Herpes Simplex and Herpes Genitalis Viruses in Etiology of Some Human Cancers* - PNAS
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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
ABSTRACT The results of complement fixation tests evidence by which virus-free experimental cancers can be
on 202 sera from people without cancer and from patients proved to have been originally induced by such DNA vi-
with cancer in 29 different areas of the body indicated that ruses as polyoma, Simian Virus 40, or certain types of
only those with nine varieties of advanced cancer (lip, adenovirus.
mouth, oropharynx, nasopharynx, kidney, urinary blad-
der, prostate, cervix uteri, and vulva-all of 56 tested) gave The evidence required for establishing DNA viruses, which
positive specific reactions with nonvirion antigens induced cause ordinary infections in animals anid humans, as etiologic
by the DNA herpes simplex (HSV 1) and herpes genitalis or inu(lUcilg agents in cancer is different from that which has
(HSV 2) viruses. None of 57 people without cancer (includ-
ing 10 with current and 18 with recurrent HSV 1 or HSV 2 established several unique RNA viruses, unassociated with
infections), none of 81 patients with 20 other varieties of ordinary infections as the (cause of various naturally occurring
advanced cancer (gum, tongue, tonsil, salivary gland, ac- malignancies of lower animals. Some DNA viruses associated
cessory sinus, epiglottis, lung-bronchus, stomach, colon, with ordinary infections (i.e., l)olyooma of mice, Simian Virus
breast, corpus uteri, ovary, testis, liver, thyroid, Wilms'
embryonal kidney, melanoma, Hodgkin's disease, acute 40 of rhesus monkeys, anad some types of human and animal
lymphocytic leukemia, and acute myelocytic leukemia), adenoviruses) can produce experimental cancers in which in-
and none of four women with early malignant changes in fectious virus, 01 infectious DNA, or any of the structural
the cervix uteri gave positive results. The seven patients (virion) antigens cannot be detected either dlirectly or by
with advanced cancer of the lip or oropharynx gave posi- their capacity to induce antibody in the tumor-bearing ani-
tive reactions with HSV 1 but not with HSV 2 nonvirion
antigens (compatible with involvement of only HSV 1), mals. There are howei-er,
in such cancers, antigens that are
all of the 13 women with advanced cancer of the cervix specific for the virus that induced the exlperimental cancer,
uteri and the one woman with advanced cancer of the vulva and tumor-bearing animals develop specific antibodies for
gave positive reactions with both HSV 1 and HSV 2 non- them (1, 2). These specific tumor antigens are identical to
virion antigens (compatible with involvement of only HSV
2), while among the 35 other positive patients only two nonvirion antigens (i.e., not structural comlpoIients of the as-
(one with cancer of the kidney and one with cancer of the sembled virus particle) that are synthesized shortly after
bladder) reacted with HSV 1 and not at all with HSV 2 hioh-multiplicitv infection of suscel)tible cultured cells. even
nonvirion antigens. Positive sera failed to react with cells when synthesis of new DNA is prevented by al)l)roi)riate in-
harvested at different times after high-multiplicity in-
fection with the DNA vaccinia virus. Massive absorption of hibitors (2-7). Phenomena associated with experimental DNA
positive sera with trypsinized, uninfected human embry- virus cancers were studied in detail with special emphasis on
onic kidney cells failed to remove, or lower the titer of, those manifestations that could be important ill the search
the HSV 1 and HSV 2 nonvirion antibodies. for evidence of the possible involvement of the well-known
All of these data taken together are interpreted as in- DNA viruses of humans, i.e., maintained in nature only by
dicating that HSV 1 and HSV 2 play an etiologic role in
certain human cancers, because they provide the kind of transmission from person to person, in human cancers (8-10).
The subsequent search for nonvirion antigens in cells infected
Abbreviations: HSV 1, herpes simplex (labialis) virus, subtype with subtypes 1 and 2 of herpes simplex virus (HSV) and
1; HSV 2, herpes simplex (genitalis) virus, subtype 2; GPK, the development of methods for identifying them, studying
guinea pig kidney; RK, rabbit kidney; HEp2, a human cell line their distinctive properties, and differentiating them from
derived from an epidermoid carcinoma of the larynx; PFU, preexisting antigens, whose concentration is increased by
plaque-forming unit. both malignant transformation and various viral infections,
*
Preliminary report of data presented at the I10th Annual have been reported (11-13).
