Phytohemagglutinin-Stimulated Immune Assay Marvin S.
Kaplan, MD;
also studied. The mean mitotic index of PHA-stimulated versus nonstimulated lymphocytes from all tumor patients was 29.2; from patients with localized tumors, 23.0; from patients with metastatic tumors, 26.0; and from controls, 29.4. In the presence of sera from tumor patients, the mitotic index in all tumor lymphocytes was 47.1; from localized tumor patients, 53.7; from metastatic tumor patients, 43.8; and from control lymphocytes, 47.4. No substantial difference in mitotic index was detected in normal compared to tumor patient lymphocytes with or without normal or tumor serum. was
Studi es specific lymphocyte-mediated immunity
of the immune response in cancer patients have tumor antigens and shown the presence of to these antigens. Pro¬ gression of the malignancy, however, indicates that there may be a defect in the immune response related to the failure of lymphocyte reactivity or interference with this cellular reactivity by substances in the sera. These obser¬ vations have been reported predominantly in patients with melanoma, neuroblastoma, and sarcoma.1 Accepted
publication Feb 24, 1975. Department of Surgery, University of California at Irvine (Drs. Kaplan and Lundak and Mr. Kummerfeld), and the Veterans Administration Hospital, Long Beach, Calif (Drs. Kaplan and Lundak, Ms. Mino, and Mr. Kummerfeld). Read before the annual meeting of the Southern California Chapter of the American College of Surgeons, Santa Barbara, Calif, Jan 17, 1975. Reprint requests to Department of Surgery, 5901 E Seventh St, Long Beach, CA 90801 (Dr. Kaplan). From the
for
in Colorectal Carcinoma Patients
Frances O. Mino, MS; Kenneth B.
Tritiated thymidine incorporation by peripheral human lymphocytes stimulated with phytohemagglutinin (PHA) was investigated in 18 colorectal carcinoma patients (seven with localized tumor and 11 with metastatic tumors). The effect of sera obtained from these patients on lymphocytes from control patients
Response
Kummerfeld; Robert L. Lundak, PhD
There is increased interest in lymphocyte reactivity to nonspecific mitogens in patients with colorectal cancer. This is due to the difficulty in utilizing specific assays be¬ cause of problems involving antigen isolation or in vitro culture of colorectal cancer cells or both. Several studies of phytohemagglutinin (PHA) stimu¬ lated reactivity of lymphocytes from patients with various cancers (including cancer of the gastrointestinal tract) have reported conflicting results concerning the presence of a nonspecific defect in the lymphocytes and/or the pres¬ ence of an inhibiting factor in the sera of tumor patients to tritiated thymidine incorporation in vitro. We have studied the response of lymphocytes from a group of patients with and without colorectal cancer to PHA stimulation in the presence or absence of control sera or tumor sera as assayed by tritiated thymidine in¬ corporation to resolve these conflicting reports. PATIENTS AND METHODS Patient Selection
Eighteen colorectal cancer patients (age 47 to 86 years, 11 with metastatic and seven with localized cancer) and 18 controls (13 pa¬ tients, age 39 to 61 years, with nonneoplastic disease and five hos¬ pital staff members, age 21 to 48 years) were studied. Informed consent was obtained from all patients after the nature of the pro¬ cedure had been fully explained. In Vitro
Lymphocyte Culture obtained by venipuncture;
10 ml was Peripheral blood was clotted and 30 ml was heparinized. Lymphocyte suspensions were prepared from the heparinized blood by the Ficoll-Hypaque gradient method described by Wybran et al.' All cells were washed twice with phosphate buffered saline and cultured in trip¬ licate in Roswell Park Memorial Institute (RPMI) medium 1640
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/28/2015
Peripheral
30 ml
vs
Patients
Blood Obtained From
Sera Collected
Ficoll-Hypaque Separation
-,
1
90%
Lymphocytes Culture (2 ml) 1-3
+
RPMI 1640
106 Lymphocytes
«
From Control or Tumor Patients
+
Penicillin/Streptomycin/Levoglutamide
Control or Tumor Sera
Fetal Calf Sera
PHA
68-hr Incubation at 37 + 1 Microcurie
C in Mixture of 5%
4 hr
C02
Lymphocytes
Tritiated Thymidine
Harvested on Glass Filters
and 95% Air
Liquid -Scintillation Fluid for Counting
Experimental plan.
