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In vitro lymphocyte response of patients with uterine cancer as related to clinical stage and radiotherapy
GYNECOLOGIC ONCOLOGY 3,
191-200
(1975)
In Vitro Lymphocyte Response of Patients with Uterine Cancer as Related to Clinical Stage and Radiotherapy 1 V E R N O N K . JENKINS,
PH.D., 2 M A R V I N
H . OLSON,
M.D.,
H O W A R D N . ELLIS, B . S . , AND E . A R C H E R D I L L A R D , JR., M . D .
Departments of Radiology and Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Texas 77550 Received March 24, 1975 Thirty-one patients with uterine cancer in different clinical stages were tested to determine numbers and immunologic responsiveness of their lymphocytes in peripheral-blood cultures to mitogenic stimulation. Seventeen patients were tested also at the end of radiotherapy and 16 patients were tested 3-6 months after radiotherapy to determine effects of radiation on responsiveness. The mean responses of the total group of untreated patients to phytohemagglutinin and concanavalin A (primarily T-cell mitogens) were only 59% and 56% of the responses of healthy controls. Eleven patients in Stage I had responses near control values, but six Stage IV patients had only 27 and 44% of the control responses. Mean response to pokeweed mitogen (primarily a B-cell mitogen) in cultures of untreated patients was not significantly less than control response. Responsiveness to all three mitogens declined in cultures of patients in all stages during radiotherapy to about 10% of control values and remained near 25% of control values 3-6 months after treatment. Decreased responsiveness of patients after therapy possibly results from radiation death of circulating lymphocytes. Reduced responsiveness after therapy for patients in early disease stages is probably not indicative of poor prognosis since excellent survival rates are reported for such patients.
INTRODUCTION There is increasing evidence that immunologic mechanisms involving both humoral and cell-mediated immunity play an important role in neoplastic disease [1,2]. Evidence that cancer patients may have impaired cellular immunity, which could influence prognosis [3,4], gives importance to assessment of immunologic competence in patients in relation to tumor histology, clinical stage of disease, and effects of therapeutic procedures. Immunologic responsiveness has been assessed in vivo by measurement of skin-test reaction rates [5] and in vitro by blastogenic responses of lymphocytes to the nonspecific mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) [6,7,8] or specific antigens and allogeneic lymphocytes [7]. In this study, immunologic responsiveness of patients with neoplasms of the uterine cervix or endometrium was assessed by quantitation of responses of their Supported by Public Health Service Research Grant No. CA-13435 from the National Cancer Institute. 2 Address correspondence to V. K. Jenkins, Ph.D., Department of Radiology, The University of Texas Medical Branch, Galveston, Texas 77550. 191 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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lymphocytes to PHA, Con A, and PWM in whole-blood cultures. In view of the excellent survival rate reported for such patients treated with radiation in the early clinical stage and the relatively poorer rates for patients treated in advanced stages [9], these studies were undertaken to correlate, if possible, in vitro lymphocyte responses with clinical stage of disease before, during, and after radiation therapy. Before treatment, responses of the lymphocytes of Stage I patients to all three mitogens were near control values. In contrast, cultures from patients with advanced lesions generally had smaller responses to PHA and Con A than controls tested at the same time. These findings correlate with the expected excellent survival rate for the Stage I patients and the relatively poorer survival rate for Stage IV patients. During radiation treatment, however, responses to all three mitogens decreased to about 10% of the control responses for patients regardless of stage at the time of treatment. Mean survival rates of patients after radiotherapy, therefore, appear to be independent of lymphocyte responsiveness measured after treatment. MATERIALS AND METHODS
Patient Staging and Radiotherapy Procedures Thirty-one patients with carcinoma of the uterus were tested to determine numbers and responsiveness of their peripheral-blood lymphocytes to mitogenic stimulation before initiation of radiation therapy. Eleven of the patients were in clinical Stage I, eight were in Stage II, six were in Stage III, and six were in Stage IV, according to the recommendation for classification and staging of the International Federation of Gynecology and Obstetrics [10]. Responses of patient blood samples were compared with concomitantly tested control samples taken from relatives of the patients near the same age, laboratory personnel, or other volunteers in normal health. The patients were treated with photon radiation of 32 MeV energy through two lateral and one anterior fields. Twenty-one of the patients were treated by external beam radiation only to a tumor dose of 6000-6400 rads, and 10 of the patients were given 4500-5000 rads external radiation and an intracavitary application of radium to produce a final tumor dose of 6000-6400 rads. Twenty-three of the 31 patients were tested again near mid-treatment, 17 of the patients were tested also within 1-2 hr after the final treatment, and 14 of the original patients and 2 additional patients treated in the same manner were tested once or twice 3-6 months after treatment.
Techniques for Whole-blood Culture For preparation of cultures, 5 ml of peripheral blood were withdrawn by venipuncture from patients and control individuals on the same day. Clotting was prevented by addition of preservative-free heparin, and total white cell numbers per cubic millimeter were determined in an electronic cell counter. Blood smears were prepared, and differential counts were made by standard methods. The blood cells then were diluted and cultured and the lymphocytes stimulated to undergo blastic transformation according to methods described
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previously [7,11,12]. Briefly, the whole blood was diluted 1:20 with cell culture medium (RPMI 1640), and 3-ml aliquots of the cell suspension containing 150/~1 of whole blood were set up in triplicate in 16 × 125-ram plastic culture tubes for each mitogen used. The culture tubes were capped tightly and incubated for 7 days at 37°C, a time period previously shown to be within peak response for P H A and Con A stimulation [7,11,12 ]. The stimulated cultures contained 10/zg of purified P H A (mitogenic-hemagglutinin activity ratio approximately 100: l, Burroughs Wellcome, Research Triangle Park, North Carolina), 10 tzg of Con A, or 300/~g of PWM.
