Reaction of Antigens Isolated from Herpes Simplex Virus Transformed Cells with Sera of Squamous Cell Carcinoma Patients1

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[CANCERRESEARCH36,4394-4401,December1976] Reaction of Antigens Isolated from Herpes Simplex Virus transformed Cells with Sera of Squamous Cell Carcinoma Patients1

Mary F. D. Notter and John J. Docherty2

Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

SUMMARY we examined the reactive patterns of cancer patient sera with antigens isolated from cells transformed by these vi Antigens isolated from herpes simplex virus type 1, muses. Our studies reveal a positive correlation between herpes simplex virus type 2, on cytomegalovinus-trans sera of patients with diagnosed squamous cell carcinoma formed hamster cells were tested against 66 semafrom non and antigens isolated from both HSV-1- and HSV-2-trans cancer individuals or patients with different types of cancer. farmed cells. By use of the microcomplement fixation procedure to quan tify all antigen-antibody interactions, it was observed that MATERIALS AND METHODS 94% (p < 0.001) of all semafrom patients with squamous cell carcinoma reacted with antigens from herpes simplex virus Cells. Hamster cells transformed by HSV-1 [14-012-8-1; type 1-transformed cells, while 84% (p < 0.001) of the same (9)], HSV-2 [333-8-9 (8)], on cytomegalovirus [CX-90-3B, T2 sena reacted with antigen preparations from herpes simplex (2)] were acquired from F. Rapp, M. S. Hershey Medical virus type 2-transformed cells. When semafrom patients with Center, Hershey, Pa., while cell cultures of normal non adenocancinoma, sarcoma, liposarcoma, and melanoma transformed hamster cells were prepared from 13-day-old were tested against these antigens, there was no significant embryos (Lakeview Hamster Colony, Newfield, N. J.). All difference in their reactivity from sera of noncancem pa cells were propagated and maintained in Dulbecco's modi tients. When sera from all individuals (normal and cancer) fied Eagle's medium; 10% fetal calf serum; 0.075% NaHCO3; were tested against antigens from cytomegalovirus-trans penicillin, 100 units/mI; and streptomycin, 10 pg/mI. formed cells, no significant reaction pattern developed. Tumor Antigen Preparation. Antigens extracted from all These studies are the first to describe the isolation of a cells, bath normal and transformed, were prepared from reactive tumor-associated protein from herpes simplex vi cultures of the different cell types used. Monolayer cultures mus-transfommedcells. of each cell type were rinsed 3 times with cold (4Â°)Eagle's basal medium containing 0.11% NaHCO3, scraped into the same medium, and recovered by centnifugation. The cell INTRODUCTION pellet was resuspended as a 10% solution in cold 0.85% NaCI solution, and the cells were subjected to repeated A variety of evidence has accumulated which associates sequential sonic disruption (3, 2, 3, 4 mm at 4Â°,20kc MSE members of the Herpesvirus group with different cancers in disintegrator) followed by ultracentnifugation (100,000 x many species (23). Of particular interest are studies that 4Â°).Thesupemnatants were combined, and protein concen suggested a relationship between HSV-13 and human oral tration was determined by the method of Lowry et al. (17), cancers (30) on HSV-2 and human uterine cervical carci after which this crude extract was fractionated by discontin noma (19). Although many of these studies are semoepide uous gradient polyacrylamide electrophoresis (12, 29). Sol miological (1, 26), the oncogenic capability of both HSV-1 uble antigens in gel region B (lower 45% of gel) were eluted and HSV-2 has been demonstrated in vitro by transforming and concentrated by Diaflo filtration (UM1Ofilter, 4Â°)diluted cells with inactivated on temperature-sensitive mutants of 100-fold with cold 0.85% NaCI solution, and reconcentrated the virus (8, 9, 14, 24, 28). Considering the relationship of to 1.25 mg/mI. The final concentrate was used in the MCF HSV-1 and HSV-2 to certain carcinomas of man and the test as described below. The tumor antigen fraction iso availability of cells transformed in vitro by HSV-1 or HSV-2, lated from HSV-1-transfommed cells is referred to as M-1, while antigens from HSV-2-transformed cells are referred to , Supported by Contract NO1-CB-43984 from the National Cancer Institute. as M-2. Antigens from cytomegalovmmus-transformed cells 2 To whom requests for reprints should be addressed, at the Department of Microbiology and Cell Biology, Pennsylvania State University, 5101 Frear are designated Cx90, and extracts of nontransfommed ham Building, University Park, Pa. 16802. stemembryo cells were not labeled and served the dual pun 3 The abbreviations used are: HSV-1 , herpes simplex virus type I ; HSV-2, pose of fetal antigen preparation and tissue controls in all herpes simplex virus type 2; MCF, microcomplement fixation; M-1, tumor antigen from HSV-1-transformed cells; M-2, tumor antigen from HSV-2-trans assays. formed cells; Cx90, cell extract from cytomegalovirus-transformed cells; Virus Antigen Preparation. Vinion antigens were pre HEL, cell extract from human embryonic lung cells; HSV, herpes simplex virus. pared by infecting HEL cells with HSV-1 (Seibert strain) or Received June 11, 1976; accepted August 17, 1976. HSV-2 (316-D strain) at a multiplicity of infection of 5. After

