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Lymphocyte Stimulation in Urologic Cancer Patients
THE
J OURKAL
Vol. 112, Sepl em hc·r
OF UROLOGY
Copyright © 1974 by The Williams & Wilkins Co.
Printed in
LYMPHOCYTE STIMULATION IN UROLOGIC CANCER PATIENTS WILLIAM J. CATALONA, JOHN L. TARPLEY, PAUL B. CHRETIEN
AND
J. ROLAND CASTLE
From the James Buchanan Bradv Urolo{!ical Institute, The Johns Hopkins Hospital, Baltimore and Tumor Immunologv Section, Su.r{ierv Branch, National Cancer Institute, National Institutes of Health, Bethesda, MCTrvwud
Current concepts of tumor immunology suggest that the ability of host defenses to restrict tumor is vested in the cellular immune mechamediated by (T) lymphocytes. Therefore, in order for an immunologic response to be effective against cancer, T lymphocyte function must be intact. The proliferative response of lymphocytes when cultured in the presence of phytohemagglutinin (PHA) has been perhaps the most widely used in vitro assay for cellular immune competence in human disease states 1). Although lymphocyte activation PHA and does not constitute a true response, it simulates the immunoproliferative response which occurs in vivo upon antigenic stimulation. In this context it is -·,,,,.,,.,-,"' that lymphocytes from with cellular immunity also manifest reduced 1J1•uu1na responses to PHA. 1 it seems reasonable to equate the in vitro lymphocyte response to PHA with the behavior of lymphocytes in vivo. The proliferative response of to PHA has been studied in cancer patients several investigators but the results reported have been somewhat ,_, Moreover, recognition of serum factors which have the capacity to inhibit PHA-induced lymphocyte blastogenesis has beclouded the conclusions drawn from studies in which lymphocytes were cultured in the presown) serum. •- 7 In (the ence of such instances it is to determine whether the poverty of reactivity observed is caused intrinsic lymAccepted for publication March 8, 1974. Read at annual meeting of American Urological Association, St. Louis, Missouri, May 19-23, 1974. 1 Hersh, E. M. and Oppenheim, J. J.: Impaired in vitro lymphocyte transformation in Hodgkin's disease. New Engl. J. Med., 273: 1006, 1965. 'Robinson, M. R. G., Nakhla, L. S. and Whitaker, R. H.: A new concept in the management of carcinoma of the prostate. Brit. J. Ural., 43: 728, 1971. 3 Robinson, M. R. G., Nakhla, L. S. and Whitaker, R. H.: Lymphocyte transformation in carcinoma of the prostate. Brit. J. Ural., 43: 480, 1971. 'McLaughlin, A. P., III, Kessler, W. 0., Triman, K. and Gittes, R. F.: Immunologic competence in patients with urologic cancer. J. Urol., Hl: 233, 1974. 'McLaughlin, A. P., III, Kessler, W. 0., Triman, K. and Gittes, R. F.: Immune lymphocyte competence in urologic cancer: plasma inhibition of lymphocyte transformation. Surg. Forum, 24: 112, 1973. 6 Astaldi, G., Costa, G. and Air6, R.: Phytohemagglutinin in leukemi.a. Lancet, l: 1394, 1965. 7 Silk, M.: Effect of plasma from patients with carcinoma on in vitro lymphocyte transformation. Cancer, 20: 2088, 1967.
phocyte defect or by serum
effect of such serum on the lymphocytes. In our study the in vitro blood to PHA effect of patient sera on normal were determined in 53 tients. Correlations are responsiveness, tumor the effects of PATIENTS AND METHODS
Patients and controls. Blood from urologic cancer patients and 44 with comparable age distribution intrinsic lymphocyte to while from 67 urologic cancer patients and 18 controls were tested for serum effect on the of normal lymphocytes to PHA. In all instances cancer patients and controls were tested concur rently. Patients were either nre>r.n,o,,o 5 days postoperative. Nine previous radiation ing the distributions of tumor types and stages shown in the table and figures 2 and 3. Culture system. An AB positive serum prepared from healthy volunteers, was used constant reference for cellular and serum The pool was heat inactivated at 56C fm 30 minutes, passed through a 0.45 micron filter and stored in smali aliquots in gen. Heparinized whole blood was per cent methylcellulose after iron for 45 minutes at 37C in a Differential phagocytosis of iron resulted in cell suspensions containing 95 to 99 per cent mononuclear cells with 99 per cent thus obtained were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium counted in a Coulter counter. Cultures containing 5.0 times 10 5 mononuclear cells per ml. of 90 per cent RPM! 1640 and HJ cent AB pool serum were
374
CATALONA AND ASSOCIATES
FIG. 1. Appearance under light microscopy oflymphocytes undergoing blast transformation in response to PHA stimulation. Reduced from x450.
