Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3225-3229, November 1973

Herpes Simplex and Herpes Genitalis Viruses in Etiology of Some Human Cancers* (DNA tumor viruses/nonvirion antigens/complement fixation/HeLa cells/HEp2 cells)

ALBERT B. SABINt AND GIIULIO TARRO$ National Cancer Institute Frederick Cancer Research Center, Fort Detrick, Frederick, Maryland 21701

Contributed by Albert B. Sabin, July 5, 1973 The results of complement fixation tests ABSTRACT on 202 sera from people without cancer and from patients with cancer in 29 different areas of the body indicated that only those with nine varieties of advanced cancer (lip, mouth, oropharynx, nasopharynx, kidney, urinary bladder, prostate, cervix uteri, and vulva-all of 56 tested) gave positive specific reactions with nonvirion antigens induced by the DNA herpes simplex (HSV 1) and herpes genitalis (HSV 2) viruses. None of 57 people without cancer (including 10 with current and 18 with recurrent HSV 1 or HSV 2 infections), none of 81 patients with 20 other varieties of advanced cancer (gum, tongue, tonsil, salivary gland, accessory sinus, epiglottis, lung-bronchus, stomach, colon, breast, corpus uteri, ovary, testis, liver, thyroid, Wilms'

embryonal kidney, melanoma, Hodgkin's disease, acute lymphocytic leukemia, and acute myelocytic leukemia), and none of four women with early malignant changes in the cervix uteri gave positive results. The seven patients with advanced cancer of the lip or oropharynx gave positive reactions with HSV 1 but not with HSV 2 nonvirion antigens (compatible with involvement of only HSV 1), all of the 13 women with advanced cancer of the cervix uteri and the one woman with advanced cancer of the vulva gave positive reactions with both HSV 1 and HSV 2 nonvirion antigens (compatible with involvement of only HSV 2), while among the 35 other positive patients only two (one with cancer of the kidney and one with cancer of the bladder) reacted with HSV 1 and not at all with HSV 2 nonvirion antigens. Positive sera failed to react with cells harvested at different times after high-multiplicity infection with the DNA vaccinia virus. Massive absorption of positive sera with trypsinized, uninfected human embryonic kidney cells failed to remove, or lower the titer of, the HSV 1 and HSV 2 nonvirion antibodies. All of these data taken together are interpreted as indicating that HSV 1 and HSV 2 play an etiologic role in certain human cancers, because they provide the kind of Abbreviations: HSV 1, herpes simplex (labialis) virus, subtype 1; HSV 2, herpes simplex (genitalis) virus, subtype 2; GPK, guinea pig kidney; RK, rabbit kidney; HEp2, a human cell line derived from an epidermoid carcinoma of the larynx; PFU, plaque-forming unit. * Preliminary report of data presented at the I10th Annual Meeting of the National Academy of Sciences, Washington, D.C., April 24, 1973. t This study was conducted while I)r. Sabin was a Fogarty Scholar-in-Residence, Fogarty International Center for Advanced Study in the Health Sciences. Reprint requests should be sent to Dr. Sabin, Fogarty International Center, National Institutes of Health, Bethesda, \Id. 20014. t During this work Dr. Tarro was on leave from, and is currently at, Virologia Oncologica, Istituto di Clinica -Medica I, Universita di Napoli, Polichinico, 80138 Napoli, Italia.

3225

evidence by which virus-free experimental cancers can be proved to have been originally induced by such DNA viruses as polyoma, Simian Virus 40, or certain types of adenovirus.