Meeting of the National Academy of Sciences, Washington, The preceding studies provided the necessary reagents and
D.C., April 24, 1973. methodology for testing human sera for HSV nonvirion anti-
t This study was conducted while I)r. Sabin was a Fogarty bodies, as well as the important fact that sera from small
Scholar-in-Residence, Fogarty International Center for Advanced
Study in the Health Sciences. Reprint requests should be sent numbers of randomly selected adults and from people with
to Dr. Sabin, Fogarty International Center, National Institutes recent HSV 1 or HSV 2 infections had no HSV nonvirion
of Health, Bethesda, \Id. 20014. antibodies demonstrable by comllement fixation (11, 13).
t During this work Dr. Tarro was on leave from, and is currently Thus, we could proceed with the final Chase of the plan (8-10)
at, Virologia Oncologica, Istituto di Clinica -Medica I, Universita to explore the possible role of HSV 1 and HSV 2 in human
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di Napoli, Polichinico, 80138 Napoli, Italia. cancer by determining whether or not the sera of certain
32253226 Medical Sciences: Sabin and Tarro Proc. Nat. Acad. Sci. USA 70 (1973)
TABLE 1. Complement fixation tests w ith HSV 1 and HSV 2 scribed (11-13). Virus stocks were prepared in HEp2 cells
nonvirion antigens and sera of people without cancer in the logarithmic phase of multiplication, which is optimum
for obtaining high yields of virus.
No.
positive with Preparation of HEp2 Cells for Absorption of Virion Anti-
bodies. We wanted to obtain large numbers of infected cells
No. HSV HSV for storage at 40 to inactivate the nonvirion antigens which in
Group Zsted
te 1 2 HEp2 cells are at peak titers at the end of the infectious pro-
Current herpesvirus infections 10 0 0 cess (11, 13). Accordingly, densely packed cell sheets, at 7-8
HSV 1, 4; HSV 2,6. days after planting 4 X 106 cells per 32-ounce bottle, are in-
Recurrent herpetic lesions 18 0 0 fected with about 5 plaque-forming units (PFU)/cell. The
Labial, 7; genital (HSV 2), 1 1. same procedure of harvesting, freezing, and sonicating is
Pregnant women 16 0 0 followed as for preparation of virus stocks except that the 10%
Last trimester, 9; postpartum, 7.
Random adults 13 0 0 cell suspension is prepared in Eagle's basal medium without
Total 57 0 0 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°.
cancer patients might exhibit a specifiic reactivity with HSV Preparation of Nonvirion Antigens in GPK and HEp2 Cells.
nonvirion antigens that would not be found
i in the absence of After the medium was decanted from GPK confluent mono-
the special cancer or cancers even amorig people of comparable layers grown for 6-7 days in 32-ounce bottles, 10 ml of Eagle's
age with frequent recurring infections f or decades. basal medium without serum or phenol red, containing pref-
MATERIALS AND MIETHODS erably about 2 X 108 PFU of HSV, are added. After incuba-
ETHD tion for 3 hr at 370, the cells are scraped into the medium and
Cell Lines and Their Cultivation. H]
onp2 cells were used for
cells
preparation of HSV 1 and HSV 2 virin on antigens for absorp-
used for
centrifuged as for preparation of stock virus, except that
basal medium without serum or phenol red is used to make
tion of sera and also for preparation of nonvirion antlgens.