Evaluation of Lymphocyte Response Transformation of the lymphocytes in response to the mitogens was assessed by a [3H]TdR incorporation method [12]; 1.0/~Ci of [3H]TdR (2.0 Ci/mM) in 0.2 ml of medium was added to each culture 24 hours before harvest. At the end of the 7-day culture period, individual cell suspensions were filtered through glass-fiber filter discs (Grade 934 AN, Reeve Angel, Clifton, New Jersey) to trap the cells on the surface. The cells were washed several times with distilled water, acetic acid, and distilled water. Then the discs were placed in the bottom of scintillation counting vials and allowed to dry. Scintillation counting fluid was added to the vials, and the counts per minute from the labeled D N A trapped on the glass-fiber filters were determined in a liquid scintillation spectrophotometer. The number of disintegrations per minute (dpm) was calculated from the counts. The statistical significance of the difference between D P M means of various groups was determined by Student's t test. RESULTS
Lymphocyte Response and Number in Relation to Clinical Stage The mean responses to P H A and Con A of the total group of patients tested before radiation therapy were only 59% and 56% of the responses of concomitantly tested control individuals (P < 0.01, Table 1). On the other hand, the TABLE 1 LYMPHOCYTE REACTIVITY OF UTERINE CANCER PATIENTS BEFORE RADIOTHERAPY IN RELATION TO CLINICAL STAGE Mean lymphocyte response dpm x 103 ± SE/culture (No. individuals tested) PHA
Con A
PWM
Clinical stage of neoplasm
Patients
Controls
Patients
Controls
Patients
Controls
1 II III IV I-IV combined
253-+48(1l) 193 ±26'~(8) 213 ± 31 b (6) 93 -+ 4 V (6) 199 ± 22 ~ (31)
397_+60(11) 319+32(8) 326 ± 42 (6) 343 ± 55 (6) 336 ± 16 (31)
68_+19(11) 43___11~(8) 39 ± 7 (6) 34 ± 13 (6) 49 --- 8 a (31)
68+17(10) 110-+26(8) 100 ± 43 (5) 77 ± 22 (6) 87 -+ 12 (29)
63_+26(11) 42_+7(7) 27 ± 10 (6) 31 ± 9 (6) 44 ± 10 (30)
48_+11(1l) 65+14(7) 45 ± 17 (6) 50 ± 20 (5) 53 -+ 7 (29)
a Values for patients were significantly less than values for controls (P < 0.01). Values for patients were significantly less than values for controls (P < 0.05).
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ET
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mean response of the total group of patients to PWM was not significantly less than the control value. Six of the 31 patients tested were in advanced Stage IV and had a very poor mean response to PHA as compared to the response of concomitantly tested controls (27%, P < 0.01) and compared to the response of 11 patients in Stage I (37%, P < 0.05). The mean response to Con A of the six patients with advanced disease was apparently less (44% of control) than the response of concomitantly tested controls and compared with the response of patients in the earliest stage, but the differences were not statistically significant for the few patients tested. The mean number of lymphocytes in peripheral blood (2230/mm 3) of the 31 patients tested before initiation of radiation therapy was only sIightly (16%) but significantly less than the number in the total group of control individuals tested (2642/mm 3) (P < 0.05, Table 2). There was no evidence, however, to show that patients in the more advanced Stage IV had fewer lymphocytes than patients in earlier stages.
Effects of Radiotherapy on Numbers and Responses of Lymphocytes The effects of radiation treatment on the numbers of lymphocytes in the peripheral blood also are shown in Table 2. The mean numbers of lymphocytes declined in the patients during treatment to 24% of the control value (P < 0.01) and 28% of the pretreatment value (P < 0.01), an average loss in the patients of about 1600 cells/mm3. The change did not relate to clinical stage of disease, since the patients in Stages I and II experienced as great a decline as patients in Stages III and IV. Patients tested 3-6 months after radiotherapy had only slight recovery in the number of lymphocytes to 36% of control and 42% of pretreatment values (P < 0.01). Again, patients in earlier clinical stages did not have a significantly greater degree of recovery than patients in later stages. The mean responses to all three mitogens also declined in cultures from patients during radiation treatment. By about mid-treatment (2000-3000 rads), the mean responses in the total group of patients to all three mitogens were de-
TABLE EFFECT
2
OF T H E R A P E U T I C R A D I A T I O N ON N U M B E R S OF L Y M P H O C Y T E S I N P A T I E N T S W I T H C A R C I N O M A OF T H E U T E R U S M e a n N o . of l y m p h o c y t e s _+ SE/mm* blood (No. individuals tested) Patient s a m p l e s
Clinical stage of neoplasm I n In IV i-iv combined
2382 2213 2109 2109 2230
0 rads
2000-3000 r a d s
6000-6400 r a d s
After treatment 3-6 m o n t h s
+ 174 (11) + 236 (8) 4- 530 (6) 4- 371 (6) -+ 136 ~ (31)
1091 809 854 1017 976
596 744 439 680 623
989 932 904 884 947
_+ 216 a (9) ± 307 ~ (3) _+ 140 ~ (6) 4- 192 a (5) ± 99 a (23)
444-+ -+
99" (8) 350" (3) 2 v (2) 172 a (4) 73 a (17)
Values f o r patients w e r e significantly less than v a l u e s f o r controls ( P < .01).
44± 4-
127 ° (11) 112 ~ (6) 18 ~ (4) 108 a (3) 62 a (24)
Control samples 2581 2793 2566 2674 2642
4- 129 (38) -4- 215 (20) 4- 149 (17) +_ 141 (18) -4- 78 (93)
LYMPHOCYTE
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CANCER
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PATIENTS
3
L Y M P H O C Y T E R E A C T I V I T Y OF U T E R I N E CANCER P A T I E N T S A F T E R
2000-3000 RADS
T R E A T M E N T IN R E L A T I O N TO C L I N I C A L S T A G E
Mean lymphocyte response dpm x 102 _+ SE/culture (No. individuals tested) PHA
Con A
PWM
Clinical stage
of neoplasm I II III IV I-IV combined
Patients 175 51 67 39 101
Controls
± 550 (9) ± lOs (3) -+ 27 ~ (6) ± 13 a (5) ± 25 ~ (23)
369 385 279 247 321
± 62 (9) ± 27 (3) ± 46 (6) -+ 33 (5) ± 29 (23)
Patients
Controls
48 ± 21 (9) 1 (1) 5 -+ 4 ° (5) 14 ± 6 (5) 26 ± 10a (20)
85 50 60 98 76
-+ 29 (9) - 15 (3) ± 23 (6) ± 49 (4) ± 15 (22)
Patients 29 ± 3 ± 8 ± 8 ± 16 ±
9 (9) 2" (2) 4 b (5) 4 (5) 5 a (21)
Con trois 55 34 53 54 52
± ± ± ± ±
19 (8) 0 (2) 17 (6) 35 (5) 11 (21)
Values for patients were significantly less than values for controls (P < 0.01). b Values for patients were significantly less than values for controls (P < 0.05).
pressed as compared to controls (Table 3). Patients in advanced Stage IV, again, had a smaller mean response to PHA than Stage I patients (22%, P < 0.05). Although responses to Con A and PWM apparently were smaller for patients with advanced lesions than for Stage I patients, the values for the few patients examined were not statistically different. By the end of treatment, responses for patients to all three mitogens were severely depressed (Table 4), although nearly one-half of the total number of patients were in clinical Stage I. The mean values for cultures of the total group of patients at the end of treatment in response to PHA and Con A were only 18% and 20% of the values for patients tested before treatment (P < 0.01) and only 10% of the values for controls (P < 0.01). Similarly, the response to PWM was reduced to 11% of the value for patients before treatment (P < 0.01) and 9% of the value for controls (P < 0,01). There were no differences in responses to either of the mitogens of the cultures from patients in different clinical stages at the end of treatment. Responses to mitogens of blood samples from patients tested 3-6 months after
TABLE 4 L Y M P H O C Y T E R E A C T I V I T Y OF U T E R I N E C A N C E R P A T I E N T S A F T E R 6 0 0 0 - 6 4 0 0
RADS
T R E A T M E N T IN R E L A T I O N TO C L I N I C A L S T A G E
Mean lymphocyte response dpm x 103 _+ SE/culture (No. individuals tested) PHA
Con A
PWM
Clinical stage
of neoplasm I II lU IV I-IV combined
Patients 44 30 31 25 35
--_ 17a (8) -4- 220 (3) --_ 400 (2) -+ 22 b (4) -+ I0 a (17)
Controls 343 287 340 372 339
± 34 (8) --_ 91 (3) ± 70 (2) -+ 112 (4) -+ 29 (17)
Patients 15 ± 7 ° (7) 6 ± 3 (3) 5 -+ 6 (2) 5 -+ 3 b (4) 10 _+ 3 ~ (16)
Controls 102 112 105 74 97
± 34 (7) --- 60 (3) --- 49 (2) -- 28 (4) -- 18 (16)
Values for patients were significantly less than values for controls (P < 0.01). 0 Values for patients were significantly less than values for controls (P < 0.05).
