Znt. J. Cancer: 19, 43-48 (1977)
A MODIFIED LEUKOCYTE ADHERENCE INHIBITION TEST IN THE LABORATORY INVESTIGATION OF GASTROINTESTINAL CANCER J. C. RUTHERFORD, B. A. J. WALTERS, G. CAVAYE and W. J. HALLIDAY The Repatriation General Hospital, Greenslopes, the Department of Surgery, Univeristy of' Queensland, and the Department of Microbiology, University of Queensland, Brisbane, Queensland, Australia A modification of the leukocyte adherence inhibition (LAI) technique has been used to test 60 hospital patients with gastrointestinal symptoms, for specific immunoreactivity against extracts of tumours of colon, pancreasand stomach. The modifiedtechnique employed mononuclear cells and soluble tumour extracts in glass test tubes, the non-adherent cells being enumerated in an electronic counter. Laboratory tests were completed before the eventual diagnosis was known. Groups of patients with adenocarcinoma of colon or rectum (11). carcinoma of pancreas (3) and adenocarcinoma of stomach (6) were clearly distinguishedfrom each other and from patients with non-malignant diseases or with neoplasms of different histological type.
Patients with malignant neoplasms of the gastrointestinal tract are reported to display cell-mediated immune reactivity to antigenic determinants present in their tumours. This has been detected by a variety of in vitro techniques including lymphocyte cytotoxicity (Hellstrom et al., 1970; Nairn et al., 1971; Vose et al., 1975), leukocyte migration inhibition (Guillou and Giles, 1973; Bull, 1973; Mc Illmurray et al., 1974; Armitstead and Gowland, 19756) and leukocyte adherence inhibition (Maluish and Halliday, 1974; Halliday et al., 1974, 1975). The leukocyte adherence inhibition (LAI) test, originally described by Halliday and Miller (1972), has attracted particular attention because of its claims to simplicity and specificity. This test is based on the observation that, when blood leukocytes from a patient with cancer are mixed with the corresponding tumour extract, the tendency of the leukocytes to adhere to glass is inhibited. Serum from these patients may interfere with or block the inhibition. Modifications of the original LA1 technique have recently been published. These have attempted to make the method more reproducible and objective, and have included preliminary purification of mononuclear cells (Powell et al., 1979, use of an electronic cell counter (Lampert and Dietmair, 1973), and glass tubes instead of haemocytometers to provide the solid surface for leukocyte adherence (HolBn et al., 1974; Grosser and Thomson, 1975). The present study incorporates all of the above modifications of technique and reports the results of LA1 tests in determining specific anti-tumour immunoreactivity in 60 patients with gastrointestinal disease.
METHODS
Patients Sixty patients being investigated at the Repatriation General Hospital for gastrointestinal symptoms were studied for LA1 reactivity with a panel of three soluble tumour extracts. None of the patients at the time of the initial study were receiving X-irradiation, chemotherapeutic agents or steroids. With many of the subjects a definitive diagnosis had not been made at the time of testing, and in cases where a malignancy was suspected the laboratory was not informed. Blood specimens were always taken before surgery, to avoid possible immunosuppressive effects. Separation of mononuclear leukocytes A 25 ml sample of heparinized venous blood was allowed to stand in a glass bottle for 30 min and the leukocyte-rich plasma was aspirated. This was then diluted 1/3 with Eagle's basal medium containing 10% foetal calf serum (inactivated) and 0.28 mg/ml sodium bicarbonate. This medium was used throughout the procedure. The diluted plasma was then layered on 3ml of Ficoll-Hypaque of density 1.077 g/ml contained in 15 ml plastic disposable centrifuge tubes (Kayline Plastics, Plympton, South Australia). The tubes were centrifuged for 30 min with an interface force of 400 g at room temperature (Boyum, 1968). The mononuclear cells at the interface were collected, by means of a Pasteur pipette, with most of the Ficoll-Hypaque above the erythrocyte pellet. The suspension was diluted 1/5 with medium and centrifuged at 400 g for 10 min at room temperature. The cells were then washed three more times and resuspended at a concentration of approximately 4 x lo' cells/ml of medium. Tumour extracts The method of Halliday et al. (1974) was used to prepare soluble tumour extracts for the test. Extracts were prepared from adenocarcinoma of the colon, adenocarcinoma of the stomach and adenocarcinoma of the pancreas (from patients other than those studied by the LA1 test). The tissue used was from the interior of the tumour, which had been stored at -40" C before use.
