Immunity in Colonic Tumor Patients after Operation: Determination
by Leukocyte-migration
Inhibition *
A. K. HOUSE, M.S., F.R.C.S.E., F.R.A.C.S.,t S. WISNIEWSKI, M.B.B.S., B. MED. SCI., + T. L. WOODINGS, B. ScI.w
From the University of Western Australia, Perth Medical Centre, Shenton Park, Western Australia
t u m o r rejection.10 T h e in-vitro test of leukocyte-migration inhibition 2 measures the latter type of i m m u n e response. Further, the results in vitro have been shown to correlate with results of skin patch testing in vivo. 13 In vitro leukocyte-migration inhibition has been used to demonstrate cellular i m m u n i t y to t u m o r antigen in tumor-bearing individuals11, ~4 and animals,a but m a n y of these observations have been made in studies of small numbers of subjects with a variety of tumors. This study was planned to verify, by determination of leukocyte-migration inhibition, that cellular i m m u n i t y was present in patients with colonic tumors, and aimed to show the change that surgery induced in this immunity.
THE PROGRESSION O[ a malignant t u m o r depends u p o n the inherent characteristics of the t u m o r and the response of the host. T h e latter is now thought to depend on an immunologic mechanism that many surgeons believe may be adversely altered by extensive ablative surgery or radiotherapy. These surgeons have shown that their more conservative surgical approach has not been to the detriment of patient survival and has reduced surgical morbidity. These observations have mainly been concerned with carcinoma of the breast,S, 8 but m a n y colorectal surgeons are now considering suitable rectal tumors for local excision, Is and some, in association with radiotherapists, I2 are using local irradiation. T h e i m m u n e response of the individual to his t u m o r is mediated by lymphocytes, which mature either as bone marrowderived cells capable of producing humoral antibodies or as thymus-processed cells capable of initiating cell-mediated graft or
Materials and .Methods
Preparation of Tumor Antigen and Normal Colonic Antigen T h e resected specimen was collected in saline solution at operation. Macroscopic examination was completed and a piece of the tumor and a piece of normal tissue were each placed in 20 ml of physiologic saline solution containing 500 mg of both ampicillin and cloxacillin. T h e specimens were stored at 4 C until they could be washed three times by vigorous shaking in 50 ml of sterile saline solution, minced with scissors, and then homogenized in 30 ml
* Read at the meeting of the Section of Proctology, Royal Society of Medicine, Section of Colonic and Rectal Surgery, Royal Australasian College of Surgeons, and the American Society of Colon and Rectal Surgeons, Washington, D. C., May 22 to 25, 1974. t Senior Lecturer in Surgery. ~. Tutor in Surgery. w Medical Statistician. Address reprint requests to: Dr. House, Sir Charles Gairdner Hospital, Perth Medical Centre, Shenton Park, Western Australia 6008.
100 Dis. Col. & Rect. March 1975
Volume 18 Number 2
Volume 18 Number 2
I M M U N I T Y IN C O L O N I C T U M O R
Hanks' balanced salt solution. T h e homosenates were centrifuged at 1,000 r p m for 10 minutes and the supernatants collected as antigens. These solutions were used in tissue culture at 100 and 300 ~g/ml by protein estimation. Antigens not in immediate use were stored at --25 C until needed.
Method of Leukocyte Migration Inhibition T h e technique was similar to that used by Bendixen and S0borg. 2 T w e n t y milliliters of beparinized venous blood were 9 collected in the first, second, third, fourth, and later postoperative weeks from each patient studied and from a matched control. T h e blood was allowed to sediment for an hour. T h e leukocyte-rich supernatant was transferred to a centrifuge tube and centrifuged at 1,000 r p m for five minutes. T h e cell pellet was washed twice in tissue culture medium (TCM) 199 (Burroughs Wellcome), then resuspended in T C M 199-4-10 per cent fetal calf serum (FCS) to give a cell concentration between 5 and 6 X 107 cells/ml. T h i s suspension was placed in uniform-bore hematocrit tubes, one end was sealed by heating, and the cells were spun into a pellet by centrifugation at 3,000 r p m for 10 minutes. T h e pellet was cut from the tube and placed in 1-ml-capacity .tissue culture chambers. T h e chambers were arranged in groups of five with five cultures in each group: 1) Control chambers: T C M - 1 - 1 0 per cent fetal calf serum (FCS). 2-5) Treatment chambers: 2) T C M -}- 10 per cent FCS -}- 100 ug/ml t u m o r antigen; 3) T C M -4- 10 per cent FCS -]- 300 ~g/ml tumor antigen; 4) T C M - } - 1 0 per cent FCS-t-100 ug/ml normal colonic antigen; 5) T C M - 4 - 1 0 per cent FCS-}-300 ~g/ml normal colonic antigen. T h e cultures were incubated for 20 hours at 37 C and then the area over which the cells had migrated was projected through a projection microscope and traced on trans-
PATIENTS
TABLE 1.
