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[CANCER RESEARCH 40, 461 2-461 6. December 1980] 0008-5472/80/0040-OOOOS02.00 Prediction of Survival in Gynecological Cancer Based on Immunological Tests1 Irene J. Check,2'3 Robert L. Hunter,2 Karen D. Rosenberg, and Arthur L. Herbst Departments of Pathology ¡I.J. C., R. L. H.. K. D. R.I and Obstetrics and Gynecology ¡A.L. H.¡.University of Chicago. Chicago. Illinois ABSTRACT Patients with advanced cancer have a depressed immunological function. We performed a battery of tests on peripheral blood samples from 42 patients with gynecological cancer to determine the extent to which this depression was due to abnormal lymphocyte function, as compared to changes in the number of lymphoid cells in the peripheral blood or in the efficiency of purification of cells in Ficoll:Hypaque gradients in preparation for testing. The percentage of lymphocytes in the gradient-derived cell suspension (% LG) and the absolute lymphocyte count were more informative than mitogen stimu lation, mixed leukocyte culture, and T- and B-cell measure ments. Both values decreased significantly with the advancing stage of cancer, and we were able to predict survival of patients with uniform stage of disease. The % LG correlated with survival better than did any other test when multivariate analy ses of all test combinations were performed. Low values of % LG reflected both the depressed lymphocyte counts and the altered buoyant density of the leukocytes of many patients with advanced cancer. A large portion of the depression in other immune function tests was statistically attributed to changes in % LG and the lymphocyte counts. We concluded that these two simple measurements provide valuable information about patients with gynecological cancer. direct measurement of the appropriate factors would provide simpler and more reliable clinical tests. To evaluate this prob lem, we carried out a battery of immune function tests on patients with gynecological cancer. Total and differential blood counts and the proportion of lymphocytes in all cell suspen sions prepared by density gradient centrifugation were deter mined at the same time. The results of these assays were analyzed to assess which tests correlated with the stage and prognosis of the patients. We found that the absolute lympho cyte count and the proportion of lymphoid cells in the density gradient-purified cell suspensions predicted survival better than did any of the subsequent tests of lymphocyte function. MATERIALS AND METHODS Patient Population. Blood was obtained from 42 patients with gynecological cancer at the Chicago Lying-in Hospital. Twenty patients had cervical cancer (Stage I, 5 patients; Stage II, 7 patients; Stage III, 5 patients; Stage IV, 3 patients), 13 patients had ovarian cancer (Stage I, 1 patient; Stage II, 1 patient; Stage III, 8 patients; Stage IV, 3 patients), 4 patients had endometrial cancer (Stage I, 1 patient; Stage IV, 3 pa tients), 4 patients had leiomyosarcoma (Stage III, 1 patient; Stage IV, 3 patients), and 1 patient had vulvar cancer (Stage I). The stage of disease was based on the criteria of the Interna tional Federation of Obstetrics and Gynecology (19). The data INTRODUCTION are presented according to stage of Cancer at the time of testing and with respect to survival after testing. Seventeen of Although many investigators have found depressed immune the patients had not had previous therapy (Stage I, 8 patients; function in patients with advanced gynecological cancer (4, Stage II, 5 patients; Stage III, 2 patients; and Stage IV, 2 10, 21, 28, 30), the relative value of the various assays and patients). Two patients with Stage II cervical cancer had un their prognostic significance for individual patients remain un dergone radiotherapy. The other patients were being treated certain. The extent to which the observed depression in ¡mwith the standard combination chemotherapy for their tumor munological tests represents functional deficits has not been type, and the blood samples were obtained just before the start separated from that which results from variation in the numbers of a new cycle of therapy. All tests were performed on coded of the peripheral blood cells. In patients with cancer, the samples without knowledge of the diagnoses. Comparisons differential WBC counts, especially the absolute lymphocyte were made among groups of patients with gynecological can counts, range widely (10, 24, 30). Furthermore, most tests of cer. immune function require density gradient-purified cell suspen Immunological Tests. Venous blood was drawn into sterile sions. Since many physiological factors affect the buoyant siliconized 20-ml vacutainer tubes containing 40 mg disodium density of leukocytes, the composition of these cell suspen EDTA. Total WBC and leukocyte differential counts were done sions is quite variable, especially in patients with cancer (9, with the Hemalog D automated differential cell analyzer (Tech11). nicon, Tareytown, N. Y.), which utilizes cytochemistry and If either of these factors contributes substantially to the continuous flow analysis to count a sample of 10,000 cells outcome of immune function assays, one might expect that (18). Leukocytes were separated on Ficoll and sodium diatrizoate 1Supported by Grant CA 14599 from the National Cancer Institute and by the (Hypaque) gradients (3) as recommended by WHO (1 ). Smears Mothers' Aid Research Fund of the Chicago Lying-in Hospital. Part of this work of the gradient cell suspension were prepared by centrifugation was presented at the meeting of the Federation of American Societies for Experimental Biology in Anaheim. Calif.. April 1980. and stained for peroxidase by the method of Kaplow (16). The 2 Present address: Department of Pathology and Laboratory Medicine. Emory proportion of lymphocytes (peroxidase-negative cells) was cal University Medical School. Atlanta. Ga. 30322 ' To whom requests for reprints should be addressed. culated from a total count of 200 cells. Differential counts of Received May 27. 