The evidence required for establishing DNA viruses, which cause ordinary infections in animals anid humans, as etiologic or inu(lUcilg agents in cancer is different from that which has established several unique RNA viruses, unassociated with ordinary infections as the (cause of various naturally occurring malignancies of lower animals. Some DNA viruses associated with ordinary infections (i.e., l)olyooma of mice, Simian Virus 40 of rhesus monkeys, anad some types of human and animal adenoviruses) can produce experimental cancers in which infectious virus, 01 infectious DNA, or any of the structural (virion) antigens cannot be detected either dlirectly or by their capacity to induce antibody in the tumor-bearing aniin such cancers, mals. There are howei-er, antigens that are specific for the virus that induced the exlperimental cancer, and tumor-bearing animals develop specific antibodies for them (1, 2). These specific tumor antigens are identical to nonvirion antigens (i.e., not structural comlpoIients of the assembled virus particle) that are synthesized shortly after hioh-multiplicitv infection of suscel)tible cultured cells. even when synthesis of new DNA is prevented by al)l)roi)riate inhibitors (2-7). Phenomena associated with experimental DNA virus cancers were studied in detail with special emphasis on those manifestations that could be important ill the search for evidence of the possible involvement of the well-known DNA viruses of humans, i.e., maintained in nature only by transmission from person to person, in human cancers (8-10). The subsequent search for nonvirion antigens in cells infected with subtypes 1 and 2 of herpes simplex virus (HSV) and the development of methods for identifying them, studying their distinctive properties, and differentiating them from preexisting antigens, whose concentration is increased by both malignant transformation and various viral infections, have been reported (11-13). The preceding studies provided the necessary reagents and methodology for testing human sera for HSV nonvirion antibodies, as well as the important fact that sera from small numbers of randomly selected adults and from people with recent HSV 1 or HSV 2 infections had no HSV nonvirion antibodies demonstrable by comllement fixation (11, 13). Thus, we could proceed with the final Chase of the plan (8-10) to explore the possible role of HSV 1 and HSV 2 in human cancer by determining whether or not the sera of certain

3226

Medical Sciences: Sabin and Tarro

Proc. Nat. Acad. Sci. USA 70 (1973)

TABLE 1. Complement fixation tests w ith HSV 1 and HSV 2 nonvirion antigens and sera of people without cancer No. positive with HSV HSV

No.

Group Current herpesvirus infections HSV 1, 4; HSV 2,6. Recurrent herpetic lesions Labial, 7; genital (HSV 2), 1 1. Pregnant women Last trimester, 9; postpartum, 7. Random adults Total

Zsted

1

2

10

0

0

18

0

0

16

0

0

13 57

0

0

0

0

te

patients might exhibit a specifiic reactivity with HSV in the absence of nonvirion antigens that would not be found i the special cancer or cancers even amorig people of comparable age with frequent recurring infections f or decades. MATERIALS AND MIETHODS cells were used for Cell Lines and Their Cultivation. H] preparation of HSV 1 and HSV 2 virin on antigens for absorption of sera and also for preparation of Very large quantities of HEp2 cells weere obtained from Flow Laboratories, Rockville, Md. either fiully grown in 32-ounce prescription bottles in 90% minimum IEagle's medium + 10% fetal-bovine serum without antibiotic s, or usually as freshly trypsinized cells which we grew oursselves in Eagle's basal medium (GIBCO powder rehydrate d) + 10% unheated, regular calf serum + 0.11% NaHCOa3 for initial growth and 0.22% NaHCO3 later + penicillin ('200 units/ml), streptomycin (0.2 mg/ml), and gentamycin (0.05 mg/ml). Primary GPK cultures were used only for proeparation of HSV nonvirion antigens. Kidneys of 2- to 3-dr Ly-old guinea pigs were trypsinized in warmed 0.25% trypsini in phosphate-buffered saline (20 ml/g of kidney) and thoroughly washed twice with large quantities of cold growth medium in a refrigerated centrifuge. The volume of cell pack was dettermined by centrifugation at 600 rpm for 10 min, and 40 ml c5f a 1:400 suspension by volume prepared in warm growth mediurn was seeded per 32ounce prescription bottle. The growlth medium consisted of 85% of 0.5% lactalbumin hydrolysatEe in Earle's salt solution without NaHCO3 (dehydrated prodiuct of GIBCO, Grand Island, N.Y. 14072) + 5% tryptose phosphate broth + 10% fetal-bovine serum (selected for abse nce of toxicity) heated at 560 for 30 min + 0.035% NaHCO3 + penicillin (200 units/ ml), streptomycin (0.2 mg/ml), and ge ntamycin (0.05 mg/ml). Medium is changed at 2 days (withi 0.035% NaHCO3), at 3-4 days (0.11% NaHCO3), and at 5-6 days (0.22% NaHCO3). Excellent, fully confluent mlonolayers are ready for use 6-7 days after cells are planted iwhen the procedure described is strictly followed. Secondaryr cultures prepared from monolayers of young (150-200 g) RE< cell cultures were used for plaque titration of the potency of the HSV used for highmultiplicity infection of cells for p antigens. Viruses Used, Preparation, and St 'orage. Sabin "Schooler" (HSV 1) and Sabin "Justin" (HSV 2) strains of HSV and the VL strain of vaccinia virus used in tthis work have been de-