]0 1
Details of Subjects under Review
N o r m a l subjects
84
Patients
31
Observations on patients
84
T u m o r site Rectum L. colon R. colon
12 11 8
Spread Local + confined to the m u c o s a Serosa L y m p h a t i c + distant
15 5 11
Survivors
23
parent paper. T h e area on the paper was measured by planimetry. Tile migration index (MI) could have been expressed as: mean treatment area, but this did not give any indication of the variation mean control area in the replicate unpaired measurements, and hence a method modified from that of Jureziz et al.7 was used. T h e nl treatment areas and n., control areas gave nln2 quotients. T h e M I and its confidence interval from the nln., quotients was calculated: MI - - median
treatment area control area
T h e results obtained in patients were compared with those obtained in normal controI individuals by applying Student's t test to the migration indices. A computer program (Fortran IV) was written to make the calculations.
Subjects T h e subjects of the study (Table 1) included 31 patients, from w h o m 84 series of observations were obtained. Twelve patients had rectal tumors, and 11 and eight had tumors of the left and right halves of the colon, respectively. T h e disease had not spread in 15, involved the serosa in five and involved lymph nodes or other organs in 11. Twenty-three patients survived, five
t 02
HOUSE, E T AL.
Dis. CoL & Reef. March 1975 MINI~IJM MIGRATION INDEX. PATIENT, 300 ~Jg/ml T @ ANTIGEN.
MIGRATION IN[]EX. 300ug/ml TUMOUR ANTIGEN A. and NORMAL COLON
cf CONTROL INHIBITION> 3 0 ~
1.284
1.2
1.0
1-0
0.8
0-8
0.6 84
06
9
9
;. 9
0.4
0.4
0.2
0-2
t=5-84
e
Q Q
9
0
t
Fie. 1. Mean and standard deviation for migration indices in each group of 84 observations. Closed symbols represent tumor antigens and open symbols, normal colonic antigens in culture. Squares represent normal subjects and triangles, patients. There is no statistically significant difference between groups.
d i e d of u n r e l a t e d causes, a n d three d i e d of t h e i r disease. E i g h t y - f o u r series of observations were o b t a i n e d from 84 c o n t r o l ind i v i d u a l s , m a t c h e d .to the cancer p a t i e n t s by b l o o d g r o u p a n d age. Each h a d a n o p e r a t i o n of s i m i l a r severity to t h a t of the c o r r e s p o n d i n g cancer p a t i e n t . T h e times after o p e r a t i o n at w h i c h the tests were carr i e d o u t were the same in the two groups. Results T h e m e a n m i g r a t i o n indices o b t a i n e d for all c u l t u r e s w i t h n o r m a l colonic a n d t u m o r a n t i g e n s in the m e d i a of m i g r a t i n g n o r m a l subjects' cells a n d p a t i e n t s ' teukocytes are r e c o r d e d in F i g u r e 1. T h e m e a n a n d s t a n d a r d d e v i a t i o n are i n d i c a t e d , a n d Stud e n t ' s t test is used to c o m p a r e each g r o u p
ns
ns
i
2
t~2.,16
3
t=2.95
~,
I~.