Patients 5 --+ 30 (7) 4 -- 5 (3) 4 ± 1~ (2) 6 ± 5 a (4) 5 ± 2 a (16)
Controls 79 53 49 30 58
-+ 29 (7) ± 47 (3) -+ i (2) + 3 (4) ± 14 (16)
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TABLE 5 LYMPHOCYTE REACTIVITY OF UTERINE CANCER PATIENTS TESTED 3--6 MONTHS AFTER 6 0 0 0 - 6 4 0 0 PADS TREATMENT Mean lymphocyte response dpm x 103 _+ SE/culture (No. individuals samples tested) PHA
Con A
PWM
Clinical stage of neoplasm
Patients
Controls
Patients
Controls
Patients
Controls
I II III IV I-IV combined
110-+ 24a (11) 34 +- 16a (6) 85 -+ 39 b (4) 44 _+ 34b (3) 78 -+ 15~ (24)
272-+ 30 (10) 323 -+ 20 (6) 260 -+ 67 (4) 317_+101(3) 289 -+ 19 (23)
28-+ 5~ (11) 10 -+ 4" (6) 14 + 6 b (4) 4_+2(3) 18 -+ 3a (24)
96 -+ 2 (10) 175 -- 21 (5) 109 -+ 45 (4) 85"+39(3) 115 _+ 15 (22)
17 -+ 4 (11) 6 -+ 2 b (6) 4 +- 1(4) 7_+ 5°(3) 11 -+ 2 ~ (24)
39 -+ 15 (10) 86 -+ 32 (6) 53 +- 27 (4) 33_+8(3) 53 _+ 11 (23)
Values for patients were significantly less than values for controls (P < 0.01). Values for patients were significantly less than values for controls (P < 0.05).
treatment remained depressed and were only slightly, if any, higher than responses of patients at the end of treatment (Table 5). Responses of the total group of patients to PHA, Con A, and PWM remained at only 39, 37, and 25% of the pretreatment values (P < 0.05) and 27, 16, and 21% of values for controls (P < 0.05). DISCUSSION
Patients with carcinoma of the uterus tested before radiation treatment generally had smaller P H A and Con A responsiveness than healthy individuals, and further, patients with advanced lesions tended to respond less than patients with Stage I lesions (Table 1). On the other hand, the patients had responses to PWM that were similar to responses of controls, and patients in the advanced stage did not have a significantly smaller response than patients in earlier stages. P H A and Con A responses are considered generally to reflect thymus-dependent or T-cell immunity, although Con A has also been reported to stimulate bone-marrowderived lymphocytes (B-cells), and PWM is primarily, though not exclusively, a B-cell mitogen [6,8,13,14]. It appears, therefore, that these patients had an overall reduced T-cell immunity, the greatest deficit occurring in patients with the most advanced lesions and the smallest deficit occurring in patients with small localized lesions. On the other hand, the patients were probably not deficient in B-cell immunity. These data are consistent with reports in the literature indicating that a deficit in cell-mediated immunity (T-cells) is more readily detected and at an earlier stage of neoplastic progression than humoral immunity (B-cells) [ 1,15]. Reduced responses to P H A in patients with advanced nonlymphoid neoplasms have also been reported previously [4,16-19], but Mekori et al. [18] did not find reduced responses in patients with advanced genitourinary lesions. Although the total group of patients with uterine cancer had a smaller mean number of peripheral blood lymphocytes (84% of control), the deficit in number of cells alone did not account totally for the reduced responses to P H A and Con
L Y M P H O C Y T E RESPONSE IN U T E R I N E CANCER P A T I E N T S
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A (59 and 56% of control). Moreover, patients with advanced disease had much smaller PHA and Con A responsiveness (27 and 44% of controls) and a near normal mean number of lymphocytes. It appears, therefore, that the whole-blood method of measuring reaction of lymphocytes to mitogens detects deficiencies that cannot be recognized from differential cell counts alone. This is not unexpected since peripheral blood lymphocytes comprise a heterogeneous population of cells with different functions. One or more mechanisms might explain the reduced T-cell response in these patients. One possibility is that some inhibitory factor, as described by Catalona et al. [16] and Whittaker et al. [19], is present in the sera of the patients and interferes with T-cell response in vitro. Studies to test this possibility have not been done as yet on these patients. Another possibility is that the patients, especially those with advanced lesions, have reduced numbers of circulating T-cells capable of mitogenic response. Whole-blood culture methods for assessing lymphocyte responses have several advantages over methods using isolated lymphocytes: (a) 5 ml of blood are more easily obtained and can be obtained more frequently than larger volumes (30-50 ml), (b) there are greater reproducibility and better correlation with skin test data [7,12], (c) there is avoidance of uncontrolled variables such as possible damage to cells during isolation, (d) there is indication (theoretically) of quantitative response of the individual rather than qualitative response of a given number of lymphocytes, which may vary in number and heterogeneity, and (e) a greater number of cultures can be tested from a single blood sample, especially from patients with a reduced number of lymphocytes following therapy. Some criticisms of the whole-blood technique are (a) augmentation of lymphocyte response by erythrocytes, (b) interference with response by polymorphonuclear cells, and (c) a long culture period relative to generation cycle. It is unlikely that the presence of erythrocytes was a serious problem in this study since the purified form of PHA was used and it has been shown that the response of cultures of lymphocytes to purified PHA was not altered when erythrocyte membranes were added [20]. Although polymorphonuclear leukocytes were reported to increase transformation of lymphocytes [21], Park and Good [22] did not find any major effect of granulocytes on stimulation of lymphocytes in their whole-blood culture system. The 7-day harvest time used in this study was shown previously to be within the peak of PHA and Con A responses for wholeblood cultures from healthy individuals [7,12] and from patients with breast cancer [23]. During radiation treatment the number of lymphocytes in the patients decreased to about one-fourth of the number in control individuals or to about 28% of the number in patients before treatment and remained at only 36% of control value 3-6 months after treatment. This effect is similar to effects on lymphocyte numbers found in radiation-treated patients with breast and lung cancer [11,23]. It is likely that the decrease in number of circulating lymphocytes resulted from death of radiosensitive lymphocytes circulating through the field of treatment. The area of treatment was confined to the pelvic region and did not include segments of the thymus or thoracic duct, which may contribute to the deficit noted in patients treated to the upper thorax. Similarly, the mean responses to mitogens of cells in cultures of a given volume of blood (150 kd)