cancer

ETHD onp2

cells

used for

nonvirion antlgens.

scribed (11-13). Virus stocks were prepared in HEp2 cells in the logarithmic phase of multiplication, which is optimum for obtaining high yields of virus.

Preparation of HEp2 Cells for Absorption of Virion Antibodies. We wanted to obtain large numbers of infected cells for storage at 40 to inactivate the nonvirion antigens which in HEp2 cells are at peak titers at the end of the infectious process (11, 13). Accordingly, densely packed cell sheets, at 7-8 days after planting 4 X 106 cells per 32-ounce bottle, are infected with about 5 plaque-forming units (PFU)/cell. The same procedure of harvesting, freezing, and sonicating is followed as for preparation of virus stocks except that the 10% cell suspension is prepared in Eagle's basal medium without serum or phenol red instead of in demineralized water, which is necessary to avoid marked anticomplementary activity, and storage is at 4° (±0.50) instead of at -80°.

Preparation of Nonvirion Antigens in GPK and HEp2 Cells. After the medium was decanted from GPK confluent monolayers grown for 6-7 days in 32-ounce bottles, 10 ml of Eagle's basal medium without serum or phenol red, containing preferably about 2 X 108 PFU of HSV, are added. After incubation for 3 hr at 370, the cells are scraped into the medium and

centrifuged as for preparation of stock virus, except that basal medium without serum or phenol red is used to make the 10% suspension. Moreover, after it was frozen and thawed, the suspension was not sonicated directly in the cup but put into a stoppered lusteroid tube which rested in water in the cup (because the volume was often too small and also because that is the way we did it before), and the sonicated suspension was frozen uncentrifuged at -80°. For maximum concentration of nonvirion antigens for complement fixation tests, the uncentrifuged suspension was used the following day or at most within 4 days. When HEp2 cells were used, they were grown and infected as for virus stock and harvested after 24 hr when the cytopathic effect is complete and the concentration of nonvirion antigens is highest (11, 13). The remainder of the process was as for GPK cells. An aliquot of the same lot of uninfected GPK or HEp2 cells was processed in preciselv the same manner to provide the control antigen for the complement fixation test.

Monitoring of Nonvirion and Virion Antisera. Sabin "Schooler" HSV 1 guinea pig 71 serum (11) and Sabin "Justin" HSV 2 guinea pig 91 serum (13) were used, after proper virion absorption, to measure the concentration of HSV 1 and HSV 2 nonvirion antigens, respectively. While HSV 1 nonvirion guinea pig serum detected nonvirion antigens induced only by HSV 1 strains, HSV 2 serum detected nonvirion antigens induced not only by HSV 2 but also by HSV 1 (13). A hyperimmunized rabbit serum prepared by one of us (A.B.S.) in 1958 against the "Schooler" HSV 1 strain, containing an especially broad spectrum of HSV virion antibodies, measured equally well the concentration of virion complement fixation antigen produced by HSV 1 and HSV 2. Human Sera. Most of the sera from patients with advanced provided by Dr. Jack Gruber, Chief, Office of Program Resources and Logistics, National Cancer Institute

cancer were

that was made at the M. D. Anderson Hospital, Houston, Texas in 1967 and stored at - 700 in aliquot amounts since then. Each of these sera came from

largely from a collection

Proc. Nat. Acad. Sci. USA 70

(1978)