TIME
FaG. 2. Minimum migration indices for patients who had tumor antigen in culture, recorded according to the weeks after operation in which they occurred. Each value is compared with the mean migration index obtained from control subjects. Inhibition greater than 30 per cent was statistically significant (t + 5:84). The mean value for each week is also compared with the control value (A) and the difference shown along the time axis. of results w i t h the m i g r a t i o n of n o r m a l subjects' leukocytes in m e d i u m c o n t a i n i n g t u m o r a n t i g e n . T h e r e is n o statistically significant difference b e t w e e n t h e groups. I m m u n i t y is i n d i c a t e d b y i n h i b i t i o n of migration. Hence, the minimum migration i n d e x for each p a t i e n t is r e c o r d e d (Fig. 2) in r e l a t i o n to the time a f t e r o p e r a t i o n at w h i c h it occurred. T h e s e m i n i m u m m i g r a t i o n indices were c o m p a r e d w i t h those obt a i n e d for n o r m a l subjects' cells m i g r a t i n g in m e d i u m c o n t a i n i n g t u m o r a n t i g e n . T h e c o m p a r i s o n s were m a d e for the collective results in each p o s t o p e r a t i v e p e r i o d a n d for each p a t i e n t ' s m i n i m u m m i g r a t i o n . Comp a r e d as g r o u p s , the p a t i e n t s ' m i g r a t i o n indices in the first a n d s e c o n d weeks after o p e r a t i o n were n o t d i f f e r e n t f r o m those of
Volume 18 Number 2
IMMUNITY IN COLONIC T U M O R PATIENTS
the normal subjects. T h e r e were significant differences between normal and patient cell migrations in the third, fourth, and later weeks postoperatively. Patient cell migration inhibition by tumor antigen greater than 30 per cent was highly significantly different from control cell migration in medium containing the same antigen. Patients whose cells showed this extent of migration inhibition by tumor antigen were considered immune to this antigen. It is apparent from Figure 2 that this immunity may occur at any time after operation. Figures 3 and 4 show the vari"~. ability in the immunity that was seen m different individuals. T h e clinical and pathologic details re-
lating to patients with statistically significant inhibition of leukocyte migration in T C M containing tumor antigen are shown i n T a b l e 2. T h e cells of Patient 16 were not significantly inhibited in migration by tumor antigen. This individual was the one of three in the series of 31 to die of metastatic disease who did show evidence of immunity. Discussion T h e results obtained in the study of 31 patients with colonic and rectal tumors showed half the patients to have immunity at some time in the postoperative period. This was fewer than might have been expected from the work of others, t, 1L 14 who
MIGRATIONINDEX
MIGRATIONINDEX PATIENT5"
103
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1.2,
1,0 .
.
.
.
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I I
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Fzo. 3, Migration indices of one patient, T h e minimum migration index occurred in the third week after operation. Inhibition of this patient's leukocyte migration by tumor antigen was transitory,
o,i
1rIME ;
2'
V
/
~
'[
' later
Fro. 4. Migration indices of one patient. T h e minimum migration index occurred in the third and fifth weeks, but there was significant inhibition of leukocyte migration by tumor antigen at each observation.
104
HOUSE,
ET
AL.
Dis. Col. &Rect. March 197.5
A
,,a
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Volume18 Number2
IMMUNITY IN COLONIC TUMOR PATIENTS
investigated other tumor types. These investigators found a greater proportion of tumor-bearing subjects to have immunity when studied by the same test system. T h e findings of this study, that half the patients were immune, agree with those of Guillou and Giles 6 and are more consistent with the expected clinical outcome for the patients, who, if all had well-developed immunity, should survive longer. Serum factors or blocking antibodies 4, 6 may be responsible for the apparent discrepancy -between the results reported from different centers and for the apparently low incidence of tumor immunity seen in this group of colonic tumor patients. T h e immunity found in the patients of this series varied in the time of appearance after operation and in its duration. Some subjects had brief and others prolonged immune responses to their tumor antigens. Patients with breast tumors and melanomas have been shown to have depressed immune behavior for two weeks after operations on their tumors. 4 Hence, surgery may account for the depressed early responses seen in some individuals of this series, but another explanation is needed for the disappearance of immunity seen in the later weeks after operations in others. T h e latter patients m a y have developed humoral blocking factors similar to those shown by Cochran et al. 4 and Guillou and Giles 6 to be present in breast and colonic tumor patients. Correlation of the i n - v i t r o findings with the clinical and pathologic state showed patients with cellular immunity to be equally distributed among those with localized and those with metastatic disease. Lewis 9 observed localized or regional melanoma to be associated with humoral antibodies that disappeared as the tumor spread widely. Hence, the cellular immune response against colonic antigen in subjects with disseminated disease may differ from