Very large quantities of HEp2 cells weere obtained from Flow
the 10% suspension. Moreover, after it was frozen and
thawed, the suspension was not sonicated directly in the cup
Laboratories, Rockville, Md. either fiully grown in 32-ounce but put into a stoppered lusteroid tube which rested in water
prescription bottles in 90% minimum IEagle's medium + 10% in the cup (because the volume was often too small and also
fetal-bovine serum without antibiotic s, or usually as freshly because that is the way we did it before), and the sonicated
trypsinized cells which we grew oursselves in Eagle's basal suspension was frozen uncentrifuged at -80°. For maximum
medium (GIBCO powder rehydrate d) + 10% unheated, concentration of nonvirion antigens for complement fixation
regular calf serum + 0.11% NaHCOa3 for initial growth and tests, the uncentrifuged suspension was used the following day
0.22% NaHCO3 later + penicillin ('200 units/ml), strepto- or at most within 4 days. When HEp2 cells were used, they
mycin (0.2 mg/ml), and gentamycin (0.05 mg/ml). Primary were grown and infected as for virus stock and harvested after
GPK cultures were used only for proeparation of HSV non- 24 hr when the cytopathic effect is complete and the concen-
virion antigens. Kidneys of 2- to 3-dr Ly-old guinea pigs were tration of nonvirion antigens is highest (11, 13). The remain-
trypsinized in warmed 0.25% trypsini in phosphate-buffered der of the process was as for GPK cells. An aliquot of the same
saline (20 ml/g of kidney) and thoroughly washed twice with lot of uninfected GPK or HEp2 cells was processed in pre-
large quantities of cold growth medium in a refrigerated centri- ciselv the same manner to provide the control antigen for the
fuge. The volume of cell pack was dettermined by centrifuga- complement fixation test.
tion at 600 rpm for 10 min, and 40 ml c5f a 1:400 suspension by
volume prepared in warm growth mediurn was seeded per 32- Monitoring of Nonvirion and Virion Antisera. Sabin
ounce prescription bottle. The growlth medium consisted of "Schooler" HSV 1 guinea pig 71 serum (11) and Sabin
85% of 0.5% lactalbumin hydrolysatEe in Earle's salt solution "Justin" HSV 2 guinea pig 91 serum (13) were used, after
without NaHCO3 (dehydrated prodiuct of GIBCO, Grand proper virion absorption, to measure the concentration of
Island, N.Y. 14072) + 5% tryptose phosphate broth + 10% HSV 1 and HSV 2 nonvirion antigens, respectively. While
fetal-bovine serum (selected for abse nce of toxicity) heated HSV 1 nonvirion guinea pig serum detected nonvirion anti-
at 560 for 30 min + 0.035% NaHCO3 + penicillin (200 units/ gens induced only by HSV 1 strains, HSV 2 serum detected
ml), streptomycin (0.2 mg/ml), and ge ntamycin (0.05 mg/ml). nonvirion antigens induced not only by HSV 2 but also by
Medium is changed at 2 days (withi 0.035% NaHCO3), at HSV 1 (13). A hyperimmunized rabbit serum prepared by
3-4 days (0.11% NaHCO3), and at 5-6 days (0.22% Na- one of us (A.B.S.) in 1958 against the "Schooler" HSV 1
HCO3). Excellent, fully confluent mlonolayers are ready for strain, containing an especially broad spectrum of HSV
use 6-7 days after cells are planted iwhen the procedure de- virion antibodies, measured equally well the concentration
scribed is strictly followed. Secondaryr cultures prepared from of virion complement fixation antigen produced by HSV 1 and
monolayers of young (150-200 g) RE< cell cultures were used HSV 2.
for plaque titration of the potency of the HSV used for high- Human Sera. Most of the sera from patients with advanced
multiplicity infection of cells for p cancer wereprovided by Dr. Jack Gruber, Chief, Office of
antigens. Program Resources and Logistics, National Cancer Institute
Viruses Used, Preparation, and St 'orage. Sabin "Schooler" largely from a collection that was made at the M. D. An-
(HSV 1) and Sabin "Justin" (HSV 2) strains of HSV and the derson Hospital, Houston, Texas in 1967 and stored at - 700
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VL strain of vaccinia virus used in tthis work have been de- in aliquot amounts since then. Each of these sera came fromProc. Nat. Acad. Sci. USA 70 (1978) Herpesviruses 1 and 2 and Human Cancers 3227
a person with histologically confirmed malignancy and evi- plement fixation test (11) was used. Each complement fixation
dence of metastasis (25 g or more of primary and metastatic test always had positive and negative controls: human sera
tumor). Some of the random sera from people without cancer from people without cancer or with different types of cancer,
were from family or other matched controls of these patients. uninfected cell suspensions representing aliquots of those
We thank Dr. Brian E. Henderson of the Department of used for preparation of nonvirion antigens and similarly and
Pathology, University of Southern California School of Medi- concurrently processed and stored, HSV 1 and HSV 2 con-
cine, Los Angeles, Calif., for the following 13 cancer sera used centrated virion antigens, titrations to determine the concen-
in the present tests: two nasopharynx from Chinese patients, tration of the nonvirion and virion antigens in the prepara-
one lip, two corpus uteri, five ovary, and three testis. Dr. tions used for the test, titrations of the absorbed monitoring
John L. Sever and Dr. David A. Fucillo, Perinatal Research HSV 1 and HSV 2 guinea pig antisera, and, of course, simul-
Branch, National Institute of Neurological Diseases and taneous complement controls on all the reagents in the test.