?HA-induced lymphocyte reactivity and serum effect on lymphocyte reactivity in 9 previously irradiated urologic cancer patients Tumor Type
Bladder Bladder Bladder Bladder Bladder Kidney Prostate Prostate Prostate Mean
Standard error
Tumor Stage
P2 P3 P3 P4 P4 Metastatic IV IV IV
PHAInduced Lympho-
Serum Effect on
cyte*
LRit
232 140 161 39 75 24 46 62 70
0.957 0.990 1.056 1.254 0.968 0.726 0.980 0.989 1.126
94.3 22.9
1.005 0.047
* Tritiated thymidine incorporation (disintegrations per minute times 10' per culture). t LRI-lymphocyte reactivity index
the cultures had incubated for 54 hours. After a labeling period of 3 hours the cultures were frozen in liquid nitrogen. The nucleoprotein was precipitated with cold 5 per cent trichloroacetic acid (TCA) and the precipitate was washed 3 times with TCA, a fourth time with saline, and was solubilized in 0.5 ml. soluenet. The soluene was neutralized with 20 lambda of glacial acetic acid and the nucleoprotein was transferred to scintillation counting vials by 3 washes with 5 ml. portions of toluene-liquiflor:j: scintillation fluid. The incorporation of 3 H Tdr by the lymphocytes was quantitated in a model 3303 Packard Tri-Carb scintillation counter and the absolute activity was expressed in disintegrations per minute (dpm). Expression of results. The intrinsic lymphocyte reactivity to PHA was expressed as the peak numerical increment in dpm above control levels (unstimulated cultures) regardless of the PHA concentration at which it occurred. The rationale for this form of data expression derives from the fact that it yielded a coefficient of variation (standard deviation expressed as a percentage of the mean) in the control population of 25.1 per cent. The same data expressed as an activity ratio (stimulated "H Tdr incorporation divided by unstimulated 3H Tdr incorporation) yielded a coefficient of variation of 68.3 per cent in the controls. Serum effect on lymphocyte reactivity. Normal donor lymphocytes were cultured in test serum (cancer patient or healthy control) and in AB positive pool serum, respectively. The 3H Tdr incorporation of this lymphocyte population in the test serum was compared to the 3 H Tdr incorporation of the same lymphocyte population in the AB positive pool serum. The result was expressed as the ratio: lymphocyte reactivity index (LRI) equals peak 3 H Tdr incorporation in test serum (dpm)/ peak 3H Tdr incorporation in AB positive pool serum (dpm). Statistical methods. Differences observed in intrinsic lymphocyte reactivity and serum effect on lymphocyte reactivity between healthy controls and urologic cancer patients were evaluated by Student's t test. RESULTS
( 'H Tdr in patient serum ) . 'H Tdr in AB+ pool
tohemagglutinin-P* reconstituted in saline and used in dilutions of 25, 12.5, 6.25, 3.125 mcg. protein per ml. culture was added to the cultures and the cultures were incubated at 37C in a humidified 95 per cent air-5 per cent carbon dioxide atmosphere. Cultures with each PHA dose and control cultures without PHA were always prepared in triplicate. Tritiated thymidine ( 3H Tdr) (300 mCi. perm. mole, 30 micro Ci. per ml. culture) was added after
* Difeo Laboratories, Detroit, Michigan.
Intrinsic lymphocyte reactivity of urologic cancer patients (fig. 2). The mean lymphocyte reactivity ("H Tdr incorporation per culture) of 10 patients with stage Pl or P2 bladder tumors was 162.9 times 10 3 dpm,§ which did not differ significantly from the mean lymphocyte reactivity of the 44 healthy controls (157.6 dpm, p greater than 0.20). However, the mean lymphocyte reactivity of 10 patients with stages P3 and P4 bladder tumors was significantly impaired relative to controls (100.2 dpm, p less than .001). The difference be-
t Packard Instrument Co., Inc. :j: New England Nuclear, Boston, Massachusetts. § All lymphocyte reactivity values referred to hereinafter are xl0 3 •
LYMPHOCYTE STIMULATION IN UROLOGIC CANCER PATIENTS PHA-STIMULATED LYMPHOCYTE BLASTOGENESIS IN UROLOGIC CANCER PATIENTS I Peak Stimulation - Control I
220
~ Meon±SE
200
160
'=' X
:,e
/40
~
CONTROLS n°44
CL 0
I
n,3
BLADDER P1a P1 nc/0
120
I
100
II
PROSTATE PROSTATE* ]][ HIT n°4 n°19 BLADDER* P3 8 P4 nc 10
80
I I
KIDNEY
I localized I
60
TESTIS n°2
I I
KIDNEY
Imetastatic I
IRRADIATED PATIENTS" n,9
n°5
* P<0.01
FIG. 2. Comparison of PHA-induced tritiated thymidine incorporation by peripheral blood lymphocytes from 53 urologic cancer patients and 44 age-matched healthy controls. Differences were evaluated by Student's t test.
EFFECT OF UROLOGIC CANCER SERA ON THE REACTIVITY OF NORMAL LYMPHOCYTES
~
1.20
X
w
~ 1.10 >>-
>
:s"' ~
t;
I I I
CONTROLS nc/8
e=
TESTIS (local) nc 2
1.00
0 I
CL
PROSTATE I-ill n°6
"I '" " ! I
PROSTATE* Ill n' 27
"'~ 0.90
I
P1 8 P1 n' 10
BLADDER P3 fl P4 n°9
TESTIS
I metoslotic) n°4
KIDNEY (localized) n°3
~ Meon±SE KIDNEY
I metastatic I
0.80
* P <0.01
n°5
FIG. 3. Comparison of effects of sera from 67 urologic cancer patients and 18 healthy controls on PHA-induced tritiated thymidine incorporation of allogeneic lymphocytes. Differences were evaluated by Student's t test.
