, .. Neutralizing Antibody to Herpes Simplex Virus Type 1 in Patients with Oral Cancer 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 Herpes simplex virus type 1 (HSV-1), type 2, and measles virus was IQeasured in the serum of patients with oral cancer, patients with oral leukoplakia, 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 antigens 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 infections have not been The 102 patients in this study were drawn from pa­ tested, and other viruses 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 laboratory 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 squamous cell carcinoma 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 disease 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 infection; 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, palate, 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 ulcers, ( lichen pla,nus, or mucous membrane pemphigoid. 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 ANTIBODIES 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.