Received: July 20, 1976 and in revised form September 15, 1976.
44
RUTHERFORD ET AL.
After testing for lack of non-specific reactivity with mononuclear cells from normal volunteers, the extracts were stored in 0.2 ml amounts at -18°C. They were diluted 1/5 with medium on the day of the test so that the final concentration in the test was 1/20.
An arithmetic mean was calculated for each pair of duplicate mixtures; the individual values did not differ from the mean by more than 6 %. The percent LA1 was calculated for each combination of leukocytes and extract by the formula: Mean % adherence with no extract - Mean "/, . _ adherence with extract x 100. Percentage LA1 = Mean % adherence with no extract Values of percentage LA1 for the series of patients and each extract were examined statistically by the Mann-Whitney test (Snedecor and Cochran, 1972).
Counting cells
Counts were made in the Coulter F N particle counter (Coulter Electronics Ltd., Bedfordshire, England), after 1/500 dilution and lysis of erythrocytes as for routine counting of leukocytes. The leukocyte adherence inhibition test
Equal volumes (0.1 ml) of mononuclear cell suspension (4 x lo6 cells) and diluted tumour extract were mixed, and medium was added to make the final volume 0.4 ml. Duplicate mixtures were made of each cell suspension with the three different tumour extracts (stomach, pancreas and colon) and controls (without extract) in small plastic-capped tubes. The tubes were incubated at 37" C for 30 min with periodic shaking. After incubation the contents of the tubes were removed into plastic cups and cells were counted in subsamples of 0.02 ml. The remaining cell mixtures were aspirated with Pasteur pipettes and placed at the bottom of Kimax screw-capped test tubes (Owens-Illinois Glass Company, Toledo, Ohio). These tubes were then incubated at 37" C for 2 h in an almost horizontal position so that the contents ran along the top three-quarters of the tube. After incubation, the tubes were gently raised to the vertical position, the medium and non-adherent cells at the bottom mixed by repeated pipetting, and the cells were recounted. The percentage adherence of cells was determined for each mixture by the formula: final count Percentage adherence = 100 x 100. initial count ~
RESULTS
The percentage of glass-adherent cells in the control mixtures (no tumour extract) ranged between 28.1 % and 93.5%, the mean being 66.6 *14.7%. These normal adherences were used as the basis for calculating the effect of added tumour extracts. For the sake of brevity, actual cell counts are not reported in full, but the calculation is explained for a single patient in Table 1. Results in Table I1 show the percentage adherence of the 60 patients' leukocytes with the three tumour extracts used. Patients are numbered and grouped in Table 11 according to the eventual clinical diagnosis. It can be seen that, in general, patients with a particular tumour had a decreased leukocyte adherence when tested with the extract corresponding to that tumour. Conversely, patients with nonmalignant conditions usually showed lower inhibitory effects of tumour extracts on their leukocytes. This relationship between cellular reactivity ( % LAI) and presence and type of tumour is further summarized in Figure 1. All 60 patients were tested with the colon carcinoma extract but only 57 with the pancreatic and 47 with the stomach carcinoma extracts.
TABLE 1 EXAMPLE OF CALCULATION OF RESULTS FOR PATIENT NO. 1 (ADENOCARCINOMA OF COLON) Tumour extract used
None Colon Pancreas Stomach
1
Initial count
A= B A B A
B A B
12,389 12,633 12,08 I 1237 1 12,809 13,021 12,757 12,912
'
Final count
"/. Adherence
3,729 2,41 I 9,797 11,154 3,497 2,851 2,564 3,603
69.9 80.9 18.9 13.3 72.7 78.1 19.9 72.1
Cells pe r pl,as read from particle counter. - A and B are duplicate mixtures.
a
~
~ % LA1 ~
75.4 16.1
78.6
75.4
0.0
76.0
-0.8
~
~
45
LA1 TEST IN GASTROINTESTINAL CANCER TABLE 11 EFFECT OF TUMOUR EXTRACTS O N LEUKOCYTE ADHERENCE AS RELATED TO EVENTUAL CLINICAL DIAGNOSIS
Patient NO.