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from the patients declined during radiation treatment until at the end of treatment the responses to all three mitogens were reduced to about 10% of control responses. The capacity of the patients' blood samples to react to mitogen stimulation remained depressed (16-27% of controls) during the 3-6-month follow-up period. Although patients in the earlier stage (Stage I) of disease development had better T-cell responses before treatment than patients in Stage IV, there was no indication that such patients had better responses after treatment. It is likely that after radiotherapy the depressed responsiveness of the patients' blood lymphocytes to the three mitogens resulted from a decreased number of cells capable of a blastogenic response rather than a release of factors into the sera that prevent the response. Studies to determine whether inhibitory factors are present in the sera of treated patients have not been completed. Radiation therapy has been shown previously to reduce numbers and mitogenic responsiveness of lymphocytes in patients with several different histological types of neoplasms [4,11,23,24]. In contrast, some investigators using mitogens or skin-test antigens have reported minimal or no depression of cellmediated immunity in patients with nonlymphoid neoplasms after radiotherapy [5,25,26]. Inconsistencies in the data on effects of radiation on patient immunity have caused investigators to question which of the several methods used are most likely to indicate capacity of the individual to mount an immunologic response [1]. Whether the in vitro correlate of overall patient immunity, as measured by mitogenic stimulation in whole-blood cultures, is a quantitative measure that indicates capacity of individual patients to react immunologically to specific tumor antigens and whether these indications are significant in predicting clinical outcome for cancer patients are conjectural. Although radiotherapy reduced lymphocyte numbers and responsiveness drastically in these patients and the deficit remained for at least 3-6 months after treatment, it is unlikely that these effects are indicative of an overall poor prognosis. The suppressive effects on immunologic response were similar in patients of all clinical stages, and yet survival rates after radiotherapy are very different for patients treated at different clinical stages of disease. The 10-yr rate of survival reported for patients after megavoltage therapy is 90% for Stage I patients and decreases to 14% for Stage IV patients [9]. Whether patients in earlier clinical stages that have good prognosis recover their lymphocyte function more fully than patients in more advanced stages in succeeding months remains to be determined. Since patients with localized lesions have an excellent survival rate after radiotherapy, it appears the treatment is effectively destroying the lesion, and a concomitant depression in immunity by the treatment may be of little significance. On the other hand, depressed immunologic competence before or after treatment in patients with lesions extended beyond the treatment area may contribute to failure to control the disease. REFERENCES 1. Harris, J., and Copeland, D. Impaired immunoresponsiveness in tumor patients, Ann. N . Y . Acad. Sci. 230, 56-85 (1974).
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2. Hellstrom, I., Hellstrom, K. E., Sjorgren, H. O., and Warner, G. A. Demonstration of cellmediated immunity to human neoplasms of various histological types, Int. J. Cancer 7, 1-16 (1971). 3. Eilber, F. R., and Morton, D. L. Impaired immunologic reactivity and recurrence following cancer surgery, Cancer 25, 362-367 (1970). 4. O'Toole, C., Perlmann, P., Unsgaard, B., Moberger, G., and Edsmyr, F. Cellular immunity to human urinary bladder carcinoma: I. Correlation to clinical stage and radiotherapy, Int. J. Cancer 10, 77-91 (1972). 5. Gross, L., Manfredi, O. L., and Protos, A. A. Effect of Cobalt-60 irradiation upon cell-mediated immunity, Radiology 106, 653-655 (1973). 6. Byrd, W., Boehmer, H., and Rouse, B. T. The role of the thymus in maturational developments of phytohemagglutinin and pokeweed mitogen responsiveness, Cell, lmmunol. 6, 12-24 (1973). 7. 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, J. Lab. Clin. Med. 82, 500-513 (1973). 8. 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). 9. Fletcher, G. H. Female pelvis, in Textbook o f radiotherapy (G. H. Fletcher, Ed.), Lea & Febiger, Philadelphia, 2nd ed. pp. 620-665 (1973). 10. 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. Obstet. 9, 172-179 (1971). 11. 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). 12. Pauly, J. L., and Sokal, J. E. A simplified technique for in vitro studies of lymphocyte reactivity, Proc. Soc. Exp. Biol. Med. 140, 40-44 (1972). 13. Andersson, J., Moller, G., and Sjoberg, O. B lymphocytes can be stimulated by concanavalin A in the presence of humoral factors released by T cells, Eur. J. lmmunol. 2, 99-101 (1972). 14. Janossy, G., and Greaves, M. F. Lymphocyte activation: I. Response of T and B lymphocytes to phytomitogens, Clin. Exp. lmmunol. 9, 483-498 (1971). 15. Curtis, J. E., Hersh, E. M., Harris, J. E., McBride, C., and Freireich, E. J. The human primary immune response to keyhole limpet haemocyanin: Inter-relationships of delayed hypersensitivity, antibody response and in vitro blast transformation, Clin. Exp. lmmunol. 6, 473-491 (1970). 16. 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). 17. Gatti, R. A., Garrioch, D. B., and Good, R. A. Depressed PHA responses in patients with nonlymphoid malignancies, in Proc. Fifth Leukocyte Cul. Conf. (J. E. Harris, Ed.), Academic Press, New York, pp. 339-358 (1970). 18. Mekori, T., Sher, S., and Robinson, E. Suppression of the mitogenic response to phytohemagglutinin in malignant neoplasia: Correlation with clinical stage of therapy, J. Nat. Cancer Inst. 52, 9-12 (1974). 19. Whittaker, M. G., Rees, K., and Clark, C. G. Reduced lymphocyte transformation in breast cancer, Lancet i, 892-893 (1971). 20. Johnson, R. A., Smith, T. K., and Kirkpatrick, C. H. Augmentation of phytohemagglutinin mitogenic activity of erythrocyte membranes, Cell. Immunol. 3, 186-197 (1972). 21. Gough, J., Elves, M. M., and Israels, M. G. G. The formation of macrophages from lymphocytes in vitro, Exp. Cell Res. 38, 465-482 (1964). 22. Park, B. H., and Good, R. A. A new micromethod for evaluating lymphocyte responses to phytohemagglutinin: Quantitative analysis of the function of thymus-dependent cells, Proc. Nat. Acad. Sci. 69, 371-373 (1972). 23. Jenkins, V. K., Olson, M. H., Cooley, R. N., and Ellis, H. N. Effect of therapeutic radiation on peripheral blood lymphocytes in patients with carcinoma of the breast, Acta Radiol. (in press).
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24. Thomas, J. W., Coy, P., Lewis, H. S., and Yuen, A. Effect of therapeutic irradiation on lymphocyte transformation in lung cancer, Cancer 27, 1046-1050 (1972). 25. Clement, J. A., and Kramer, S. Immunocompetence in patients with solid tumors undergoing cobalt 60 irradiation, Cancer 34, 193-196 (1974). 26. 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).