Stroke, supplied the sera from women before and after early The sera in any one test were scrambled and numbered by a
manifestations of malignancy of the cervix uteri, and also the code contained in a sealed envelope which was not opened
sera from pregnant women without cancer. We especially until after the results had been recorded.
thank Dr. A. J. Nahmias, Department of Pediatrics, Emory RESULTS
University School of Medicine, Atlanta, Ga., for sera on pa-
tients with recurrent HSV 2 infections he had been studying People Without Cancer. The data in Table 1 show that none
for many years. These male and female patients ranged in of the sera from 57 people without cancer had nonvirion anti-
age from 20-64 years (six of them 52 years or older) with re- bodies at the 1:4 dilution tested. The results on the 10 people
curring lesions on the penis (two), vulva (two), buttocks with current or very recent HSV 1 or HSV 2 infections have
(five), thigh (one), and finger (one). Four of these patients been reported (11, 13). Available evidence suggests that iii
have had recurrent lesions for 15, 15, 20, and 23 years, re- recurrent HSV lesions, which appear at the same site year
spectively, three with four to six recurrences per year and after year, there is derepression and full expression of total
one with 20-30 per year. Among the others there were five viral genome which persists in a repressed state in the cells of
with 10-25 recurrences per year. Sera from people with herpes the affected area (14). The completely negative results ob-
labialis were received from different sources and were from tained with sera from 18 people with long histories of recur-
people over 35 years of age. rent HSV 1 and HSV 2 infections indicate, therefore, that
special conditions are required for production and persistence
Absorption of Virion Antibody from Human and Guinea Pig of antibodies for the nonvirion HSV antigens. The negative
Sera before Tests for Nonvirion Antibody. The basic procedures results obtained with the sera of 16 pregnant women indicate
for absorption were described (3, 11). Although storage at that the nonvirion HSV antigens we used to test the sera from
about 40 had been found to eliminate HSV 1 and HSV 2 cancer patients are free from carcinoembryonic antigens for
nonvirion reactivity from infected HEp2 cell suspensions be- which such sera may have antibodies. The sera from 13
tween 7 and 9 days and between 12 and 15 days, respectively random adults included a serum (kindly supplied by Dr. Paul
(11, 13), when 0.1-ml quantities were being tested, the very Gerber, Bureau of Biologics, Food and Drug Administration,
much larger quantities (100-times or more) required for ab- National Institutes of Health, Bethesda, AMd.) that has been
sorption of the human and guinea pig sera might still contain extensively used in studies on human Epsteiin-Barr DNA
enough nonvirion antigen at 14 days to reduce the titer of virus because of its broad spectrum of antibodies against
nonvirion antibody in the absorbed sera. Moreover, the orig- "early" and "late" antigens of Epstein-Barr virus.
