Vol. 13, No. 5 Printed in U.S.A.

INFECTION AND IMMUNITY, May 1976, p. 1382-1386

Copyright CiD 1976 American Society for Microbiology

Antinuclear Antibodies and Elevated Anti-Epstein-Barr Virus Titers in Cancer Patients N. KELLETT McCORMICK,' K. J. McCORMICK,2 AND J. J. TRENTIN*

Division of Experimental Biology, Baylor College of Medicine, Houston, Texas 77030

Received for publication 19 September 1975

One and one-half percent of human sera from patients seen at a clinic for treatment of cancer contai dA antibodies to the nuclei of chick kidney cells by indirect immunofluorescence tests. In the group of sera containing antinuclear antibodies, the geometric mean titer to Epstein-Barr virus (EBV) capsid antigen was significantly elevated. Sera obtained from normal adults or from patients with similar histological types of tumnors that possessed no antinuclea antibodies contained lower levels of anti-EBV antibodies. The elevated titers to EBV were correlated with the presence or absence of antinuclear antibodies and not with a particular type or site of neoplastic disease.

While surveying human sera (K. J. McCormick, N. K. McCormick, W. A. Stenback, and J. J. Trentin, Proc. Am. Assoc. Cancer Res. 14:89, 1973) for antibodies to the T antigen of CELO virus (an avian adenovirus reported to tiansform human cells in vitro 13D), we found a small percentage of sera from cancer patients that contained antibodies to the nuclei of chick kidney (CK) cells. Antinuclear antibodies (ANA) have been found in sera from individuals with either infectious mononucleosis (IM) (22) or nasopharyngeal carcinoma (NPC) (33), both of which are associated with Epstein-Barr virus (EBV) (8). ANA, however, have not been demonstrated in sera from cases of Burkitt's lymphoma (BL) (34), the African lymphoma consistently producing high antibody titers to EBV. Patients with diseases characterized by immunological malfunction, such as systemic lupus erythematosus (SLE) (9, 10) or sarcoidosis (19, 31), also produce elevated titers of antibodies to EBV capsid antigen. We therefore wished to determine whether cancer patients with obvious immunological disorders, as evidenced bv the presence of ANA to CK nuclei, also possess increased antibody titers to this EBV antigen when compared with individuals with a similar type of tumor but without ANA. MATERIALS AND METHODS Cells. Primary CK cells were prepared by the method of Anderson et al. (2). The HR-1K lymphoblastoid cell line (18) derived from the P3J line of Burkitt's lymphoma cells was used as the source of Present address: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Tex. 77030. 2 Present address: St. Joseph's Hospital Laboratory for Cancer Research, The Stehlin Foundation for Cancer Research, Houston, Tex. 77002.

EBV capsid antigen (13). The BALB/c 3T3 cell line of murine fibroblasts (30) was used to confirm the presence of ANA in human sera. The preparation of CK cells containing CELO T antigen was described (1). Sera. Nonselected sera from cancer patients were obtained from the M. D. Anderson Hospital and Tumor Institute, Houston, Tex., through the courtesy of G. M. Britten. Diagnoses were obtained from J. G. Sinkovics. Sera from cases of IM, Hodgkin's disease (HD), and BL were obtained from D. M. Mumford, M. A. South, and G. Klein, respectively. Normal sera were obtained from employees and students at Baylor College of Medicine. Immunofluorescence tests. Cells were grown or smeared (for HR-1K cells) on cover slips, washed three times with phosphate-buffered saline (PBS), air dried, and fixed with acetone for 10 min. Fixed slides were stored at -80 C. Sera were diluted 1:4 before testing for ANA. Fourfold dilutions (initial, 1:10) were used to titrate antibodies to EBV. A semimicro procedure for indirect immunofluorescence (26) was used. In brief, cover slips were incubated with the serum for 1 h at 37 C. After washing the cover slip with PBS, appropriately diluted goat antihuman immunoglobulin G, H- and L-chain specific (Hyland, Costa Mesa, Calif.), was added and incubated for an additional hour at 37 C. The preparations were then washed three times with PBS, dipped in distilled water, blotted dry, and mounted in glycerol-PBS (1:1). Statistics. The distribution of titers was compared by X2 tests. Differences in geometric mean titers (GMT) were analyzed by the t test.