1
2 3 4 5
6 7 8 9 10 I1 12 13 14 15
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
56 57 58 59 60 I
Age (years)
74 56 45 64 60 83 62 76 75 77 65 57 81 64 78 50 65 76 72 79 56 66 63 85 56 64 68 81 60 57 65 60 52 64 58 82 65 67 62 74 65 56 60 57 46 56 44 65 62 60 28 84 54 62 86 70 73 70
70 45
Mean % adherence with tumour extracts
sex
None
Colon
Pancreas
Stomach
M M F M M M M F F M M M M M M M M M M M M M M M M M M F M M M M M M M M F F M F M F M M M M M
75.4 82.4 78.9 64.0 69.1 65.8 71.2 73.0 86.1 73.0 77.1 65.3 88.0 61.8 58.9 83.9 56.2 83.2 86.7 69.6 72.7 66.2 68.4 73.4 44.8 78.9 75.7 60.0 71.6 71.5 50.2 49.4 55.4 81.1 48.4 86.9 46.0 28.1 48.4 47.6 67.0 64.2 65.5 29.4 68.9 57.5 62.3 72.8 56.2 92.8 73.2 89.7 72.4 93.5 55.9 78.8 44.6 54.0 43.3 59.2
16.1 67.3 48.6 30.9 50.5 53.6 48.4 45.1 67.3 48.8 46.7 66.5 89.2 68.7 53.1 75.1 51.6 81.3 86.5 74.4 82.6 64.8 59.3 73.7 57.5 77. I 70.1 50.4 77.1 63.5 60.0 57.6 53.3 80.5 48.2 16.7 51.2 30.4 51.9 57.9 67.4 68.2 60.5 33.1 67.7 60.4 67.0 74.7 58.0 87.4 66.4 86.6 66.8 79.9 66.7 72.1 50.7 48.8 43.3 62.4
75.4 78.2 78.7 66.4 60.5 73.8 70.8 90.6 79.9 84. I 85.1 57.2 91.5 48.4 63.6 75.9 59.5 77.3 ND 75.9 82.4 68.5 53.4 71.5 ND 72.1 38.7 33.8 66.3 68.5 71.5 46.0 ND 89.7 63.8 71.6 51.3 32.4 59.5 61.7 62.2 66.3 57.6 26.0 74.9 56.4 50.8 74.9 55.6 92.0 74.5 88.6 72.7 95.1 58.2 59.6 33.9 60.2 50.8 50.8
76.0 83.2 ND ND 56.7 71.3 78.9 70.0 81.8 78.7 84.2 64.2 87.1 68.4 66.0 80.3 55.2 74.7 69.9 55.4 ND 20.4 51.9 48.3 25.6 83.5 70.0 ND 68.3 70.4 61.2 70.1 58.8 63.0 52.0 ND 47.6 28.2 ND 44.1 ND 64.0 58.2 29.2 65.6 67.6 ND 67.7 ND 89.5 69.7 91.9 82.8 89.3 ND 64.5 ND ND 56.1 ND
F F
M M M M M M M F M
M M
Well differentiated. - ND = not done.
Eventual diagnosis
Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma rectum Adenocarcinoma colon (removed 3 years) Adenocarcinoma colon (removed 2 years) Adenocarcinoma colon (removed 1 year) Adenocarcinoma colon (removed 1 year) Adenocarcinoma colon (removed 3 years) Adenocarcinoma colon (removed 4 years) Squamous cell carcinoma colon (secondary) Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Mucus-secreting adenocarcinoma stomach Anaplastic adenocarcinoma stomach Carcinoma pancreas Carcinoma pancreas Carcinoma pancreas Gastric ulcer Gastric ulcer Gastric ulcer Gastric ulcer Reflux gastritis Reflux gastritis Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Pancreatitis Polyposis coli Duodenal ulcer Duodenal ulcer Duodenal ulcer Haemorrhoids Reflux alkaline gastritis Lymphoma Small bowel obstruction Chronic gastritis Secondary malignancy (no apparent primary) Rectal bleed; investigation negative Change in bowel habits; investigation negative Change in bowel habits; investigation negative Change in bowel habits; investigation negative Abdominal pain; investigation negative Abdominal pain; investigation negative
46
RUTHERFORD ET AL.