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24 hr, newly synthesized infectious virus and accompanying known negative serum, serum controls, tissue antigen con infected membranes were separated from large cell debris trols, complement controls, and Kalmer (16) saline con by differential centrifugation. The partially purified virus trols. These precautions were sufficient to establish anti plus infected cell membranes were pelleted by centnifuga complementary activity of any reagent used in each test. tion (100,000 x g, 1 hr) and washed repeatedly with Kol Readings of percentage hemolysis of each microtiter well men's (16) saline. The virus pellet and membranes were solu were made using a test reading mirror which held 1 dispos bilized by extensive sonic disruption, the protein cancentra able plate and 1 row of 12 microtiter wells cut from a tion was determined, and the extract was stored at â€”60Â°.disposable plate containing 11 color standards from 0 to Uninfected HEL cells processed as described above served 100% hemolysis. A positive MCF reaction is defined as the as noninfected tissue culture control for vimion antigen in highest serum dilution that resulted in greater than 10% the MCF test. fixation of complement oventhe controls. It should be noted Test Sera. A total of 66 coded sera were obtained from that 10% is a minimum value and was selected in order to patients at the M. S. Hershey Medical Center, The Pennsyl provide sera from noncancer patients every opportunity to vania State University, College of Medicine, Hershey, Pa., react with these antigen preparations. However, the results and the Surgery Branch of the National Cancer Institute, to be presented were also analyzed at the 20 to 40% fixation NIH. These sena included samples from patients diagnosed level, and the data did not change significantly. Clinical as squamous cell carcinoma of uterine cervix, vulva, larynx, information for each serum was withheld until all sera were palate, lip, and tonsil; also included were senafrom patients completely and repeatedly analyzed (a minimum of 3 times with adenocarcinoma of breast, lung, cervix, endometnium, with each serum for all antigens). These results were statis and ovary, as well as semafrom individuals without cancer. tically processed by the x2 or Student's t test. When received, all sera were heat inactivated (56Â°,30mm), diluted with Kolmer's (16) saline, and stored at â€”20Â°. RESULTS MCF. M-1, M-2, Cx90, and fetal antigens were used in a modified MCF test (22) at a concentration of 40 pg/mI (1.0 The results of this study begin with reactive patterns of pg/reaction volume), while vinion antigens were used at a antigens M-1, M-2, Cx90, HSV-1, and HSV-2 with semafrom concentration of 20 @g/ml(0.5jig/reaction volume). Stand patients with squamous cell carcinoma of the uterine cervix ardization of all reagents (hemolysin, complement, and an (Tables 1, 5, and 6). When these patients' semawere reacted tigens) was performed on a regular basis as well as with with tumor antigens M-1 on M-2, 20 of 22 [91%, p < 0.001 each new lot of antigen. A disposable â€œUâ€•platecontaining (Tables 1 and 5)] and 19 of 22 [86%, p < 0.001 (Tables 1 and 96 wells was used to examine simultaneously several dilu 5)], respectively, reacted in a positive fashion. In contrast, tions of each unknown serum, 1 known positive serum, 1 the reactivity of these semawith extracts of Cx90 was only