RESULTS One and one-half percent of sera (37/2,397) collected from patients at a hospital for treatment of cancer contained antibodies detectable on CELO-infected CK cells by the indirect immunofluorescence test. Although the fluores382

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heavily granular and distributed that of normal (t test, P < 0.01) or ANA-negathroughout the nucleus, resembling the T anti- tive tumor sera (t test, P < 0.005). Sera from 68 gen of CELO virus (1), it resulted from ANA to 100% of patients with ANA, IM, HD, or BL since each of the 37 sera was also positive on had elevated anti-EBV titers (-1:160). Overall, uninfected CK cells. ANA were detected only in the GMT fell into three categories: (i) low, apsera from certain cancer patients. Sera from 262 proximately 1:50, found in normal or ANA-negnormal adults, 10 patients with IM, 12 patients ative tumor sera; (ii) intermediate, 1:143 to with HD, and 5 patients with BL, thus repre- 1:211, in HD, IM, or ANA-positive tumor sera; senting both benign and malignant lymphopro- and (iii) high, 1:490, in BL. liferative diseases, contained no ANA to the Anti-EBV titers in sera from ANA-positive nuclei of CK cells. cancer patients with similar histological types To confirm the ANA activity, we tested 28 of tumors were then compared (Table 2). ApANA-positive and 20 ANA-negative sera on proximately 68% of sera from ANA-positive murine cells. Most ANA-positive sera (26/28) groups had elevated anti-EBV titers. Only 28%k also combined with the nuclei of murine 3T3 of ANA-negative sera possessed elevated anticells. Although the staining of 3T3 nuclei had EBV titers. Although the GMT of ANA-posieither a diffuse or peripheral distribution, the tive sera were consistently higher than those of majority (69%) of the positive sera produced the ANA-negative sera, only ANA-positive sera peripheral stain. For the purpose of this paper, from adenocarcinoma patients had a signifiwe will be concerned only with results of ANA cantly (P < 0.005) higher GMT (1:278) than was tests on CK cells. found in ANA-negative sera either from other The 28 ANA-positive sera were assayed for adenocarcinoma patients (1:31) or from patients antibodies to the capsid antigen (13) of EBV with other malignant diseases combined (1:48). (Table 1). Control sera (ANA negative) were The distribution of elevated titers in ANA-posiobtained from patients with HD, IM, BL, and tive sera from patients with adenocarcinomas ANA-negative malignant diseases, as well as was, of course, also significantly increased (X2, from normal adults. The latter population was P < 0.05) from that in ANA-negative sera from divided into two groups: group A, aged 20 to 50 adenocarcinomas. The presence of ANA and years; and group B, aged 54 to 68 years. Alelevated anti-EBV titers in sera from cases of though the percentage (42%) of normal individ- adenocarcinoma were unrelated to any specific uals in group A with antibodies to EBV was tissue origin of the malignant growth. significantly lower (X2 test, P < 0.005) than that When data were analyzed according to site of in any other group, the GMT and distribution tumor without regard to histological type (Taof titers in sera of the younger EBV-positive ble 3), groups of ANA-positive sera always posnormal individuals were similar to those in sessed higher GMT. However, differences in older normal adults (group B) and in ANA- GMT between ANA-positive and ANA-neganegative tumor patients used as controls. The tive groups with the same tumor site were stamajority (89%) of ANA-positive sera from tu- tistically significant only in patients with tumor patients contained anti-EBV antibodies mors of the gastrointestinal or genitourinary with GMT of 1:143, a significant elevation from tracts. cence was

TABLE 1. Distribution of antibodies to EBV in sera from normal individuals and patients with diseases related or unrelated to EBV Gr Group oup

Normal, Group Aa

No.% Posi-

tested tested

tive te

No. of sera of indicated titer

____________-1160 Range of titer

.1:160 1:101:101:40

of sera %osea

GMT of positive sera

.1:10

57 42 33 16 8 1:10-1:640 33 1:53 13 93 1 7 5 1:10-1:640 38 1:51 28 89 3 8 17 1:10-1:2560 68 1:143& 21 100 0 14 7 1:10-1:160 33 1:48 10 100 0 2 8 1:10-1:640 80 1:211 HD 12 91 1 3 8 1:40-1:640 72 1:200 BL 5 100 0 0 5 1:160-1:2560 100 1:490 Age range, 20 to 50 years. Age range, 54 to 68 years. 'GMT is significantly elevated when compared with normal sera (t test, P < 0.01) or ANA-negative sera from tumorous patients (t test, P < 0.005). GMT is significantly lower than sera from BL patients (t test, p < 0.01).

Group Bb Tumorous (ANA+) Tumorous (ANA-) IM

INFECT. IMMUN.