Adenocarcinoma of colon or rectum All the patients with this tumour (and none without) had LA1 values of 18.3% or greater. The three patients with LA1 values just under 18.3% were Nos. 23, 28 and 54, all of whom suffered from malignancies of other types (Table 11). In the group with low reactivity were six (Nos. 12-17) who had colon carcinomas removed 1-4 years previously, indicating loss of detectable immune response after removal of the antigenic stimulus. These patients were thoroughly examined and no evidence of recurrence was detected. Pancreatic carcinoma Although the number of patients diagnosed in this group was small, two of the three were clearly separated fromlthe control group by their high LAI
A
B
C
values. One patient (No. 29) who failed to give a distinctive reaction had disseminated advanced cancer. All patients without detectable pancreatic tumours had LA1 values of 23.9% or less. Two of these, with values approaching 23.9 % (Nos. 14 and 23), had histories of other malignancies. Carcinoma of stomach Of the eight patients with diagnosed stomach carcinoma, only seven appear in Figure 1 since patient No. 21 was not tested with this extract; however, he had not reacted significantly with the other extracts. Six of those tested gave high values of percentage LA1 (18.9% or higher), with slight overlap of the control group. The three patients with marginal reactivity (Nos. 5, 34 and 56) were suffering from, respectively, carcinoma of colon, reflux gastritis and abdominal pain with change in bowel habits (the last two patients are still under investigation for possible tumours). When the patients examined with each extract were arranged in rank order of LA1 reactivity, there was for each tumour type a statistically significant difference between the group with tumours and those without (p
A MODIFIED LEUKOCYTE ADHERENCE INHIBITION TEST IN THE LABORATORY INVESTIGATION OF GASTROINTESTINAL CANCER J. C. RUTHERFORD, B. A. J. WALTERS, G. CAVAYE and W. J. HALLIDAY The Repatriation General Hospital, Greenslopes, the Department of Surgery, Univeristy of' Queensland, and the Department of Microbiology, University of Queensland, Brisbane, Queensland, Australia A modification of the leukocyte adherence inhibition (LAI) technique has been used to test 60 hospital patients with gastrointestinal symptoms, for specific immunoreactivity against extracts of tumours of colon, pancreasand stomach. The modifiedtechnique employed mononuclear cells and soluble tumour extracts in glass test tubes, the non-adherent cells being enumerated in an electronic counter. Laboratory tests were completed before the eventual diagnosis was known. Groups of patients with adenocarcinoma of colon or rectum (11). carcinoma of pancreas (3) and adenocarcinoma of stomach (6) were clearly distinguishedfrom each other and from patients with non-malignant diseases or with neoplasms of different histological type.
Patients with malignant neoplasms of the gastrointestinal tract are reported to display cell-mediated immune reactivity to antigenic determinants present in their tumours. This has been detected by a variety of in vitro techniques including lymphocyte cytotoxicity (Hellstrom et al., 1970; Nairn et al., 1971; Vose et al., 1975), leukocyte migration inhibition (Guillou and Giles, 1973; Bull, 1973; Mc Illmurray et al., 1974; Armitstead and Gowland, 19756) and leukocyte adherence inhibition (Maluish and Halliday, 1974; Halliday et al., 1974, 1975). The leukocyte adherence inhibition (LAI) test, originally described by Halliday and Miller (1972), has attracted particular attention because of its claims to simplicity and specificity. This test is based on the observation that, when blood leukocytes from a patient with cancer are mixed with the corresponding tumour extract, the tendency of the leukocytes to adhere to glass is inhibited. Serum from these patients may interfere with or block the inhibition. Modifications of the original LA1 technique have recently been published. These have attempted to make the method more reproducible and objective, and have included preliminary purification of mononuclear cells (Powell et al., 1979, use of an electronic cell counter (Lampert and Dietmair, 1973), and glass tubes instead of haemocytometers to provide the solid surface for leukocyte adherence (HolBn et al., 1974; Grosser and Thomson, 1975). The present study incorporates all of the above modifications of technique and reports the results of LA1 tests in determining specific anti-tumour immunoreactivity in 60 patients with gastrointestinal disease.