Table1 Reaction pattern of sera from female patients with squamous cell carcinoma of the uterine cervix when exposed to antigens extracted from HSV-1(M-1), HSV-2(M-2), and cytomegalovirus-transformed cells, as wellas HSV-1and HSV-2 virion antigensMCF titers0Sample HSV-2952Age (yr) Race Term M-1 M-2 Cx90 HSV-1 56 C'@ Preoperative 8 16 32 256 256 1094 60 C Postoperative 4 8 0 64 64 226 49 C Postoperative 4 16 0 64 64 316 56 C Postoperative 4 2 0 64 64 855 46 C Preoperative 8 8 0 128 128 1028 46 C Preoperative 4 8 0 32 32 729 58 C Preoperative 32 32 0 1024 512 944 49 N Preoperative 8 8 0 64 64 75623 53 C Preoperative 8 8 0 4 0 73434 71 C Preoperative 4 4 0 64 64 79621 57 C Preoperative 8 0 0 128 32 73251 45 C Preoperative 2 4 0 32 64 73604 59 C Preoperative 4 4 0 64 32 75416 29 C Preoperative 4 4 0 128 128 76234 77 C Preoperative 4 16 0 512 512 80352 58 C Preoperative 4 16 0 64 64 78757 48 C Postoperative 4 4 0 64 64 85091 56 C Preoperative 4 2 0 64 256 82736 58 C Preoperative 0 4 0 64 32 84956 61 C Preoperative 0 0 0 128 256 73765 66 C Preoperative 8 16 0 32 64 84512 53 0 Preoperative 4 0 0 128 64

a Numerical titers express the reciprocal of the highest dilution of serum that fixed greater than 10% of complement when comparedto controls. Cell preparations(extract from Syrian hamsterembryocells and extract from HELcells) that were usedas tissue controls commonly fixed 0 to 30%of complement. S C, Caucasian; N, negroid; 0, oriental.

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4.5% (Tables 1 and 5). Most of these sena, when tested with for M-1 is high due to one serum, No. 962, which reacted at tumor antigen M-1, reacted at dilutions of <8 with a mean a dilution of 128 (Table 2)]. When tested against vinion titer for the group of 5.91 Â±1.31 [p < 0.003 (Tables 1 and antigens, titers of 105.6 Â±2.30 (p < 0.001) for HSV-1 and 6)]. When titered with M-2, a greater number of these sena 100 Â±2.87 (p < 0.003) for HSV-2 were realized (Tables 2 and reacted at dilutions of 8 on >8, and the average titer for the 6). set was 8.18 Â±1.61 [p < 0.001 (Tables 1 and 6)1. Titers of These studies were continued with sera of patients with these senato vinion antigens yielded values of 144.18 Â±2.28 cancers other than the squamous cell type. These included (p < 0.01)for HSV-1 and 128.00 Â±2.18 (p < 0.001)for HSV-2 sarcoma, melanoma. liposarcama, adenocarcinoma of the (Tables 1 and 6). breast, lung, and genitalia, as well as carcinoma of the Interestingly, a similar response was observed with sara endometnium. As presented in Tables 3 and 5, 6 of 15 (40% from patients with squamous cell carcinoma of other sites not significant) reacted with M-1, 7 of 15 (47% not signifi (palate, larynx, tonsil, head-neck, vulva, and lip), as 10 of 10 cant) reacted with M-2, and 2 of 15 (13% not significant) [100%, p < 0.001 (Tables 2 and 5)] reacted with M-1 , and 8 reacted with Cx90 antigens. The mean titers of these sara to of 10 [80%, p < 0.001 (Tables 2 and 5)] reacted with M-2. M-1 and M-2 ranged from 1.73 Â±0.62 to 2.93 Â±1.15, which When tested with Cx90 antigens, none of the 10 sara tested did not differ significantly from control values (Tables 3 and (Tables 2 and 5) reacted. The titers of these sera for M-1 and 6). However, when these sera were tested against vinion M-2 were 16.60 Â±11.75 (p < 0.10) and 6.80 Â±2.03 (p < antigens (Table 6), the mean titer to HSV-1 was 138.67 Â± 0.007), respectively (Table 6). [The mean value presented 2.32 (p < 0.001) and 92.8 Â±2.24(p < 0.002) to HSV-2. These