tween patients with advanced bladder carcinoma and controls was also significant (97.8 dpm, p less than .02) when 4 irradiated patients were excluded (See table). Lymphocyte reactivity was impaired in both patients with localized prostatic carcinoma (stages I to HI-121.5 dpm, p less than .10) and those with metastases present (stage IV -113 .9 dpm, p less than .01). This difference also remained significant (124.1 dpm, p less than .02) when 3 irradiated patients were excluded (See table). Patients with localized renal cell carcinoma manifested normal intrinsic lymphocyte reactivity (184.0 dpm, p greater than .20), while those with metastases had lower lymphocyte reactivity (112.0
dpm, p less than 0.10). However, the difference between controls and cancer patients in the latter group was diminished (134.0 dpm, p less than .20) when the one previously irradiated patient was excluded (See table). Only 2 patients with germinal testis tumors were tested. One with a localized tumor had an assay score of 184 dpm, while the other patient who had metastatic disease had an assay score of 144 Effect of radiation therapy (table and fig. 2). group, previously irradiated patients manifested markedly impaired intrinsic lymphocyte (94.3 dpm, p less than .01). Serum effect on lymphocyte reactivity (figs. 3 and 4). The effects of sera from 18 healthy, age
376
CATALONA AND ASSOCIATES
matched controls on the reactivity of allogeneic (another person's) lymphocytes were compared to the effects of cancer sera on the same lymphocyte population, using the AB positive serum pool as a constant reference. Results were expressed as a lymphocyte reactivity index (LRI). The mean LRI for the control population was 1.100. Only patients with metastatic prostatic carcinoma had a mean LRI which was significantly lower than that of controls (LRI = 0.979, p less than .02). However, in all patient groups studied, a lower mean LRI was observed among patients with more advanced disease (fig. 3). Serum-mediated inhibition of lymphocyte reactivity was arbitrarily considered to be significant in patients with LRI's of less than 0.950, since nearly 90 per cent of healthy controls tested had LRI's above this value. Using this arbitrary classification serum inhibition was observed in 9 per cent of bladder cancer patients, 29 per cent of prostatic cancer patients, 33 per cent of renal cell carcinoma patients and none of the patients tested with germinal testis tumors (fig. 4). DISCUSSION
The significance of the immunoproliferative response derives from the fact that by this process a population of immunologically committed lymphocytes is formed. An understanding of the mechanism underlying the apparent failure of this fundamental response in urologic cancer patients may afford important insights into the biology of urologic cancer. Robinson and associates studied prostatic carcinoma patients and reported that lymphocyte responsiveness to PHA was impaired only in patients with metastases present. 3 McLaughlin and associates demonstrated impaired lymphocyte reactivity in patients with a variety of urologic tumors but were unable to demonstrate a correlation between PHA responsiveness and tumor stage. 4 In both studies lymphocytes were cultured in the presence of autologous serum. Thus, it was not possible to determine whether the impairment of lymphocyte responsiveness was caused by an intrinsic cellular abnormality or by the presence of serum inhibitors. Addressing themselves to this issue in a subsePERCENTAGE OF PATIENTS WITH SERUM INHIBITION
CONTROLS N, I 8
11%
PROSTATE N,34 Kl ONEY
N,9
BLADDER
N,22
TESTIS
N,7
29% 33%
9% 0%
FIG. 4. Comparison of respective proportions of patients having significant serum inhibition of lymphocyte reactivity (lymphocyte reactivity index less than 0.950) in patient groups studied.
quent communication, McLaughlin and associates reported that depressed lymphocyte reactivity to PHA in 19 cancer patients tested could be restored to normal by culturing the cells in the presence of non-cancer plasma. 5 They suggested, therefore, that the impairment of lymphocyte reactivity in urologic cancer patients was caused solely by serum-mediated inhibition and not an intrinsic cellular defect. A relevant feature of our study is the demonstration of an intrinsic lymphocyte defect in a significant proportion of urologic cancer patients. In our assay system cancer lymphocytes, washed 3 times and cultured in the presence of pooled AB positive serum from healthy volunteers, manifested significantly impaired reactivity to PHA. The relationship between lymphocyte reactivity to PHA and tumor stage also contrasts with previous reports. 3 • 4 We found lymphocyte reactivity to be normal in patients with early bladder cancer but significantly impaired among patients with advanced bladder cancer. A similar relationship was observed in patients with renal cell carcinoma, although statistical significance was not achieved. In contrast, lymphocyte reactivity was substantially impaired in patients with localized prostatic carcinoma as well as those with advanced tumors (fig. 2). In general, the patterns of immunologic reactivity found in this study are in concert with the results of parallel studies from our laboratory using dinitrochlorobenzene (DNCB) contact sensitization •·10 and the T rosette assay 11 to quantify cellular immune competence in urologic cancer patients. Therefore, it seems reasonable to infer that in our patient population there is a significant intrinsic deficiency of T lymphocyte function. This defect has been observed infrequently in patients with early bladder or renal cell carcinoma but has been found in a significant proportion of patients with clinically localized prostatic carcinoma. Too few patients with germinal testis tumors have been studied to warrant comment. The proliferative response of lymphocytes to PHA was noted to be impaired in a majority of patients who had received radiation therapy. However, most of these patients also had advanced tumors and the small number of irradiated patients in each group precluded meaningful statistical analysis. Reports on the effects of radiation therapy on FHA-induced lymphocyte reactivity are conflict' Catalana, W. J., Sample, W. F. and Chretien, P. B.: Lymphocyte reactivity in cancer patients: correlation with tumor histology and clinical stage. Cancer, 31: 65, 1973. 'Catalana, W. J., Chretien, P. B. and Trahan, E. E.: Abnormalities of cell-mediated immunocompetence in genitourinary cancer. J. Urol., lll: 229, 1974. 1 °Catalona, W. J. and Chretien, P. B.: Correlation among host immunocompetence and tumor stage, tumor grade and vascular permeation in transitional carcinoma. J. Urol., 110: 526, 1973. 11 Catalana, W. J ., Potvin, C. and Chretien, P. B.: T lymphocytes in bladder and prostatic cancer patients. J. Urol., 112: 378, 1974.