METHODS
Patients Sixty patients being investigated at the Repatriation General Hospital for gastrointestinal symptoms were studied for LA1 reactivity with a panel of three soluble tumour extracts. None of the patients at the time of the initial study were receiving X-irradiation, chemotherapeutic agents or steroids. With many of the subjects a definitive diagnosis had not been made at the time of testing, and in cases where a malignancy was suspected the laboratory was not informed. Blood specimens were always taken before surgery, to avoid possible immunosuppressive effects. Separation of mononuclear leukocytes A 25 ml sample of heparinized venous blood was allowed to stand in a glass bottle for 30 min and the leukocyte-rich plasma was aspirated. This was then diluted 1/3 with Eagle's basal medium containing 10% foetal calf serum (inactivated) and 0.28 mg/ml sodium bicarbonate. This medium was used throughout the procedure. The diluted plasma was then layered on 3ml of Ficoll-Hypaque of density 1.077 g/ml contained in 15 ml plastic disposable centrifuge tubes (Kayline Plastics, Plympton, South Australia). The tubes were centrifuged for 30 min with an interface force of 400 g at room temperature (Boyum, 1968). The mononuclear cells at the interface were collected, by means of a Pasteur pipette, with most of the Ficoll-Hypaque above the erythrocyte pellet. The suspension was diluted 1/5 with medium and centrifuged at 400 g for 10 min at room temperature. The cells were then washed three more times and resuspended at a concentration of approximately 4 x lo' cells/ml of medium. Tumour extracts The method of Halliday et al. (1974) was used to prepare soluble tumour extracts for the test. Extracts were prepared from adenocarcinoma of the colon, adenocarcinoma of the stomach and adenocarcinoma of the pancreas (from patients other than those studied by the LA1 test). The tissue used was from the interior of the tumour, which had been stored at -40" C before use.
Received: July 20, 1976 and in revised form September 15, 1976.
44
RUTHERFORD ET AL.
After testing for lack of non-specific reactivity with mononuclear cells from normal volunteers, the extracts were stored in 0.2 ml amounts at -18°C. They were diluted 1/5 with medium on the day of the test so that the final concentration in the test was 1/20.
An arithmetic mean was calculated for each pair of duplicate mixtures; the individual values did not differ from the mean by more than 6 %. The percent LA1 was calculated for each combination of leukocytes and extract by the formula: Mean % adherence with no extract - Mean "/, . _ adherence with extract x 100. Percentage LA1 = Mean % adherence with no extract Values of percentage LA1 for the series of patients and each extract were examined statistically by the Mann-Whitney test (Snedecor and Cochran, 1972).