supplemented with 1% levoglutamide (Gibco) 10,000 units penicil¬ and 10,000/^g streptomycin sulfate (Gibco) per 100 ml media,
lin,
and 10% fetal calf sera (Gibco) at a concentration of 1 to 3 x 10" cells per milliliter in 16 125-mm capped plastic tubes (Falcon). Tumor or normal sera (0.2 ml) was added to the appropriate cul¬ tures at time zero. After four hours, 0.025 ml of PHA-P (Difco) per milliliter of media was added to each culture. Cells were incu¬ bated for three days in a mixture of 5% carbon dioxide and 95% air at 37 C using airtight plexiglass boxes. The cultures were pulsed with microcurie tritiated thymidine (Amersham-Searle) for four hours and harvested by vacuum fil¬ tration on glass fiber filters (Reeves Angel 934 AH) with precipi¬ tation by cold trichloroacetic acid. Tritiated thymidine incorpora¬ tion was measured by liquid scintillation (Figure).
Study of Inhibitory Serum Factors The suppressive activity of sera from colorectal cancer patients was investigated for its ability to inhibit PHA-induced DNA syn¬ thesis by lymphocytes from colorectal and control patients as com¬ to the effect of control sera thesis. Normal or tumor sera (0.2 ml) 2-ml cultures at time zero.
pared
Statistical
were
compared.
Lymphocyte Reactivity
10 ml Clotted
Heparinized
metastatic tumors
on
was
PHA-induced DNA syn¬ added to the appropriate
Analysis
The mitotic index of each culture was calculated by dividing the average counts per minute of the PHA-stimulated lymphocytes by the average counts per minute of the nonstimulated lymphocytes. The mean mitotic index of each group studied was calculated and compared using the Student t test.
RESULTS The range of mitotic indices of "tumor" versus "control" lymphocytes and lymphocytes from patients with localized
The mitotic indices of PHA-stimulated lymphocytes from 18 control patients varied between 0.7 and 116 (mean, 29.2). The comparative mitotic indices of PHAstimulated lymphocytes from 18 patients with colorectal carcinoma varied between 2.0 and 60 (mean, 24.9). Statis¬ tical comparison of these values yielded a value of >.30. The mean mitotic index for lymphocytes from patients with localized carcinoma cultured was 23.1 (range, 4 to 52) in media. Lymphocytes from patients with metastatic car¬ cinoma showed a mean mitotic index of 26.0 (range, 4 to 52) in media. Comparison of these mitotic indices yielded a value of >.35. Effect of Adding Sera Obtained From Controls or Patients With Colorectal Carcinoma
With the addition of autologous sera, the mitotic index for control lymphocytes ranged between 1 and 111 (mean, 39.6). The addition of sera obtained from the human con¬ trols to the cultured lymphocytes from patients with can¬ cer resulted in the varying of the mitotic index between 9 and 155 (mean, 48.0). Comparisons of these mitotic indices yielded a value of >.25. The lymphocytes from patients with localized tumor showed a mean mitotic index of 48.4 (range, 9 to 155). Lymphocytes from patients with meta¬ static cancer showed a mean mitotic index of 47.7 (range, 11 to 148). Comparison of these mitotic indices yielded a value of >.50. With the addition of sera from cancer patients, the mitotic index for control lymphocytes ranged between 1 and 206 (mean, 47.4). The addition of this sera to the cul¬ tured lymphocytes from colorectal cancer patients re¬ sulted in a mitotic index varying between 2 and 104 (mean, 47.1). Comparison of these mitotic indices yielded a value of >.50. The lymphocytes from patients with local¬ ized tumor showed a mean mitotic index of 53.6 (range, 2 to 88). Similarly treated lymphocytes from patients with metastatic cancer showed a mean mitotic index of 43.8 (range, 5 to 104). Comparison of these mitotic indices yielded a value of >.25. COMMENT
The results of 19 other studies of
lymphocyte inhibition
to PHA stimulation and/or the presence of
inhibition of PHA-stimulated rized in the Table.'21
lymphocytes
serum
factor
are summa¬
Lymphocyte Reactivity Six studies support the finding of a lymphocyte defect in cancer patients. Two positive studies were based on blast transformation. Fisher et al (1972)," using tritiated thymidine incorporation in 76 patients, showed no statis¬ tical difference nor did Vetto et al (1974, 26 patients).'Other well-controlled studies by Lejtenyi et al (1971, 21 patients)" and Oishi et al (1973, 46 patients)17 showed no
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/28/2015
