2073 Lymphocyte Subpopulations in Patients with Multiple Primary Tumors Eliezer Robinson, M.D.1 Ruth Segal, B.Sc.1 Lily Struminger, M.Sc.1 David Faraggi, Ph.D.1 Ruchama El’ad-Yarum, Tamar Mekori, Ph.D.1 BACKGROUND. Cancer patients with single tumors live longer today due to earlier 2 B.Sc. 1 Northern Israel Oncology Center, Rambam Medical Center and Faculty of Medicine, TechnionIsrael Institute of Technology, Haifa, Israel. 2 University of Haifa, Haifa, Israel. detection and improved treatment methods. For this reason, the authors see more patients who develop a second primary tumor. The etiology of the second tumor can be the same as the first, whether treatment-induced or unknown. The prognoses of these patients usually depend on the behavior of the second tumor. METHODS. The authors investigated the lymphocyte subset in 88 of the more than 750 patients listed in the tumor registry at their treatment center who had at least one carcinoma of the breast or colon and a second primary of the same or another site. Mononuclear cells were obtained from heparinized blood by the standard fractionation Hypaque gradient centrifugation technique. Helper and suppressor cells were identified by using three murine monoclonal antibodies: CD3 for mature T lymphocytes, CD4 for helper inducer cells, and CD8 for suppressor cytotoxic cells. T-cell subset distribution was evaluated with flow cytometry. RESULTS. Most values of CD3, CD4, and CD4/CD8 were lower in patients than in healthy controls. The values of CD4 and CD4/CD8 were lower in patients who had a second tumor in the colon rather than in the breast. CONCLUSIONS. As tumors in patients with a second primary sometimes recur or the patient develops a third primary, the authors are prospectively following their patients to see whether those with immunosuppression have a greater tendency to develop recurrent disease or a third primary. Cancer 1999;85:2073– 6. © 1999 American Cancer Society. KEYWORDS: lymphocyte subsets, lymphocyte subpopulation, multiple primary tumors, second primary tumor. C The authors thank Mrs. M. Perlmutter for her assistance in the preparation of this article. Address for reprints: Eliezer Robinson, M.D., Northern Israel Oncology Center, Rambam Medical Center, P.O. Box 3595, Haifa 31034, Israel. Received February 1, 1999; accepted February 4, 1999. © 1999 American Cancer Society ancer patients with single tumors live longer today due to earlier detection and improved treatment methods. For this reason, we see more patients who develop a second primary tumor. The etiology of the second tumor can be the same as the first, whether treatmentinduced or unknown. The prognosis of these patients usually depends on the behavior of the second tumor. Many authors, including us, have reported on the immune status of cancer patients and its relation to the prognosis.1–3 We have a multiple primary tumor registry at our treatment center that contains over 750 patients. In this study, we investigated the lymphocyte subset in 88 of these patients with at least 1 breast or colon carcinoma and a second primary at the same or another site. PATIENTS AND METHODS Patient Characteristics Fifty-two patients with breast carcinoma and 36 with colon carcinoma who had a metachronous second primary solid tumor, according to the definition of Warren and Gates, at the same site or at 2074 CANCER May 1, 1999 / Volume 85 / Number 9 another site were included in this study. Twenty-one patients had breast carcinoma as a first primary tumor and another tumor as a second primary (br1x). Fourteen patients had the first tumor at any site and breast carcinoma as the second one (x1br). Twenty-nine patients had bilateral breast tumors (br1br) and 16 had a single breast tumor (br). The mean age (6 standard deviation) of the healthy controls was 39.8 6 10 years, of the patients with single breast carcinoma 60.8 6 19 years, and of the patients with multiple primaries 67.0 6 9 years. Twelve patients had colon carcinoma and a second primary tumor at another site (col1x). In 19 patients, colon carcinoma was the second tumor (x1col), and 5 patients had two primaries in the colon (col1col). (Eight patients had br1col and four had col1br; these were included in the abovenoted numbers.) Ten men and 20 women served as controls. All patients registered in the tumor registry at least 1 year before the diagnosis of the second tumor were included. Patients were