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In Vitro Lymphocyte Response of Patients with Uterine Cancer as Related to Clinical Stage and Radiotherapy 1 V E R N O N K . JENKINS,
PH.D., 2 M A R V I N
H . OLSON,
M.D.,
H O W A R D N . ELLIS, B . S . , AND E . A R C H E R D I L L A R D , JR., M . D .
Departments of Radiology and Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Texas 77550 Received March 24, 1975 Thirty-one patients with uterine cancer in different clinical stages were tested to determine numbers and immunologic responsiveness of their lymphocytes in peripheral-blood cultures to mitogenic stimulation. Seventeen patients were tested also at the end of radiotherapy and 16 patients were tested 3-6 months after radiotherapy to determine effects of radiation on responsiveness. The mean responses of the total group of untreated patients to phytohemagglutinin and concanavalin A (primarily T-cell mitogens) were only 59% and 56% of the responses of healthy controls. Eleven patients in Stage I had responses near control values, but six Stage IV patients had only 27 and 44% of the control responses. Mean response to pokeweed mitogen (primarily a B-cell mitogen) in cultures of untreated patients was not significantly less than control response. Responsiveness to all three mitogens declined in cultures of patients in all stages during radiotherapy to about 10% of control values and remained near 25% of control values 3-6 months after treatment. Decreased responsiveness of patients after therapy possibly results from radiation death of circulating lymphocytes. Reduced responsiveness after therapy for patients in early disease stages is probably not indicative of poor prognosis since excellent survival rates are reported for such patients.
INTRODUCTION There is increasing evidence that immunologic mechanisms involving both humoral and cell-mediated immunity play an important role in neoplastic disease [1,2]. Evidence that cancer patients may have impaired cellular immunity, which could influence prognosis [3,4], gives importance to assessment of immunologic competence in patients in relation to tumor histology, clinical stage of disease, and effects of therapeutic procedures. Immunologic responsiveness has been assessed in vivo by measurement of skin-test reaction rates [5] and in vitro by blastogenic responses of lymphocytes to the nonspecific mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) [6,7,8] or specific antigens and allogeneic lymphocytes [7]. In this study, immunologic responsiveness of patients with neoplasms of the uterine cervix or endometrium was assessed by quantitation of responses of their Supported by Public Health Service Research Grant No. CA-13435 from the National Cancer Institute. 2 Address correspondence to V. K. Jenkins, Ph.D., Department of Radiology, The University of Texas Medical Branch, Galveston, Texas 77550. 191 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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lymphocytes to PHA, Con A, and PWM in whole-blood cultures. In view of the excellent survival rate reported for such patients treated with radiation in the early clinical stage and the relatively poorer rates for patients treated in advanced stages [9], these studies were undertaken to correlate, if possible, in vitro lymphocyte responses with clinical stage of disease before, during, and after radiation therapy. Before treatment, responses of the lymphocytes of Stage I patients to all three mitogens were near control values. In contrast, cultures from patients with advanced lesions generally had smaller responses to PHA and Con A than controls tested at the same time. These findings correlate with the expected excellent survival rate for the Stage I patients and the relatively poorer survival rate for Stage IV patients. During radiation treatment, however, responses to all three mitogens decreased to about 10% of the control responses for patients regardless of stage at the time of treatment. Mean survival rates of patients after radiotherapy, therefore, appear to be independent of lymphocyte responsiveness measured after treatment. MATERIALS AND METHODS
Patient Staging and Radiotherapy Procedures Thirty-one patients with carcinoma of the uterus were tested to determine numbers and responsiveness of their peripheral-blood lymphocytes to mitogenic stimulation before initiation of radiation therapy. Eleven of the patients were in clinical Stage I, eight were in Stage II, six were in Stage III, and six were in Stage IV, according to the recommendation for classification and staging of the International Federation of Gynecology and Obstetrics [10]. Responses of patient blood samples were compared with concomitantly tested control samples taken from relatives of the patients near the same age, laboratory personnel, or other volunteers in normal health. The patients were treated with photon radiation of 32 MeV energy through two lateral and one anterior fields. Twenty-one of the patients were treated by external beam radiation only to a tumor dose of 6000-6400 rads, and 10 of the patients were given 4500-5000 rads external radiation and an intracavitary application of radium to produce a final tumor dose of 6000-6400 rads. Twenty-three of the 31 patients were tested again near mid-treatment, 17 of the patients were tested also within 1-2 hr after the final treatment, and 14 of the original patients and 2 additional patients treated in the same manner were tested once or twice 3-6 months after treatment.
Techniques for Whole-blood Culture For preparation of cultures, 5 ml of peripheral blood were withdrawn by venipuncture from patients and control individuals on the same day. Clotting was prevented by addition of preservative-free heparin, and total white cell numbers per cubic millimeter were determined in an electronic cell counter. Blood smears were prepared, and differential counts were made by standard methods. The blood cells then were diluted and cultured and the lymphocytes stimulated to undergo blastic transformation according to methods described
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previously [7,11,12]. Briefly, the whole blood was diluted 1:20 with cell culture medium (RPMI 1640), and 3-ml aliquots of the cell suspension containing 150/~1 of whole blood were set up in triplicate in 16 × 125-ram plastic culture tubes for each mitogen used. The culture tubes were capped tightly and incubated for 7 days at 37°C, a time period previously shown to be within peak response for P H A and Con A stimulation [7,11,12 ]. The stimulated cultures contained 10/zg of purified P H A (mitogenic-hemagglutinin activity ratio approximately 100: l, Burroughs Wellcome, Research Triangle Park, North Carolina), 10 tzg of Con A, or 300/~g of PWM.