Counting cells
Counts were made in the Coulter F N particle counter (Coulter Electronics Ltd., Bedfordshire, England), after 1/500 dilution and lysis of erythrocytes as for routine counting of leukocytes. The leukocyte adherence inhibition test
Equal volumes (0.1 ml) of mononuclear cell suspension (4 x lo6 cells) and diluted tumour extract were mixed, and medium was added to make the final volume 0.4 ml. Duplicate mixtures were made of each cell suspension with the three different tumour extracts (stomach, pancreas and colon) and controls (without extract) in small plastic-capped tubes. The tubes were incubated at 37" C for 30 min with periodic shaking. After incubation the contents of the tubes were removed into plastic cups and cells were counted in subsamples of 0.02 ml. The remaining cell mixtures were aspirated with Pasteur pipettes and placed at the bottom of Kimax screw-capped test tubes (Owens-Illinois Glass Company, Toledo, Ohio). These tubes were then incubated at 37" C for 2 h in an almost horizontal position so that the contents ran along the top three-quarters of the tube. After incubation, the tubes were gently raised to the vertical position, the medium and non-adherent cells at the bottom mixed by repeated pipetting, and the cells were recounted. The percentage adherence of cells was determined for each mixture by the formula: final count Percentage adherence = 100 x 100. initial count ~
RESULTS
The percentage of glass-adherent cells in the control mixtures (no tumour extract) ranged between 28.1 % and 93.5%, the mean being 66.6 *14.7%. These normal adherences were used as the basis for calculating the effect of added tumour extracts. For the sake of brevity, actual cell counts are not reported in full, but the calculation is explained for a single patient in Table 1. Results in Table I1 show the percentage adherence of the 60 patients' leukocytes with the three tumour extracts used. Patients are numbered and grouped in Table 11 according to the eventual clinical diagnosis. It can be seen that, in general, patients with a particular tumour had a decreased leukocyte adherence when tested with the extract corresponding to that tumour. Conversely, patients with nonmalignant conditions usually showed lower inhibitory effects of tumour extracts on their leukocytes. This relationship between cellular reactivity ( % LAI) and presence and type of tumour is further summarized in Figure 1. All 60 patients were tested with the colon carcinoma extract but only 57 with the pancreatic and 47 with the stomach carcinoma extracts.
TABLE 1 EXAMPLE OF CALCULATION OF RESULTS FOR PATIENT NO. 1 (ADENOCARCINOMA OF COLON) Tumour extract used
None Colon Pancreas Stomach
1
Initial count
A= B A B A
B A B
12,389 12,633 12,08 I 1237 1 12,809 13,021 12,757 12,912
'
Final count
"/. Adherence
3,729 2,41 I 9,797 11,154 3,497 2,851 2,564 3,603
69.9 80.9 18.9 13.3 72.7 78.1 19.9 72.1
Cells pe r pl,as read from particle counter. - A and B are duplicate mixtures.
a
~
~ % LA1 ~
75.4 16.1
78.6
75.4
0.0
76.0
-0.8
~
~
45
LA1 TEST IN GASTROINTESTINAL CANCER TABLE 11 EFFECT OF TUMOUR EXTRACTS O N LEUKOCYTE ADHERENCE AS RELATED TO EVENTUAL CLINICAL DIAGNOSIS
Patient NO.
1
2 3 4 5
6 7 8 9 10 I1 12 13 14 15
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
56 57 58 59 60 I
Age (years)
74 56 45 64 60 83 62 76 75 77 65 57 81 64 78 50 65 76 72 79 56 66 63 85 56 64 68 81 60 57 65 60 52 64 58 82 65 67 62 74 65 56 60 57 46 56 44 65 62 60 28 84 54 62 86 70 73 70
70 45
Mean % adherence with tumour extracts
sex
None
Colon
Pancreas
Stomach
M M F M M M M F F M M M M M M M M M M M M M M M M M M F M M M M M M M M F F M F M F M M M M M
75.4 82.4 78.9 64.0 69.1 65.8 71.2 73.0 86.1 73.0 77.1 65.3 88.0 61.8 58.9 83.9 56.2 83.2 86.7 69.6 72.7 66.2 68.4 73.4 44.8 78.9 75.7 60.0 71.6 71.5 50.2 49.4 55.4 81.1 48.4 86.9 46.0 28.1 48.4 47.6 67.0 64.2 65.5 29.4 68.9 57.5 62.3 72.8 56.2 92.8 73.2 89.7 72.4 93.5 55.9 78.8 44.6 54.0 43.3 59.2