Studies
by Other Investigators Type of
Author
Whittaker et al (1971)3
Sample
et al (1971) Oishl et al (1973) ' Suciu-Foca et al (1973)4
Golubetal (1969)6
No. of Patients
Cancer Studied Studied Immune Defects in Patients With Various Carcinomas Breast 30 Lymphocyte transformation Tritiated thymidine Miscellaneous 120 Tritiated thymidine Miscellaneous 46 Miscellaneous 60 Lymphocyte transformation Tritiated thymidine Lung, gastrointestinal tract, miscellaneous
Tritiated thymidine
Mekori et al (1974)'
Tritiated thymidine
Gastrointestinal tract, miscellaneous Gastrointestinal tract, miscellaneous
Nelson (1969)' Fisher et al (1972)»
Tritiated thymidine
Lejtenyi
et al (1971)'5 Budda et al (1974)5 Vetro et al (1974)«
Tritiated thymidine Tritiated thymidine Tritiated thymidine
Steward
Tritiated thymidine
pulmonary
et
al (1974)7
Chretian and Ketcham
(1973)" Robinson et al (1974)J1
Twomey
et al
(1974)8
Tritiated thymidine
Tritiated thymidine Tritiated thymidine
Gastrointestinal tract, miscellaneous Gastrointestinal tract, breast —; not
defect. Only Buda et al (1974) | showed lymphocyte inhibi¬ tion in 85 patients. Chretien and Ketcham (1973)'s showed a defect for patients with squamous cell carcinoma of the head and neck but not for adenocarcinoma patients. Mekori et al (1974, 14 patients)" showed a defect in patients with metastatic cancer only. In our study, although the mean value of normal lym¬ phocyte reactivity (29.1) was slightly higher than the reac¬ tivity of tumor patients (24.9), this was not a significant difference (P>.30). Our wide range of normal values coin¬ cides with that of Mclntyre and Cole-2 for normal lympho¬ cyte reactivity, although it differs with the controls of other authors in studies of PHA responsiveness in tumor patients.512 There was a rather large variation in both normal and in tumor patients' lymphocytes as is reflected by the standard deviation of ± 17.3 for tumor patients. Based on our data, we conclude that the lymphocytes from patients with colorectal carcinoma are not inhibited in their response to a nonspecific mitogen, PHA.
Lymphocytes'
Responsiveness*
-for metastatic disease only
15
Gastrointestinal tract, blast Gastrointestinal tract Gastrointestinal tract Gastrointestinal tract, miscellaneous Gastrointestinal tract, blast Squamous cell carcinoma, adenocarcinoma
Effect believed present by author, +; effect believed absent,
Serum Factors Present Inhibiting
+
3 20 2 12
Immune Defects in Patients With Colorectal Carcinoma Blast transformation Gastrointestinal tract 20 Blast transformation 17 Gastrointestinal tract, miscellaneous 35 Blast transformation 7 Gastrointestinal tract, miscellaneous 13 Tritiated thymidine Gastrointestinal tract, 1
Silk (1967)2»
Inhibited to PHA Stimulation*
Assay
Gatti et al (1970)18
Manousos et al (1973)' Al-Sarraf et al (1971)
Lymphocytes
26 50 21
32 6 20 23 12 38 19 5
0
+ for squamous cell carcinoma for adenocarcinoma
+
—
15 10 9
investigated, 0. Presence of Serum Factors
Inhibiting Lymphocyte Response Eleven studies support the finding of a serum factor inhibiting lymphocyte response. Positive studies by Whittaker et al (1971, 30 patients with breast cancer)3 and Suciu-Foca et al (1973, 60 patients with miscellaneous can¬ cers)4 used lymphocyte transformation assays. Using tritiated thymidine incorporation, Nelson (1969, 16 patients)13 and Golub et al (1969, ten patients)" found no serum effect, but Buda et al (1974),5 Vetto et al (1974),12 and Steward et al (1974)7 did find such an effect. There are four studies of PHA-stimulated thymidine in¬ corporation by lymphocytes from patients with colorectal or other gastrointestinal tract cancer. Vetto et al found sera that blocked tritiated thymidine uptake in lympho¬ cytes from four of six patients with colorectal cancer, but only one example of his data is presented. Buda et al con¬ sidered blocking to be present if only a 33% decreased in-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/28/2015
corporation was found in autologous sera and if only a 15% decrease was found in control lymphocytes treated with tumor sera. Steward et al showed a 20% decrease in thymi¬ dine incorporation in ten of 23 colorectal patients but no blocking of control lymphocytes by sera from cancer patients. No statistically significant inhibitory effect was ob¬ served when sera from tumor patients was added to con¬ trol patient lymphocytes or tumor patient lymphocytes.