McCORMICK, McCORMICK, AND TRENTIN

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TABLE 2. Distribution of anti-EBV titers in sera according to the type of tumor and presence or absence of ANA No. of sera of

indicated titer Serum

Type of malignancy

Range

No. tested

1:10-

% of sera

.o1:160

GMT

1:40- 1:160

1:110 66 1:10-1:2560 4 2 6 Lymphoid 1:278" 84 1:160-1:640 5 0 5 Adenocarcinoma 1:119 57 1:10-1:640 8 6 14 Squamous carcinoma 1:143 68 1:10-1:2560 17 8 25 Total 1:28 20 1:10-1:160 1 4 5 Lymphoid ANA1:31 0 1:10-1:40 0 5 5 Adenocarcinoma 1:67 45 1:10-1:160 5 11 6 Squamous carcinoma 1:48 28 1:10-1:160 6 15 21 Total GMT is significantly elevated when compared with ANA-negative sera from adenocarcinoma patients (t test, P < 0.005) or to all ANA-negative sera combined (t test, P < 0.05).

ANA+

TABLE 3. Distribution of anti-EBV titers in sera according to the site of malignancy and presence or absence of ANA No. of sera of indicated titer Serum

Site of tumor

Range

No. tested 1:10-

% of sera

.1:160

GMT

1:160

1:40

1:110 66 1:10-1:2560 4 2 6 Lymphoid 1:50 33 1:10-1:160 2 4 6 Mammary 1:160 66 1:40-1:640 2 1 3 Skin-soft tissue 1:254a 83 1:40-1:640 1 5 6 Gastrointestinal 1:320" 100 4 1:160-1:640 4 0 Genitourinary 1:28 60 1:40-1:640 3 2 5 Lymphoid ANA1:10 0 0 1 1 Mammary 1:63 50 1:10-1:160 3 3 6 Skin-soft tissue 1:20 0 1:10-1:40 2 0 2 Gastrointestinal 1:40 28 1:10-1:160 2 5 7 Genitourinary gastrointeswith patients from GMT is significantly elevated when compared with ANA-negative sera tinal tumors (t test, P < 0.025). bGMT is significantly elevated when compared with ANA-negative sera from patients with genitourinary tumors (t test, P < 0.005) or with all ANA-negative sera combined (t test, P < 0.005). ANA+

DISCUSSION An increase in both the percentage of positive and titer of antibodies to EBV, the etiological agent of IM (15, 27), has also been associated with a variety of malignant diseases such as BL (14), NPC (6, 17), HD (20, 24), and lymphocytic lymphomas and leukemias (16, 21, 23, 25). Sera from other neoplastic conditions have not been reported to have elevated titers to EBV. The highest titers of antibody are present in patients with BL and NPC; intermediate titers are present in IM and the remaining malignant diseases (16, 23). No increases (7, 12) in antiEBV titers were reported in other malignant diseases of lymphoproliferative, carcinomatous, or sarcomatous origin. Since patients with IM or NPC (22, 33), both associated with EBV, may produce ANA, we studied anti-EBV titers in cancer patients with or without ANA to nuclei of CK cells. The GMT sera

of antibodies in sera from patients negative for ANA was similar to that in normal sera. In contrast, sera from patients who produced ANA showed elevated titers to EBV. The GMT obtained in this study correspond to those found in patients with IM, HD, SLE, (9, 10), or sarcoidosis (19, 31). The elevated titers appeared to be associated with the presence of ANA since: (i) sera of normal individuals produced GMT similar to those of cancer patients lacking ANA; (ii) the GMT of ANA-positive sera from patients with similar histological types of tumors were consistently elevated; and (iii) higher GMT were found in sera containing ANA when compared with the GMT of sera from ANA-negative patients with tumors of the same organ. The elevated titers in ANA-positive sera were not the result of age differences between cancer patients and normal controls. Although cancer occurs predominantly in older age groups, normal sera of group B (mean age, 61 years) pos-

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NUCLEAR AND EBV ANTIBODIES IN CANCER

sessed neither ANA nor elevated GMT to EBV, but, as expected, showed a higher incidence of antibodies to EBV than did the younger group A (93% versus 42%). ANA to CK nuclei were not observed in sera from normal individuals or patients with EBVassociated benign or malignant diseases. Our inability to demonstrate ANA in sera from IM (22) may be related to the time at which serum samples were obtained during the course of the disease. However, in patients with NPC, the presence of ANA is unrelated to the treatment or clinical course of the disease (34). ANA are reported to appear in 40 to 50% of cases of NPC; in 5 to 10% of patients with malignant diseases of the hematopoietic organs; and in 20 to 25% of patients with other types of cancer (34). Perhaps a greater number of sera might have been positive if 3T3 cells had also been used as antigen. We observed that 3 of 20 sera that were negative for ANA on CK cells were positive when tested on the murine cells. In regard to the patterns of fluorescence observed, only a heavy speckled appearance was detected in the nuclei of CK cells. However, the same sera produced peripheral or homogenous patterns in murine cells. Different patterns of ANA staining occur when mouse or rat kidney cells are used (5), and the pattern may be related to the nuclear antigen detected. The antigens involved are deoxyribonucleic acid and soluble nucleoprotein, particulate nucleoprotein, or saline-soluble components of the nucleus. Recently, Yasuda and Yoshida (32) purified an acidic protein containing ribonucleic acid that is involved in staining by sera from patients with NPC. Although the nuclear antigens were not characterized in our study, there was no correlation between the staining pattern on murine cells and the titer of antibodies to EBV in ANA-positive sera. Titers of ANA in cancer patients (34) are usually low (1:10 to 1:160), and Evans and Rothfield (9) found no correlation between presence and titer of ANA and elevated anti-EBV titers in individual patients with SLE. The increase in anti-EBV titers of sera containing ANA to CK cells could result from (i) activation of latent EBV; (ii) an increased susceptibility to EBV; (iii) cross-reactivities with antigens of other herpesviruses (11, 28); or (iv) a nonspecific response of a malfunctioning immune system. ANA have been shown to appear in mice after infection with type C leukemia viruses (4). Although ANA may be found in sera from cases of IM and NPC, BL patients have not been shown to produce ANA (34), suggesting that ANA is not directly related to infection by EBV. Since intermediately ele-