105
humoral immune behavior in advanced melanoma. T h e tumors in Lewis' study appeared to overcome the host response, but this in,teraction in the colonic tumor patient was less clear, possibly because the observations of the present study began randomly during the natural course of the disease. An ongoing study, comparing the survivals of those individuals who do and those who do not have immunity to their early neoplasms, will determine the influence that cellular immunity has on tumor progression. Summary T u m o r immunity in 31 colonic tumor patients was studied by determining in v i t r o leukocyte-migration inhibition. In tissue culture 30 per cent inhibition of patient leukocyte migration by tumor antigen could be demonstrated in half of the patients, suggesting that they were immune. This immunity was not related to the clinical or pathologic stage of the disease and varied in different patients. It was transient in some and persistent in others. In addition, a positive response at a fixed time after operation was not found. Acknowledgment The authors are grateful to the Cancer Council of Western Australia, and the Arnold Yeldham and Mary Raine Research Foundations, for financial support, to the Royal Perth and Sir Charles Gairdher Hospitals for travel grants, and to their colleagues for allowing study of patients in their care. They also thank laboratory assistants Sue Barnett and Clare Hormer. References 1. Andersen V, Bendixen G, Schi0dt T: An in vitro demonstration of cellular immunity against autologous mammary carcinoma in man: Preliminary report. Acta Med Scand 186: 101, 1969 2. Bendixen G, SCborg M: A leucocyte migration technique for in vitro detection of cellular (delayed type) hypersensitivity in man. Dan Med Bull 16: 1, 1969
106
HOUSE, ET AL.
3. Bloom BR, Bennett B, Oettgen HF, et al: Demonstration of delayed hypersensitivity to soluble antigens of chemically induced tumors by inhibition of macrophage migration. Proc Natl Acad Sci USA 64: 1176, 1969 4. Cochran AJ, Spilg WGS, Mackie RM, et al: Postoperative depression of tumour-directed cell-mediated immunity in patients with malignant disease. Br Med J 4: 67, 1972 5. Crile G Jr: Results of simple mastectomy without irradiation in the treatment of operative stage I cancer of the breast. Ann Surg 168: 330, 1968 6. Guillou PJ, Giles GR: Inhibition of leucocyte migration by turnout-associated antigens of the colon and rectum. Gut 14: 733, 1973 7. Jureziz RE, Thor DE, Dray S: Transfer with RNA extracts of the cell migration inhibition correlate of delayed hypersensitivity in the guinea pig. J Immunol 101: 823, 1968 8. Kaae S, Johansen H: Simple versus radical mastectomy in primary breast cancer, Prognostic Factors in Breast Cancer. Edited by AP Forrest, PB Kunkler. Edinburgh, E. & S. Livingstone Ltd., 1968, p 93 9. Lewis MG: Immunological studies in patients
10.
11.
12. 13.
14.
15.
Dis. Col. & Rect. March 1975
with malignant melanoma: The role of circulating antibody. Melanoma and skin cancer. Proc. Int. Cancer Conf. Edited by W H McCarthy, VC Blight. Sydney, Australia, Government Printers, 1972, p 233 Roitt IM, Greaves MF, Torrigiani G, et al: The cellular basis of immunological responses: A synthesis o[ some current views. Lancet 2: 367, 1969 Segall A, Weiler O, Genin J, et al: In vitro study of cellular immunity against autochthonous human cancer. Int J Cancer 9: 417, 1972 Sischy B, Remington JH: Endocavitary contact radiation for carcinoma of the rcctum (in press) S~borg M: In vitro detection of cellular hypersensitivity in man: Specific migration inhibition of white blood cells from Brucella-posi. tire persons. Acta Med Scand 182: 167, 1967 Wolberg WH, Goelzer ML: In vitro assay of cell mediated immunity in human cancer: Definition of leukocyte migration inhibition factor. Nature (Lond) 229: 632, 1971 York Mason A: Carcinoma o[ the lower twothirds of the rectum (in press)
by Leukocyte-migration
Inhibition *
A. K. HOUSE, M.S., F.R.C.S.E., F.R.A.C.S.,t S. WISNIEWSKI, M.B.B.S., B. MED. SCI., + T. L. WOODINGS, B. ScI.w
From the University of Western Australia, Perth Medical Centre, Shenton Park, Western Australia
t u m o r rejection.10 T h e in-vitro test of leukocyte-migration inhibition 2 measures the latter type of i m m u n e response. Further, the results in vitro have been shown to correlate with results of skin patch testing in vivo. 13 In vitro leukocyte-migration inhibition has been used to demonstrate cellular i m m u n i t y to t u m o r antigen in tumor-bearing individuals11, ~4 and animals,a but m a n y of these observations have been made in studies of small numbers of subjects with a variety of tumors. This study was planned to verify, by determination of leukocyte-migration inhibition, that cellular i m m u n i t y was present in patients with colonic tumors, and aimed to show the change that surgery induced in this immunity.
