patients with leukoplakia showed appearances that var- ied from hyperkeratosis in some cases to severe dyspla- sia in others. However, no association between HSV-1 3 • antibody titer and histologic appearance was found. In YEARS • patients with carcinoma there was no association be-

2 tween degree of differentiation of the tumor and HSV- •• 1 neutralization titer . • • Discussion • • • ,o.s • • The titer of neutralizing antibody to HSV -1 is influ- enced by several factors. People who have never ~ad a •, 1/HSV-2 Titer primary infection do not have neutralizing antibody. I ~5 •• Those who have had a primary infection do have an- tibody, and those repeatedly exposed to virus ,

as from recurrent infections, have antibody of higher ( 4 • titers. 15 In animals, malignant cells transformed by HSV also induce an antibody response. 16 In this study we excluded any individuals with a history of recurrent ( 3 • • herpetic infection; therefore, HSV -1 antibody differ- \ YEARS ences were likely to be the result of exposure to HSV I antigens, either from symptomless reactivation of a la- 2 • • tent infection or from malignant cells expressing HSV ( • antigens. Because there is extensive antigenic cross- reactivity between HSV-1 and HSV-2, we tested the • •• ~ • • • specificity of the antibody by measuring antibody to \ ,0.5 • HSV-2 and to measles virus as controls. Measles virus was chosen because it is unrelated to the herpesviruses, \I

1/~leasles Titer has not been associated with any human , and I is widespread in the population. I FIG. 3. Tumor-free su!Vival time in patients who had been treated for oral cancer, shown with respect to their neutralizing antibody titer HSV -1 antibody titers were significantly higher in to HSV-1 (top) ~ HSV-2 (center), ·and measles virus (bottom). controls who smoked than in controls who did not. This J ' ..

.

No. II HERPES ANTIBODIES IN ORAL CANCER Shillitoe et a/. 2319

confirms the earlier observations of Smith et a/., 17 who of patients,9 being higher when epithelial dysplasia is used an immunofluorescent technique. In addition, we present. Although Lehner et a/. 2 found increased cell­ found that the II/I index was lower in smokers than mediated immune responses to HSV-1 in patients with in nonsmokers, showing that the difference between the dysplasia, we found no association between HSV-1 an­ groups was due probably to antibody to HSV-1 rather tibody titer and the histologic appearances. Continued than to HSV-2. The difference was not the result of any observation of these patients will show if the rate of nonspecific increase in immunoglobulin levels, because malignant change is associated with high or low anti­ the smokers bad lower levels of antibody to measles body titers, or with changes in the titer. virus (caused perhaps by the immunosuppressive effects This study provides further evidence to support a link of smoking18 ). The reason for the increased HSV-1 an­ between HSV -1 and oral cancer, although the high tibody level in smokers is unknown, although it bas been of antibody in noncancer patients, including suggested that epithelium made hyperkeratotic by ci­ nonsmokers, makes careful interpretation of the data garette smoke could serve as a focus of HSV-1 repli­ necessary. More specific information may come from cation.17 An alternative explanation is that an irritant prospective studies, studies of antibody to purified HSV effect of cigarette smoke reactivates a latent HSV-1 antigens, and studies of immunoglobulin classes of an­ infection, leading to symptomless shedding of the virus tibody. These are now in progress. into the mouth with boosting of the antibody titer. Shed­ ding of HSV onto mucous membranes can occur with­ out signs or symptoms, 19·20 although it is unknown REFERENCES whether this happens more frequently in the mouth of I. Shillitoe EJ, Silverman S Jr. Oral cancer and herpes simplex smokers. Preliminary evidence in animals bas shown virus-a review. Oral Surg 1979; 48:216-224. that polycyclic aromatic hydrocarbons related to those 2. Lehner T, Wilton JMA, Shillitoe EJ, Ivanyi L. Cell-mediated of cigarette smoke can induce HSV-1 shedding in immunity and antibodies to herpesvirus hominis type I in oral leu­ 21 koplakia and _carcinoma. Br J Cancer 1973; 27:351-361. mice. If the oral epithelium is exposed simultaneously 3. LehnerT, Shillitoe EJ, Wilton JMA, Ivanyi L. Cell-mediated to HSV-1 and cigarette smoke, this might be a potent il:nmunity to Herpesvirus type I in carcinoma and pre-cancerous le­ carcinogenic combination. Southam et a/. 22 have shown sions. Br J Cancer (Suppl) 1973; 28:128-134. 4. Sabin AB, Tarro G. Herpes simplex and herpes genitalis viruses a cocarcinogenic interaction between HSV and a hy­ in etiology of some human cancer. Proc Nat/ Acad Sci USA 1973; drocarbon carcinogen, and numerous other virus-chem­ 70:3225-3229. ical combinations are more carcinogenic than either 5. Hollinshead AC, Lee 0, Chretien PB, Tarpley JL, Rawls WE, 23 24 Adam E. Antibodies to herpesvirus nonvirion antigens in squamous agent alone. • carcinomas. Science 1973; 182:713-715. Patients with untreated oral cancer were smokers in 6. Sabin AB. Herpex simplex-genitalis virus nonvirion antigens and all but one case, and had antibody titers to HSV-1 their implication in certain human cancers: unconfirmed. Proc Nat/ Acad Sci USA 1974; 71:3248-3252. within the range of the controls who smoked. However, 7. Shillitoe EJ, 'Tarro G, Lehner T. Cell-mediated immunity to those with larger tumors had higher HSV-1 antibody herpes simplex virus types I and 2 antigens in leukoplakia and car­ titers. This is parallel to the situation in cervical car­ cinoma in man. Oncology 1976; 33:192-195. 8. Notter MFD, Docherty JJ. Comparative diagnostic aspects of cinoma, where patients with more advanced tumors herpes simplex virus tumor-associated antigens. J Nat/ Cancer lnst have higher antibody levels to HSV-2,25·26 and might 1976; 57:483-488. be due to HSV antigen expression by tumor cells. Mter 9. WHO Collaborating Centre for Oral Precancerous . Definition of leukoplakia and related lesions: an aid to studies on oral treatment for oral cancer, long-term survivors had precancer. Oral Surg 1978; 46:518-539. higher HSV-1 titers than short-term survivors. This 10 . Wynder EL. Etiological aspects of squamous cancers of the association was not found with measles antibodies and head and neck. JAMA 1971; 215:452-462. 11. American Joint Committee for Cancer Staging and End-Re­ therefore does not reflect nonspecific immunocompe­ sults Reporting. Manual for Staging of Cancer, 1977. Chicago: tence. Two explanations are possible: either HSV-1 an­ AJCCS, 1977; 27-48. tibody levels fall after removal of the tumor and then 12. Rawls WE, Iwamoto K, AdamE, Melnick JL. Measurement of antibodies to herpesvirus types 1 and 2 in human sera. J lmmunol rise progressively, or else antibody levels are stable and 1970; 104:599-606. patients with higher titers have a better prognosis. A 13. Reed LJ, Muench H. A simple method of estimating fifty per prospective study is now in progress to find out which cent endpoints. Am J Hygiene 1938; 27:493-497. 14. Zar JH. Biostatistical Analysis. Englewood Clift's, NJ: Pren­ explanation is correct, but it may be relevant that in tice-Hall, 1974; 105-106 (sec. 9.3), 243-244 (sec. 18.6). , antibodies to HSV-2 rise in those pa­ 15. Lopez C, O'Reilly RJ. Cell-mediated immune responses in re­ tients who remain tumor free after treatment.25·26 current herpesvirus infections. J lmmunol 1977; 118:895-902. 16. Dull' R, Rapp F. Oncogenic transformation of hamster embryo Leukoplakia transforms into oral cancer in some cells after exposure to inactivated herpes simplex virus type I. J Virol cases. Reported transformation rates range from 3-30% 1973; 12:209-217. \ '·