Evaluation of Lymphocyte Response Transformation of the lymphocytes in response to the mitogens was assessed by a [3H]TdR incorporation method [12]; 1.0/~Ci of [3H]TdR (2.0 Ci/mM) in 0.2 ml of medium was added to each culture 24 hours before harvest. At the end of the 7-day culture period, individual cell suspensions were filtered through glass-fiber filter discs (Grade 934 AN, Reeve Angel, Clifton, New Jersey) to trap the cells on the surface. The cells were washed several times with distilled water, acetic acid, and distilled water. Then the discs were placed in the bottom of scintillation counting vials and allowed to dry. Scintillation counting fluid was added to the vials, and the counts per minute from the labeled D N A trapped on the glass-fiber filters were determined in a liquid scintillation spectrophotometer. The number of disintegrations per minute (dpm) was calculated from the counts. The statistical significance of the difference between D P M means of various groups was determined by Student's t test. RESULTS
Lymphocyte Response and Number in Relation to Clinical Stage The mean responses to P H A and Con A of the total group of patients tested before radiation therapy were only 59% and 56% of the responses of concomitantly tested control individuals (P < 0.01, Table 1). On the other hand, the TABLE 1 LYMPHOCYTE REACTIVITY OF UTERINE CANCER PATIENTS BEFORE RADIOTHERAPY IN RELATION TO CLINICAL STAGE Mean lymphocyte response dpm x 103 ± SE/culture (No. individuals tested) PHA
Con A
PWM
Clinical stage of neoplasm
Patients
Controls
Patients
Controls
Patients
Controls
1 II III IV I-IV combined
253-+48(1l) 193 ±26'~(8) 213 ± 31 b (6) 93 -+ 4 V (6) 199 ± 22 ~ (31)
397_+60(11) 319+32(8) 326 ± 42 (6) 343 ± 55 (6) 336 ± 16 (31)
68_+19(11) 43___11~(8) 39 ± 7 (6) 34 ± 13 (6) 49 --- 8 a (31)
68+17(10) 110-+26(8) 100 ± 43 (5) 77 ± 22 (6) 87 -+ 12 (29)
63_+26(11) 42_+7(7) 27 ± 10 (6) 31 ± 9 (6) 44 ± 10 (30)
48_+11(1l) 65+14(7) 45 ± 17 (6) 50 ± 20 (5) 53 -+ 7 (29)
a Values for patients were significantly less than values for controls (P < 0.01). Values for patients were significantly less than values for controls (P < 0.05).
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mean response of the total group of patients to PWM was not significantly less than the control value. Six of the 31 patients tested were in advanced Stage IV and had a very poor mean response to PHA as compared to the response of concomitantly tested controls (27%, P < 0.01) and compared to the response of 11 patients in Stage I (37%, P < 0.05). The mean response to Con A of the six patients with advanced disease was apparently less (44% of control) than the response of concomitantly tested controls and compared with the response of patients in the earliest stage, but the differences were not statistically significant for the few patients tested. The mean number of lymphocytes in peripheral blood (2230/mm 3) of the 31 patients tested before initiation of radiation therapy was only sIightly (16%) but significantly less than the number in the total group of control individuals tested (2642/mm 3) (P < 0.05, Table 2). There was no evidence, however, to show that patients in the more advanced Stage IV had fewer lymphocytes than patients in earlier stages.
Effects of Radiotherapy on Numbers and Responses of Lymphocytes The effects of radiation treatment on the numbers of lymphocytes in the peripheral blood also are shown in Table 2. The mean numbers of lymphocytes declined in the patients during treatment to 24% of the control value (P < 0.01) and 28% of the pretreatment value (P < 0.01), an average loss in the patients of about 1600 cells/mm3. The change did not relate to clinical stage of disease, since the patients in Stages I and II experienced as great a decline as patients in Stages III and IV. Patients tested 3-6 months after radiotherapy had only slight recovery in the number of lymphocytes to 36% of control and 42% of pretreatment values (P < 0.01). Again, patients in earlier clinical stages did not have a significantly greater degree of recovery than patients in later stages. The mean responses to all three mitogens also declined in cultures from patients during radiation treatment. By about mid-treatment (2000-3000 rads), the mean responses in the total group of patients to all three mitogens were de-
TABLE EFFECT
2
OF T H E R A P E U T I C R A D I A T I O N ON N U M B E R S OF L Y M P H O C Y T E S I N P A T I E N T S W I T H C A R C I N O M A OF T H E U T E R U S M e a n N o . of l y m p h o c y t e s _+ SE/mm* blood (No. individuals tested) Patient s a m p l e s
Clinical stage of neoplasm I n In IV i-iv combined
2382 2213 2109 2109 2230
0 rads
2000-3000 r a d s
6000-6400 r a d s
After treatment 3-6 m o n t h s
+ 174 (11) + 236 (8) 4- 530 (6) 4- 371 (6) -+ 136 ~ (31)
1091 809 854 1017 976
596 744 439 680 623
989 932 904 884 947
_+ 216 a (9) ± 307 ~ (3) _+ 140 ~ (6) 4- 192 a (5) ± 99 a (23)
444-+ -+
99" (8) 350" (3) 2 v (2) 172 a (4) 73 a (17)
Values f o r patients w e r e significantly less than v a l u e s f o r controls ( P < .01).
44± 4-
127 ° (11) 112 ~ (6) 18 ~ (4) 108 a (3) 62 a (24)
Control samples 2581 2793 2566 2674 2642
4- 129 (38) -4- 215 (20) 4- 149 (17) +_ 141 (18) -4- 78 (93)
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3
L Y M P H O C Y T E R E A C T I V I T Y OF U T E R I N E CANCER P A T I E N T S A F T E R
2000-3000 RADS
T R E A T M E N T IN R E L A T I O N TO C L I N I C A L S T A G E
Mean lymphocyte response dpm x 102 _+ SE/culture (No. individuals tested) PHA
Con A
PWM
Clinical stage
of neoplasm I II III IV I-IV combined
Patients 175 51 67 39 101
Controls
± 550 (9) ± lOs (3) -+ 27 ~ (6) ± 13 a (5) ± 25 ~ (23)
369 385 279 247 321
± 62 (9) ± 27 (3) ± 46 (6) -+ 33 (5) ± 29 (23)
Patients
Controls
48 ± 21 (9) 1 (1) 5 -+ 4 ° (5) 14 ± 6 (5) 26 ± 10a (20)
85 50 60 98 76
-+ 29 (9) - 15 (3) ± 23 (6) ± 49 (4) ± 15 (22)
Patients 29 ± 3 ± 8 ± 8 ± 16 ±
9 (9) 2" (2) 4 b (5) 4 (5) 5 a (21)
Con trois 55 34 53 54 52
± ± ± ± ±
19 (8) 0 (2) 17 (6) 35 (5) 11 (21)
Values for patients were significantly less than values for controls (P < 0.01). b Values for patients were significantly less than values for controls (P < 0.05).
pressed as compared to controls (Table 3). Patients in advanced Stage IV, again, had a smaller mean response to PHA than Stage I patients (22%, P < 0.05). Although responses to Con A and PWM apparently were smaller for patients with advanced lesions than for Stage I patients, the values for the few patients examined were not statistically different. By the end of treatment, responses for patients to all three mitogens were severely depressed (Table 4), although nearly one-half of the total number of patients were in clinical Stage I. The mean values for cultures of the total group of patients at the end of treatment in response to PHA and Con A were only 18% and 20% of the values for patients tested before treatment (P < 0.01) and only 10% of the values for controls (P < 0.01). Similarly, the response to PWM was reduced to 11% of the value for patients before treatment (P < 0.01) and 9% of the value for controls (P < 0,01). There were no differences in responses to either of the mitogens of the cultures from patients in different clinical stages at the end of treatment. Responses to mitogens of blood samples from patients tested 3-6 months after
TABLE 4 L Y M P H O C Y T E R E A C T I V I T Y OF U T E R I N E C A N C E R P A T I E N T S A F T E R 6 0 0 0 - 6 4 0 0
RADS
T R E A T M E N T IN R E L A T I O N TO C L I N I C A L S T A G E
Mean lymphocyte response dpm x 103 _+ SE/culture (No. individuals tested) PHA
Con A
PWM
Clinical stage
of neoplasm I II lU IV I-IV combined
Patients 44 30 31 25 35
--_ 17a (8) -4- 220 (3) --_ 400 (2) -+ 22 b (4) -+ I0 a (17)
Controls 343 287 340 372 339
± 34 (8) --_ 91 (3) ± 70 (2) -+ 112 (4) -+ 29 (17)
Patients 15 ± 7 ° (7) 6 ± 3 (3) 5 -+ 6 (2) 5 -+ 3 b (4) 10 _+ 3 ~ (16)
Controls 102 112 105 74 97
± 34 (7) --- 60 (3) --- 49 (2) -- 28 (4) -- 18 (16)
Values for patients were significantly less than values for controls (P < 0.01). 0 Values for patients were significantly less than values for controls (P < 0.05).