The absence of this serum inhibition in these studies may be due to technique or the actual absence of this effect. Additional experiments were done in which fetal calf sera was deleted in the tubes containing control human sera or tumor sera. Preliminary data indicate no signifi¬ cant inhibition. Based on our study, we conclude that thymidine uptake
of lymphocytes secondary to PHA stimulation is similar in lymphocytes from patients with colorectal cancers and controls and the uptake is not inhibited by substance pres¬ ent in tumor
sera.
Demonstration of an immune defect in the lymphocytes of patients with gastrointestinal tract carcinoma could de¬ pend on other immune indexes, such as the absence of rec¬ ognition of specific tumor antigens. Lejtenyi et al have shown that lymphocytes from patients with gastrointesti¬ nal tract cancer do not react to carcinoembryonic anti¬ gen.15 There may be inhibition of the effector limb of the immune response inhibiting specific cytotoxic function of the lymphocyte, perhaps by antigen-antibody complexes or by prevention of the release of lymphokines from effec¬ tor cells.
References 1. Pilch YH, Golub SH: Lymphocyte-mediated immune responses in neoplasia. Am J Clin Pathol 62:184-211, 1974. 2. Wybran J, Levin AS, Spitler LE, et al: Rosette-forming cells, immunologic deficiency diseases and transfer factor. N Engl J Med 288:710-713,
1973. 3. Whittaker MG, Rees K, Clark CG: Reduced lymphocyte transformation in breast cancer. Lancet 1:892-893, 1971. 4. Suciu-Foca N, Buda J, McManus J, et al: Impaired responsiveness of lymphocytes and serum-inhibitory factors in patients with cancer. Cancer Res 33:2373-2377, 1973. 5. Buda JA, Suciu-Foca N, Reemtsma KB: Impaired cell-mediated immunity in patients with cancer. Surg Forum 15:89-91, 1974. 6. Golub EK, Israsena T, Quatrale AC, et al: Effect of serum from cancer patients on homologous lymphocyte cultures. Cancer 23:306-308, 1969. 7. Steward AM, Kupchik HZ, Zamcheck N: Circulating carcinoembryonic antigen levels and serum suppression of phytohemagglutinin-stimulated lymphocyte DNA synthesis: An inverse correlation in the cancer patient. J Natl Cancer Inst 53:3-9, 1974. 8. Twomey PL, Catalona WJ, Chretian PB: Cellular immunity in cured cancer patients. Cancer 33:435-440, 1974. 9. Mekori T, Sher S, Robinson E: Suppression of the mitogenic response to phytohemagglutinin in malignant neoplasia: Correlation with clinical stage and therapy. J Natl Cancer Inst 52:9-12, 1974. 10. Al-Sarraf M, Sardesai S, Vaitkevicius VK: Effect of syngeneic and allogeneic plasma on lymphocytes from cancer patients, patients with non\x=req-\ neoplastic diseases and normal subjects. Cancer 27:1426-1432, 1971. 11. Manousos ON, Economidou J, Pathouli CH, et al: Disturbance of cell\x=req-\ mediated immunity in patients with carcinoma of colon and rectum. Gut 14:739-742, 1973. 12. Vetto RM, Burger DR, Lilley DP: Evaluation of immune status in tumor patients. Arch Surg 108:558-560, 1974.