1980; accepted September 8. 1980. 100 parallel Wright:Giemsa-stained slides showed that the 4612 CANCER RESEARCH VOL. 40 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. Immunological nonlymphoid (peroxidase-positive) cells were predominantly monocytes, with increasing numbers of neutrophils, and im mature myeloid cells in cell suspensions with the lowest pro portions of lymphocytes. The 2 stains gave comparable lym phocyte proportions. In healthy donors, the median percentage of lymphocytes was 55%, with 95% of the values between 40 and 80%. Lymphocyte stimulation assays were set up in triplicate in flat-bottomed 96-well microtiter plates (Linbro Scientific, Inc., Hamden, Conn.) as described previously (20). Cells (2.5 x 105) were incubated in 200-fil cultures using Roswell Park Memorial Institute Tissue Culture Medium 1640 (Grand Island Biological Co., Grand Island, N. Y.) containing 7.5% heatinactivated AB-positive normal human serum. The following final concentrations of mitogens were used: Con A,410 fig/ml, (Grade IV; Sigma Chemical Co., St. Louis, Mo.); PWM, 1:80 (Grand Island Biological Co.); and PHA, 1:800 (DIFCO Labo ratories, Inc., Detroit, Mich.). MLC contained 2.5 x 10s pooled irradiated (3000 rads) cells from 3 normal unrelated donors in place of mitogen with additional control cultures containing medium in place of patient cells. Medium control cultures contained patient cells but no mitogen or allogeneic cells. The cultures were incubated at 37° in a humidified CO2 incubator (Forma Scientific, Marietta, Ohio). The incubation period was 3 days for Con A-, PWM-, and PHA-stimulated cultures and 5 days for MLC and a second set of PWMstimulated cultures. They were then pulsed with 1 ftCi [methyl3H]thymidine (specific activity, 20 Ci/mmol; Amersham/Searle Corp., Arlington Heights, III.) and incubated an additional 4 hr. The cells were harvested, and radioactivity was counted as described previously (20). The radioactivity that was incorpo rated by medium control cultures was subtracted from that incorporated by stimulated cultures. Their dpm (1520 ±1638, S.D., of all 42 patients on Day 3; 1900 ± 2565 on Day 5) showed no significant differences among patients with different extents of disease. We obtained 5 to 10 x 108 lymphoid cells from normal individuals by leukopheresis and then froze them. An aliquot of these "control cells" was thawed and tested in parallel with all patient samples (20). Statistics. Changes in test values with stage of disease or tumor cell type were evaluated using analysis of variance. The relationships between different tests were determined by the Pearson correlation coefficients. The frequency of abnormal results among groups of patients was assessed using contin gency table analysis with the x2 test- Survival curves and median survival times were determined by life table analysis (15). The ability of the tests to predict survival was determined by comparing the life table survival curves of groups of patients with different test values using Lee-Desu statistics (17). The nonparametric multivariate regression model of Cox (8) was used to predict survival of individual patients having specified test values. This statistical procedure uses test and survival data from all available patients to predict the survival of a patient with particular test values. The significance levels of the Cox regression were calculated from the x2 values obtained by maximizing log likelihood ratios. The coefficients (/8's) derived 4 The abbreviations used are: Con A, concanavalin A; PWM. pokeweed mitogen; PHA, phytohemagglutinin; MLC, mixed leukocyte culture; % LG, per centage of lymphocytes in gradient interface. DECEMBER 1980 Tests in Gynecological Cancer from the Cox equation were used to calculate the magnitude of the upward or downward shift in the survival curves expected from a specified change in test values. RESULTS A battery of immunological tests was done on each of 42 patients admitted to this hospital with gynecological cancer (Table 1). We found that the absolute lymphocyte count and the percentage of lymphocytes in the Ficoll:Hypaque gradientpurified cell suspensions decreased most significantly in pa tients with advanced cancer. The results of the lymphocyte stimulation tests using mitogens or MLC also decreased with stage of disease. The total WBC, neutrophil, and monocyte counts and the percentages of sheep erythrocytes, active sheep erythrocytes, or sheep erythrocytes sensitized with an