a person with histologically confirmed malignancy and evidence of metastasis (25 g or more of primary and metastatic tumor). Some of the random sera from people without cancer were from family or other matched controls of these patients. We thank Dr. Brian E. Henderson of the Department of Pathology, University of Southern California School of Medicine, Los Angeles, Calif., for the following 13 cancer sera used in the present tests: two nasopharynx from Chinese patients, one lip, two corpus uteri, five ovary, and three testis. Dr. John L. Sever and Dr. David A. Fucillo, Perinatal Research Branch, National Institute of Neurological Diseases and Stroke, supplied the sera from women before and after early manifestations of malignancy of the cervix uteri, and also the sera from pregnant women without cancer. We especially thank Dr. A. J. Nahmias, Department of Pediatrics, Emory University School of Medicine, Atlanta, Ga., for sera on patients with recurrent HSV 2 infections he had been studying for many years. These male and female patients ranged in age from 20-64 years (six of them 52 years or older) with recurring lesions on the penis (two), vulva (two), buttocks (five), thigh (one), and finger (one). Four of these patients have had recurrent lesions for 15, 15, 20, and 23 years, respectively, three with four to six recurrences per year and one with 20-30 per year. Among the others there were five with 10-25 recurrences per year. Sera from people with herpes labialis were received from different sources and were from people over 35 years of age. Absorption of Virion Antibody from Human and Guinea Pig Sera before Tests for Nonvirion Antibody. The basic procedures for absorption were described (3, 11). Although storage at about 40 had been found to eliminate HSV 1 and HSV 2 nonvirion reactivity from infected HEp2 cell suspensions between 7 and 9 days and between 12 and 15 days, respectively (11, 13), when 0.1-ml quantities were being tested, the very much larger quantities (100-times or more) required for absorption of the human and guinea pig sera might still contain enough nonvirion antigen at 14 days to reduce the titer of nonvirion antibody in the absorbed sera. Moreover, the original observations (11, 13) were not made under conditions of strict temperature control (an ordinary refrigerator that was opened many times each day had been used); we could not assume that under the conditions of storage used in the present work (an infrequently used walk-in refrigerator carefully controlled and monitored at 40 + 0.50) the nonvirion antigens would be inactivated at the same rate. We indeed found that the standard guinea pig sera gave much lower nonvirion antibody titers when they were absorbed with HEp2 cell suspensions stored at the strictly controlled 40 temperature for 2-4 weeks, while storage for 5-8 weeks yielded higher titers of nonvirion antibody without loss of capacity to remove the virion antibody. A mixture of equal parts of HSV 1 and HSV 2 antigens stored at +40 was used for absorption of virion antibodies from human sera and concurrently for each test from the standard HSV 1 and HSV 2 guinea pig monitoring sera.

Complement Fixation Test. The amount of complement required for the 1.5 exact units to be used in the test was determined by a preliminary titration of complement in the presence of 0.1 ml of the 10% uncentrifuged cell-suspension antigens and of the absorbed human sera that are diluted 1 :4 during virion absorption. A described procedure for the com-