inal observations (11, 13) were not made under conditions of
strict temperature control (an ordinary refrigerator that was People with Cancer. The data in Table 2 on sera from 137
opened many times each day had been used); we could not as- patients with advanced cancer in 29 different sites of the body
sume that under the conditions of storage used in the present are remarkable because uniformly positive results were ob-
work (an infrequently used walk-in refrigerator carefully con- tained with 56 sera from patients with the nine listed varieties
trolled and monitored at 40 + 0.50) the nonvirion antigens of cancer, and uniformly negative results were obtained in
would be inactivated at the same rate. We indeed found that simultaneous tests with the same reagents with the sera from
the standard guinea pig sera gave much lower nonvirion anti- 81 patients with 20 other varieties of cancer. The fact that
body titers when they were absorbed with HEp2 cell suspen- the sera of all seven patients with advanced cancers of the lip
sions stored at the strictly controlled 40 temperature for 2-4 and oropharynx, areas commonly infected by HSV 1, reacted
weeks, while storage for 5-8 weeks yielded higher titers of only with HSV 1 and not at all with HSV 2 nonvirion antigens
nonvirion antibody without loss of capacity to remove the (Table 3) is remarkably in accord with the observations that
virion antibody. A mixture of equal parts of HSV 1 and HSV high-multiplicity infection of tissue culture cells with HSV 1
2 antigens stored at +40 was used for absorption of virion strains has induced antigens which on hyperimmunization of
antibodies from human sera and concurrently for each test guinea pigs produced antibodies that react with HSV 1 and
from the standard HSV 1 and HSV 2 guinea pig monitoring not with HSV 2 nonvirion antigens (13). These results sug-
sera. gest the possibility of a special involvement of HSV 1 in these
cancers, unlike that associated with ordinary primary and
Complement Fixation Test. The amount of complement re- recurrent infections with this virus. Similarly, the fact that
quired for the 1.5 exact units to be used in the test was de- the sera of all 14 patients with advanced carcinoma of the
termined by a preliminary titration of complement in the cervix uteri or vulva, areas commonly infected by HSV 2,
presence of 0.1 ml of the 10% uncentrifuged cell-suspension reacted with both HSV 1 and HSV 2 nonvirion antigens is
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antigens and of the absorbed human sera that are diluted 1 :4 remarkably in accord with the observations that high-multi-
during virion absorption. A described procedure for the com- plicity infection of tissue cultures with HSV 2, which has in-3228 Medical Sciences: Sabin and Tarro Proc. Nat. Acad. Sci. USA 70 (1973)
TABLE 2. Complement fixation tests with HSV 1 and HSV 2 TABLE 4. Complementfixation antibody titers of positive human
antigens and sera of patients with advanced cancer in
nonvirion cancer sera with HSV 1 and HSV 2 nonvirion antigens
different parts of the body
CF antibody titers* of individual sera in
All in this group All in this group
positive* negative* Site No. corresponding order with
of cancer tested HSV 1 HSV 2
No. No.
Site of cancer tested Site of cancer tested 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
Lip 2 Gum 1 Vulva 1 4 8
Mouth 1 Tongue 2 Prostate 8 8, 6, 44, 4,4, 4, 4 8, 4, 4,4,4 4,4, 0?
Oropharynx 5 Tonsil 2 Bladder 8 12, 88, 6, 6, 4, 4, 4 16, 4, 0, 0?, 4, 4, 8, 8
Nasopharynx 10 Salivary gland 2 Kidney 8 6, 4, 4, 4, 4, 4,4,4 8,88,8, 16, 4,0? 0
Kidney 8 Accessory sinus 3 Nasopharynx 10 8, 8, 8, 8, 8 6, 6, 6, 6, 4
Bladder 8 Epiglottis 1 4,4,4,4,44 4,4,4,4,0?
Prostate 8 Lung and bronchus 7 Oropharynx 5 6, 4, 4, 4, 4 ,0, 0, 0, 0
Cervix uteri 13 Stomach 5 Mouth 1 4 0?
Vulva 1 Colon 9 Lip 2 4,4 0, 0
Breast 5
Corpus uteri 5 * Titer = dilution at which there was no hemolysis or less than
Ovary 5 50% of erythrocytes were hemolyzed. Titers of 6 or 12 signify 50%
Testis 3 hemolysis at dilutions of 1:8 or 1:16 and no hemolysis or less
Liver 4 than 50% hemolysis at 1:4 and 1:8, respectively. 0? = about
Thyroid 4 50% hemolysis at 1:4 dilution with complete hemolysis in all
Kidney-embryonal, Wilms' 4 controls. 0 = complete hemolysis (usually) or occasionally in-
Melanoma 4 complete hemolysis with a residue of less than 33% of erythro-
Hodgkin's lymphoma 5 cytes at 1:4 dilution.