tibody and complement-rosetting lymphocytes did not change significantly with stage of disease (data not shown). None of these tests changed significantly with the cell type of the tumor (data not shown). The significance levels in Table 1 are con servative estimates since they do not take into account the ranking of the stages. The extent to which the depressed lymphocyte function resulted from the depressed absolute lymphocyte counts or % LG was evaluated by using a series of correlation analyses. The lymphocyte stimulation data, obtained after 3 days of culture with mitogens, correlated significantly with the total lymphocyte count (r = 0.56, 0.54, and 0.60 for Con A, PWM, and PHA with p values <0.001 for each) and with the values of % LG (r = 0.53, 0.53, and 0.49 for Con A, PWM, and PHA with p values <0.001 for each). The % LG was correlated highly with the total lymphocyte count (r = 0.67). In view of the large experimental errors inherent in lymphocyte stimulation tests, these correlation values suggest that most of the depres sion observed in 3-day stimulation tests was due to depression in absolute lymphocyte count and % LG rather than to changes in the capacity of individual lymphocytes to respond. The lymphocyte stimulation data obtained after 5 days of culture also correlated significantly with the % LG but to a lesser extent (r values from 0.27 to 0.34). The tests which changed with stage of disease were then analyzed to determine if any of them had prognostic value for individual patients. The broad range of test values in each stage of disease is illustrated for % LG in Chart 1. We consid ered whether test values might affect the prognosis of patients with a particular stage of disease and evaluated this possibility using the life table regression model of Cox (8). Five of the tests predicted the survival of patients with ad vanced gynecological cancer significantly better than did the stage of disease alone, Table 2. The % LG and the Day 3 PWM response were the most significant individual tests. In multivariate analysis, however, only % LG retained signif icance in all combinations. The lymphocyte count and the PWM responses did not predict survival significantly when % LG was factored out. This constitutes statistical evidence that % LG measured the more basic independent parameter which pre dicted survival of this group of patients. The prediction of survival achieved with the combination of % LG and the MLC response was more significant than that achieved by either test alone, p = 0.0009. No other combina tion of tests improved the prediction of % LG alone. This 4613 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. /. J. Check et al. Mean values (±S.D.) of blood counts and immunological (cells/cu of pa mm)7620 tients881412WBC StageIIIIIIIVPcNo. Table 1 test results in gynecological 3)a%LG64 cancer patients grouped according to stage of cancer stimulation tests (dpm x 10 (cells/cu mm)2138 2484b5621 ± 10341572 ± 22716504 ± 847923 ± 43065242 ± 384690 ± A164 2155± 4497± 77192± 751 ± 1338+1635 ± 6866± 82127± 7886 30 ± 4078± 88127± 5888± 4440.0003Lymphocyte ± 190.004PWM-3153 ± 620.01PHA224 ± 30000.51Lympocytes ± a [3HJThymidine incorporation, 370.03Con ± 65165± 7692± 921 ± 5147± 06 6653± 4730± 540.01MLC86 ± 610.06PWM-5147 ± 310.06 ± Con A. PWM-3, and PHA assays were harvested after 3 days of culture, and PWM-5 and MLC were harvested after 5 days of culture. b Mean ±S.D. c Difference among stages, analysis of variance. IOO- 80- o 60~ .£ S 40o. 20- I I X S Clinical I ffl I n? Stage at Time of Testing Chart 1. Relation between % LG and stage of gynecological cancer. The proportion of lymphocytes present in cell suspensions prepared by FicollrHypaque gradient separation of blood from 42 patients with gynecological cancer is compared to the stage of cancer at time of testing. Each point represents one patient, and the bars represent the mean values for each stage (significance of decrease in % LG with stage is p = 0.004). Fifty-eight % of patients with Stage III and IV disease, in contrast to 6% of patients with Stage I and II disease, had less than 35% LG (x2 = 11.1 ; p < 0.001 ). In patients with Stage III and IV cancer, % LG correlated with survival time (p = 0.005; Cox analysis) as in Table 2 and Chart 2. Table 2 Survival prediction using immunological parameters in Stage III and IV gynecological cancer: magnitude of change in test values required for 50% decrease in death rate multiplier Test results of 26 patients with Stage III or IV gynecological cancer were evaluated to determine whether the magnitude of the test values was related to the survival times after testing. The significance of the relation between each test and survival time (p) and the increase in test value required for a 50% decrease in the death rate multiplier were calculated using Cox multivariate regression analysis. Test Required increase in test value % LGLymphocyte countPWM-5PWM-3MLC13%407 4614 mm86,600 cells/cu dpm35,000 dpm35.000 dpm0.0050.030.020.0050.02 suggests that the MLC test measured a parameter of lympho cyte function with predicted survival and was not dependent upon the % LG. Since all but 4 of the patients with advanced cancer had undergone some form of chemotherapy or radiotherapy, we considered