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Lymphocyte response and radiation therapy for patients with gynecologic cancer
GYNECOLOGIC
ONCOLOGY
Lymphocyte
9, 209-219 (1980)
Response and Radiation Therapy Gynecologic Cancer1
VERNON K. JENKINS, PH.D., MARVIN H. OLSON, M.D., Departments
of Radiology
and Medical
for Patients with
E. ARCHER DILLARD, JR., M.D.,* AND ROBERT R. PERRY, PH.D.
*Obstetrics and Branch, Galveston,
Gynecology, Texas
the
University
of Texas
77550
Received June 18, 1979 To relate radiotherapy, lymphocyte responses, and clinical course for patients with gynecologic lesions, responses to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) were measured in whole-blood cultures before and after radiotherapy. For 82 patients tested before radiotherapy, lymphocyte counts and responses to PHA, Con A, and PWM were 79, 62, 55, and 67% of control values, respectively. For 29 patients who died within 18 months, the pretreatment lymphocyte count was 80% and mitogenic responses were about one-half of values for 37 patients who lived beyond 18 months. During radiotherapy lymphocyte counts declined to about 30% and mitogenic responses declined to about 10% of control values. For patients who lived beyond 18 months, PWM and Con A responses recovered to pretreatment values within 13-18 months and lymphocyte counts recovered within 19-24 months. PHA response, however, failed to recover above 70% of the pretreatment response. In contrast, patients who died within 18 months had no increase in lymphocyte counts or mitogenic responses after treatment. In general, depressed pretreatment responses to PHA and Con A correlated with a poor clinical course and responses near control levels indicated a good clinical course.
INTRODUCTION
Immunological mechanisms involving recognition and reaction to cancer cells by the host may play an important role in the neoplastic process [l-3]. Evidence that some patients with neoplastic lesions have suppressed cellular immunity which could indicate poor prognosis despite definitive therapy [4-61 gives importance to assessment of immunologic competence before treatment in relation to clinical progress of the patients. There is also considerable concern that treatment with surgery, radiotherapy, and/or chemotherapy for patients with cancer may further suppress natural immunity and afford increased chance for growth of residual or metastatic tumor cells [7-121. Recovery of immunological responsiveness, on the other hand, has been correlated with a very good clinical course [5,13,14]. In this study, immunologic responsiveness of patients with neoplasms of I Supported by Public Health Service Grant CA-13435 from the National Cancer Institute. 209 0090-8258/80/020209-11$01.00/O Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
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gynecologic structures was assessed by measurement of numbers and responses of their lymphocytes to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) in whole-blood cultures, before, during, and at various periods of time after radiation therapy. Lymphocyte numbers and responses to mitogens for patients who had a poor clinical course after treatment were compared with values for patients who responded well to radiation therapy to determine whether their lymphocyte responses before or after treatment were correlated with clinical outcome. In view of an excellent rate of survival for patients treated with radiation in early clinical stages and the relatively poorer rates for patients treated in advanced stages [ 151, longevity of the patients and lymphocyte responses were also considered in relation to clinical stage at the time of treatment. SUBJECTS AND METHODS
Clinical staging for the patients was made according to the recommendation for classification and staging of the International Federation of Gynecology and Obstetrics [ 161. Eighty-two patients were entered into this study and were tested prior to radiotherapy (Table 1); 74 patients were subsequently evaluated near midtreatment, 55 patients were tested at the end of treatment, and 66 samples from 20 patients were assessed more than 24 months after treatment. Values obtained from the patients were compared with values for control samples taken usually on the same day. Control samples were taken from relatives of the patients at or near the same age, laboratory personnel, or other volunteers in normal health, but it was not possible to use the same control each time a patient was HISTOLOGIC
TYPE,
Histologic type
SITE,
TABLE AND STAGE OF LESIONS
1 IN PATIENTS
Primary site of tumor
WITH GYNECOLOGIC
No. of patients in clinical stages I
Adenocarcinoma
Adenoacanthoma Adenoid cystic carcinoma Squamous cell carcinoma Carcinoma (NOS)
Ovary Corpus uteri, endometrium, myometrium, cervix uteri, vagina Corpus uteri, endometrium, myometrium Ovary Corpus uteri, endometrium, myometrium cervix uteri, vagina Corpus uteri, endometrium, myometrium, cervix uteri, vagina
Stromal cell carcinoma n NOS, not otherwise specified.
CANCER
II
III
IV
1 14
3
2
3 2
4 2
1 1
II 1
1 1
1
1
1
16
6
7
1 1
1
LYMPHOCYTE
RESPONSE
AND
RADIOTHERAPY
211
tested. Informed consent was obtained from patients or control individuals after the nature of the procedure was fully explained. The patients were treated with external photon radiation of 32 meV energy usually through two lateral and one anterior fields, by external radiation followed by one or two intracavitary applications of radium, or by external radiation within 8 weeks before or after hysterectomy. For external radiation treatment, a daily dose of 160 to 200 rad was given for 5 days each week for 5 to 7 weeks. Twenty-nine of the patients were given 4500- to 6000-rad external radiation before or after hysterectomy and 31 of the patients received 4000- to 5000-rad external radiation and an intracavitary application(s) to produce a final dose of about 8000 rad to point A and 6000 rad to point E. Point E is defined as a point 5 cm to the right or left of the midline on the ileopectineal line. Twenty-two patients were treated to 5000- to 6000-rad tumor dose by external radiation only. For preparation of cultures 5 ml of peripheral blood were obtained by venipuncture from patients before, during or after treatment and from healthy control individuals. Preservative-free heparin was added to prevent clotting and the total number of white cells was determined in an electronic cell counter. Differential cell counts were made from blood smears by standard methods. The whole blood was diluted 1:20 with culture medium and 3-ml cultures containing 150 ~1 of blood were incubated at 37°C with mitogens to stimulate the lymphocytes to undergo blastic transformation according to methods previously described [ 17-201. Transformation of the lymphocytes in response to the mitogens was assessed by “HTdR incorporation [20]; 1.O &i of 3HTdR (2.0 CilmM) in 0.2 ml of culture medium was added to each culture 24 hr before the end of the 7-day culture period. For harvest the cell suspensions were filtered through glass-fiber disks (grade 934AN, Reeve Angel) in a multiple automated sample harvester. The number of disintegrations per minute (dpm) was calculated from the counts per minute for each culture as was determined in a liquid scintillation counter. The statistical significance of the difference between mean dpm or mean number of lymphocytes of the various groups was determined by the Wilcoxon (Mann-Whitney) sum of ranks comparison [21]. A probability of less than 0.05 for groups being the same population was considered to indicate a significant difference. RESULTS
The mean number of lymphocytes in the peripheral blood of the total group of patients tested prior to radiotherapy was 7% of the number for controls and responses per culture to PHA, Con A, and PWM were 62, 55, and 67% of control values, respectively (P < 0.002, Fig. 1). Mean numbers of lymphocytes and mean responses to mitogens in whole-blood cultures for the total group of patients declined during radiation treatment to values at the end of treatment that were 28% (lymphocytes), 12% (PHA), 9% (Con A), and 11% (PWM) of control values (P < 0.001, Fig. 1). Mean numbers of lymphocytes gradually increased after treatment and the numbers for patients tested at 19-24 months or more than 24 months after treatment approached the number for the total group of patients tested before radiation treatment; however, patient values were still only 58 and 68% of the number for the controls (P < 0.001). Responses to mitogens increased
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slowly after radiotherapy and by 13-18 months after treatment PWM and Con A responses were not significantly less than pretreatment values, but PWM and Con A values even for patients tested beyond 24 months did not exceed pretreatment values. PHA responses for the patients did not recover to the pretreatment level, even though maximal recovery (about 70% of pretreatment response) occurred by 13-18 months after treatment (Fig. 1).