Acute lymphocytic leukemia 5
Acute myelocytic leukemia 5 The following control tests were done to establish the speci-
Total patients 56 Total patients 81 ficity of the antibodies in the positive cancer sera for non-
virion antigens coded by HSV genetic material: (a) 10 Posi-
* With HSV 1 only or with HSV 1 and HSV 2. tive sera (two each of nasopharynx, bladder, kidney, prostrate,
and cervix uteri) failed to react with cells, harvested 10 min
duced only HSV 2 nonvirion antigens demonstrable by com- and 6 hr after high-multiplicity infection with the DNA vac-
plement fixation has, nevertheless, on hyperimmunization of cinia virus, which produces, within 10 min (12), a transitory
guinea pigs produced antibodies for both HSV 2 and HSV 1 marked rise in concentration of a preexisting normal antigen
nonvirion antigens (13). In these cancers, the results suggest that is also present in greater concentration in almost all
the possibility of a special involvement of HSV 2, unlike transformed and malignant human cells; (b) the antibodies in
that associated with ordinary primary and recurrent infections the same 10 sera were not removed or diminished in titer by
with this virus. The words "suggest the possibility" are used massive absorption with freshly harvested HEp2 cells; and (c)
here instead of "indicate" to emphasize the need for estab- the antibodies in four positive sera (two each of kidney and
lishing, by appropriate tests, that the nonvirion antibodies cervix uteri) were not removed or diminished in titer by mas-
in these patients are actually in response to neoantigens coded sive absorption with freshly trypsinized cells from the kidneys
by HSV genetic information and not equally well by any other of 12- to 14-week-old human embryos. These results, taken
DNA virus or in response to antigens, coded by the host's together with the other manifestations of specificity (Tables
DNA, which may be repressed except during embryonic 2 and 3), strongly support the conclusion that the antibodies
development and differentiation, e.g., the different varieties we demonstrated in the positive human cancer sera were in-
of so-called carcinoembryonic antigens. deed against neoantigens coded in one group of cancers (oro-
pharynx and lip) by HSV 1 and in the other group of cancers
TABLE 3. Complement fixation reactivity of positive cancer
(cervix uteri, vulva, and with a few possible exceptions also
sera with HSV 1 and HSV 2 nonvirion antigens
prostate, bladder, kidney, and nasopharynx) by HSV 2. It is,
therefore, all the more intriguing why all five oropharyngeal
No. positive with cancers are so strictly associated only with HSV 1 while at
least nine of ten (and perhaps all) nasopharyngeal cancers are
Site of cancer No. tested HSV 1 HSV 2 so definitely associated with HSV 2.
Cervix 13 13 13 The results in Table 4, showing the titers of the HSV 1 and
Vulva 1 1 1 HSV 2 nonvirion antibodies for each positive serum, are of
Prostate 8 8 7 + 1? interest especially because of the large number of sera that
Bladder 8 8 6+ 1? were positive only at the 1:4 dilution. Had we started our
Kidney 8 8 6 + 1? tests with a 1:10 dilution of serum and used the micro com-
Nasopharynx 10 10 9 + 1? plement fixation procedure, as was done in the negative tests
Oropharynx 5 5 0 with various adenovirus T (tumor) antigens and unabsorbed
Mouth 1 1 0?
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sera (15) from the same collection that we used, 59%/ of our 56
Lip 2 2 0
positive sera with titers of 1: 6 or less and perhaps also 32% ofProc. Nat. Acad. Sci. USA 70 (1973) Herpesviruses 1 and 2 and Human Cancers 3229
the sera with titers of 1:8 would have yielded negative results We thank Miss Louise Malan, Mr. Willis Foster, and during
in our macro complement fixation test, in which four-times the latter part of the program Dr. Ann Boyd for their dedicated
help with the tremendous amount of laboratory work. Special
more serum is used than in the micro test. Low antibody re- thanks are due to Dr. John Landon, Director for Science, Fred-
sponses by tumor-bearing animals to nonvirion antigens are erick Cancer Research Center operated by Litton Bionetics, Inc.
not unusual with some of the experimentally produced DNA for his help in the rapid mobilization of laboratory facilities,
virus malignancies (9). The negative results that we ob- equipment, and manpower.
tained with the sera of four women with early, limited, malig-
nant changes in the cervix uteri, in contradistinction to the 1. Huebner, R. J., Rowe, W. P., Turner, H. C. & Lane, W. T.