Herpesviruses 1 and 2 and Human Cancers

3227

plement fixation test (11) was used. Each complement fixation test always had positive and negative controls: human sera from people without cancer or with different types of cancer, uninfected cell suspensions representing aliquots of those used for preparation of nonvirion antigens and similarly and concurrently processed and stored, HSV 1 and HSV 2 concentrated virion antigens, titrations to determine the concentration of the nonvirion and virion antigens in the preparations used for the test, titrations of the absorbed monitoring HSV 1 and HSV 2 guinea pig antisera, and, of course, simultaneous complement controls on all the reagents in the test. The sera in any one test were scrambled and numbered by a code contained in a sealed envelope which was not opened until after the results had been recorded. RESULTS People Without Cancer. The data in Table 1 show that none of the sera from 57 people without cancer had nonvirion antibodies at the 1:4 dilution tested. The results on the 10 people with current or very recent HSV 1 or HSV 2 infections have been reported (11, 13). Available evidence suggests that iii recurrent HSV lesions, which appear at the same site year after year, there is derepression and full expression of total viral genome which persists in a repressed state in the cells of the affected area (14). The completely negative results obtained with sera from 18 people with long histories of recurrent HSV 1 and HSV 2 infections indicate, therefore, that special conditions are required for production and persistence of antibodies for the nonvirion HSV antigens. The negative results obtained with the sera of 16 pregnant women indicate that the nonvirion HSV antigens we used to test the sera from cancer patients are free from carcinoembryonic antigens for which such sera may have antibodies. The sera from 13 random adults included a serum (kindly supplied by Dr. Paul Gerber, Bureau of Biologics, Food and Drug Administration, National Institutes of Health, Bethesda, AMd.) that has been extensively used in studies on human Epsteiin-Barr DNA virus because of its broad spectrum of antibodies against "early" and "late" antigens of Epstein-Barr virus. People with Cancer. The data in Table 2 on sera from 137 patients with advanced cancer in 29 different sites of the body are remarkable because uniformly positive results were obtained with 56 sera from patients with the nine listed varieties of cancer, and uniformly negative results were obtained in simultaneous tests with the same reagents with the sera from 81 patients with 20 other varieties of cancer. The fact that the sera of all seven patients with advanced cancers of the lip and oropharynx, areas commonly infected by HSV 1, reacted only with HSV 1 and not at all with HSV 2 nonvirion antigens (Table 3) is remarkably in accord with the observations that high-multiplicity infection of tissue culture cells with HSV 1 strains has induced antigens which on hyperimmunization of guinea pigs produced antibodies that react with HSV 1 and not with HSV 2 nonvirion antigens (13). These results suggest the possibility of a special involvement of HSV 1 in these cancers, unlike that associated with ordinary primary and recurrent infections with this virus. Similarly, the fact that the sera of all 14 patients with advanced carcinoma of the cervix uteri or vulva, areas commonly infected by HSV 2, reacted with both HSV 1 and HSV 2 nonvirion antigens is remarkably in accord with the observations that high-multiplicity infection of tissue cultures with HSV 2, which has in-

Medical Sciences: Sabin and Tarro

3228

Proc. Nat. Acad. Sci. USA 70

TABLE 2. Complement fixation tests with HSV 1 and HSV 2 antigens and sera of patients with advanced cancer in different parts of the body

nonvirion

All in this group positive* Site of

cancer

All in this group negative*

No. tested

Lip Mouth Oropharynx Nasopharynx Kidney Bladder Prostate Cervix uteri Vulva

Site of

2

No. tested

cancer

Gum Tongue Tonsil Salivary gland Accessory sinus Epiglottis Lung and bronchus Stomach

1 5

10 8 8 8 13

1 2 2 2 3 1 7 5 9 5 5 5 3 4 4 4 4

Colon Breast

1

Corpus uteri Ovary Testis Liver Thyroid Kidney-embryonal, Wilms' Melanoma

Total patients *

56

Hodgkin's lymphoma Acute lymphocytic leukemia Acute myelocytic leukemia Total patients

5 5 5

81

With HSV 1 only or with HSV 1 and HSV 2.

duced only HSV 2 nonvirion antigens demonstrable by complement fixation has, nevertheless, on hyperimmunization of guinea pigs produced antibodies for both HSV 2 and HSV 1 nonvirion antigens (13). In these cancers, the results suggest the possibility of a special involvement of HSV 2, unlike that associated with ordinary primary and recurrent infections with this virus. The words "suggest the possibility" are used here instead of "indicate" to emphasize the need for establishing, by appropriate tests, that the nonvirion antibodies in these patients are actually in response to neoantigens coded by HSV genetic information and not equally well by any other DNA virus or in response to antigens, coded by the host's DNA, which may be repressed except during embryonic development and differentiation, e.g., the different varieties of so-called carcinoembryonic antigens. TABLE 3. Complement fixation reactivity of positive sera with HSV 1 and HSV 2 nonvirion antigens

cancer

No. positive with

Site of cancer

No. tested

HSV 1

HSV 2

Cervix Vulva Prostate Bladder Kidney Nasopharynx Oropharynx Mouth Lip

13 1 8

13 1 8 8 8 10 5 1

13 1 7 + 1? 6+ 1? 6 + 1? 9 + 1? 0 0? 0

8

8 10 5 1 2

2

(1973)