2Reaction Table pattern of serafrom patients with squamouscell carcinomaof anatomicalsites other than the uterine cervix whenexposedto antigensextractedfrom HSV-1-(M-1),HSV-2-(M-2),and cytomegalovirus-transformedcellsaswell as HSV-1and HSV-2virion antigensMCF titersâ€• Age Site of carci SampleHSV-2364 (yr) Race Sex noma Term M-1 M-2 Cx90 HSV-1 46 C' M Palate Preoperative 4 4 0 64 64 945 62 C M Larynx Preoperative 4 16 0 64 64 1300 43 C M Larynx Preoperative 8 16 0 64 64 962 61 C M Larynx Postoperative 128 16 0 512 512 1090 61 C M Tonsil Preoperative 4 4 0 64 64 1092 70 C M Tonsil Preoperative 2 2 0 64 64 1538 65 C M Head,neck Preoperative 4 0 0 64 64 78910 75 C F Vulva Preoperative 4 8 0 32 32 79859 68 C F Vulva Preoperative 4 0 0 64 8 J-DU-1 54 C M Lip Postoperative 4 2 0 64 64

a Numerical titers express the reciprocal of the highest dilution of serum that fixed greater than 10% of complement when comparedto controls. Cell preparations(extract from Syrian hamsterembryo cells and extract from HELcells) that were usedas tissue controls commonly fixed 0 to 30%of complement. b c, Caucasian.

Table3 antigensextractedReaction patterns of sera from patients with cancers other than squamous cell type when exposed to from HSV-1-(M-1),HSV-2-(M-2),and cytomegalovirus-transformedcellsasvirionantigens.MCF well as HSV-1and HSV-2

titersâ€• Age SampleHSV-2651 (yr) Race Sex Diagnosis Site M-1 M-2 Cx90 HSV-1 641522 66 C F Adenocarcinoma Breast 4 8 0 64 161790 57 C F Adenocarcinoma Breast 0 0 2 32 161523 57 C F Adenocarcinoma Breast 0 0 2 16 6483714 68 N M Adenocarcinoma Lung 8 8 0 128 51283707 53 C F Adenocamcinoma Endometnium 4 0 0 512 6480620 50 C F Adenocarcinoma Endometnium 0 2 0 64 3281 68 C F Adenocarcinoma Cervix 2 4 0 64 648871836 64 C F Adenocarcinoma Ovary 0 0 0 256 64814701 63 C F Adenocarcinoma Ovary 4 0 0 256 25675773 67 C F Carcinoma Endometnium 0 0 0 512 64722 47 C F Carcinoma Endometnium 4 2 0 64 81539 63 C F Liposarcoma 0 0 0 8 12883723 56 C F Sarcoma 0 16 0 64 8853 69 C F Sarcoma-Uterus 0 4 0 8 62 C M Melanoma 0 0 0 32 32

a Numerical titers express the reciprocal of the highest dilution of serum that fixed greater than 1 0% of complement when comparedto controls. Cell preparations(extract from Syrian hamsterembryo cells and extract from HELcells) that were used as tissue controls commonly fixed 0 to 30% of complement. S @,Caucasian; N, negroid.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. Reaction of Herpes-transformed Cell Antigens with Cancer Sara values to vinion antigens differed significantly from control antigens, 2 of 19 reacted [11%; mean titer, 0.32 Â±0.23 noncancer patients. (Tables 4 to 6)]. Nonetheless, although these sara were Of critical importance to these studies was the reaction generally unreactive against tumor antigens M-1 and M-2, pattern of sara from noncancem patients with these antigen the majority had titers to HSV-1 vinion antigens of 60.63 Â± preparations. When 19 sena of this group were tested, 3 1.72 and to HSV-2 vinion antigens of 70.32 Â±1.95 (Table 6). reacted with M-1 [16%; mean titer, 1.05 Â±0.84 [Tables 4 to While the titers of normal senato vinion antigens were sign if 6)J and 3 reacted with M-2 (16%; mean titer, 1.16 Â±0.86; icantly lower than titers of cancer sara to the same antigens, Tables 4 to 6). If these normal sara were tested with Cx90 we were unable to develop a predictable relationship be