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vated titers to EBV are also present in a bacterial disease, lepromatous leprosy, in which cellmediated immunity is depressed (29), we assume that a nonspecific immunological disorder characterized by ANA formation is also responsible for the elevated EBV titers. Serological studies utilizing other herpesviruses as antigen should indicate whether or not an elevated titer is specific for only the one human herpesvirus (EBV) or represents a general immunological stimulation to latent herpesviruses. ACKNOWLEDGMENTS This work was supported by Public Health Service grants CA 11363, CA 12093, and K6 CA 14219 (from the National Cancer Institute) and grant RR 00259 (from the Division of Research Resources). LITERATURE CITED 1. Anderson, J., K. J. McCormick, W. A. Stenback, and J. J. Trentin. 1971. The development of chick-embryolethal-orphan (CELO) virus T and V antigens in lytically infected chick kidney cells. Int. J. Cancer 7:5964. 2. Anderson, J., K. J. McCormick, W. A. Stenback, and J. J. Trentin. 1971. A plaque assay for chick-embryolethal-orphan (CELO) virus. Proc. Soc. Exp. Biol. Med. 136:499-502. 3. Anderson, J., V. J. Yates, V. Jasty, and L. 0. Mancini. 1969. The in vitro transformation by an avian adenovirus (CELO). III. Human amnion cell cultures. J. Natl. Cancer Inst. 43:575-580. 4. Cannat, A., and B. Varet. 1972. Induction of antinuclear antibodies in (C x B6)F, mice inoculated with Graffi and Rauscher leukemogenic viruses. Proc. Soc. Exp. Biol. Med. 141:1077-1080. 5. Cleymaet, J. E., and R. M. Nakamura. 1972. Indirect immunofluorescent antibody tests: comparison of sensitivity and specificity of different substrates. Am. J. Clin. Pathol. 58:388-393. 6. DeSchryver, A., A. Friberg, Jr., G. Klein, W. Henle, G. Henle, G. de The, P. Clifford, and H. C. Ho. 1969. Epstein-Barr virus-associated antibody patterns in carcinoma of the post nasal space. Clin. Exp. Immunol. 5:443-459. 7. DeSchryver, A., G. Klein, G. Henle, W. Henle, H. M. Cameron, L. Santesson, and P. Clifford. 1972. EB virus associated serology in malignant disease: antibody levels to viral capsid antigens (VCA), membrane antigens (MA) and early antigens (EA) in patients with various neoplastic conditions. Int. J. Cancer 9:353-364. 8. Epstein, M. A., B. G. Achong, and Y. M. Barr. 1964. Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet i:702-703. 9. Evans, A. S., and N. F. Rothfield. 1973. EB virus and other viral antibodies in systemic lupus erythematosus. Lancet i:1127-1128. 10. Evans, A. S., N. F. Rothfield, and J. C. Niederman. 1971. Raised antibody titers to EB viruo in systemic lupus erythematosus. Lancet i:167-168. 11. Evans, D. L., J. WN. Barnett, J. M. Bowen, and L. Dmochowski. 1972. Antigenic relationship between the herpesviruses of infectious bovine rhinotracheitis, Marek's disease, and Burkitt's lymphoma. J. Virol. 10:277-287. 12. Gahrton, G., B. Wahren, D. Killander, and G. E. Foley. 1971. Epstein-Barr and other herpesvirus antibodies in children with acute leukemia. Int. J. Cancer 8:242-244.

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Antinuclear antibodies and elevated anti-Epstein-Barr virus titers in cancer patients.

Vol. 13, No. 5 Printed in U.S.A. INFECTION AND IMMUNITY, May 1976, p. 1382-1386 Copyright CiD 1976 American Society for Microbiology Antinuclear An...
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