TABLE 4. Complementfixation antibody titers of positive human cancer sera with HSV 1 and HSV 2 nonvirion antigens CF antibody titers* of individual sera in corresponding order with Site No. of cancer tested HSV 1 HSV 2 Cervix uteri 13 32, 6, 4,4 4, 4, 4 32, 16, 8, 8, 8, 6, 4 4,4,4,4,4,4 4,4,4,4,4,4 Vulva 1 4 8 Prostate 8 8, 6, 44, 4,4, 4, 4 8, 4, 4,4,4 4,4, 0? Bladder 8 12, 88, 6, 6, 4, 4, 4 16, 4, 0, 0?, 4, 4, 8, 8 Kidney 8 6, 4, 4, 4, 4, 4,4,4 8,88,8, 16, 4,0? 0 Nasopharynx 10 8, 8, 8, 8, 8 6, 6, 6, 6, 4

4,4,4,4,44 Oropharynx Mouth Lip

5 1 2

6, 4, 4, 4, 4 4

4,4

4,4,4,4,0? ,0, 0, 0, 0 0? 0, 0

Titer = dilution at which there was no hemolysis or less than 50% of erythrocytes were hemolyzed. Titers of 6 or 12 signify 50% hemolysis at dilutions of 1:8 or 1:16 and no hemolysis or less than 50% hemolysis at 1:4 and 1:8, respectively. 0? = about 50% hemolysis at 1:4 dilution with complete hemolysis in all controls. 0 = complete hemolysis (usually) or occasionally incomplete hemolysis with a residue of less than 33% of erythrocytes at 1:4 dilution. *

The following control tests were done to establish the specificity of the antibodies in the positive cancer sera for nonvirion antigens coded by HSV genetic material: (a) 10 Positive sera (two each of nasopharynx, bladder, kidney, prostrate, and cervix uteri) failed to react with cells, harvested 10 min and 6 hr after high-multiplicity infection with the DNA vaccinia virus, which produces, within 10 min (12), a transitory marked rise in concentration of a preexisting normal antigen that is also present in greater concentration in almost all transformed and malignant human cells; (b) the antibodies in the same 10 sera were not removed or diminished in titer by massive absorption with freshly harvested HEp2 cells; and (c) the antibodies in four positive sera (two each of kidney and cervix uteri) were not removed or diminished in titer by massive absorption with freshly trypsinized cells from the kidneys of 12- to 14-week-old human embryos. These results, taken together with the other manifestations of specificity (Tables 2 and 3), strongly support the conclusion that the antibodies we demonstrated in the positive human cancer sera were indeed against neoantigens coded in one group of cancers (oropharynx and lip) by HSV 1 and in the other group of cancers (cervix uteri, vulva, and with a few possible exceptions also prostate, bladder, kidney, and nasopharynx) by HSV 2. It is, therefore, all the more intriguing why all five oropharyngeal cancers are so strictly associated only with HSV 1 while at least nine of ten (and perhaps all) nasopharyngeal cancers are so definitely associated with HSV 2. The results in Table 4, showing the titers of the HSV 1 and HSV 2 nonvirion antibodies for each positive serum, are of interest especially because of the large number of sera that were positive only at the 1:4 dilution. Had we started our tests with a 1:10 dilution of serum and used the micro complement fixation procedure, as was done in the negative tests with various adenovirus T (tumor) antigens and unabsorbed sera (15) from the same collection that we used, 59%/ of our 56

positive sera with titers of 1: 6 or less and perhaps also 32% of

Proc. Nat. Acad. Sci. USA 70

(1973)

the sera with titers of 1:8 would have yielded negative results in our macro complement fixation test, in which four-times more serum is used than in the micro test. Low antibody responses by tumor-bearing animals to nonvirion antigens are not unusual with some of the experimentally produced DNA virus malignancies (9). The negative results that we obtained with the sera of four women with early, limited, malignant changes in the cervix uteri, in contradistinction to the positive results on all 13 women with advanced cancer of the cervix uteri, is an indication that the HSV nonvirion antibodies appear after extensive and perhaps continuous antigenic stimulation. Huebner (16) reported the disappearance of nonvirion antibodies after surgical excision of experimental adenovirus-induced tumors in hamsters and their reappearance after regrowth of the tumor. DISCUSSION