LYMPHOCYTE
RESPONSE AND RADIOTHERAPY
213
Fourteen of the 82 patients tested before radiotherapy had stage IV lesions and had PHA responses that were 40% (P < 0.02) of responses for 32 patients in stage I, 50% (P < 0.02) of responses for 22 patients in stage II, and Con A responses that were 51 and 56% (P < 0.02) of response for patients in stages I and II (Fig. 2). There were apparently fewer lymphocytes and a reduced PWM response in stage IV patients compared to patients in stages I and II, but the differences were not significant. The differences related to clinical stage did not persist at the end of treatment or at l-3 months after treatment. Of the 82 patients tested before treatment, 37 were known alive longer than 18 months, 29 were dead within 18 months, and 16 were known alive less than 18 months or have been lost to follow-up. Lymphocyte numbers and responses for patients who lived beyond 18 months after treatment were compared with values for patients who died within 18 months after treatment (Fig. 3). Pretreatment numbers of lymphocytes and responses to PHA, Con A, and PWM for patients who died within 18 months of treatment were 80% (P < 0.02), 48% (P < O.OOl), 56% (P < 0.003), and 58% (P = 0.07), respectively, of pretreatment values for patients who lived longer than 18 months after treatment. The number of lymphocytes and responses to Con A and PWM for the two groups of patients were not different at the end of treatment, but the PHA response for patients who died within 18 months was only 47% (P < 0.02) of the responses for patients who lived beyond 18 months. No significant recovery in responses to mitogens occurred for patients who died within 18 months. whereas PWM and Con A responses in-
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FIG. 2. Mean numbers and mitogenic responses of lymphocytes in relation to clinical stage: open bars indicate values for patients before radiotherapy, diagonally striped bars indicate values at the end of treatment, and shaded bars indicate values l-3 months after treatment. Numbers of blood samples tested are given in parentheses and standard errors are indicated.
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creased in patients who lived beyond 18 months to near pretreatment values by 13-18 months after treatment. PHA responses, although usually greater for patients who survived beyond 18 months than for patients who died within 18 months, failed to recover to pretreatment values (Fig. 3), The mean pretreatment response to PHA for I1 patients in clinical stages I and II combined who died within 18 months of treatment was 59% (P < 0,003) of the PHA response for 3 1 stage I and II patients who lived beyond 18 months (Fig. 4).
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Similarly, the pretreatment response to PHA for 18 patients in stages III and IV who died within 18 months was 50% (P < 0.02) of the value for six patients in stages III and IV who lived beyond 18 months. At the end of treatment and up to 7- 12 months after treatment responses to PHA for patients who lived beyond 18 months were not significantly different from values for patients of similar stages who died within 18 months. There were no differences in Con A responses between those patients in stages I and II or III and IV who lived beyond 18 months and patients of similar stages who died within 18 months. In Table 2 are given numbers and percentages of patients who survived beyond 18 months in relation to their pretreatment response to PHA and in relation to clinical stage. None of 13 patients with very poor PHA responses (< 99 x 101 dpmkulture) lived beyond 18 months, whereas, 11 of 12 patients (92%) with good responses (> 300 x 103 dprnkulture) lived beyond I8 months. Patients with stages I, II, III, and IV lesions had survival rates beyond 18 months of 86, 60, 36, and 15%, respectively. DISCUSSION
Responses of peripheral blood lymphocyte to in vitro stimulation by PHA and Con A are generally considered to reflect thymus-dependent or T-cell immunity, although Con A has been reported to stimulate bone marrow-derived lymphocytes
216
JENKINS TABLE
NUMBERS
18
AND PERCENTAGES OF PATIENTS WITH MONTHS IN RELATION TO PRETREATMENT
ET AL. 2 GYNECOLOGIC CANCER WHO LIVED BEYOND PHA RESPONSE AND CLINICAL STAGE
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x x x x
103- 99 103-199 103-299 103-399 x 103-536 x lOa-
x x x x x x
103 103 103 103 lo3 lo3
I
II
Oil 416 717 414 414 19122
O/l 317 8110 112 12/20
(86%)
(6W
III
IV
o/3 l/4 l/2 212 4111
O/8 214 O/l 2113
(36%)
(15%)
All stages combined o/13 (0%) 10/2 1 (48%) 16/20 (80%) l/8 (88%) 4/4 (100%)