Patients 5 --+ 30 (7) 4 -- 5 (3) 4 ± 1~ (2) 6 ± 5 a (4) 5 ± 2 a (16)
Controls 79 53 49 30 58
-+ 29 (7) ± 47 (3) -+ i (2) + 3 (4) ± 14 (16)
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TABLE 5 LYMPHOCYTE REACTIVITY OF UTERINE CANCER PATIENTS TESTED 3--6 MONTHS AFTER 6 0 0 0 - 6 4 0 0 PADS TREATMENT Mean lymphocyte response dpm x 103 _+ SE/culture (No. individuals samples tested) PHA
Con A
PWM
Clinical stage of neoplasm
Patients
Controls
Patients
Controls
Patients
Controls
I II III IV I-IV combined
110-+ 24a (11) 34 +- 16a (6) 85 -+ 39 b (4) 44 _+ 34b (3) 78 -+ 15~ (24)
272-+ 30 (10) 323 -+ 20 (6) 260 -+ 67 (4) 317_+101(3) 289 -+ 19 (23)
28-+ 5~ (11) 10 -+ 4" (6) 14 + 6 b (4) 4_+2(3) 18 -+ 3a (24)
96 -+ 2 (10) 175 -- 21 (5) 109 -+ 45 (4) 85"+39(3) 115 _+ 15 (22)
17 -+ 4 (11) 6 -+ 2 b (6) 4 +- 1(4) 7_+ 5°(3) 11 -+ 2 ~ (24)
39 -+ 15 (10) 86 -+ 32 (6) 53 +- 27 (4) 33_+8(3) 53 _+ 11 (23)
Values for patients were significantly less than values for controls (P < 0.01). Values for patients were significantly less than values for controls (P < 0.05).
treatment remained depressed and were only slightly, if any, higher than responses of patients at the end of treatment (Table 5). Responses of the total group of patients to PHA, Con A, and PWM remained at only 39, 37, and 25% of the pretreatment values (P < 0.05) and 27, 16, and 21% of values for controls (P < 0.05). DISCUSSION
Patients with carcinoma of the uterus tested before radiation treatment generally had smaller P H A and Con A responsiveness than healthy individuals, and further, patients with advanced lesions tended to respond less than patients with Stage I lesions (Table 1). On the other hand, the patients had responses to PWM that were similar to responses of controls, and patients in the advanced stage did not have a significantly smaller response than patients in earlier stages. P H A and Con A responses are considered generally to reflect thymus-dependent or T-cell immunity, although Con A has also been reported to stimulate bone-marrowderived lymphocytes (B-cells), and PWM is primarily, though not exclusively, a B-cell mitogen [6,8,13,14]. It appears, therefore, that these patients had an overall reduced T-cell immunity, the greatest deficit occurring in patients with the most advanced lesions and the smallest deficit occurring in patients with small localized lesions. On the other hand, the patients were probably not deficient in B-cell immunity. These data are consistent with reports in the literature indicating that a deficit in cell-mediated immunity (T-cells) is more readily detected and at an earlier stage of neoplastic progression than humoral immunity (B-cells) [ 1,15]. Reduced responses to P H A in patients with advanced nonlymphoid neoplasms have also been reported previously [4,16-19], but Mekori et al. [18] did not find reduced responses in patients with advanced genitourinary lesions. Although the total group of patients with uterine cancer had a smaller mean number of peripheral blood lymphocytes (84% of control), the deficit in number of cells alone did not account totally for the reduced responses to P H A and Con
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A (59 and 56% of control). Moreover, patients with advanced disease had much smaller PHA and Con A responsiveness (27 and 44% of controls) and a near normal mean number of lymphocytes. It appears, therefore, that the whole-blood method of measuring reaction of lymphocytes to mitogens detects deficiencies that cannot be recognized from differential cell counts alone. This is not unexpected since peripheral blood lymphocytes comprise a heterogeneous population of cells with different functions. One or more mechanisms might explain the reduced T-cell response in these patients. One possibility is that some inhibitory factor, as described by Catalona et al. [16] and Whittaker et al. [19], is present in the sera of the patients and interferes with T-cell response in vitro. Studies to test this possibility have not been done as yet on these patients. Another possibility is that the patients, especially those with advanced lesions, have reduced numbers of circulating T-cells capable of mitogenic response. Whole-blood culture methods for assessing lymphocyte responses have several advantages over methods using isolated lymphocytes: (a) 5 ml of blood are more easily obtained and can be obtained more frequently than larger volumes (30-50 ml), (b) there are greater reproducibility and better correlation with skin test data [7,12], (c) there is avoidance of uncontrolled variables such as possible damage to cells during isolation, (d) there is indication (theoretically) of quantitative response of the individual rather than qualitative response of a given number of lymphocytes, which may vary in number and heterogeneity, and (e) a greater number of cultures can be tested from a single blood sample, especially from patients with a reduced number of lymphocytes following therapy. Some criticisms of the whole-blood technique are (a) augmentation of lymphocyte response by erythrocytes, (b) interference with response by polymorphonuclear cells, and (c) a long culture period relative to generation cycle. It is unlikely that the presence of erythrocytes was a serious problem in this study since the purified form of PHA was used and it has been shown that the response of cultures of lymphocytes to purified PHA was not altered when erythrocyte membranes were added [20]. Although polymorphonuclear leukocytes were reported to increase transformation of lymphocytes [21], Park and Good [22] did not find any major effect of granulocytes on stimulation of lymphocytes in their whole-blood culture system. The 7-day harvest time used in this study was shown previously to be within the peak of PHA and Con A responses for wholeblood cultures from healthy individuals [7,12] and from patients with breast cancer [23]. During radiation treatment the number of lymphocytes in the patients decreased to about one-fourth of the number in control individuals or to about 28% of the number in patients before treatment and remained at only 36% of control value 3-6 months after treatment. This effect is similar to effects on lymphocyte numbers found in radiation-treated patients with breast and lung cancer [11,23]. It is likely that the decrease in number of circulating lymphocytes resulted from death of radiosensitive lymphocytes circulating through the field of treatment. The area of treatment was confined to the pelvic region and did not include segments of the thymus or thoracic duct, which may contribute to the deficit noted in patients treated to the upper thorax. Similarly, the mean responses to mitogens of cells in cultures of a given volume of blood (150 kd)