THE PROGRESSION O[ a malignant t u m o r depends u p o n the inherent characteristics of the t u m o r and the response of the host. T h e latter is now thought to depend on an immunologic mechanism that many surgeons believe may be adversely altered by extensive ablative surgery or radiotherapy. These surgeons have shown that their more conservative surgical approach has not been to the detriment of patient survival and has reduced surgical morbidity. These observations have mainly been concerned with carcinoma of the breast,S, 8 but m a n y colorectal surgeons are now considering suitable rectal tumors for local excision, Is and some, in association with radiotherapists, I2 are using local irradiation. T h e i m m u n e response of the individual to his t u m o r is mediated by lymphocytes, which mature either as bone marrowderived cells capable of producing humoral antibodies or as thymus-processed cells capable of initiating cell-mediated graft or
Materials and .Methods
Preparation of Tumor Antigen and Normal Colonic Antigen T h e resected specimen was collected in saline solution at operation. Macroscopic examination was completed and a piece of the tumor and a piece of normal tissue were each placed in 20 ml of physiologic saline solution containing 500 mg of both ampicillin and cloxacillin. T h e specimens were stored at 4 C until they could be washed three times by vigorous shaking in 50 ml of sterile saline solution, minced with scissors, and then homogenized in 30 ml
* Read at the meeting of the Section of Proctology, Royal Society of Medicine, Section of Colonic and Rectal Surgery, Royal Australasian College of Surgeons, and the American Society of Colon and Rectal Surgeons, Washington, D. C., May 22 to 25, 1974. t Senior Lecturer in Surgery. ~. Tutor in Surgery. w Medical Statistician. Address reprint requests to: Dr. House, Sir Charles Gairdner Hospital, Perth Medical Centre, Shenton Park, Western Australia 6008.
100 Dis. Col. & Rect. March 1975
Volume 18 Number 2
Volume 18 Number 2
I M M U N I T Y IN C O L O N I C T U M O R
Hanks' balanced salt solution. T h e homosenates were centrifuged at 1,000 r p m for 10 minutes and the supernatants collected as antigens. These solutions were used in tissue culture at 100 and 300 ~g/ml by protein estimation. Antigens not in immediate use were stored at --25 C until needed.
Method of Leukocyte Migration Inhibition T h e technique was similar to that used by Bendixen and S0borg. 2 T w e n t y milliliters of beparinized venous blood were 9 collected in the first, second, third, fourth, and later postoperative weeks from each patient studied and from a matched control. T h e blood was allowed to sediment for an hour. T h e leukocyte-rich supernatant was transferred to a centrifuge tube and centrifuged at 1,000 r p m for five minutes. T h e cell pellet was washed twice in tissue culture medium (TCM) 199 (Burroughs Wellcome), then resuspended in T C M 199-4-10 per cent fetal calf serum (FCS) to give a cell concentration between 5 and 6 X 107 cells/ml. T h i s suspension was placed in uniform-bore hematocrit tubes, one end was sealed by heating, and the cells were spun into a pellet by centrifugation at 3,000 r p m for 10 minutes. T h e pellet was cut from the tube and placed in 1-ml-capacity .tissue culture chambers. T h e chambers were arranged in groups of five with five cultures in each group: 1) Control chambers: T C M - 1 - 1 0 per cent fetal calf serum (FCS). 2-5) Treatment chambers: 2) T C M -}- 10 per cent FCS -}- 100 ug/ml t u m o r antigen; 3) T C M -4- 10 per cent FCS -]- 300 ~g/ml tumor antigen; 4) T C M - } - 1 0 per cent FCS-t-100 ug/ml normal colonic antigen; 5) T C M - 4 - 1 0 per cent FCS-}-300 ~g/ml normal colonic antigen. T h e cultures were incubated for 20 hours at 37 C and then the area over which the cells had migrated was projected through a projection microscope and traced on trans-
PATIENTS
TABLE 1.