13. Nelson HS: Delayed hypersensitivity in cancer patients: Cutaneous and in vitro lymphocyte response to specific antigens. J Natl Cancer Inst 42:765-770, 1969. 14. Fisher B, Saffer EA, Fisher ER: Studies concerning the regional lymph node in cancer III response of regional lymph node cells from breast and colon cancer patients to PHA stimulation. Cancer 30:1202-1215, 1972. 15. Lejtenyi MC, Freedman SO, Gold P: Response of lymphocytes from patients with gastrointestinal cancer to the carcinoembryonic antigen of the human digestive system. Cancer 28:115-120, 1971. 16. Sample WF, Gertner HR, Chretien PB: Inhibition of phytohemagglutinin-induced in vitro lymphocyte transformation by serum from patients with carcinoma. J Natl Cancer Inst 46:1291-1297, 1971. 17. Oishi N, Torikawa C, Ochiai H, et al: Relationship of C-reactive protein in autologous plasma of cancer patients and choline phosphate in lymphocyte responses to phytohemagglutinin. J Reticuloendothel Soc 14:242\x=req-\
249, 1973.
18. Gatti RA, Gurrioch DB, Good RS: Depressed PHA response in patients with non-lymphoid malignancies, in Harris JE (ed): Leucocyte Culture Conference: Fifth Proceedings. Ottawa, Academic Press Inc, 1970, pp 339-358. 19. Chretien PB, Ketcham AS: Quantitation of immunologic defects in cancer patients, in National Cancer Conference: Seventh Proceedings. Philadelphia, JB Lippincott Co, 1973, pp 187-192. 20. Silk M: Effect of plasma from patients with carcinoma of in vitro lymphocyte transformation. Cancer 20:2088-2089, 1967. 21. Robinson E, Sher S, Mekori T: Lymphocyte stimulation by phytohemagglutinin and tumor cells of malignant effusions. Cancer Res 34:1549\x=req-\ 1551, 1974. 22. McIntyre OR, Cole AF: Variation in the response of normal lymphocytes to PHA. Int Arch Allergy 35:105-118, 1969.
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/28/2015
Kaplan, MD;
also studied. The mean mitotic index of PHA-stimulated versus nonstimulated lymphocytes from all tumor patients was 29.2; from patients with localized tumors, 23.0; from patients with metastatic tumors, 26.0; and from controls, 29.4. In the presence of sera from tumor patients, the mitotic index in all tumor lymphocytes was 47.1; from localized tumor patients, 53.7; from metastatic tumor patients, 43.8; and from control lymphocytes, 47.4. No substantial difference in mitotic index was detected in normal compared to tumor patient lymphocytes with or without normal or tumor serum. was
Studi es specific lymphocyte-mediated immunity
of the immune response in cancer patients have tumor antigens and shown the presence of to these antigens. Pro¬ gression of the malignancy, however, indicates that there may be a defect in the immune response related to the failure of lymphocyte reactivity or interference with this cellular reactivity by substances in the sera. These obser¬ vations have been reported predominantly in patients with melanoma, neuroblastoma, and sarcoma.1 Accepted
publication Feb 24, 1975. Department of Surgery, University of California at Irvine (Drs. Kaplan and Lundak and Mr. Kummerfeld), and the Veterans Administration Hospital, Long Beach, Calif (Drs. Kaplan and Lundak, Ms. Mino, and Mr. Kummerfeld). Read before the annual meeting of the Southern California Chapter of the American College of Surgeons, Santa Barbara, Calif, Jan 17, 1975. Reprint requests to Department of Surgery, 5901 E Seventh St, Long Beach, CA 90801 (Dr. Kaplan). From the
for
in Colorectal Carcinoma Patients
Frances O. Mino, MS; Kenneth B.