whether therapy might account for the test results. The test results did not correlate with either the type of therapy or the length of time from the start of therapy. For example, 2 of the patients with extremely low values of % LG (7 and 24%) had not been treated, so that the depressed % LG values could not have been caused by therapy. On the other hand, 2 of the patients with over 60% LG had been diagnosed over 2 years ago so that therapy did not necessarily depress the % LG. To assess the potential clinical value of the statistically significant tests, we evaluated the magnitude of the change predicted by each test. The relative magnitude of the effects of these tests on survival is best shown by comparing the death rates of patients with different test values. Table 2 shows the increase in each test value that predicts or is correlated with a 50% decrease in the death rate multiplier of patients with Stage III and IV gynecological cancer. Although each of the tests affected the death rate, % LG was the most sensitive test, requiring only a 13% change for a 50% decrease in the death rate. We also examined the magnitude of the effect of % LG by calculating the survival curves predicted for patients with par ticular test values. Thus, Chart 2 shows the effects of different values of % LG on survival of patients with advanced gyneco logical cancer. The predicted median survival time of patients with 20% LG was only 2.6 months, while that of patients with 50% LG was greater than 9 months. The overall median survival time for all these patients was 4.9 months. The effect of % LG on the projected survival of individual patients was verified by stratifying the patients into 2 groups, those who had high (greater than 35%) and low (less than 35%) values of % LG, and comparing the Kaplan-Meier esti mates of the survival curves within each stratum. The survival curves were significantly different, with a median survival time of 3.2 months for the 15 patients with less than 35% LG and over 9 months for the 11 patients with higher values (Chart 3). DISCUSSION In this study, we explored the relationships among various tests of peripheral blood cells and assessed their relative ability to predict survival in patients with gynecological cancer. CANCER RESEARCH VOL. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. 40 Immunological 1.0 .9 .8 I 7 E 6 3 .5 C o tj -^ o .2- I 23456789 Months after testing Chart 2. Survival curves predicted for patients with Stage III or IV gynecolog ical cancer having different values of % LG. The curves were obtained by using the Cox analysis of test values and survival times that were observed in 26 patients with Stage III or IV disease. The significance of the relation between % LG and survival time, p = 0.005. > 35 % LG P =.01 8 Months after testing Chart 3. Kaplan-Meier life table survival curves for patients with Stage III or IV gynecological cancer having greater than 35% LG (11 patients) or less than 35% LG (15 patients). . survival curve for all 26 patients. Other investigators have observed that both lymphocyte number and results of immunological function tests correlate with the prognosis of patients with gynecological cancer (10, 21 ). Previous workers have also reported that gradient-derived cell suspensions from patients with cancer are frequently "con taminated" by large numbers of nonlymphoid cells (2, 9, 11, 27). We verified that many patients with advanced gynecological cancer have depressed lymphocyte function. In addition, we determined to what extent the abnormalities in lymphocyte function tests reflect changes in the functional capacity of the lymphocytes and to what extent they reflect differences in absolute lymphocyte count or variations in the efficiency of purification of the lymphoid cells used in the assays. Each of these 3 factors contributed to the results. Primarily, however, the results of the lymphocyte function tests in our patients reflected the variation in lymphoid cell purification, as DECEMBER Tests in Gynecological Cancer indicated by the % LG. Both life table and Cox analysis clearly demonstrated the predictive value of % LG in patients with advanced gynecological cancer. To illustrate how each test predicts survival of patients with comparable extents of dis ease, these analyses were reported on patients with advanced disease. Including patients with Stage I or II disease produced results with even greater statistical significance. The ability of % LG values to predict the survival of patients with advanced cancer suggests that the factors which influence the composition of the cell suspensions are fundamentally related to the disease process. Variation in absolute lympho cyte count among patients accounted for 45% of the variation in % LG (r = 0.67), and changes in lymphocyte recovery accounted for an additional 13% (r = 0.36). Increased reten tion of the nonlymphoid cells, especially immature myeloid cells, accounted for the remainder of the decreased % LG in patients with advanced cancer. The % LG did not correlate with the type of therapy, although the number of patients in each category was too small for statistical evaluation. We also found no relationship between % LG and the cell type of the tumor, and we have reported similar