LYMPHOCYTE STIMULATION IN UROLOGIC CANCER PATIENTS
ing. An increased proliferative response to PHA was reported by McCredie and associates, 12 while Stjemsward 13 and Campbell 1 4 and their associates found lymphocyte reactivity to be diminished in previously irradiated patients. In a previous study we found that radiation therapy for urologic cancer produces a reduction in the percentage of T lymphocytes in the peripheral blood and it is principally the T cell population which responds to PHA. 1 ' Thus, it appears that the impaired lymphocyte reactivity observed in irradiated patients may be caused in part by the adverse effects of irradiation on circulating lymphocytes. The existence of serum factors which have an inhibitory effect on the proliferative response of lymphocytes has been demonstrated previously by a number of investigators. ,-s In our study only sera from patients with metastatic prostatic carcinoma had a statistically significant inhibitory effect on the response of normal lymphocytes to PHA, although sera from patients with all varieties of advanced urologic cancer were generally more inhibitory than sera from their counterparts with early cancer. The low over-all proportion of bladder cancer patients manifesting serum inhibitory effects (2 of 22) militates against serum factors being the principal mechanism underlying impaired lymphocyte reactivity in these patients. However, among prostatic cancer patients and perhaps renal cell carcinoma patients as well, 2 µv,,~une mechanisms exist whereby lymphocyte might be impaired: 1) an intrinsic defect or 2) serummediated inhibition. Previous studies have shown that certain serum alpha 2 globulins have the to inhibit PHA-induced lymphocyte proliferation. 16 These alpha 2 globulins are present in normal sera but are increased in human and experimental cancer. 17 • 18 Further, a significant correlation between serum inhibitory effects and serum alpha globulin levels have been demonstrated in patients 12 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, 1972. 13 Stjemsward, J .. Jondal, M., Vanky, F., Wigzell, H. and Sealy, R.: Lymphopenia and change in distribution of human B and T lymphocytes in peripheral blood induced by irradiation for mammary carcinoma. Lancet, l: 1352, 1972. 1 ' Campbell, A. C., Hersey, P., Harding, B., Hollingsworth, P. M., Skinner, J. and Maclennan, I. C. M.: Effects of anti-cancer agents on immunologic status. Brit. J. Cancer, suppl. l, 28: 254, 1973. 15 Catalona, W. J., Potvin, C. and Chretien, P. B.: Effect of radiation therapy for urologic cancer on circulating thymus-derived lymphocytes. J. Urol., 112: 261, 1974. 16 Cooperband, S. R., Davis, R. C., Schmid, K. and Mannick, J. A.: Competitive blockade of lymphocyte stimulation by a serum immunoregulatory alpha globulin (IRA). Transplant. Proc., 1: 516, 1969. 17 Bogen, A. E., Brown, M. D., Neville, G. A. and Gray, M.: Primary (methylcholanthrene-induced) fibrosarcomas and glycoprotein synthesis. Cancer Res., 27: 230, 1967. 18 Macbeth, R. A. and Bekesi, J. G.: Plasma glycoproteins in malignant disease. Arch. Surg., 88: 633, 1964.
with colon cancer. 19 In this connection it is relevant that Ablin and associates reported increased lating levels of alpha 2 globulin in prostatic carcinoma which were more those with advanced tumors. 20 cently reported increased ribonuclease activity in the sera of urologic cancer Although ribonuclease is known to mrmosuppressive properties we found no tion between S-R Nase and serummediated inhibition of lymphocyte PHA. Another variety of serum inhibitory factors been demonstrated which is capable of specific lymphocyte-mediated tumor eel! destruction.2 2 These are believed to be antigen· antibody complexes and may exert their directly on lymphocytes. 22 Thus, while it is possible that the serum inhibi tory effects demonstrated in our may mediated by one of the factors previously discussed the available data do not further tion at this time. SUMMARY
The proliferative response of phytohemagglutinin stimulation and cancer sera on normal lymphocyte determined in 53 urologic cancer tested in non-cancer sera with early bladder cancer renal cell carcinoma but was impaired in their counterparts with advanced tumors. contrast, was impaired in localized those with impaired reactivity. Statistically significant serum-mediated inhibition of normal reactivity was demonstrated among patients with metastatic pros tatic carcinoma, although a strong trend observed in with metastatic renal noma. The results suggest that PHA-induced cyte stimulation can be impaired mechanisms in different tumor types: 1) an intrinsic cellular defect and 2) a serum-mediated tion. Ms.
Brown
technical assistance.
19 Hsu, C. C. and LoGerfo, P.: Correlation between serum alpha-globulin and plasma inhibitory effect PHA-stimulated lymphocytes in colon cancer patients. Proc. Soc. Exp. Biol. Med., rn9: 575, 1972. 211 Ablin, R. J., Gonder, M. J. and Soanes, W. Serum proteins in prostatic cancer. I. tween clinical stage and level. J. 21 Catalona, W. J ., Chretien, P. B., and Tarpley, J. L.: Serum ribonuclease in cancer: relation to host immunocompetence. Urology, 577, 1973. 22 Sjogren, H. 0., Hellstrom, I.. Bansal, S. Hellstrom, K. E.: Suggestive evidence that antibodies" of tumor-bearing individuals may be gen-antibody complexes. Proc. Nat. Acad. Sci., 68: 1372, 1971.