Tritiated thymidine incorporation by peripheral human lymphocytes stimulated with phytohemagglutinin (PHA) was investigated in 18 colorectal carcinoma patients (seven with localized tumor and 11 with metastatic tumors). The effect of sera obtained from these patients on lymphocytes from control patients
Response
Kummerfeld; Robert L. Lundak, PhD
There is increased interest in lymphocyte reactivity to nonspecific mitogens in patients with colorectal cancer. This is due to the difficulty in utilizing specific assays be¬ cause of problems involving antigen isolation or in vitro culture of colorectal cancer cells or both. Several studies of phytohemagglutinin (PHA) stimu¬ lated reactivity of lymphocytes from patients with various cancers (including cancer of the gastrointestinal tract) have reported conflicting results concerning the presence of a nonspecific defect in the lymphocytes and/or the pres¬ ence of an inhibiting factor in the sera of tumor patients to tritiated thymidine incorporation in vitro. We have studied the response of lymphocytes from a group of patients with and without colorectal cancer to PHA stimulation in the presence or absence of control sera or tumor sera as assayed by tritiated thymidine in¬ corporation to resolve these conflicting reports. PATIENTS AND METHODS Patient Selection
Eighteen colorectal cancer patients (age 47 to 86 years, 11 with metastatic and seven with localized cancer) and 18 controls (13 pa¬ tients, age 39 to 61 years, with nonneoplastic disease and five hos¬ pital staff members, age 21 to 48 years) were studied. Informed consent was obtained from all patients after the nature of the pro¬ cedure had been fully explained. In Vitro
Lymphocyte Culture obtained by venipuncture;
10 ml was Peripheral blood was clotted and 30 ml was heparinized. Lymphocyte suspensions were prepared from the heparinized blood by the Ficoll-Hypaque gradient method described by Wybran et al.' All cells were washed twice with phosphate buffered saline and cultured in trip¬ licate in Roswell Park Memorial Institute (RPMI) medium 1640
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/28/2015
Peripheral
30 ml
vs
Patients
Blood Obtained From
Sera Collected
Ficoll-Hypaque Separation
-,
1
90%
Lymphocytes Culture (2 ml) 1-3
+
RPMI 1640
106 Lymphocytes
«
From Control or Tumor Patients
+
Penicillin/Streptomycin/Levoglutamide
Control or Tumor Sera
Fetal Calf Sera
PHA
68-hr Incubation at 37 + 1 Microcurie
C in Mixture of 5%
4 hr
C02
Lymphocytes
Tritiated Thymidine
Harvested on Glass Filters
and 95% Air
Liquid -Scintillation Fluid for Counting
Experimental plan.
supplemented with 1% levoglutamide (Gibco) 10,000 units penicil¬ and 10,000/^g streptomycin sulfate (Gibco) per 100 ml media,
lin,
and 10% fetal calf sera (Gibco) at a concentration of 1 to 3 x 10" cells per milliliter in 16 125-mm capped plastic tubes (Falcon). Tumor or normal sera (0.2 ml) was added to the appropriate cul¬ tures at time zero. After four hours, 0.025 ml of PHA-P (Difco) per milliliter of media was added to each culture. Cells were incu¬ bated for three days in a mixture of 5% carbon dioxide and 95% air at 37 C using airtight plexiglass boxes. The cultures were pulsed with microcurie tritiated thymidine (Amersham-Searle) for four hours and harvested by vacuum fil¬ tration on glass fiber filters (Reeves Angel 934 AH) with precipi¬ tation by cold trichloroacetic acid. Tritiated thymidine incorpora¬ tion was measured by liquid scintillation (Figure).
Study of Inhibitory Serum Factors The suppressive activity of sera from colorectal cancer patients was investigated for its ability to inhibit PHA-induced DNA syn¬ thesis by lymphocytes from colorectal and control patients as com¬ to the effect of control sera thesis. Normal or tumor sera (0.2 ml) 2-ml cultures at time zero.
pared
Statistical
were
compared.
Lymphocyte Reactivity
10 ml Clotted
Heparinized
metastatic tumors
on
was
PHA-induced DNA syn¬ added to the appropriate
Analysis
The mitotic index of each culture was calculated by dividing the average counts per minute of the PHA-stimulated lymphocytes by the average counts per minute of the nonstimulated lymphocytes. The mean mitotic index of each group studied was calculated and compared using the Student t test.