Table 4 Reaction patterns of sera from noncancer patients when exposed to antigens extracted from HSV-1-(M-1), HSV-2-(M-2), and cytomegalovirus-transformed cells, as wellas HSV-1 and HSV-2 virion antigensMCF titersâ€•Sample

HSV-21548 Age Race Sex M-1 M-2 Cx9O HSV-1 46 C F 16 4 0 64 64 3618 0 0 0 0 8 3619 37 C M 0 0 0 0 0 3233 52 C F 0 0 0 16 16 3236 33 C F 0 0 0 256 256 3378 0 16 0 128 128 11111 23 C F 0 0 0 64 64 782 28 C F 0 0 0 64 32 542 29 C M 0 0 0 16 128 621 22 C F 0 0 0 64 64 549 21 C F 2 0 0 0 0 325 42 C F 0 0 0 16 32 496 22 C F 0 0 2 128 32 0069 24 C M 0 0 0 0 0 0125 30 N M 0 0 4 64 256 166 25 C M 0 0 0 0 0 0567 26 C M 0 0 0 16 0 0999 26 C M 0 0 0 0 32 255 25 0 F 2 2 0 256 256 a Numerical titers express the reciprocal of the highest dilution of serum that fixed greater than 10%of complementwhen comparedto controls. Cell preparations(extract from Syrian hamster embryo cells and extract from HEL cells) that were used as tissue controls commonly fixed 0 to 30% of complement. b C, Caucasian; N, Negroid; 0, Oriental.

Table 5 Reactivepatternsof serafrom patients with or without cancerwhenexposedto antigenextractsof HSV-1(M-1),HSV-2 (M-2), or cytomegalovirus-(Cx90) transformed cells All antigen-antibody reactions were determined by the MCF procedure, and a serum was considered positive if it fixed greater than 10% of the availablecontrols.M-1 complement when compared to Cx90No. M-2

positive/no. No. positive/ No. positive/ pSquamousGroup tested pa no. tested p no. tested cell carci- 20/22 (91)b <0.001 19/22 (86) <0.001 1/22 (4.5) NSC noma;cervixSquamous cell carci- 10/10 (100) <0.001 8/10 (80) <0.001 0/10 (0) NS noma; head, neck, vulvaTotal

squamous cell 30/32 (94) <0.001 27/32 (84) <0.001 1/32 (3.1) NS carcinomaOther cancers: mela- 6/15 (40) NS 7/15 (47) NS 2/15 (13) NS noma, sarcoma, adenocarcinomaNormals 3/19 (16) 3/19 (16) 2/19 (11) a Determined by x2. b Numbers in parentheses, percentages.

C NS, no significant difference from controls.

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M. F. D. Notter and J. J. Docherty