(B-cells), and PWM may stimulate B-cells and under certain conditions T-cells [22-251. The patients with gynecologic lesions tested prior to radiotherapy had deficits in immunity as indicated by responses to PHA, Con A, and PWM in whole-blood cultures (Fig. 1). Furthermore, patients in advanced stage IV had responses to PHA and Con A that were about one-half the responses of patients in stages I and II (Fig. 2). The deficits in mitogenic responses for the total group of patients, which were S-67% of values for controls, were similar to the deficits reported previously for a smaller group of patients with similar lesions [201. Deficits for the patients may be accounted for only partly by the reduced lymphocyte count which was 7% of the count for controls. Since the lymphocyte pool is comprised of a heterogeneous population of cells with differing functions, it is not unreasonable to surmise that reduced responsiveness reflects fewer cells in subpopulations that are capable of mitogenic responses. Another possible mechanism for reduced responsiveness of lymphocytes is that some inhibitory factor(s) as described by Catalona et af. [ 11 and Whittaker et al. [26] is present in the sera of these patients and interferes with the mitogenic response in vitro. At the end of radiotherapy the number of lymphocytes for the total group of patients was reduced to about one-third of the number for controls, and responses to mitogens were reduced to about 10% of control values (Fig. 1) which shows that radiation not only reduced the number of lymphocytes in a given volume of blood but also reduced the numbers in subpopulations that are responsive to mitogenic stimulation. Reduced responses to mitogens noted at the end of radiotherapy remained until 13-18 months after treatment at which time Con A and PWM responses recovered to near pretreatment values. PHA responses, however, failed to increase to more than about 70% of pretreatment values even for patients tested longer than two years after radiotherapy and even though lymphocyte numbers recovered to pretreatment values. Radiation therapy has been shown previously to reduce numbers and mitogenic responses of lymphocytes in patients with different types and location of neoplasms [6,17,19,20,27-301. In contrast, some investigators who have used mitogens in vitro or skin test reactions to measure delayed hypersensitivity have reported minimal or no depression of
LYMPHOCYTE
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217
cell-mediated immunity after radiotherapy for patients with nonlymphoid neoplasms [31-331. Inconsistencies in data on effects of radiotherapy on patient immunity have caused investigators to question which of several methods used is most likely to indicate the capacity of individuals to mount an immunological response. Whether the in vitro assessment of overall immunity in these patients as measured by mitogenic stimulation in whole-blood cultures is a quantitative measure of the capacity of individual patients to react immunologically to specific tumor antigens is conjecture. Of importance however, is whether the suppressed responses recognizable by this technique before or at time periods after radiotherapy may be correlated with clinical outcome for the patients. Twenty-nine patients who died within 18 months after treatment had PHA and Con A responses before treatment that were about one-half the values for 37 patients who lived beyond 18 months (Fig. 3). The relatively poor response to T-cell mitogens for the patients who had a poor clinical course even after definitive radiotherapy indicate that suppressed response before treatment is correlated with a poor prognosis. In view of a 90%, IO-year survival rate for stage I patients and decreased rates of survival with advanced stages to 14% for stage IV patients treated with megavoltage radiotherapy [151, an obvious question is whether the reduced responses recognized before treatment merely reflect advanced clinical stage, in which case such measurements would add little to prediction of clinical outcome. Of 42 patients with localized lesions (stages I and II) who were at risk for at least I8 months after radiotherapy, 31 survived beyond 18 months and 11 patients died within 18 months (Fig. 4). The mean response to PHA for patients who died within 18 months was only 5% (P < 0.003) of the response for patients who lived beyond 18 months and indicates that some patients staged clinically as having localized lesions have suppressed T-cell response and a poor clinical course. Similarly, of 24 patients with more advanced lesions (stages III and IV), 18 died within 18 months and their mean PHA response was only 50% (P < 0.02) of the response for 6 patients who lived beyond 18 months (Fig. 4). These data are consistent with other reports that impaired cellular immunity before surgery [41 or radiotherapy [ 131 is indicative of a poor clinical course. The report by Dellon et al. [131 also showed that patients with bronchogenic or esophageal carcinoma who had a superior clinical course after radiotherapy experienced an abrupt rise in T-cell levels within 30 days after therapy. In contrast, our data showed slow recovery in T-cell responses, maximal recovery of responses at 13-18 months after radiotherapy, and failure of PHA responses to recover to more than about 70% of pretreatment response even for patients tested more than 2 years after radiotherapy. The significance of failure to recover PHA response even for patients with a good clinical course is not evident, although it may be that the treatment is effectively destroying the lesion and minimal lymphocyte reactivity is sufficient to prevent recurrence. In contrast, suppressed responses to mitogens before treatment and failure to recover responses significantly after treatment for patients with a poor clinical course may indicate occult metastases, regardless of clinical stage, and may contribute to failure to control the disease. It is desirable for treatment planning to know whether measurement of lymphocyte responses made for individual patients prior to treatment or within a few
218
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ET AL.