]0 1
Details of Subjects under Review
N o r m a l subjects
84
Patients
31
Observations on patients
84
T u m o r site Rectum L. colon R. colon
12 11 8
Spread Local + confined to the m u c o s a Serosa L y m p h a t i c + distant
15 5 11
Survivors
23
parent paper. T h e area on the paper was measured by planimetry. Tile migration index (MI) could have been expressed as: mean treatment area, but this did not give any indication of the variation mean control area in the replicate unpaired measurements, and hence a method modified from that of Jureziz et al.7 was used. T h e nl treatment areas and n., control areas gave nln2 quotients. T h e M I and its confidence interval from the nln., quotients was calculated: MI - - median
treatment area control area
T h e results obtained in patients were compared with those obtained in normal controI individuals by applying Student's t test to the migration indices. A computer program (Fortran IV) was written to make the calculations.
Subjects T h e subjects of the study (Table 1) included 31 patients, from w h o m 84 series of observations were obtained. Twelve patients had rectal tumors, and 11 and eight had tumors of the left and right halves of the colon, respectively. T h e disease had not spread in 15, involved the serosa in five and involved lymph nodes or other organs in 11. Twenty-three patients survived, five
t 02
HOUSE, E T AL.
Dis. CoL & Reef. March 1975 MINI~IJM MIGRATION INDEX. PATIENT, 300 ~Jg/ml T @ ANTIGEN.
MIGRATION IN[]EX. 300ug/ml TUMOUR ANTIGEN A. and NORMAL COLON
cf CONTROL INHIBITION> 3 0 ~
1.284
1.2
1.0
1-0
0.8
0-8
0.6 84
06
9
9
;. 9
0.4
0.4
0.2
0-2
t=5-84
e
Q Q
9
0
t
Fie. 1. Mean and standard deviation for migration indices in each group of 84 observations. Closed symbols represent tumor antigens and open symbols, normal colonic antigens in culture. Squares represent normal subjects and triangles, patients. There is no statistically significant difference between groups.
d i e d of u n r e l a t e d causes, a n d three d i e d of t h e i r disease. E i g h t y - f o u r series of observations were o b t a i n e d from 84 c o n t r o l ind i v i d u a l s , m a t c h e d .to the cancer p a t i e n t s by b l o o d g r o u p a n d age. Each h a d a n o p e r a t i o n of s i m i l a r severity to t h a t of the c o r r e s p o n d i n g cancer p a t i e n t . T h e times after o p e r a t i o n at w h i c h the tests were carr i e d o u t were the same in the two groups. Results T h e m e a n m i g r a t i o n indices o b t a i n e d for all c u l t u r e s w i t h n o r m a l colonic a n d t u m o r a n t i g e n s in the m e d i a of m i g r a t i n g n o r m a l subjects' cells a n d p a t i e n t s ' teukocytes are r e c o r d e d in F i g u r e 1. T h e m e a n a n d s t a n d a r d d e v i a t i o n are i n d i c a t e d , a n d Stud e n t ' s t test is used to c o m p a r e each g r o u p
ns
ns
i
2
t~2.,16
3
t=2.95
~,
I~.