(1963) Proc. Nat. Acad. Sci. USA 50, 379-389.
positive results on all 13 women with advanced cancer of the 2. Sabin, A. B. & Koch, M. A. (1963) Proc. Nat. Acad. Sci.
cervix uteri, is an indication that the HSV nonvirion anti- USA 50, 407-417.
bodies appear after extensive and perhaps continuous anti- 3. Sabin, A. B. & Koch, M. A. (1964) Proc. Nat. Acad. Sci.
genic stimulation. Huebner (16) reported the disappearance USA 52, 1131-1138.
of nonvirion antibodies after surgical excision of experimental 4. Rapp, F., Kitahara, T., Butel, J. S. & Melnick, J. L. (1964)
Proc. Nat. Acad. Sci. USA 52, 1138-1142.
adenovirus-induced tumors in hamsters and their reap- 5. Pope, J. H. & Rowe, W. P. (1964) J. Exp. Med. 120, 577-
pearance after regrowth of the tumor. 588.
6. Hoggan, M. D., Rowe, W. P., Black, P. H. & Huebner, R. J.
DISCUSSION (1965) Proc. Nat. Acad. Sci. USA 53, 12-19.
7. Rapp, F., Butel, J. S., Feldman, L. A., Kitahara, T. &
The results presented here provide strong evidence for an Melnick, J. L. (1965) J. Exp. Med. 121, 935-944.
etiologic role of HSV 1 and HSV 2 in at least nine varieties of 8. Sabin, A. B. (1967) Memoires de L'Academie Royale de
human cancer. Relevant data in support of this conclusion are Medicine de Belgiques, Ie Sgrie 6, 61-81.
contained in a recent publication (17), which reported that a 9. Sabin, A. B. (1967) in Specific Tumor Antigens, UICC Mono-
human carcinoma of the cervix uteri that had no infectious graph Series, ed. Harris, R. J. C. (Munksgaard, Copen-
hagen), Vol. 2, pp. 251-264, 328-332.
virus contained HSV messenger RNA and also a fragment 10. Sabin, A. B. (1968) Cancer Res. 28, 1849-1858.
of HSV DNA linked to host DNA. Moreover, it was shown 11. Tarro, G. & Sabin, A. B. (1970) Proc. Nat. Acad. Sci. USA
that "about half of the viral DNA template transcribed in 65, 753-760.
the cervical tumor belongs to the set of sequences that are 12. Tarro, G. & Sabin, A. B. (1970) Proc. Nat. Acad. Sci. USA
67, 731-737.
shared in common between HSV 1 and HSV 2." Another re- 13. Tarro, G. & Sabin, A. B. (1973) Proc. Acad. Sci. USA 70,
cent publication (18) reported positive complement fixation 1032-1036.
reactions of nonvirion antigens in HEp2 cells infected with an 14. Roizman, B. (1966) in Perspectives in Virology, ed. Pollard,
HSV 2 strain and also of a special fraction from human car- M. (Harper & Row, New York, N. Y.), Vol. 4, pp. 283-304.
15. Gilden, R. V., Kern, J., Lee, Y. K., Rapp, F., Melnick,
cinoma of the cervix uteri with our HSV 1 guinea pig 71 J. L., Riggs, J. L., Lennette, E. H., Zbar, B., Rapp, H. J.,
serum which did not react with nonvirion antigens in cells Turner, H. C. & Huebner, R. J. (1970) Amer. J. Epidemiol.
infected with three HSV 2 strains (13); then these-reported 91, 500-509.
tests cannot be properly evaluated. The procedures reported 16. Huebner, R. J. (1967) in Perspectives in Virology, ed. Pol-
in the present and preceding communications (11-13) can now lard, M. (Academic Press, New York), Vol. 5, pp. 147-166.
17. Frenkel, N., Roizman, B., Cassai, E. & Nahmias, A. (1972)
be used for more extensive investigations of the role of HSV Proc. Nat. Acad. Sci. USA 69, 3784-3789.
and other DNA viruses in various human cancers. 18. Hollinshead, A. C. & Tarro, G. (1973) Science 179, 698-700.
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