J OURKAL
Vol. 112, Sepl em hc·r
OF UROLOGY
Copyright © 1974 by The Williams & Wilkins Co.
Printed in
LYMPHOCYTE STIMULATION IN UROLOGIC CANCER PATIENTS WILLIAM J. CATALONA, JOHN L. TARPLEY, PAUL B. CHRETIEN
AND
J. ROLAND CASTLE
From the James Buchanan Bradv Urolo{!ical Institute, The Johns Hopkins Hospital, Baltimore and Tumor Immunologv Section, Su.r{ierv Branch, National Cancer Institute, National Institutes of Health, Bethesda, MCTrvwud
Current concepts of tumor immunology suggest that the ability of host defenses to restrict tumor is vested in the cellular immune mechamediated by (T) lymphocytes. Therefore, in order for an immunologic response to be effective against cancer, T lymphocyte function must be intact. The proliferative response of lymphocytes when cultured in the presence of phytohemagglutinin (PHA) has been perhaps the most widely used in vitro assay for cellular immune competence in human disease states 1). Although lymphocyte activation PHA and does not constitute a true response, it simulates the immunoproliferative response which occurs in vivo upon antigenic stimulation. In this context it is -·,,,,.,,.,-,"' that lymphocytes from with cellular immunity also manifest reduced 1J1•uu1na responses to PHA. 1 it seems reasonable to equate the in vitro lymphocyte response to PHA with the behavior of lymphocytes in vivo. The proliferative response of to PHA has been studied in cancer patients several investigators but the results reported have been somewhat ,_, Moreover, recognition of serum factors which have the capacity to inhibit PHA-induced lymphocyte blastogenesis has beclouded the conclusions drawn from studies in which lymphocytes were cultured in the presown) serum. •- 7 In (the ence of such instances it is to determine whether the poverty of reactivity observed is caused intrinsic lymAccepted for publication March 8, 1974. Read at annual meeting of American Urological Association, St. Louis, Missouri, May 19-23, 1974. 1 Hersh, E. M. and Oppenheim, J. J.: Impaired in vitro lymphocyte transformation in Hodgkin's disease. New Engl. J. Med., 273: 1006, 1965. 'Robinson, M. R. G., Nakhla, L. S. and Whitaker, R. H.: A new concept in the management of carcinoma of the prostate. Brit. J. Ural., 43: 728, 1971. 3 Robinson, M. R. G., Nakhla, L. S. and Whitaker, R. H.: Lymphocyte transformation in carcinoma of the prostate. Brit. J. Ural., 43: 480, 1971. 'McLaughlin, A. P., III, Kessler, W. 0., Triman, K. and Gittes, R. F.: Immunologic competence in patients with urologic cancer. J. Urol., Hl: 233, 1974. 'McLaughlin, A. P., III, Kessler, W. 0., Triman, K. and Gittes, R. F.: Immune lymphocyte competence in urologic cancer: plasma inhibition of lymphocyte transformation. Surg. Forum, 24: 112, 1973. 6 Astaldi, G., Costa, G. and Air6, R.: Phytohemagglutinin in leukemi.a. Lancet, l: 1394, 1965. 7 Silk, M.: Effect of plasma from patients with carcinoma on in vitro lymphocyte transformation. Cancer, 20: 2088, 1967.
phocyte defect or by serum
effect of such serum on the lymphocytes. In our study the in vitro blood to PHA effect of patient sera on normal were determined in 53 tients. Correlations are responsiveness, tumor the effects of PATIENTS AND METHODS
Patients and controls. Blood from urologic cancer patients and 44 with comparable age distribution intrinsic lymphocyte to while from 67 urologic cancer patients and 18 controls were tested for serum effect on the of normal lymphocytes to PHA. In all instances cancer patients and controls were tested concur rently. Patients were either nre>r.n,o,,o 5 days postoperative. Nine previous radiation ing the distributions of tumor types and stages shown in the table and figures 2 and 3. Culture system. An AB positive serum prepared from healthy volunteers, was used constant reference for cellular and serum The pool was heat inactivated at 56C fm 30 minutes, passed through a 0.45 micron filter and stored in smali aliquots in gen. Heparinized whole blood was per cent methylcellulose after iron for 45 minutes at 37C in a Differential phagocytosis of iron resulted in cell suspensions containing 95 to 99 per cent mononuclear cells with 99 per cent thus obtained were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium counted in a Coulter counter. Cultures containing 5.0 times 10 5 mononuclear cells per ml. of 90 per cent RPM! 1640 and HJ cent AB pool serum were
374
CATALONA AND ASSOCIATES
FIG. 1. Appearance under light microscopy oflymphocytes undergoing blast transformation in response to PHA stimulation. Reduced from x450.