The results presented here provide strong evidence for an etiologic role of HSV 1 and HSV 2 in at least nine varieties of human cancer. Relevant data in support of this conclusion are contained in a recent publication (17), which reported that a human carcinoma of the cervix uteri that had no infectious virus contained HSV messenger RNA and also a fragment of HSV DNA linked to host DNA. Moreover, it was shown that "about half of the viral DNA template transcribed in the cervical tumor belongs to the set of sequences that are shared in common between HSV 1 and HSV 2." Another recent publication (18) reported positive complement fixation reactions of nonvirion antigens in HEp2 cells infected with an HSV 2 strain and also of a special fraction from human carcinoma of the cervix uteri with our HSV 1 guinea pig 71 serum which did not react with nonvirion antigens in cells infected with three HSV 2 strains (13); then these-reported tests cannot be properly evaluated. The procedures reported in the present and preceding communications (11-13) can now be used for more extensive investigations of the role of HSV and other DNA viruses in various human cancers.

Herpesviruses 1 and 2 and Human Cancers

3229

We thank Miss Louise Malan, Mr. Willis Foster, and during the latter part of the program Dr. Ann Boyd for their dedicated help with the tremendous amount of laboratory work. Special thanks are due to Dr. John Landon, Director for Science, Frederick Cancer Research Center operated by Litton Bionetics, Inc. for his help in the rapid mobilization of laboratory facilities, equipment, and manpower. 1. Huebner, R. J., Rowe, W. P., Turner, H. C. & Lane, W. T. (1963) Proc. Nat. Acad. Sci. USA 50, 379-389. 2. Sabin, A. B. & Koch, M. A. (1963) Proc. Nat. Acad. Sci. USA 50, 407-417. 3. Sabin, A. B. & Koch, M. A. (1964) Proc. Nat. Acad. Sci. USA 52, 1131-1138. 4. Rapp, F., Kitahara, T., Butel, J. S. & Melnick, J. L. (1964) Proc. Nat. Acad. Sci. USA 52, 1138-1142. 5. Pope, J. H. & Rowe, W. P. (1964) J. Exp. Med. 120, 577588. 6. Hoggan, M. D., Rowe, W. P., Black, P. H. & Huebner, R. J. (1965) Proc. Nat. Acad. Sci. USA 53, 12-19. 7. Rapp, F., Butel, J. S., Feldman, L. A., Kitahara, T. & Melnick, J. L. (1965) J. Exp. Med. 121, 935-944. 8. Sabin, A. B. (1967) Memoires de L'Academie Royale de Medicine de Belgiques, Ie Sgrie 6, 61-81. 9. Sabin, A. B. (1967) in Specific Tumor Antigens, UICC Monograph Series, ed. Harris, R. J. C. (Munksgaard, Copenhagen), Vol. 2, pp. 251-264, 328-332. 10. Sabin, A. B. (1968) Cancer Res. 28, 1849-1858. 11. Tarro, G. & Sabin, A. B. (1970) Proc. Nat. Acad. Sci. USA 65, 753-760. 12. Tarro, G. & Sabin, A. B. (1970) Proc. Nat. Acad. Sci. USA 67, 731-737. 13. Tarro, G. & Sabin, A. B. (1973) Proc. Acad. Sci. USA 70, 1032-1036. 14. Roizman, B. (1966) in Perspectives in Virology, ed. Pollard, M. (Harper & Row, New York, N. Y.), Vol. 4, pp. 283-304. 15. Gilden, R. V., Kern, J., Lee, Y. K., Rapp, F., Melnick, J. L., Riggs, J. L., Lennette, E. H., Zbar, B., Rapp, H. J., Turner, H. C. & Huebner, R. J. (1970) Amer. J. Epidemiol. 91, 500-509. 16. Huebner, R. J. (1967) in Perspectives in Virology, ed. Pollard, M. (Academic Press, New York), Vol. 5, pp. 147-166. 17. Frenkel, N., Roizman, B., Cassai, E. & Nahmias, A. (1972) Proc. Nat. Acad. Sci. USA 69, 3784-3789. 18. Hollinshead, A. C. & Tarro, G. (1973) Science 179, 698-700.