TIME
FaG. 2. Minimum migration indices for patients who had tumor antigen in culture, recorded according to the weeks after operation in which they occurred. Each value is compared with the mean migration index obtained from control subjects. Inhibition greater than 30 per cent was statistically significant (t + 5:84). The mean value for each week is also compared with the control value (A) and the difference shown along the time axis. of results w i t h the m i g r a t i o n of n o r m a l subjects' leukocytes in m e d i u m c o n t a i n i n g t u m o r a n t i g e n . T h e r e is n o statistically significant difference b e t w e e n t h e groups. I m m u n i t y is i n d i c a t e d b y i n h i b i t i o n of migration. Hence, the minimum migration i n d e x for each p a t i e n t is r e c o r d e d (Fig. 2) in r e l a t i o n to the time a f t e r o p e r a t i o n at w h i c h it occurred. T h e s e m i n i m u m m i g r a t i o n indices were c o m p a r e d w i t h those obt a i n e d for n o r m a l subjects' cells m i g r a t i n g in m e d i u m c o n t a i n i n g t u m o r a n t i g e n . T h e c o m p a r i s o n s were m a d e for the collective results in each p o s t o p e r a t i v e p e r i o d a n d for each p a t i e n t ' s m i n i m u m m i g r a t i o n . Comp a r e d as g r o u p s , the p a t i e n t s ' m i g r a t i o n indices in the first a n d s e c o n d weeks after o p e r a t i o n were n o t d i f f e r e n t f r o m those of
Volume 18 Number 2
IMMUNITY IN COLONIC T U M O R PATIENTS
the normal subjects. T h e r e were significant differences between normal and patient cell migrations in the third, fourth, and later weeks postoperatively. Patient cell migration inhibition by tumor antigen greater than 30 per cent was highly significantly different from control cell migration in medium containing the same antigen. Patients whose cells showed this extent of migration inhibition by tumor antigen were considered immune to this antigen. It is apparent from Figure 2 that this immunity may occur at any time after operation. Figures 3 and 4 show the vari"~. ability in the immunity that was seen m different individuals. T h e clinical and pathologic details re-
lating to patients with statistically significant inhibition of leukocyte migration in T C M containing tumor antigen are shown i n T a b l e 2. T h e cells of Patient 16 were not significantly inhibited in migration by tumor antigen. This individual was the one of three in the series of 31 to die of metastatic disease who did show evidence of immunity. Discussion T h e results obtained in the study of 31 patients with colonic and rectal tumors showed half the patients to have immunity at some time in the postoperative period. This was fewer than might have been expected from the work of others, t, 1L 14 who
MIGRATIONINDEX
MIGRATIONINDEX PATIENT5"
103
PATIENT ").
1.2,
1,0 .
.
.
.
0'8,
0"8,
I I
\
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0'6"
I
0'4" I
"
i
]TIME '~
2
3
4
Later
Fzo. 3, Migration indices of one patient, T h e minimum migration index occurred in the third week after operation. Inhibition of this patient's leukocyte migration by tumor antigen was transitory,
o,i
1rIME ;
2'
V
/
~
'[
' later
Fro. 4. Migration indices of one patient. T h e minimum migration index occurred in the third and fifth weeks, but there was significant inhibition of leukocyte migration by tumor antigen at each observation.
104
HOUSE,
ET
AL.