?HA-induced lymphocyte reactivity and serum effect on lymphocyte reactivity in 9 previously irradiated urologic cancer patients Tumor Type
Bladder Bladder Bladder Bladder Bladder Kidney Prostate Prostate Prostate Mean
Standard error
Tumor Stage
P2 P3 P3 P4 P4 Metastatic IV IV IV
PHAInduced Lympho-
Serum Effect on
cyte*
LRit
232 140 161 39 75 24 46 62 70
0.957 0.990 1.056 1.254 0.968 0.726 0.980 0.989 1.126
94.3 22.9
1.005 0.047
* Tritiated thymidine incorporation (disintegrations per minute times 10' per culture). t LRI-lymphocyte reactivity index
the cultures had incubated for 54 hours. After a labeling period of 3 hours the cultures were frozen in liquid nitrogen. The nucleoprotein was precipitated with cold 5 per cent trichloroacetic acid (TCA) and the precipitate was washed 3 times with TCA, a fourth time with saline, and was solubilized in 0.5 ml. soluenet. The soluene was neutralized with 20 lambda of glacial acetic acid and the nucleoprotein was transferred to scintillation counting vials by 3 washes with 5 ml. portions of toluene-liquiflor:j: scintillation fluid. The incorporation of 3 H Tdr by the lymphocytes was quantitated in a model 3303 Packard Tri-Carb scintillation counter and the absolute activity was expressed in disintegrations per minute (dpm). Expression of results. The intrinsic lymphocyte reactivity to PHA was expressed as the peak numerical increment in dpm above control levels (unstimulated cultures) regardless of the PHA concentration at which it occurred. The rationale for this form of data expression derives from the fact that it yielded a coefficient of variation (standard deviation expressed as a percentage of the mean) in the control population of 25.1 per cent. The same data expressed as an activity ratio (stimulated "H Tdr incorporation divided by unstimulated 3H Tdr incorporation) yielded a coefficient of variation of 68.3 per cent in the controls. Serum effect on lymphocyte reactivity. Normal donor lymphocytes were cultured in test serum (cancer patient or healthy control) and in AB positive pool serum, respectively. The 3H Tdr incorporation of this lymphocyte population in the test serum was compared to the 3 H Tdr incorporation of the same lymphocyte population in the AB positive pool serum. The result was expressed as the ratio: lymphocyte reactivity index (LRI) equals peak 3 H Tdr incorporation in test serum (dpm)/ peak 3H Tdr incorporation in AB positive pool serum (dpm). Statistical methods. Differences observed in intrinsic lymphocyte reactivity and serum effect on lymphocyte reactivity between healthy controls and urologic cancer patients were evaluated by Student's t test. RESULTS
( 'H Tdr in patient serum ) . 'H Tdr in AB+ pool
tohemagglutinin-P* reconstituted in saline and used in dilutions of 25, 12.5, 6.25, 3.125 mcg. protein per ml. culture was added to the cultures and the cultures were incubated at 37C in a humidified 95 per cent air-5 per cent carbon dioxide atmosphere. Cultures with each PHA dose and control cultures without PHA were always prepared in triplicate. Tritiated thymidine ( 3H Tdr) (300 mCi. perm. mole, 30 micro Ci. per ml. culture) was added after
* Difeo Laboratories, Detroit, Michigan.
Intrinsic lymphocyte reactivity of urologic cancer patients (fig. 2). The mean lymphocyte reactivity ("H Tdr incorporation per culture) of 10 patients with stage Pl or P2 bladder tumors was 162.9 times 10 3 dpm,§ which did not differ significantly from the mean lymphocyte reactivity of the 44 healthy controls (157.6 dpm, p greater than 0.20). However, the mean lymphocyte reactivity of 10 patients with stages P3 and P4 bladder tumors was significantly impaired relative to controls (100.2 dpm, p less than .001). The difference be-
t Packard Instrument Co., Inc. :j: New England Nuclear, Boston, Massachusetts. § All lymphocyte reactivity values referred to hereinafter are xl0 3 •
LYMPHOCYTE STIMULATION IN UROLOGIC CANCER PATIENTS PHA-STIMULATED LYMPHOCYTE BLASTOGENESIS IN UROLOGIC CANCER PATIENTS I Peak Stimulation - Control I
220
~ Meon±SE
200
160
'=' X
:,e
/40
~
CONTROLS n°44
CL 0
I
n,3
BLADDER P1a P1 nc/0
120
I
100
II
PROSTATE PROSTATE* ]][ HIT n°4 n°19 BLADDER* P3 8 P4 nc 10
80
I I
KIDNEY
I localized I
60
TESTIS n°2
I I
KIDNEY
Imetastatic I
IRRADIATED PATIENTS" n,9
n°5
* P<0.01
FIG. 2. Comparison of PHA-induced tritiated thymidine incorporation by peripheral blood lymphocytes from 53 urologic cancer patients and 44 age-matched healthy controls. Differences were evaluated by Student's t test.
EFFECT OF UROLOGIC CANCER SERA ON THE REACTIVITY OF NORMAL LYMPHOCYTES
~
1.20
X
w
~ 1.10 >>-
>
:s"' ~
t;
I I I
CONTROLS nc/8
e=
TESTIS (local) nc 2
1.00
0 I
CL
PROSTATE I-ill n°6
"I '" " ! I
PROSTATE* Ill n' 27
"'~ 0.90
I
P1 8 P1 n' 10
BLADDER P3 fl P4 n°9
TESTIS
I metoslotic) n°4
KIDNEY (localized) n°3
~ Meon±SE KIDNEY
I metastatic I
0.80
* P <0.01
n°5
FIG. 3. Comparison of effects of sera from 67 urologic cancer patients and 18 healthy controls on PHA-induced tritiated thymidine incorporation of allogeneic lymphocytes. Differences were evaluated by Student's t test.