weeks after treatment can be used to indicate the clinical course for the patient. Although minimal or no recovery in responses that can be correlated with survival or clinical stage occurred within a year after treatment, pretreatment responses to PHA were related to rate of survival and clinical stage for individual patients. Increased responses to PHA were, in general, directly correlated with rate of survival. Clinical stage at the time of treatment was inversely related to survival (Table 2). None of 13 patients who had very poor responses to PHA (< 100 x 1V dpmculture), 2 of whom had localized lesions clinically, lived beyond 18 months. On the other hand, 11 of 12 patients, 2 of whom had more advanced lesions, with PHA responses that exceeded the approximate overall mean for all healthy controls tested (- 300 x 103 dpm/culture), lived beyond 18 months. Both clinical stage and pretreatment response to PHA may be important in predicting the outcome for these patients. Patients who are recognized to have a poor probability of being successfully treated with conventional radiotherapy, or surgery and radiotherapy, may be good candidates for combination of conventional therapy and immunotherapy. REFERENCES 1. Catalona, W. J., Sample, W. F., and Chretien, P. B. Lymphocyte reactivity in cancer patients: Correlation with tumor histology and clinical stage, Cancer 31, 65-71 (1973). 2. Harris, J., and Copeland, D. Impaired immunoresponsiveness in tumor patients, Ann. N. Y. Acad. Sci. 230, 56-85 (1974). 3. Hellstrom, I., Hellstrom, K. E., and Sjorgren, H. O., et al. Demonstration of cell-mediated immunity to human neoplasms of various histological types, Inr. J. Cancer 7, l-16 (1971). 4. Chretien, P. B., Crowder, W. L., and Gertner, H. R., er al. Correlation of preoperative lymphocyte reactivity with the clinical course of cancer patients, Surg. Gynecol. Obsret. 136,380-384 (1973). 5. Eilber, F. R., and Morton, D. L. Impaired immunologic reactivity and recurrence following cancer surgery, Cancer 25, 362-367 (1970). 6. O’Toole, C., Perlmann, P., and Unsgaard, B., er al. Cellular immunity to human urinary bladder carcinoma: I. Correlation to clinical stage and radiotherapy, Inr. J. Cancer 10, 77-91 (1972). 7. Dao, T. L., and Kovaric, J. Incidence of pulmonary and skin metastases in women with breast cancer who received post-operative irradiation, Surgery 52, 203-212 (1962). 8. Deodhar, S. D., Crile, G., Jr., and Esselstyn, C. B., Jr. Study of the tumor cell-lymphocyte interaction in patients with breast cancer, Cancer 29, 1321-1325 (1972). 9. Fisher, B., Slack, N. H., and Cavanaugh, P. J., er al. Post-operative radiotherapy in treatment of breast cancer: Results of the NSABP clinical trial, Ann. Surg. 172, 71 l-732 (1970). 10. Meyer, K. K. Radiation-induced lymphocyte-immune deficiency: A factor in the increased visceral metastases and decreased hormonal responsiveness of breast cancer, Arch. Surg. 101, 114-121 (1970). 11. Stjernsward, J., Jondal, M., and Vanky, F., et al. Lymphopenia and change in distribution of human B and T lymphocytes in peripheral blood induced by irradiation for mammary carcinoma, Lancer 1, 1352-1356 (1972). 12. Hancock, B. W., Bruce, L., and Heath, J., et al. The effects of radiotherapy on immunity in patients with localized carcinoma of the cervix uteri, Cancer 43, 118-123 (1979). 13. Dellon, A. L., Potvin, C., and Chretien, P. B. Thymus-dependent lymphocyte levels during radiation therapy for bronchogenic and esophageal carcinoma: Correlations with clinical course in responders and nonresponders, Amer. J. Roenrgenol. Radium Ther. Nucl. Med. 123,500-5 11 (1975). 14. Hersh, E. M., Whitecar, J. P., Jr., and McCredie, K. B., et al. Chemotherapy, immunocompetence, immunosuppression and prognosis in acute leukemia, N. Engl. J. Med. 285, 1211-1216 (1971).
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15. Fletcher, G. H. Female pelvis, in Textbook ofradiotherapy (G. H. Fletcher, Ed.), Lea & Febiger, Philadelphia, 2nd ed., pp. 620-665 (1973). 16. Kottmeier, H. L. Report presented by the Cancer Committee of the General Assembly of F.I.G.O., New York, April 1970, Int. J. Gynaecol. Obsrer. 9, 172-179 (1971). 17. Jenkins, V. K., Olson, M. H., and Cooley, R. N., ef al. Effect of therapeutic radiation on peripheral blood lymphocytes in patients with carcinoma of the breast, Acta Radio/. 14, 385-395 (1975). 18. Pauly, J. L., Sokal, J. E., and Han, T. Whole-blood culture technique for functional studies of lymphocyte reactivity to mitogens, antigens and homologous lymphocytes, 1. Lob. Clin. Med. 82, 500-513 (1973). 19. Jenkins, V. K., Ray, P., and Ellis, H. N., et a/. Lymphocyte response in patients with head and neck cancer-Effects of clinical stage and radiotherapy, Arch. Otolaryngo/. 102,596-600 (1976). 20. Jenkins, V. K., Olson, M. H., and Ellis, H. N., et a/. In vitro lymphocyte response of patients with uterine cancer as related to clinical stage and radiotherapy, Gynecol. On&. 3, 191-200 (1975). 21. Burington, R. S., and May, D. C., Jr. Handbook of probability and statistics with rabies, McGraw-Hill, New York, 2nd ed., pp. 265-267 (1970). 22. Andersson, J., Moller, G., and Sjoberg, 0. B lymphocytes can be stimulated by Concanavalin A in the presence of humoral factors released by T cells, Eur. J. Immunol. 2, 99-101 (1972). 23. Byrd, W., Boehmer, H., and Rouse, B. T. The role of the thymus in maturational developments of phytohemagglutinin and pokeweed mitogen responsiveness, Cell. Immunol. 6, 12-24 (1973). 24. Janossy, G., and Greaves, M. F. Lymphocyte activation: I. Response of T and B lymphocytes to phytomitogens, Clin. Exp. Immunol. 9, 483-498 (1971). 25. Weksler, M. E., Bodine, S., and Rommer, J. Response of lymphocytes to plant lectins: I. A thymic-dependent lymphoid population responsive to pokeweed mitogen, Immunology 16, 281-290 (1974). 26. Whittaker, M. G., Rees, K., and Clark, C. G. Reduced lymphocyte transformation in breast cancer, Lancer 1, 892-893 (1971). 27. Chee, C. A., Illbery, P. L. T., and Rickinson, A. B. Depression of lymphocyte replicating ability in radiotherapy patients, Brit. J. Radio/. 47, 37-43 (1974). 28. Cosimi, A. B., Brunsterrer, F. H., and Kemmerer, W. T., et (I/. Cellular immune competence of breast cancer patients receiving chemotherapy, Arch. Surg. 107, 531-535 (1973). 29. Gatti, R. A., Garrioch, D. B., and Good, R. A. Depressed PHA responses in patients with nonlymphoid malignancies, in Proceedings of the jifth Ieukocyfe culture conference (J. E. Harris, Ed.), Academic Press, New York, pp. 339-358 (1970). 30. Jenkins, V. K., Olson, M. H., and Ellis, H. N. In vitro methods of assessing lymphocyte transformation in patients undergoing radiotherapy for bronchogenic cancer, Tex. Rep. Biol. Med. 31, 19-28 (1973). 31. Gross, L., Manfredi, 0. L., and Protos, A. A. Effect of Cobalt-60 irradiation upon cell-mediated immunity, Radiology 106, 653-655 (1973). 32. Clement, J. A., and Kramer, S. Immunocompetence in patients with solid tumors undergoing Cobalt-60 irradiation, Cuncer 34, 193-196 (1974). 33. McCredie, J. A., Inch, W. R., and Sutherland, R. M. Effect of postoperative radiotherapy on peripheral blood lymphocytes in patients with carcinoma of the breast, Cancer 29, 349-356 (1972).