Dis. Col. &Rect. March 197.5
A
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Volume18 Number2
IMMUNITY IN COLONIC TUMOR PATIENTS
investigated other tumor types. These investigators found a greater proportion of tumor-bearing subjects to have immunity when studied by the same test system. T h e findings of this study, that half the patients were immune, agree with those of Guillou and Giles 6 and are more consistent with the expected clinical outcome for the patients, who, if all had well-developed immunity, should survive longer. Serum factors or blocking antibodies 4, 6 may be responsible for the apparent discrepancy -between the results reported from different centers and for the apparently low incidence of tumor immunity seen in this group of colonic tumor patients. T h e immunity found in the patients of this series varied in the time of appearance after operation and in its duration. Some subjects had brief and others prolonged immune responses to their tumor antigens. Patients with breast tumors and melanomas have been shown to have depressed immune behavior for two weeks after operations on their tumors. 4 Hence, surgery may account for the depressed early responses seen in some individuals of this series, but another explanation is needed for the disappearance of immunity seen in the later weeks after operations in others. T h e latter patients m a y have developed humoral blocking factors similar to those shown by Cochran et al. 4 and Guillou and Giles 6 to be present in breast and colonic tumor patients. Correlation of the i n - v i t r o findings with the clinical and pathologic state showed patients with cellular immunity to be equally distributed among those with localized and those with metastatic disease. Lewis 9 observed localized or regional melanoma to be associated with humoral antibodies that disappeared as the tumor spread widely. Hence, the cellular immune response against colonic antigen in subjects with disseminated disease may differ from
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humoral immune behavior in advanced melanoma. T h e tumors in Lewis' study appeared to overcome the host response, but this in,teraction in the colonic tumor patient was less clear, possibly because the observations of the present study began randomly during the natural course of the disease. An ongoing study, comparing the survivals of those individuals who do and those who do not have immunity to their early neoplasms, will determine the influence that cellular immunity has on tumor progression. Summary T u m o r immunity in 31 colonic tumor patients was studied by determining in v i t r o leukocyte-migration inhibition. In tissue culture 30 per cent inhibition of patient leukocyte migration by tumor antigen could be demonstrated in half of the patients, suggesting that they were immune. This immunity was not related to the clinical or pathologic stage of the disease and varied in different patients. It was transient in some and persistent in others. In addition, a positive response at a fixed time after operation was not found. Acknowledgment The authors are grateful to the Cancer Council of Western Australia, and the Arnold Yeldham and Mary Raine Research Foundations, for financial support, to the Royal Perth and Sir Charles Gairdher Hospitals for travel grants, and to their colleagues for allowing study of patients in their care. They also thank laboratory assistants Sue Barnett and Clare Hormer. References 1. Andersen V, Bendixen G, Schi0dt T: An in vitro demonstration of cellular immunity against autologous mammary carcinoma in man: Preliminary report. Acta Med Scand 186: 101, 1969 2. Bendixen G, SCborg M: A leucocyte migration technique for in vitro detection of cellular (delayed type) hypersensitivity in man. Dan Med Bull 16: 1, 1969
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3. Bloom BR, Bennett B, Oettgen HF, et al: Demonstration of delayed hypersensitivity to soluble antigens of chemically induced tumors by inhibition of macrophage migration. Proc Natl Acad Sci USA 64: 1176, 1969 4. Cochran AJ, Spilg WGS, Mackie RM, et al: Postoperative depression of tumour-directed cell-mediated immunity in patients with malignant disease. Br Med J 4: 67, 1972 5. Crile G Jr: Results of simple mastectomy without irradiation in the treatment of operative stage I cancer of the breast. Ann Surg 168: 330, 1968 6. Guillou PJ, Giles GR: Inhibition of leucocyte migration by turnout-associated antigens of the colon and rectum. Gut 14: 733, 1973 7. Jureziz RE, Thor DE, Dray S: Transfer with RNA extracts of the cell migration inhibition correlate of delayed hypersensitivity in the guinea pig. J Immunol 101: 823, 1968 8. Kaae S, Johansen H: Simple versus radical mastectomy in primary breast cancer, Prognostic Factors in Breast Cancer. Edited by AP Forrest, PB Kunkler. Edinburgh, E. & S. Livingstone Ltd., 1968, p 93 9. Lewis MG: Immunological studies in patients
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with malignant melanoma: The role of circulating antibody. Melanoma and skin cancer. Proc. Int. Cancer Conf. Edited by W H McCarthy, VC Blight. Sydney, Australia, Government Printers, 1972, p 233 Roitt IM, Greaves MF, Torrigiani G, et al: The cellular basis of immunological responses: A synthesis o[ some current views. Lancet 2: 367, 1969 Segall A, Weiler O, Genin J, et al: In vitro study of cellular immunity against autochthonous human cancer. Int J Cancer 9: 417, 1972 Sischy B, Remington JH: Endocavitary contact radiation for carcinoma of the rcctum (in press) S~borg M: In vitro detection of cellular hypersensitivity in man: Specific migration inhibition of white blood cells from Brucella-posi. tire persons. Acta Med Scand 182: 167, 1967 Wolberg WH, Goelzer ML: In vitro assay of cell mediated immunity in human cancer: Definition of leukocyte migration inhibition factor. Nature (Lond) 229: 632, 1971 York Mason A: Carcinoma o[ the lower twothirds of the rectum (in press)