relationships between % LG and survival in newly di agnosed untreated patients with lung cancer (6) and in patients with head and neck cancer (7). The factors that influence the sedimentation of nonlymphoid cells in gradients need further study. Immune complexes, which are present in the sera of some cancer patients (25, 29), may bind to the Fc receptors on granulocytes (26) and change their buoyant density. Other serum factors, such as cytotaxins pro duced by endotoxin activation, alter the sedimentation prop erties of human granulocytes and may occur in some patients with gynecological cancer (5). The systematic change in % LG may partially explain the conflicting results of lymphocyte subclass determinations in cancer patients in work reported previously (13, 23, 30). Ficoll: Hypaque gradients are used to prepare lymphocyte-enriched cell suspensions for nearly all immunological studies. The interpretation of the test depends on whether lymphocytes are distinguished from nonlymphoid cells. We performed several rosette tests to measure lymphocyte subclasses in these pa tients and found the same subclass composition in patients with different stages of disease when we controlled for changes in % LG. We also observed that decreases in the fraction of lympho cytes in the gradient correlated with decreased activity in lymphocyte function tests. The tests are especially vulnerable to the suppressive effects of macrophages and granulocytes. These cells can inhibit lymphocyte function by producing prostaglandin E2 (12, 14) or hydrogen peroxide (22). Since a decreased proportion of lymphocytes at the gradient interface implies an increased percentage of suppressive cells in these cell suspensions, changes in % LG would be expected to correlate with decreased lymphocyte function in vitro. In fact, the observation that patients with advanced cancer show in creased suppressive activity in lymphocyte function assays may primarily reflect alterations in gradient-derived cell sus pensions. Similarly, recent findings of impaired lymphocytemediated cytotoxicity concomitant with enhanced macrophage-mediated cytotoxicity in cancer patients might also sim ply result from the decreased % LG (4). Comparing the results of various functional assays before and after removing the 1980 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. 4615 /. J. Check et al. nonlymphoid cells from the cell suspensions would address this question. However, available procedures for accomplishing this reduce the lymphocyte yield and introduce other artifacts (1). The high correlation value between % LG and stage of cancer supports our view that alteration in % LG is a primary phenomenon that may reflect in vivo immune function. Altered in vitro suppressor or cytotoxic activities may be secondary phenomena, although they may also be biologically significant. The relative ability of the various assays to predict survival also supports this view. All of the lymphocyte stimulation tests decreased in patients with advanced cancer. However, the prognostic component of these tests primarily paralleled the decrease in % LG. Thus, once the effect of % LG was factored out by multivariate analysis, the MLC response, only, signifi cantly predicted survival in advanced disease. We have shown that the % LG is strongly related to the basic disease process as judged by its ability to predict patient survival. In our view, these results offer a new approach to understanding the interaction between the immune system and cancer. Both the WBC counts and the % LG are more funda mental parameters than is lymphocyte stimulation, and both are easier to measure. Achieving a better understanding of how the cancer process affects the number of WBC and their sedimentation behavior should shed new light on the altered immune status of cancer patients and may tell us something about the mechanism of cancer itself. We are currently pursu ing these issues. ACKNOWLEDGMENTS We thank T. Coley. A. Matutis. and R Merker for expert technical assistance; P. Weiser for clinical information; and T. Karrison for help with statistical analyses. REFERENCES 1. Aiuti, F.. Cerottini, J-C.. Coombs. R. R. A., Cooper. M., Dickler. H. B.. Froland, S.. Fudenberg, H. H.. Greaves. M. F.. Grey. H. M., Kunkel. H. G.. Natvlg, J., Preud'homme. J. L. Rabellino. E.. Ritts. R. E.. Rowe. D. S., 2. 3. 4. 5. 6. Seligmann, M.. Slegai. F. 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Decline of natural nonselective cell-mediated cytotoxicity in patients with tumor progression. Cancer Res., 37. 413-418. 1977. 29. Theofilopoulos, A. N.. Andrews. B. S., Urist. M. M., Morton, D. L., and Dixon. F. J. The nature of immune complexes in human cancer sera. J. Immunol., Õ79. 657-663. 1977. 30. Wanebo, H. J. Immunologie testing as a guide to cancer management. Surg. Clin. N. Am., 59: 323-347, 1979. CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. VOL. 40 Prediction of Survival in Gynecological Cancer Based on Immunological Tests Irene J. Check, Robert L. Hunter, Karen D. Rosenberg, et al. Cancer Res 1980;40:4612-4616. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/40/12/4612 Sign up to receive free email-alerts related to this article or journal. 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