tween patients with advanced bladder carcinoma and controls was also significant (97.8 dpm, p less than .02) when 4 irradiated patients were excluded (See table). Lymphocyte reactivity was impaired in both patients with localized prostatic carcinoma (stages I to HI-121.5 dpm, p less than .10) and those with metastases present (stage IV -113 .9 dpm, p less than .01). This difference also remained significant (124.1 dpm, p less than .02) when 3 irradiated patients were excluded (See table). Patients with localized renal cell carcinoma manifested normal intrinsic lymphocyte reactivity (184.0 dpm, p greater than .20), while those with metastases had lower lymphocyte reactivity (112.0
dpm, p less than 0.10). However, the difference between controls and cancer patients in the latter group was diminished (134.0 dpm, p less than .20) when the one previously irradiated patient was excluded (See table). Only 2 patients with germinal testis tumors were tested. One with a localized tumor had an assay score of 184 dpm, while the other patient who had metastatic disease had an assay score of 144 Effect of radiation therapy (table and fig. 2). group, previously irradiated patients manifested markedly impaired intrinsic lymphocyte (94.3 dpm, p less than .01). Serum effect on lymphocyte reactivity (figs. 3 and 4). The effects of sera from 18 healthy, age
376
CATALONA AND ASSOCIATES
matched controls on the reactivity of allogeneic (another person's) lymphocytes were compared to the effects of cancer sera on the same lymphocyte population, using the AB positive serum pool as a constant reference. Results were expressed as a lymphocyte reactivity index (LRI). The mean LRI for the control population was 1.100. Only patients with metastatic prostatic carcinoma had a mean LRI which was significantly lower than that of controls (LRI = 0.979, p less than .02). However, in all patient groups studied, a lower mean LRI was observed among patients with more advanced disease (fig. 3). Serum-mediated inhibition of lymphocyte reactivity was arbitrarily considered to be significant in patients with LRI's of less than 0.950, since nearly 90 per cent of healthy controls tested had LRI's above this value. Using this arbitrary classification serum inhibition was observed in 9 per cent of bladder cancer patients, 29 per cent of prostatic cancer patients, 33 per cent of renal cell carcinoma patients and none of the patients tested with germinal testis tumors (fig. 4). DISCUSSION
The significance of the immunoproliferative response derives from the fact that by this process a population of immunologically committed lymphocytes is formed. An understanding of the mechanism underlying the apparent failure of this fundamental response in urologic cancer patients may afford important insights into the biology of urologic cancer. Robinson and associates studied prostatic carcinoma patients and reported that lymphocyte responsiveness to PHA was impaired only in patients with metastases present. 3 McLaughlin and associates demonstrated impaired lymphocyte reactivity in patients with a variety of urologic tumors but were unable to demonstrate a correlation between PHA responsiveness and tumor stage. 4 In both studies lymphocytes were cultured in the presence of autologous serum. Thus, it was not possible to determine whether the impairment of lymphocyte responsiveness was caused by an intrinsic cellular abnormality or by the presence of serum inhibitors. Addressing themselves to this issue in a subsePERCENTAGE OF PATIENTS WITH SERUM INHIBITION
CONTROLS N, I 8
11%
PROSTATE N,34 Kl ONEY
N,9
BLADDER
N,22
TESTIS
N,7
29% 33%
9% 0%
FIG. 4. Comparison of respective proportions of patients having significant serum inhibition of lymphocyte reactivity (lymphocyte reactivity index less than 0.950) in patient groups studied.
quent communication, McLaughlin and associates reported that depressed lymphocyte reactivity to PHA in 19 cancer patients tested could be restored to normal by culturing the cells in the presence of non-cancer plasma. 5 They suggested, therefore, that the impairment of lymphocyte reactivity in urologic cancer patients was caused solely by serum-mediated inhibition and not an intrinsic cellular defect. A relevant feature of our study is the demonstration of an intrinsic lymphocyte defect in a significant proportion of urologic cancer patients. In our assay system cancer lymphocytes, washed 3 times and cultured in the presence of pooled AB positive serum from healthy volunteers, manifested significantly impaired reactivity to PHA. The relationship between lymphocyte reactivity to PHA and tumor stage also contrasts with previous reports. 3 • 4 We found lymphocyte reactivity to be normal in patients with early bladder cancer but significantly impaired among patients with advanced bladder cancer. A similar relationship was observed in patients with renal cell carcinoma, although statistical significance was not achieved. In contrast, lymphocyte reactivity was substantially impaired in patients with localized prostatic carcinoma as well as those with advanced tumors (fig. 2). In general, the patterns of immunologic reactivity found in this study are in concert with the results of parallel studies from our laboratory using dinitrochlorobenzene (DNCB) contact sensitization •·10 and the T rosette assay 11 to quantify cellular immune competence in urologic cancer patients. Therefore, it seems reasonable to infer that in our patient population there is a significant intrinsic deficiency of T lymphocyte function. This defect has been observed infrequently in patients with early bladder or renal cell carcinoma but has been found in a significant proportion of patients with clinically localized prostatic carcinoma. Too few patients with germinal testis tumors have been studied to warrant comment. The proliferative response of lymphocytes to PHA was noted to be impaired in a majority of patients who had received radiation therapy. However, most of these patients also had advanced tumors and the small number of irradiated patients in each group precluded meaningful statistical analysis. Reports on the effects of radiation therapy on FHA-induced lymphocyte reactivity are conflict' Catalana, W. J., Sample, W. F. and Chretien, P. B.: Lymphocyte reactivity in cancer patients: correlation with tumor histology and clinical stage. Cancer, 31: 65, 1973. 'Catalana, W. J., Chretien, P. B. and Trahan, E. E.: Abnormalities of cell-mediated immunocompetence in genitourinary cancer. J. Urol., lll: 229, 1974. 1 °Catalona, W. J. and Chretien, P. B.: Correlation among host immunocompetence and tumor stage, tumor grade and vascular permeation in transitional carcinoma. J. Urol., 110: 526, 1973. 11 Catalana, W. J ., Potvin, C. and Chretien, P. B.: T lymphocytes in bladder and prostatic cancer patients. J. Urol., 112: 378, 1974.