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. Reaction of Herpes-transformed Cell Antigens with Cancer Sara tween titers to vinion antigens and the ability to react with (3), HSV-TAA has not as yet been definitely associated with tumor antigens M-1 and M-2. either component of the virus. It is unknown at present whether M-1 and M-2 are vinion components, but since M-1 and M-2 were isolated in a manner similar to HSV-TAA and DISCUSSION also react with sera from squamous cell carcinoma patients similar to HSV-TAA (11), it is tempting to speculate that they The hypothesis suggesting that tumor antigens could be may be similar antigens. To test this possibility further we isolated from HSV-tmansformedcells was based on the accu have prepared HSV-TAA (20) and compared it with M-1 and mulation of recent information, including (a) the humonal M-2 for reactivity with various sema.When tested against 22 and cellular immune response to HSV antigens in animals sena from squamous carcinoma patients, HSV-TAA, M-1, bearing tumors induced by these cells (8, 9, 15); (b) the in and M-2 gave positive reactions with 19 of the same sera vitro demonstration of virus-specific antigens in the cyto (86% agreement). When these antigens were examined with plasm as well as plasma membrane of HSV-transformed 7 normal sera, HSV-TAA, M-1, and M-2 failed to react with 6 cells (6, 8, 9, 14, 24, 27, 28); (c) the presence of HSV-specific (85% agreement) of the same sera. However, basic differ nucleic acids in these cells (7, 10); and (d) the isolation of ences exist, since HSV-TAA is stable only for a matter of tumor-reactive proteins from cells productively infected days (20), while M-1 and M-2 remain active with squamous with HSV-1 or HSV-2 (3, 4, 11). Indeed, our results support cell carcinoma sena in excess of 3 months (M. F. D. Notten this hypothesis, as the sera of squamous cell carcinoma and J. J. Docherty, unpublished data). Furthermore, since patients preferentially reacted with our antigen prepana AG-4 and VP134 have not been extensively examined for lions M-1 and M-2. No significant reaction pattern evolved their ability to react with semafrom patients with squamous when these antigens were tested against semafrom patients cell cancers of other sites, it is difficult to correlate their with other cancer on noncancer patients. reactive pattern to M-1 and M-2. Three groups (3, 4, 11) have previously reported the suc Of particular interest were the higher complement fixa cessful isolation of diagnostically significant tumor-associ tion titers of all cancer sena to vinion antigens HSV-1 and ated antigens from cells productively infected with HSV-1 HSV-2 (Table 6). The possibility was considered that if M-1 and HSV-2. Our experimental design to this problem was and M-2 were minor antigenic components of the virus, different in that all reactive antigens were isolated from cells then they would be detected with serum of high titer to the transformed by HSV-1 or HSV-2. The rationale for this ap virus, depending on the clinical status of the patient. As proach was (a) to eliminate variability inherent in the use of presented, the titers to HSV-1 and HSV-2 of sera from squa infectious virus, including strain differences and quantita mous cell carcinoma patients, as well as those with other tive fluctuations of defective particles in virus stocks, and forms of cancer, are significantly higher than those of non (b) to increase the level of discrimination relative to the cancer patients (Table 6). Therefore, the relationship be number of viral proteins. Since these transformed cells tween titers to tumor antigens M-1 and M-2 and vimion contain limited HSV genetic (10) information and HSV pro antigens HSV-1 or HSV-2 was determined by the correlation teins (8, 9), one is not required to deal with the many viral coefficient for each cancer group and the noncancer group polypeptides that appear in the lytically infected cell (13). (Table 7). Whether M-1 and M-2 are similar to HSV tumor antigens, When M-1 on M-2 titens were compared to HSV-1 on HSV-2 AG-4 (4), HSV-TAA (11), on VP134 (3), which are isolated titers for the uterine squamous carcinoma patients, a linear from lytically infected cells, remains to be examined. Al relationship was observed, suggesting an association be though, AG-4 appears to be a minor structural component tween these 2 sets of values. When comparing these titers of the vinion (5) and VP134 a nonstmuctunalvirus polypeptide for patients with squamous carcinoma of other sites, a

Table 7 Relationship between titers to tumor antigens M-1 or M-2 and virion antigens HSV-1 or HSV-2 within the various groups of patients as determined by the correlation coefficient Antigen comparisons

HSV-2Squamous M-1+ HSV-1M-1+ HSV-2M-2 + HSV-1M-2 + carcinoma; 0.01Squamousuterine cervixp0.819Â° < 0.001â€•p0.515 < 0.02p0.716 < 0.0010.562 p < carci NSCancersnoma; other sitesp0.997 < 0.001p0.991 < 0.0010.470 NSC0.504 other than NSNoncancersquamous type0.190 NS0.213 NSâ€”0.274 NSâ€”0.070 patients0.061 6 NS0.01 NS0.276 NS0.203 NS a Correlation coefficient; as values approach + 1 or â€”1, a linear response is expected, suggesting a relationship betweenthe various antigens. b Determined by Student's t test.