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from the patients declined during radiation treatment until at the end of treatment the responses to all three mitogens were reduced to about 10% of control responses. The capacity of the patients' blood samples to react to mitogen stimulation remained depressed (16-27% of controls) during the 3-6-month follow-up period. Although patients in the earlier stage (Stage I) of disease development had better T-cell responses before treatment than patients in Stage IV, there was no indication that such patients had better responses after treatment. It is likely that after radiotherapy the depressed responsiveness of the patients' blood lymphocytes to the three mitogens resulted from a decreased number of cells capable of a blastogenic response rather than a release of factors into the sera that prevent the response. Studies to determine whether inhibitory factors are present in the sera of treated patients have not been completed. Radiation therapy has been shown previously to reduce numbers and mitogenic responsiveness of lymphocytes in patients with several different histological types of neoplasms [4,11,23,24]. In contrast, some investigators using mitogens or skin-test antigens have reported minimal or no depression of cellmediated immunity in patients with nonlymphoid neoplasms after radiotherapy [5,25,26]. Inconsistencies in the data on effects of radiation on patient immunity have caused investigators to question which of the several methods used are most likely to indicate capacity of the individual to mount an immunologic response [1]. Whether the in vitro correlate of overall patient immunity, as measured by mitogenic stimulation in whole-blood cultures, is a quantitative measure that indicates capacity of individual patients to react immunologically to specific tumor antigens and whether these indications are significant in predicting clinical outcome for cancer patients are conjectural. Although radiotherapy reduced lymphocyte numbers and responsiveness drastically in these patients and the deficit remained for at least 3-6 months after treatment, it is unlikely that these effects are indicative of an overall poor prognosis. The suppressive effects on immunologic response were similar in patients of all clinical stages, and yet survival rates after radiotherapy are very different for patients treated at different clinical stages of disease. The 10-yr rate of survival reported for patients after megavoltage therapy is 90% for Stage I patients and decreases to 14% for Stage IV patients [9]. Whether patients in earlier clinical stages that have good prognosis recover their lymphocyte function more fully than patients in more advanced stages in succeeding months remains to be determined. Since patients with localized lesions have an excellent survival rate after radiotherapy, it appears the treatment is effectively destroying the lesion, and a concomitant depression in immunity by the treatment may be of little significance. On the other hand, depressed immunologic competence before or after treatment in patients with lesions extended beyond the treatment area may contribute to failure to control the disease. REFERENCES 1. Harris, J., and Copeland, D. Impaired immunoresponsiveness in tumor patients, Ann. N . Y . Acad. Sci. 230, 56-85 (1974).
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2. Hellstrom, I., Hellstrom, K. E., Sjorgren, H. O., and Warner, G. A. Demonstration of cellmediated immunity to human neoplasms of various histological types, Int. J. Cancer 7, 1-16 (1971). 3. Eilber, F. R., and Morton, D. L. Impaired immunologic reactivity and recurrence following cancer surgery, Cancer 25, 362-367 (1970). 4. O'Toole, C., Perlmann, P., Unsgaard, B., Moberger, G., and Edsmyr, F. Cellular immunity to human urinary bladder carcinoma: I. Correlation to clinical stage and radiotherapy, Int. J. Cancer 10, 77-91 (1972). 5. Gross, L., Manfredi, O. L., and Protos, A. A. Effect of Cobalt-60 irradiation upon cell-mediated immunity, Radiology 106, 653-655 (1973). 6. Byrd, W., Boehmer, H., and Rouse, B. T. The role of the thymus in maturational developments of phytohemagglutinin and pokeweed mitogen responsiveness, Cell, lmmunol. 6, 12-24 (1973). 7. 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, J. Lab. Clin. Med. 82, 500-513 (1973). 8. 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). 9. Fletcher, G. H. Female pelvis, in Textbook o f radiotherapy (G. H. Fletcher, Ed.), Lea & Febiger, Philadelphia, 2nd ed. pp. 620-665 (1973). 10. 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. Obstet. 9, 172-179 (1971). 11. 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). 12. Pauly, J. L., and Sokal, J. E. A simplified technique for in vitro studies of lymphocyte reactivity, Proc. Soc. Exp. Biol. Med. 140, 40-44 (1972). 13. Andersson, J., Moller, G., and Sjoberg, O. B lymphocytes can be stimulated by concanavalin A in the presence of humoral factors released by T cells, Eur. J. lmmunol. 2, 99-101 (1972). 14. Janossy, G., and Greaves, M. F. Lymphocyte activation: I. Response of T and B lymphocytes to phytomitogens, Clin. Exp. lmmunol. 9, 483-498 (1971). 15. Curtis, J. E., Hersh, E. M., Harris, J. E., McBride, C., and Freireich, E. J. The human primary immune response to keyhole limpet haemocyanin: Inter-relationships of delayed hypersensitivity, antibody response and in vitro blast transformation, Clin. Exp. lmmunol. 6, 473-491 (1970). 16. 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). 17. Gatti, R. A., Garrioch, D. B., and Good, R. A. Depressed PHA responses in patients with nonlymphoid malignancies, in Proc. Fifth Leukocyte Cul. Conf. (J. E. Harris, Ed.), Academic Press, New York, pp. 339-358 (1970). 18. Mekori, T., Sher, S., and Robinson, E. Suppression of the mitogenic response to phytohemagglutinin in malignant neoplasia: Correlation with clinical stage of therapy, J. Nat. Cancer Inst. 52, 9-12 (1974). 19. Whittaker, M. G., Rees, K., and Clark, C. G. Reduced lymphocyte transformation in breast cancer, Lancet i, 892-893 (1971). 20. Johnson, R. A., Smith, T. K., and Kirkpatrick, C. H. Augmentation of phytohemagglutinin mitogenic activity of erythrocyte membranes, Cell. Immunol. 3, 186-197 (1972). 21. Gough, J., Elves, M. M., and Israels, M. G. G. The formation of macrophages from lymphocytes in vitro, Exp. Cell Res. 38, 465-482 (1964). 22. Park, B. H., and Good, R. A. A new micromethod for evaluating lymphocyte responses to phytohemagglutinin: Quantitative analysis of the function of thymus-dependent cells, Proc. Nat. Acad. Sci. 69, 371-373 (1972). 23. Jenkins, V. K., Olson, M. H., Cooley, R. N., and Ellis, H. N. Effect of therapeutic radiation on peripheral blood lymphocytes in patients with carcinoma of the breast, Acta Radiol. (in press).
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24. Thomas, J. W., Coy, P., Lewis, H. S., and Yuen, A. Effect of therapeutic irradiation on lymphocyte transformation in lung cancer, Cancer 27, 1046-1050 (1972). 25. Clement, J. A., and Kramer, S. Immunocompetence in patients with solid tumors undergoing cobalt 60 irradiation, Cancer 34, 193-196 (1974). 26. 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).