2320 CANCER June 1 1982 Vol. 49

17. Smith HG, Horowitz N, Silverman NA, Henson DE, Chretien Petropulos SF. Enhancement of responses to chemical carcinogens by PB. Humoral immunity to herpes simplex viral-induced antigens in nononcogenic viruses and antimetabolites. Prog Exp Tumor Res 1969; smokers. Cancer 1976; 38:1155-1162. 11:194-212. 18. Finkle!l JF, Hasselblad V, Riggan WB, Nelson WC, Hammer 23. Casto BC, Pieczynski WJ, Janosko N, DipaoloJA. Significance \ Dl, Newill VA. Cigarette smoking and hemagglutination inhibition of treatment interval and DNA repair in the enhancement of viral \ response to influenza after natural disease and immunization. Am Rev transformation by chemical carcinogens and mutagens. Chem Bioi Respir Dis 1971; 104:368-376. Interact 1976; 13:105-125. 19. Douglas RG Jr, Couch RB. A prospective study of chronic 24. Fisher PB, Weinstein IB, Eisenberg D, Ginsberg HS. Inter­ herpes simplex virus infection and recurrent in humans. actions between adenovirus, a tumor promoter, and chemical carcin­ J lmmuno/1970; 104:289-295. ogens in transformation of rat embryo cell cultures. Proc Nat/ Acad 20. Adam E, Kaufman RH, Mirkovic RR, Melnick JL. Persistence Sci USA 1978; 75:2311-2314. ·· of virus shedding in women after recovery from herpes 25. Heise ER, Kucera LS, Raben M, Homesley H. Serological genitalis. Obstet Gyneco/1979; 54:171-173. response patterns to herpes virus type 2 early and late antigens in 21. Kao RT, Shillitoe EJ. Interaction between herpes simplex virus cervical carcinoma patients. Cancer Res 1979; 39:4022-4026. and DMBA in epithelial tumorigenesis. J Dent Res (Special Issue A) 26. Christenson B, Espmark A. Long-term follow-up studies on 1980; 59:332 (Abstract). herpes simplex antibodies in the course of cervical cancer. Patterns 22. Southam CM, Tanaka S, Arata T, Simkovic D, Miura M, of neutralizing antibodies. Am J Epidemio/1977; 105:296-302. I

( (