ONCOLOGY
Lymphocyte
9, 209-219 (1980)
Response and Radiation Therapy Gynecologic Cancer1
VERNON K. JENKINS, PH.D., MARVIN H. OLSON, M.D., Departments
of Radiology
and Medical
for Patients with
E. ARCHER DILLARD, JR., M.D.,* AND ROBERT R. PERRY, PH.D.
*Obstetrics and Branch, Galveston,
Gynecology, Texas
the
University
of Texas
77550
Received June 18, 1979 To relate radiotherapy, lymphocyte responses, and clinical course for patients with gynecologic lesions, responses to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) were measured in whole-blood cultures before and after radiotherapy. For 82 patients tested before radiotherapy, lymphocyte counts and responses to PHA, Con A, and PWM were 79, 62, 55, and 67% of control values, respectively. For 29 patients who died within 18 months, the pretreatment lymphocyte count was 80% and mitogenic responses were about one-half of values for 37 patients who lived beyond 18 months. During radiotherapy lymphocyte counts declined to about 30% and mitogenic responses declined to about 10% of control values. For patients who lived beyond 18 months, PWM and Con A responses recovered to pretreatment values within 13-18 months and lymphocyte counts recovered within 19-24 months. PHA response, however, failed to recover above 70% of the pretreatment response. In contrast, patients who died within 18 months had no increase in lymphocyte counts or mitogenic responses after treatment. In general, depressed pretreatment responses to PHA and Con A correlated with a poor clinical course and responses near control levels indicated a good clinical course.
INTRODUCTION
Immunological mechanisms involving recognition and reaction to cancer cells by the host may play an important role in the neoplastic process [l-3]. Evidence that some patients with neoplastic lesions have suppressed cellular immunity which could indicate poor prognosis despite definitive therapy [4-61 gives importance to assessment of immunologic competence before treatment in relation to clinical progress of the patients. There is also considerable concern that treatment with surgery, radiotherapy, and/or chemotherapy for patients with cancer may further suppress natural immunity and afford increased chance for growth of residual or metastatic tumor cells [7-121. Recovery of immunological responsiveness, on the other hand, has been correlated with a very good clinical course [5,13,14]. In this study, immunologic responsiveness of patients with neoplasms of I Supported by Public Health Service Grant CA-13435 from the National Cancer Institute. 209 0090-8258/80/020209-11$01.00/O Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
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gynecologic structures was assessed by measurement of numbers and responses of their lymphocytes to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) in whole-blood cultures, before, during, and at various periods of time after radiation therapy. Lymphocyte numbers and responses to mitogens for patients who had a poor clinical course after treatment were compared with values for patients who responded well to radiation therapy to determine whether their lymphocyte responses before or after treatment were correlated with clinical outcome. In view of an excellent rate of survival for patients treated with radiation in early clinical stages and the relatively poorer rates for patients treated in advanced stages [ 151, longevity of the patients and lymphocyte responses were also considered in relation to clinical stage at the time of treatment. SUBJECTS AND METHODS
Clinical staging for the patients was made according to the recommendation for classification and staging of the International Federation of Gynecology and Obstetrics [ 161. Eighty-two patients were entered into this study and were tested prior to radiotherapy (Table 1); 74 patients were subsequently evaluated near midtreatment, 55 patients were tested at the end of treatment, and 66 samples from 20 patients were assessed more than 24 months after treatment. Values obtained from the patients were compared with values for control samples taken usually on the same day. Control samples were taken from relatives of the patients at or near the same age, laboratory personnel, or other volunteers in normal health, but it was not possible to use the same control each time a patient was HISTOLOGIC
TYPE,
Histologic type
SITE,
TABLE AND STAGE OF LESIONS
1 IN PATIENTS
Primary site of tumor
WITH GYNECOLOGIC
No. of patients in clinical stages I
Adenocarcinoma
Adenoacanthoma Adenoid cystic carcinoma Squamous cell carcinoma Carcinoma (NOS)
Ovary Corpus uteri, endometrium, myometrium, cervix uteri, vagina Corpus uteri, endometrium, myometrium Ovary Corpus uteri, endometrium, myometrium cervix uteri, vagina Corpus uteri, endometrium, myometrium, cervix uteri, vagina
Stromal cell carcinoma n NOS, not otherwise specified.