diagnosed at least 1 year before the test was performed. Most patients had had prior chemotherapy and/or radiotherapy. Blood was taken after informed consent was obtained. TABLE 1 Comparison of T-Cell Subsets in Cancer Patients and Healthy Controls Group No. of patients Mean 6 SD Median CD3 Healthy controls At least one breast tumor At least one colon tumor 30 52 34 70.03 6 14.61 60.78 6 18.67 59.80 6 18.95 CD4 Healthy controls At least one breast tumor At least one colon tumor 30 52 36 51 6 13.53 51.46c 44.83 6 20.14 41.76d 35.43 6 13.79 38.00c,d CD4/CD8 Healthy controls At least one breast tumor At least one colon tumor 30 52 36 1.41 6 0.61 1.17 6 0.7 0.91 6 0.46 73.82a,b 61.45a 65.18b 1.42e,f 0.99e 0.80f The groups with the same indices indicate that they differ significantly (a 5 0.05). a P 5 0.0214. b P 5 0.0242. c P 5 0.0001. d P 5 0.0407. e P 5 0.0222. f P 5 0.0001. Methods Twenty mL of heparinized blood was drawn from each donor. The test was carried out as described by Reinherz et al.4 Mononuclear cells were obtained by the standard fractionation Hypaque gradient centrifugation technique. Helper and suppressor cells were identified by using three murine monoclonal antibodies (Ortho Pharmaceuticals, Raritan, NJ): CD3 for mature T lymphocytes; CD4 for helper inducer cells, and CD8 for suppressor cytotoxic (method described previously5). The appropriate monoclonal antibody was added to aliquots of 1–2 3 106 cells and the cells were stained by an immune fluorescent technique using a fluorescein-labeled goat antimouse: immunoglobulin (Ig)G fluorescein isothiocyanate– conjugated goat antimouse IgG antibody (Tago Inc., Burlingame, CA).5 T-cell subsets distribution was evaluated using the flow cytometer (FacScan, Becton-Dickinson, San Jose, CA). Statistical Evaluation The Wilcoxon test was used to evaluate the differences between pairs of means. For a comparison of three or more means, the Kruskal–Wallis test was used.6 RESULTS The T-cell subsets for healthy controls and patients are shown in Table 1. The values of CD3 were statistically significantly lower in patients as compared with healthy controls (P 5 0.01). CD4 was found to be significantly higher in patients with at least one breast carcinoma than in patients with at least one colon carcinoma (44.83 6 20.14 vs. 35.43 6 13.79; P , 0.0407) (Table 1). CD4 was also significantly higher in the x1br group than in the x1col group (44.15 6 12.68 vs. 34.40 6 14.19; P , 0.0432) (Table 2). In the group with bilateral breast tumors, CD4 was significantly higher as compared with the patients with two primaries in the colon (47.71 6 21.10 vs. 28.24 6 17.15; P , 0.0239). CD4 was significantly lower in these patients than in controls (P 5 0.004) (Table 2). The CD4/CD8 ratio was lowest in patients with at least one colon carcinoma and statistically significantly higher in patients with at least one breast carcinoma, but still significantly lower than in healthy controls (Table 1). It was found to be significantly higher in x1br as compared with x1col (1.14 6 0.39 vs. 0.88 6 0.48; P , 0.0376). It was significantly lower in patients with br1x as compared with normal controls (P 5 0.0014) or patients with a single breast carcinoma (P 5 0.0046). The CD4/CD8 values of patients with x1br were significantly higher than those with br1x (P 5 0.0450). The values obtained for patients with x1col or col1col were significantly lower when compared with those for controls (P 5 0.0004 and 0.0077) (Table 2). The ratio of CD4/CD8 was higher in br1br groups than in those with two primaries in the colon (1.3 6 0.75 vs. 0.72 6 0.26), but the Lymphocyte Subpopulations/Robinson et al. 2075 TABLE 2 The Values of CD4, CD8, and CD4/CD8 Ratio in Patients with Breast Carcinoma, Patients with Colon Carcinoma, and Healthy Controls CD4 CD8 CD4/CD8 Group No. Mean 6 SD Median Mean 6 SD Median Mean 6 SD Median Healthy controls Single breast Breast 1 x x 1 breast Bilateral breast Colon 1 x x 1 colon Colon 1 colon 30 16 21 14 29 12 19 5 51.00 6 13.53 45.84 6 16.04 39.33 6 20.01 44.15 6 12.68 47.71 6 21.10 40.04 6 10.95 34.40 6 14.19 28.24 6 17.15 51.46e 44.90 36.00 43.50a 40.98b 47.70 31.80a,c 26.12b 40.20 6 16.03 33.75 6 13.95 45.34 6 21.98 40.57 6 11.77 39.95 6 15.74 45.21 6 18.64 42.27 6 16.76 39.73 6 21.54 36.24 36.00 37.40 40.89 39.40 39.91 37.88 29.90 1.41 6 0.61 1.41 6 0.62 1.00 6 0.70 1.14 6 0.39 1.30 6 0.75 1.03 6 0.50 0.88 6 0.48 0.72 6 0.26 1.42f,h,i 