C NS, not significant; correlation coefficient of tumor antigen and vimion antigen are not linearly related.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. M. F. D. Notter and J. J. Docherty similar linear relationship was observed for M-1 and HSV-1 ity between the 2 viruses. It is also noteworthy that antigens or HSV-2 but not for M-2 and HSV-1 or HSV-2. In regard to isolated from cytomegalovinus- (a member of the Herpesvi other cancers on noncancem patients, no significant mela rus group) transformed cells, as well as antigens from fetal tionship between tumor titers and vinion titers was noted. hamster cells, were uniformly unreactive with the sera. Consequently, the relationship between tumor and vimion Our preliminary data would suggest that antigens isolated titers, when compared by the correlation coefficient, ap from these HSV-tnansformed cells react with sena from pears to be also related to the clinical status of the patient squamous carcinoma patients. The precise association of (i.e., squamous carcinoma versus other cancers). M-1 and M-2 with squamous carcinoma is unknown, but However, it should be pointed out that, while the comrela there does not seem to be a relationship to age, race, and tion coefficient can predict a linear relationship between sex (Tables 1 and 2). Nonetheless, in order to clarify this titers to tumor and virus antigen within a group, individual association further, we are currently examining (a) the asso exceptions were noted. As an example, Serum 84956 (Table ciatian between titer (or reactivity) to M-1 or M-2 and the 1) did not react with M-1 or M-2 but had a titer of 128 and extent of tumor development (i.e., Stage 1 to 4 uterine 256 for HSV-1 and HSV-2, respectively. Comparatively, Se cervical cancer or head and neck cancer) and (b) the precise rum 855 (Table 1) had a similar titer to HSV-1 and HSV-2 but identification of the reactive antigen and its relationship to did react with M-1 and M-2 at a dilution of 8. Similarly, structural or nonstructural components of the virus. It is the serum from squamous carcinoma patient 952 (Table 1) had goal of these studies to provide for the continued refine titers of 8 and 16 to M-1 and M-2 and a titer of 256 for HSV-1 ment of these antigens for use in automated procedures and HSV-2. However, normal Serum 3236 (Table 4) also had providing diagnostic reagents for the rapid detection or a titer to HSV-1 and HSV-2 of 256 but was unmeactivewith M monitoring of certain forms of cancer. 1 and M-2. Nonetheless, upon further examination, our results sug ACKNOWLEDGMENTS gest that a serum capable of reacting with M-1 on M-2 also had a complement fixation titer to HSV-1 and HSV-2 (the WethankLindaS.Richardsonforexperttechnicalassistance. only exception was serum 549, Table 4). However, a serum with a complement fixation titer to HSV-1 or HSV-2 did not REFERENCES necessarily react with M-1 or M-2 unless the patient had some form of squamous cell cancer. The results are con 1. Adam, E., Kaufman, R. H., and Melnick, J. L. Seroepidemiologic Studies sistent with established theories (21, 26) in that they suggest of HerpesvirusType2andCarcinomaoftheCervix.III.Houston,Texas. that HSV-1 on HSV-2 infection in these patients precedes the Am. J. Epidemiol., 96: 427-442, 1972. 2. Albrect, T. , and Rapp, F. Malignant Transformation of Hamster Embryo malignant state. Interestingly, a similar pattern was ob Fibroblasts Following Exposure to Ultraviolet-Irradiated Human Cyto served with herpes tumor antigen VP134 in that only sara megalovirus. Virology, 55: 53-61 , 1973. that had a neutralizing titer to HSV-1 or HSV-2 were capable 3. Anzai, T., Dreesman, G. R., Courtney, R. J., Adam, E., Rawls, W. E., and Benyesh-Melnick, M. Antibody to Herpes Simplex Virus Type 2-Induced of reacting with VP134 (3). Nonstructural Proteins in Women with Cervical Cancer and in Control Consequently, the possibility remains that M-1 and M-2 Groups. J. NatI. Cancer Inst., 54: 1051-1059, 1975. 4. Aurelian, L., Schuman, B., Marcus, R. I., and Davis, H. J. Antibody to are minor antigenic components of the vimion, relatively HSV-2 Induced Tumor Specific Antigen in Serums from Patients with nonantigenic components of the vinion, or viral palypep Cervical Carcinoma. 5cience, 181: 161-163, 1973. tides produced in limited quantities in a lytic herpes infec 5. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. Reaction of Antigens Isolated from Herpes Simplex Virus-transformed Cells with Sera of Squamous Cell Carcinoma Patients

Mary F. D. Notter and John J. Docherty

Cancer Res 1976;36:4394-4401.

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