CANCER
II
III
IV
1 14
3
2
3 2
4 2
1 1
II 1
1 1
1
1
1
16
6
7
1 1
1
LYMPHOCYTE
RESPONSE
AND
RADIOTHERAPY
211
tested. Informed consent was obtained from patients or control individuals after the nature of the procedure was fully explained. The patients were treated with external photon radiation of 32 meV energy usually through two lateral and one anterior fields, by external radiation followed by one or two intracavitary applications of radium, or by external radiation within 8 weeks before or after hysterectomy. For external radiation treatment, a daily dose of 160 to 200 rad was given for 5 days each week for 5 to 7 weeks. Twenty-nine of the patients were given 4500- to 6000-rad external radiation before or after hysterectomy and 31 of the patients received 4000- to 5000-rad external radiation and an intracavitary application(s) to produce a final dose of about 8000 rad to point A and 6000 rad to point E. Point E is defined as a point 5 cm to the right or left of the midline on the ileopectineal line. Twenty-two patients were treated to 5000- to 6000-rad tumor dose by external radiation only. For preparation of cultures 5 ml of peripheral blood were obtained by venipuncture from patients before, during or after treatment and from healthy control individuals. Preservative-free heparin was added to prevent clotting and the total number of white cells was determined in an electronic cell counter. Differential cell counts were made from blood smears by standard methods. The whole blood was diluted 1:20 with culture medium and 3-ml cultures containing 150 ~1 of blood were incubated at 37°C with mitogens to stimulate the lymphocytes to undergo blastic transformation according to methods previously described [ 17-201. Transformation of the lymphocytes in response to the mitogens was assessed by “HTdR incorporation [20]; 1.O &i of 3HTdR (2.0 CilmM) in 0.2 ml of culture medium was added to each culture 24 hr before the end of the 7-day culture period. For harvest the cell suspensions were filtered through glass-fiber disks (grade 934AN, Reeve Angel) in a multiple automated sample harvester. The number of disintegrations per minute (dpm) was calculated from the counts per minute for each culture as was determined in a liquid scintillation counter. The statistical significance of the difference between mean dpm or mean number of lymphocytes of the various groups was determined by the Wilcoxon (Mann-Whitney) sum of ranks comparison [21]. A probability of less than 0.05 for groups being the same population was considered to indicate a significant difference. RESULTS
The mean number of lymphocytes in the peripheral blood of the total group of patients tested prior to radiotherapy was 7% of the number for controls and responses per culture to PHA, Con A, and PWM were 62, 55, and 67% of control values, respectively (P < 0.002, Fig. 1). Mean numbers of lymphocytes and mean responses to mitogens in whole-blood cultures for the total group of patients declined during radiation treatment to values at the end of treatment that were 28% (lymphocytes), 12% (PHA), 9% (Con A), and 11% (PWM) of control values (P < 0.001, Fig. 1). Mean numbers of lymphocytes gradually increased after treatment and the numbers for patients tested at 19-24 months or more than 24 months after treatment approached the number for the total group of patients tested before radiation treatment; however, patient values were still only 58 and 68% of the number for the controls (P < 0.001). Responses to mitogens increased
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slowly after radiotherapy and by 13-18 months after treatment PWM and Con A responses were not significantly less than pretreatment values, but PWM and Con A values even for patients tested beyond 24 months did not exceed pretreatment values. PHA responses for the patients did not recover to the pretreatment level, even though maximal recovery (about 70% of pretreatment response) occurred by 13-18 months after treatment (Fig. 1).
LYMPHOCYTE
RESPONSE AND RADIOTHERAPY
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Fourteen of the 82 patients tested before radiotherapy had stage IV lesions and had PHA responses that were 40% (P < 0.02) of responses for 32 patients in stage I, 50% (P < 0.02) of responses for 22 patients in stage II, and Con A responses that were 51 and 56% (P < 0.02) of response for patients in stages I and II (Fig. 2). There were apparently fewer lymphocytes and a reduced PWM response in stage IV patients compared to patients in stages I and II, but the differences were not significant. The differences related to clinical stage did not persist at the end of treatment or at l-3 months after treatment. Of the 82 patients tested before treatment, 37 were known alive longer than 18 months, 29 were dead within 18 months, and 16 were known alive less than 18 months or have been lost to follow-up. Lymphocyte numbers and responses for patients who lived beyond 18 months after treatment were compared with values for patients who died within 18 months after treatment (Fig. 3). Pretreatment numbers of lymphocytes and responses to PHA, Con A, and PWM for patients who died within 18 months of treatment were 80% (P < 0.02), 48% (P < O.OOl), 56% (P < 0.003), and 58% (P = 0.07), respectively, of pretreatment values for patients who lived longer than 18 months after treatment. The number of lymphocytes and responses to Con A and PWM for the two groups of patients were not different at the end of treatment, but the PHA response for patients who died within 18 months was only 47% (P < 0.02) of the responses for patients who lived beyond 18 months. No significant recovery in responses to mitogens occurred for patients who died within 18 months. whereas PWM and Con A responses in-
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Similarly, the pretreatment response to PHA for 18 patients in stages III and IV who died within 18 months was 50% (P < 0.02) of the value for six patients in stages III and IV who lived beyond 18 months. At the end of treatment and up to 7- 12 months after treatment responses to PHA for patients who lived beyond 18 months were not significantly different from values for patients of similar stages who died within 18 months. There were no differences in Con A responses between those patients in stages I and II or III and IV who lived beyond 18 months and patients of similar stages who died within 18 months. In Table 2 are given numbers and percentages of patients who survived beyond 18 months in relation to their pretreatment response to PHA and in relation to clinical stage. None of 13 patients with very poor PHA responses (< 99 x 101 dpmkulture) lived beyond 18 months, whereas, 11 of 12 patients (92%) with good responses (> 300 x 103 dprnkulture) lived beyond I8 months. Patients with stages I, II, III, and IV lesions had survival rates beyond 18 months of 86, 60, 36, and 15%, respectively. DISCUSSION