16.1 67.3 48.6 30.9 50.5 53.6 48.4 45.1 67.3 48.8 46.7 66.5 89.2 68.7 53.1 75.1 51.6 81.3 86.5 74.4 82.6 64.8 59.3 73.7 57.5 77. I 70.1 50.4 77.1 63.5 60.0 57.6 53.3 80.5 48.2 16.7 51.2 30.4 51.9 57.9 67.4 68.2 60.5 33.1 67.7 60.4 67.0 74.7 58.0 87.4 66.4 86.6 66.8 79.9 66.7 72.1 50.7 48.8 43.3 62.4
75.4 78.2 78.7 66.4 60.5 73.8 70.8 90.6 79.9 84. I 85.1 57.2 91.5 48.4 63.6 75.9 59.5 77.3 ND 75.9 82.4 68.5 53.4 71.5 ND 72.1 38.7 33.8 66.3 68.5 71.5 46.0 ND 89.7 63.8 71.6 51.3 32.4 59.5 61.7 62.2 66.3 57.6 26.0 74.9 56.4 50.8 74.9 55.6 92.0 74.5 88.6 72.7 95.1 58.2 59.6 33.9 60.2 50.8 50.8
76.0 83.2 ND ND 56.7 71.3 78.9 70.0 81.8 78.7 84.2 64.2 87.1 68.4 66.0 80.3 55.2 74.7 69.9 55.4 ND 20.4 51.9 48.3 25.6 83.5 70.0 ND 68.3 70.4 61.2 70.1 58.8 63.0 52.0 ND 47.6 28.2 ND 44.1 ND 64.0 58.2 29.2 65.6 67.6 ND 67.7 ND 89.5 69.7 91.9 82.8 89.3 ND 64.5 ND ND 56.1 ND
F F
M M M M M M M F M
M M
Well differentiated. - ND = not done.
Eventual diagnosis
Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma colon Adenocarcinoma rectum Adenocarcinoma colon (removed 3 years) Adenocarcinoma colon (removed 2 years) Adenocarcinoma colon (removed 1 year) Adenocarcinoma colon (removed 1 year) Adenocarcinoma colon (removed 3 years) Adenocarcinoma colon (removed 4 years) Squamous cell carcinoma colon (secondary) Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Adenocarcinoma stomach Mucus-secreting adenocarcinoma stomach Anaplastic adenocarcinoma stomach Carcinoma pancreas Carcinoma pancreas Carcinoma pancreas Gastric ulcer Gastric ulcer Gastric ulcer Gastric ulcer Reflux gastritis Reflux gastritis Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Diverticular disease Pancreatitis Polyposis coli Duodenal ulcer Duodenal ulcer Duodenal ulcer Haemorrhoids Reflux alkaline gastritis Lymphoma Small bowel obstruction Chronic gastritis Secondary malignancy (no apparent primary) Rectal bleed; investigation negative Change in bowel habits; investigation negative Change in bowel habits; investigation negative Change in bowel habits; investigation negative Abdominal pain; investigation negative Abdominal pain; investigation negative
46
RUTHERFORD ET AL.
Adenocarcinoma of colon or rectum All the patients with this tumour (and none without) had LA1 values of 18.3% or greater. The three patients with LA1 values just under 18.3% were Nos. 23, 28 and 54, all of whom suffered from malignancies of other types (Table 11). In the group with low reactivity were six (Nos. 12-17) who had colon carcinomas removed 1-4 years previously, indicating loss of detectable immune response after removal of the antigenic stimulus. These patients were thoroughly examined and no evidence of recurrence was detected. Pancreatic carcinoma Although the number of patients diagnosed in this group was small, two of the three were clearly separated fromlthe control group by their high LAI
A
B
C
values. One patient (No. 29) who failed to give a distinctive reaction had disseminated advanced cancer. All patients without detectable pancreatic tumours had LA1 values of 23.9% or less. Two of these, with values approaching 23.9 % (Nos. 14 and 23), had histories of other malignancies. Carcinoma of stomach Of the eight patients with diagnosed stomach carcinoma, only seven appear in Figure 1 since patient No. 21 was not tested with this extract; however, he had not reacted significantly with the other extracts. Six of those tested gave high values of percentage LA1 (18.9% or higher), with slight overlap of the control group. The three patients with marginal reactivity (Nos. 5, 34 and 56) were suffering from, respectively, carcinoma of colon, reflux gastritis and abdominal pain with change in bowel habits (the last two patients are still under investigation for possible tumours). When the patients examined with each extract were arranged in rank order of LA1 reactivity, there was for each tumour type a statistically significant difference between the group with tumours and those without (p