1.20d 0.80d,e,f 1.11e,g 1.06 0.96 0.80g,h 0.73i SD: standard deviation. The groups with the same indices indicate that they differ significantly [a 5 0.05]. a P 5 0.0432. b P 5 0.0239. c P 5 0.0004. d P 5 0.0046. e P 5 0.0450. f P 5 0.0014. g P 5 0.0376. h P 5 0.0004. i P 5 0.0077. difference was not statistically significant (P , 0.09) due to the small size of the group in col1col (5 patients only). No significant difference was found when comparing br1x with those with col1x (Table 2). There was no significant difference in the CD8 cell subset among patients with multiple primary tumors and between each group of patients and healthy controls. DISCUSSION One hundred four patients with breast or colon carcinoma were included in the study, 88 of whom had 2 metachronous primary tumors (1 of which was in the breast or colon) and 16 of whom had single breast (SB) carcinomas. Studying all patients together, we found that the CD3, CD4, and CD4/CD8 ratios were significantly lower in patients than in healthy controls (HC). We could not rule out the possibility that the difference between normal controls and patients was due to the younger age of the patients. When the patients were divided into seven subgroups according to the site of the tumor, a significant difference was found in CD4 between col1x and controls. In the CD4/CD8 ratios, the following six significant differences were found: HC.br1x; SB.br1x; x1br.br1x; x1br.x1col; HC.x1col; HC.col1col. Reviewing the literature, we found few studies on lymphocyte subsets in patients with single tumors. The mean CD4/CD8 mean helper suppressor ratio was significantly lower in patients with metastatic breast carcinoma as compared with that observed either in controls or in patients without metastasis. The results of this study suggest that a decrease in CD4/ CD8 ratios in patients with metastatic disease is due to a decrease in T helper cells.7 In breast carcinoma patients without relapse, increased CD4/CD8 ratios were found as compared with patients who had a relapse.8 Patients without axillary lymph node involvement had a higher CD4/CD8 ratio than patients with axillary metastases.9 Patients with metastatic cancer treated with a cancer vaccine (ASI) showed an increase in CD4 cells on improved survival.10 Breast carcinoma patients treated with tamoxifen showed an increase in CD4/CD8 lymphocyte subsets as compared with a control group not receiving this treatment. It seems that tamoxifen helps in the recovery of lymphocyte populations decreased by radiotherapy.11 In patients with breast carcinoma who had lymph node involvement or distant metastases, the average CD4/CD8 values were lower than in patients without metastases (difference not significant).12 In a proportion of patients with renal cell carcinoma, interferon seemed to influence the host immune system, resulting in increased CD4/CD8 ratios concomitant with tumor regression.13 In patients with cervical carcinoma, it was found that low CD4 cell counts and CD4/CD8 ratios were characteristic of patients who 2076 CANCER May 1, 1999 / Volume 85 / Number 9 had recurrent disease as compared with those who did not.14 We did not find any references to the lymphocyte subsets in patients with multiple primary tumors, but it is clear that these patients have an immune suppression similar to that of patients with single tumors, and of an even greater degree. It seems also that, in patients who have colon carcinoma as a second tumor, the immunosuppression is greater than in patients with breast carcinoma as a second primary. The CD4/CD8 was also higher in patients with a second primary in the breast and the first elsewhere as compared with the reverse. This was also in accordance with the better prognosis of the first group. Of course, we could not rule out the possibility that this was due to other causes. As tumors in patients with a second primary sometimes recur or the patient develops a third primary, we are prospectively following the patients to see whether those with immunosuppression have a greater tendency to develop recurrent disease or a third primary. In conclusion, lymphocyte subsets were studied in 88 patients with two metachronous tumors and compared with those of healthy controls and patients with a single breast carcinoma. Most values of CD3, CD4, and CD4/CD8 were lower in patients than in healthy controls. 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