Responses of peripheral blood lymphocyte to in vitro stimulation by PHA and Con A are generally considered to reflect thymus-dependent or T-cell immunity, although Con A has been reported to stimulate bone marrow-derived lymphocytes
216
JENKINS TABLE
NUMBERS
18
AND PERCENTAGES OF PATIENTS WITH MONTHS IN RELATION TO PRETREATMENT
ET AL. 2 GYNECOLOGIC CANCER WHO LIVED BEYOND PHA RESPONSE AND CLINICAL STAGE
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I
II
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IV
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O/8 214 O/l 2113
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(B-cells), and PWM may stimulate B-cells and under certain conditions T-cells [22-251. The patients with gynecologic lesions tested prior to radiotherapy had deficits in immunity as indicated by responses to PHA, Con A, and PWM in whole-blood cultures (Fig. 1). Furthermore, patients in advanced stage IV had responses to PHA and Con A that were about one-half the responses of patients in stages I and II (Fig. 2). The deficits in mitogenic responses for the total group of patients, which were S-67% of values for controls, were similar to the deficits reported previously for a smaller group of patients with similar lesions [201. Deficits for the patients may be accounted for only partly by the reduced lymphocyte count which was 7% of the count for controls. Since the lymphocyte pool is comprised of a heterogeneous population of cells with differing functions, it is not unreasonable to surmise that reduced responsiveness reflects fewer cells in subpopulations that are capable of mitogenic responses. Another possible mechanism for reduced responsiveness of lymphocytes is that some inhibitory factor(s) as described by Catalona et af. [ 11 and Whittaker et al. [26] is present in the sera of these patients and interferes with the mitogenic response in vitro. At the end of radiotherapy the number of lymphocytes for the total group of patients was reduced to about one-third of the number for controls, and responses to mitogens were reduced to about 10% of control values (Fig. 1) which shows that radiation not only reduced the number of lymphocytes in a given volume of blood but also reduced the numbers in subpopulations that are responsive to mitogenic stimulation. Reduced responses to mitogens noted at the end of radiotherapy remained until 13-18 months after treatment at which time Con A and PWM responses recovered to near pretreatment values. PHA responses, however, failed to increase to more than about 70% of pretreatment values even for patients tested longer than two years after radiotherapy and even though lymphocyte numbers recovered to pretreatment values. Radiation therapy has been shown previously to reduce numbers and mitogenic responses of lymphocytes in patients with different types and location of neoplasms [6,17,19,20,27-301. In contrast, some investigators who have used mitogens in vitro or skin test reactions to measure delayed hypersensitivity have reported minimal or no depression of
LYMPHOCYTE
RESPONSE
AND
RADIOTHERAPY
217
cell-mediated immunity after radiotherapy for patients with nonlymphoid neoplasms [31-331. Inconsistencies in data on effects of radiotherapy on patient immunity have caused investigators to question which of several methods used is most likely to indicate the capacity of individuals to mount an immunological response. Whether the in vitro assessment of overall immunity in these patients as measured by mitogenic stimulation in whole-blood cultures is a quantitative measure of the capacity of individual patients to react immunologically to specific tumor antigens is conjecture. Of importance however, is whether the suppressed responses recognizable by this technique before or at time periods after radiotherapy may be correlated with clinical outcome for the patients. Twenty-nine patients who died within 18 months after treatment had PHA and Con A responses before treatment that were about one-half the values for 37 patients who lived beyond 18 months (Fig. 3). The relatively poor response to T-cell mitogens for the patients who had a poor clinical course even after definitive radiotherapy indicate that suppressed response before treatment is correlated with a poor prognosis. In view of a 90%, IO-year survival rate for stage I patients and decreased rates of survival with advanced stages to 14% for stage IV patients treated with megavoltage radiotherapy [151, an obvious question is whether the reduced responses recognized before treatment merely reflect advanced clinical stage, in which case such measurements would add little to prediction of clinical outcome. Of 42 patients with localized lesions (stages I and II) who were at risk for at least I8 months after radiotherapy, 31 survived beyond 18 months and 11 patients died within 18 months (Fig. 4). The mean response to PHA for patients who died within 18 months was only 5% (P < 0.003) of the response for patients who lived beyond 18 months and indicates that some patients staged clinically as having localized lesions have suppressed T-cell response and a poor clinical course. Similarly, of 24 patients with more advanced lesions (stages III and IV), 18 died within 18 months and their mean PHA response was only 50% (P < 0.02) of the response for 6 patients who lived beyond 18 months (Fig. 4). These data are consistent with other reports that impaired cellular immunity before surgery [41 or radiotherapy [ 131 is indicative of a poor clinical course. The report by Dellon et al. [131 also showed that patients with bronchogenic or esophageal carcinoma who had a superior clinical course after radiotherapy experienced an abrupt rise in T-cell levels within 30 days after therapy. In contrast, our data showed slow recovery in T-cell responses, maximal recovery of responses at 13-18 months after radiotherapy, and failure of PHA responses to recover to more than about 70% of pretreatment response even for patients tested more than 2 years after radiotherapy. The significance of failure to recover PHA response even for patients with a good clinical course is not evident, although it may be that the treatment is effectively destroying the lesion and minimal lymphocyte reactivity is sufficient to prevent recurrence. In contrast, suppressed responses to mitogens before treatment and failure to recover responses significantly after treatment for patients with a poor clinical course may indicate occult metastases, regardless of clinical stage, and may contribute to failure to control the disease. It is desirable for treatment planning to know whether measurement of lymphocyte responses made for individual patients prior to treatment or within a few
218
JENKINS
ET AL.