LYMPHOCYTE STIMULATION IN UROLOGIC CANCER PATIENTS
ing. An increased proliferative response to PHA was reported by McCredie and associates, 12 while Stjemsward 13 and Campbell 1 4 and their associates found lymphocyte reactivity to be diminished in previously irradiated patients. In a previous study we found that radiation therapy for urologic cancer produces a reduction in the percentage of T lymphocytes in the peripheral blood and it is principally the T cell population which responds to PHA. 1 ' Thus, it appears that the impaired lymphocyte reactivity observed in irradiated patients may be caused in part by the adverse effects of irradiation on circulating lymphocytes. The existence of serum factors which have an inhibitory effect on the proliferative response of lymphocytes has been demonstrated previously by a number of investigators. ,-s In our study only sera from patients with metastatic prostatic carcinoma had a statistically significant inhibitory effect on the response of normal lymphocytes to PHA, although sera from patients with all varieties of advanced urologic cancer were generally more inhibitory than sera from their counterparts with early cancer. The low over-all proportion of bladder cancer patients manifesting serum inhibitory effects (2 of 22) militates against serum factors being the principal mechanism underlying impaired lymphocyte reactivity in these patients. However, among prostatic cancer patients and perhaps renal cell carcinoma patients as well, 2 µv,,~une mechanisms exist whereby lymphocyte might be impaired: 1) an intrinsic defect or 2) serummediated inhibition. Previous studies have shown that certain serum alpha 2 globulins have the to inhibit PHA-induced lymphocyte proliferation. 16 These alpha 2 globulins are present in normal sera but are increased in human and experimental cancer. 17 • 18 Further, a significant correlation between serum inhibitory effects and serum alpha globulin levels have been demonstrated in patients 12 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, 1972. 13 Stjemsward, J .. Jondal, M., Vanky, F., Wigzell, H. and Sealy, R.: Lymphopenia and change in distribution of human B and T lymphocytes in peripheral blood induced by irradiation for mammary carcinoma. Lancet, l: 1352, 1972. 1 ' Campbell, A. C., Hersey, P., Harding, B., Hollingsworth, P. M., Skinner, J. and Maclennan, I. C. M.: Effects of anti-cancer agents on immunologic status. Brit. J. Cancer, suppl. l, 28: 254, 1973. 15 Catalona, W. J., Potvin, C. and Chretien, P. B.: Effect of radiation therapy for urologic cancer on circulating thymus-derived lymphocytes. J. Urol., 112: 261, 1974. 16 Cooperband, S. R., Davis, R. C., Schmid, K. and Mannick, J. A.: Competitive blockade of lymphocyte stimulation by a serum immunoregulatory alpha globulin (IRA). Transplant. Proc., 1: 516, 1969. 17 Bogen, A. E., Brown, M. D., Neville, G. A. and Gray, M.: Primary (methylcholanthrene-induced) fibrosarcomas and glycoprotein synthesis. Cancer Res., 27: 230, 1967. 18 Macbeth, R. A. and Bekesi, J. G.: Plasma glycoproteins in malignant disease. Arch. Surg., 88: 633, 1964.
with colon cancer. 19 In this connection it is relevant that Ablin and associates reported increased lating levels of alpha 2 globulin in prostatic carcinoma which were more those with advanced tumors. 20 cently reported increased ribonuclease activity in the sera of urologic cancer Although ribonuclease is known to mrmosuppressive properties we found no tion between S-R Nase and serummediated inhibition of lymphocyte PHA. Another variety of serum inhibitory factors been demonstrated which is capable of specific lymphocyte-mediated tumor eel! destruction.2 2 These are believed to be antigen· antibody complexes and may exert their directly on lymphocytes. 22 Thus, while it is possible that the serum inhibi tory effects demonstrated in our may mediated by one of the factors previously discussed the available data do not further tion at this time. SUMMARY
The proliferative response of phytohemagglutinin stimulation and cancer sera on normal lymphocyte determined in 53 urologic cancer tested in non-cancer sera with early bladder cancer renal cell carcinoma but was impaired in their counterparts with advanced tumors. contrast, was impaired in localized those with impaired reactivity. Statistically significant serum-mediated inhibition of normal reactivity was demonstrated among patients with metastatic pros tatic carcinoma, although a strong trend observed in with metastatic renal noma. The results suggest that PHA-induced cyte stimulation can be impaired mechanisms in different tumor types: 1) an intrinsic cellular defect and 2) a serum-mediated tion. Ms.
Brown
technical assistance.
19 Hsu, C. C. and LoGerfo, P.: Correlation between serum alpha-globulin and plasma inhibitory effect PHA-stimulated lymphocytes in colon cancer patients. Proc. Soc. Exp. Biol. Med., rn9: 575, 1972. 211 Ablin, R. J., Gonder, M. J. and Soanes, W. Serum proteins in prostatic cancer. I. tween clinical stage and level. J. 21 Catalona, W. J ., Chretien, P. B., and Tarpley, J. L.: Serum ribonuclease in cancer: relation to host immunocompetence. Urology, 577, 1973. 22 Sjogren, H. 0., Hellstrom, I.. Bansal, S. Hellstrom, K. E.: Suggestive evidence that antibodies" of tumor-bearing individuals may be gen-antibody complexes. Proc. Nat. Acad. Sci., 68: 1372, 1971.