RESULTS The range of mitotic indices of "tumor" versus "control" lymphocytes and lymphocytes from patients with localized
The mitotic indices of PHA-stimulated lymphocytes from 18 control patients varied between 0.7 and 116 (mean, 29.2). The comparative mitotic indices of PHAstimulated lymphocytes from 18 patients with colorectal carcinoma varied between 2.0 and 60 (mean, 24.9). Statis¬ tical comparison of these values yielded a value of >.30. The mean mitotic index for lymphocytes from patients with localized carcinoma cultured was 23.1 (range, 4 to 52) in media. Lymphocytes from patients with metastatic car¬ cinoma showed a mean mitotic index of 26.0 (range, 4 to 52) in media. Comparison of these mitotic indices yielded a value of >.35. Effect of Adding Sera Obtained From Controls or Patients With Colorectal Carcinoma
With the addition of autologous sera, the mitotic index for control lymphocytes ranged between 1 and 111 (mean, 39.6). The addition of sera obtained from the human con¬ trols to the cultured lymphocytes from patients with can¬ cer resulted in the varying of the mitotic index between 9 and 155 (mean, 48.0). Comparisons of these mitotic indices yielded a value of >.25. The lymphocytes from patients with localized tumor showed a mean mitotic index of 48.4 (range, 9 to 155). Lymphocytes from patients with meta¬ static cancer showed a mean mitotic index of 47.7 (range, 11 to 148). Comparison of these mitotic indices yielded a value of >.50. With the addition of sera from cancer patients, the mitotic index for control lymphocytes ranged between 1 and 206 (mean, 47.4). The addition of this sera to the cul¬ tured lymphocytes from colorectal cancer patients re¬ sulted in a mitotic index varying between 2 and 104 (mean, 47.1). Comparison of these mitotic indices yielded a value of >.50. The lymphocytes from patients with local¬ ized tumor showed a mean mitotic index of 53.6 (range, 2 to 88). Similarly treated lymphocytes from patients with metastatic cancer showed a mean mitotic index of 43.8 (range, 5 to 104). Comparison of these mitotic indices yielded a value of >.25. COMMENT
The results of 19 other studies of
lymphocyte inhibition
to PHA stimulation and/or the presence of
inhibition of PHA-stimulated rized in the Table.'21
lymphocytes
serum
factor
are summa¬
Lymphocyte Reactivity Six studies support the finding of a lymphocyte defect in cancer patients. Two positive studies were based on blast transformation. Fisher et al (1972)," using tritiated thymidine incorporation in 76 patients, showed no statis¬ tical difference nor did Vetto et al (1974, 26 patients).'Other well-controlled studies by Lejtenyi et al (1971, 21 patients)" and Oishi et al (1973, 46 patients)17 showed no
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/28/2015
Studies
by Other Investigators Type of
Author
Whittaker et al (1971)3
Sample
et al (1971) Oishl et al (1973) ' Suciu-Foca et al (1973)4
Golubetal (1969)6
No. of Patients
Cancer Studied Studied Immune Defects in Patients With Various Carcinomas Breast 30 Lymphocyte transformation Tritiated thymidine Miscellaneous 120 Tritiated thymidine Miscellaneous 46 Miscellaneous 60 Lymphocyte transformation Tritiated thymidine Lung, gastrointestinal tract, miscellaneous
Tritiated thymidine
Mekori et al (1974)'
Tritiated thymidine
Gastrointestinal tract, miscellaneous Gastrointestinal tract, miscellaneous
Nelson (1969)' Fisher et al (1972)»
Tritiated thymidine
Lejtenyi
et al (1971)'5 Budda et al (1974)5 Vetro et al (1974)«
Tritiated thymidine Tritiated thymidine Tritiated thymidine
Steward
Tritiated thymidine
pulmonary
et
al (1974)7
Chretian and Ketcham
(1973)" Robinson et al (1974)J1
Twomey
et al
(1974)8
Tritiated thymidine
Tritiated thymidine Tritiated thymidine
Gastrointestinal tract, miscellaneous Gastrointestinal tract, breast —; not
defect. Only Buda et al (1974) | showed lymphocyte inhibi¬ tion in 85 patients. Chretien and Ketcham (1973)'s showed a defect for patients with squamous cell carcinoma of the head and neck but not for adenocarcinoma patients. Mekori et al (1974, 14 patients)" showed a defect in patients with metastatic cancer only. In our study, although the mean value of normal lym¬ phocyte reactivity (29.1) was slightly higher than the reac¬ tivity of tumor patients (24.9), this was not a significant difference (P>.30). Our wide range of normal values coin¬ cides with that of Mclntyre and Cole-2 for normal lympho¬ cyte reactivity, although it differs with the controls of other authors in studies of PHA responsiveness in tumor patients.512 There was a rather large variation in both normal and in tumor patients' lymphocytes as is reflected by the standard deviation of ± 17.3 for tumor patients. Based on our data, we conclude that the lymphocytes from patients with colorectal carcinoma are not inhibited in their response to a nonspecific mitogen, PHA.