weeks after treatment can be used to indicate the clinical course for the patient. Although minimal or no recovery in responses that can be correlated with survival or clinical stage occurred within a year after treatment, pretreatment responses to PHA were related to rate of survival and clinical stage for individual patients. Increased responses to PHA were, in general, directly correlated with rate of survival. Clinical stage at the time of treatment was inversely related to survival (Table 2). None of 13 patients who had very poor responses to PHA (< 100 x 1V dpmculture), 2 of whom had localized lesions clinically, lived beyond 18 months. On the other hand, 11 of 12 patients, 2 of whom had more advanced lesions, with PHA responses that exceeded the approximate overall mean for all healthy controls tested (- 300 x 103 dpm/culture), lived beyond 18 months. Both clinical stage and pretreatment response to PHA may be important in predicting the outcome for these patients. Patients who are recognized to have a poor probability of being successfully treated with conventional radiotherapy, or surgery and radiotherapy, may be good candidates for combination of conventional therapy and immunotherapy. REFERENCES 1. Catalona, W. J., Sample, W. F., and Chretien, P. B. Lymphocyte reactivity in cancer patients: Correlation with tumor histology and clinical stage, Cancer 31, 65-71 (1973). 2. Harris, J., and Copeland, D. Impaired immunoresponsiveness in tumor patients, Ann. N. Y. Acad. Sci. 230, 56-85 (1974). 3. Hellstrom, I., Hellstrom, K. E., and Sjorgren, H. O., et al. Demonstration of cell-mediated immunity to human neoplasms of various histological types, Inr. J. Cancer 7, l-16 (1971). 4. Chretien, P. B., Crowder, W. L., and Gertner, H. R., er al. Correlation of preoperative lymphocyte reactivity with the clinical course of cancer patients, Surg. Gynecol. Obsret. 136,380-384 (1973). 5. Eilber, F. R., and Morton, D. L. Impaired immunologic reactivity and recurrence following cancer surgery, Cancer 25, 362-367 (1970). 6. O’Toole, C., Perlmann, P., and Unsgaard, B., er al. Cellular immunity to human urinary bladder carcinoma: I. Correlation to clinical stage and radiotherapy, Inr. J. Cancer 10, 77-91 (1972). 7. Dao, T. L., and Kovaric, J. Incidence of pulmonary and skin metastases in women with breast cancer who received post-operative irradiation, Surgery 52, 203-212 (1962). 8. Deodhar, S. D., Crile, G., Jr., and Esselstyn, C. B., Jr. Study of the tumor cell-lymphocyte interaction in patients with breast cancer, Cancer 29, 1321-1325 (1972). 9. Fisher, B., Slack, N. H., and Cavanaugh, P. J., er al. Post-operative radiotherapy in treatment of breast cancer: Results of the NSABP clinical trial, Ann. Surg. 172, 71 l-732 (1970). 10. Meyer, K. K. Radiation-induced lymphocyte-immune deficiency: A factor in the increased visceral metastases and decreased hormonal responsiveness of breast cancer, Arch. Surg. 101, 114-121 (1970). 11. Stjernsward, J., Jondal, M., and Vanky, F., et al. Lymphopenia and change in distribution of human B and T lymphocytes in peripheral blood induced by irradiation for mammary carcinoma, Lancer 1, 1352-1356 (1972). 12. Hancock, B. W., Bruce, L., and Heath, J., et al. The effects of radiotherapy on immunity in patients with localized carcinoma of the cervix uteri, Cancer 43, 118-123 (1979). 13. Dellon, A. L., Potvin, C., and Chretien, P. B. Thymus-dependent lymphocyte levels during radiation therapy for bronchogenic and esophageal carcinoma: Correlations with clinical course in responders and nonresponders, Amer. J. Roenrgenol. Radium Ther. Nucl. Med. 123,500-5 11 (1975). 14. Hersh, E. M., Whitecar, J. P., Jr., and McCredie, K. B., et al. Chemotherapy, immunocompetence, immunosuppression and prognosis in acute leukemia, N. Engl. J. Med. 285, 1211-1216 (1971).
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AND RADIOTHERAPY
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15. Fletcher, G. H. Female pelvis, in Textbook ofradiotherapy (G. H. Fletcher, Ed.), Lea & Febiger, Philadelphia, 2nd ed., pp. 620-665 (1973). 16. Kottmeier, H. L. Report presented by the Cancer Committee of the General Assembly of F.I.G.O., New York, April 1970, Int. J. Gynaecol. Obsrer. 9, 172-179 (1971). 17. Jenkins, V. K., Olson, M. H., and Cooley, R. N., ef al. Effect of therapeutic radiation on peripheral blood lymphocytes in patients with carcinoma of the breast, Acta Radio/. 14, 385-395 (1975). 18. Pauly, J. L., Sokal, J. E., and Han, T. Whole-blood culture technique for functional studies of lymphocyte reactivity to mitogens, antigens and homologous lymphocytes, 1. Lob. Clin. Med. 82, 500-513 (1973). 19. Jenkins, V. K., Ray, P., and Ellis, H. N., et a/. Lymphocyte response in patients with head and neck cancer-Effects of clinical stage and radiotherapy, Arch. Otolaryngo/. 102,596-600 (1976). 20. Jenkins, V. K., Olson, M. H., and Ellis, H. N., et a/. In vitro lymphocyte response of patients with uterine cancer as related to clinical stage and radiotherapy, Gynecol. On&. 3, 191-200 (1975). 21. Burington, R. S., and May, D. C., Jr. Handbook of probability and statistics with rabies, McGraw-Hill, New York, 2nd ed., pp. 265-267 (1970). 22. Andersson, J., Moller, G., and Sjoberg, 0. B lymphocytes can be stimulated by Concanavalin A in the presence of humoral factors released by T cells, Eur. J. Immunol. 2, 99-101 (1972). 23. Byrd, W., Boehmer, H., and Rouse, B. T. The role of the thymus in maturational developments of phytohemagglutinin and pokeweed mitogen responsiveness, Cell. Immunol. 6, 12-24 (1973). 24. Janossy, G., and Greaves, M. F. Lymphocyte activation: I. Response of T and B lymphocytes to phytomitogens, Clin. Exp. Immunol. 9, 483-498 (1971). 25. Weksler, M. E., Bodine, S., and Rommer, J. Response of lymphocytes to plant lectins: I. A thymic-dependent lymphoid population responsive to pokeweed mitogen, Immunology 16, 281-290 (1974). 26. Whittaker, M. G., Rees, K., and Clark, C. G. Reduced lymphocyte transformation in breast cancer, Lancer 1, 892-893 (1971). 27. Chee, C. A., Illbery, P. L. T., and Rickinson, A. B. Depression of lymphocyte replicating ability in radiotherapy patients, Brit. J. Radio/. 47, 37-43 (1974). 28. Cosimi, A. B., Brunsterrer, F. H., and Kemmerer, W. T., et (I/. Cellular immune competence of breast cancer patients receiving chemotherapy, Arch. Surg. 107, 531-535 (1973). 29. Gatti, R. A., Garrioch, D. B., and Good, R. A. Depressed PHA responses in patients with nonlymphoid malignancies, in Proceedings of the jifth Ieukocyfe culture conference (J. E. Harris, Ed.), Academic Press, New York, pp. 339-358 (1970). 30. Jenkins, V. K., Olson, M. H., and Ellis, H. N. In vitro methods of assessing lymphocyte transformation in patients undergoing radiotherapy for bronchogenic cancer, Tex. Rep. Biol. Med. 31, 19-28 (1973). 31. Gross, L., Manfredi, 0. L., and Protos, A. A. Effect of Cobalt-60 irradiation upon cell-mediated immunity, Radiology 106, 653-655 (1973). 32. Clement, J. A., and Kramer, S. Immunocompetence in patients with solid tumors undergoing Cobalt-60 irradiation, Cuncer 34, 193-196 (1974). 33. McCredie, J. A., Inch, W. R., and Sutherland, R. M. Effect of postoperative radiotherapy on peripheral blood lymphocytes in patients with carcinoma of the breast, Cancer 29, 349-356 (1972).