Lymphocytes'
Responsiveness*
-for metastatic disease only
15
Gastrointestinal tract, blast Gastrointestinal tract Gastrointestinal tract Gastrointestinal tract, miscellaneous Gastrointestinal tract, blast Squamous cell carcinoma, adenocarcinoma
Effect believed present by author, +; effect believed absent,
Serum Factors Present Inhibiting
+
3 20 2 12
Immune Defects in Patients With Colorectal Carcinoma Blast transformation Gastrointestinal tract 20 Blast transformation 17 Gastrointestinal tract, miscellaneous 35 Blast transformation 7 Gastrointestinal tract, miscellaneous 13 Tritiated thymidine Gastrointestinal tract, 1
Silk (1967)2»
Inhibited to PHA Stimulation*
Assay
Gatti et al (1970)18
Manousos et al (1973)' Al-Sarraf et al (1971)
Lymphocytes
26 50 21
32 6 20 23 12 38 19 5
0
+ for squamous cell carcinoma for adenocarcinoma
+
—
15 10 9
investigated, 0. Presence of Serum Factors
Inhibiting Lymphocyte Response Eleven studies support the finding of a serum factor inhibiting lymphocyte response. Positive studies by Whittaker et al (1971, 30 patients with breast cancer)3 and Suciu-Foca et al (1973, 60 patients with miscellaneous can¬ cers)4 used lymphocyte transformation assays. Using tritiated thymidine incorporation, Nelson (1969, 16 patients)13 and Golub et al (1969, ten patients)" found no serum effect, but Buda et al (1974),5 Vetto et al (1974),12 and Steward et al (1974)7 did find such an effect. There are four studies of PHA-stimulated thymidine in¬ corporation by lymphocytes from patients with colorectal or other gastrointestinal tract cancer. Vetto et al found sera that blocked tritiated thymidine uptake in lympho¬ cytes from four of six patients with colorectal cancer, but only one example of his data is presented. Buda et al con¬ sidered blocking to be present if only a 33% decreased in-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 05/28/2015
corporation was found in autologous sera and if only a 15% decrease was found in control lymphocytes treated with tumor sera. Steward et al showed a 20% decrease in thymi¬ dine incorporation in ten of 23 colorectal patients but no blocking of control lymphocytes by sera from cancer patients. No statistically significant inhibitory effect was ob¬ served when sera from tumor patients was added to con¬ trol patient lymphocytes or tumor patient lymphocytes.
The absence of this serum inhibition in these studies may be due to technique or the actual absence of this effect. Additional experiments were done in which fetal calf sera was deleted in the tubes containing control human sera or tumor sera. Preliminary data indicate no signifi¬ cant inhibition. Based on our study, we conclude that thymidine uptake
of lymphocytes secondary to PHA stimulation is similar in lymphocytes from patients with colorectal cancers and controls and the uptake is not inhibited by substance pres¬ ent in tumor
sera.
Demonstration of an immune defect in the lymphocytes of patients with gastrointestinal tract carcinoma could de¬ pend on other immune indexes, such as the absence of rec¬ ognition of specific tumor antigens. Lejtenyi et al have shown that lymphocytes from patients with gastrointesti¬ nal tract cancer do not react to carcinoembryonic anti¬ gen.15 There may be inhibition of the effector limb of the immune response inhibiting specific cytotoxic function of the lymphocyte, perhaps by antigen-antibody complexes or by prevention of the release of lymphokines from effec¬ tor cells.
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