Int. J. Cancer: 15, 823-829 (1975)
INHIBITION OF HUMAN LEUKOCYTE MIGRATION IN AGAROSE BY KCl EXTRACTS OF CARCINOMA OF THE LUNG
A. W. BODDIE,JR., E. Carmack HOLMES, Jack A. ROTHand Donald L. MORTON Department of Surgery, School of Medicine, The Center for the Health Sciences, Los Angeles, Calif. 90027, USA; and the Veterans Hospital, Sepulreda, Calif. 91343, USA
The leukocyte migration in agarose assay recently developed by Clausen (Clausen, 1971), was used to test 22 lung cancer patients against soluble extracts of allogeneic lung cancer and allogeneic normal lung. Seventeen were inhibited to a significantly greater degree by at least one tumor extract (average migration index ( M I = 0.58) than by the corresponding normal lung extract (average M I = 0.83). DNCB-positive and D NCB-negative patients reacted with equal frequency to tumor extracts. Three patients tested against their autologous tumor and normal lung extracts were specifically inhibited by the tumor extract (average M I = 0.53) but not by the normal lung extract (average M I = 0.83). None of seven lung cancer patients tested against non-pulmonary tumor extracts was significantly inhibited (average M I = 1.1). Only 6/53 controls including patients with other tumors, patients with emphysema and age-matched non-smokers showed significant inhibition against any of the lung cancer extracts. These findings strongly suggest the presence of tumor-associated antigens in KCI-solubilized extracts of human lung cancer.
In recent years, several investigators have used the leukocyte migration inhibition assay of Serberg and Bendixen (1967) to detect tumorassociated antigens in a variety of human neoplasms including malignant melanoma and carcinoma of the breast, colon and kidney (Cochran et al., 1972, 1973; Andersen et al., 1970; McCoy et al., 1974; Bull et al., 1973; Kjaer, 1974). Efforts to further identify and purify human tumor-associated antigens have been hindered to some extent by the fact that the assay is a technically difficult one to perform and secondly by the fact that, with one exception (McCoy, 1974), most of these studies used insoluble membrane extracts which are not amenable to further purification. The leukocyte migration in agarose technique described by Clausen in 1971 has simplified the
assay (Clausen, 1971 ; Serberg and Bendixen, 1967) and has been shown to correlate well with delayed cutaneous hypersensitivity to a variety of common skin test antigens (Astor et al., 1973). Preliminary work with melanoma antigens has demonstrated the usefulness of the assay in the study of human tumor antigens (Spitler, 1974). In this study we have demonstrated cell-mediated immunity to soluble lung cancer antigens in lung cancer patients using the leukocyte migration in agarose technique. MATERIAL AND METHODS
Preparation of antigens
Soluble antigens were prepared according to the technique o f Reisfeld (Reisfeld et al., 1971; Leonard et al., 1972). Fresh tumors and control
Received : December 16, 1974.
823
BODDIE ET AL.
tissues obtained sterile from surgery or from necropsy were weighed and finely minced with a scalpel. The total number of viable cells in each specimen was determined by counting the number of cells which excluded trypan blue in a singlecell suspension of a weighed aliquot. Minced tissue was suspended in cold 3 M KCI at PH 7.4 with 1-3 x lo7 viable cells/ml and mixed for 16 to 24 h on a turning rotor at 4" C. The mixtures were centrifuged at 175,000 x g for 60 min at 4" C and the resulting supernatant was dialyzed against 200vol PBS with three changes of medium in 24 h. The dialysate was then centrifuged at 1,500x g for 15 min (in some of the later antigen preparations the dialysate was centrifuged at 136,000 x g for 15 min). The final supernatant was millipore-filtered, and its protein concentration was determined by the Biuret method. This material was used in the subsequent assays. Soluble antigen preparations with protein concentrations of 5.0 mg/ml were the most satisfactory. When mixed with leukocyte suspension in a I :10 ratio this gives a final antigen concentration of 454 yglml. More dilute preparations (227 ,ug/ml) were less reactive. More concentrated preparations sometimes reacted nonspecifically though some antigen tested in final concentrations up to 900 pg/ml showed no evidence of cellular toxicity. Soluble bacterial antigen contamination was excluded by culture of the tissue specimens and by testing the final soluble antigen preparations against 35 different antisera to common bacterial antigens in Ouchterlony gel diffusion. Aliquots of the antigen preparations were stored in liquid nitrogen at -70°C. Antigen activity did not appear to be affected by freeze-thawing. Leukocyte immobilization
Leukocyte migration in agarose was performed according to the technique of Clausen (1971). Thirty to 50 ml of blood were drawn from each patient and control into a syringe containing 3 to 5 m l of 1,000IU/ml phenol-free heparin (Bio-Heparin, Renalab, Division of Ries Biological, Los Angeles, Calif., USA). Three to 5 ml of 6 % Dextran (type 200-C mw 240,000, Sigma, St. Louis, Mo., USA) were then added and mixed thoroughly. The syringes were inverted for 45 to 60 min at 37" C to allow the red cells to sediment. The leukocyte-rich plasma was drawn off, diluted 2:l with warm Hanks' solution,
824
washed and centrifuged at 287 r g four times. The cells were counted. The percentage of white cells was determined by Wright's staining and the cells suspended to a final concentration of 2 . 2 ~ 1 0viable ~ WBCs per ml in medium 199 (Gibco, Grand Island, N.Y., USA) with 10% horse serum, 66 IU penicillin and 66 yglrnl streptomycin and 1.25 g NaNCO,/L (adjusted to PH 7.4 in a 5 % CO, atmosphere). Five /cl of test antigen, the normal tissue antigen and a saline control were added to separate test tubes, mixed with 50pI of cell suspension and allowed to incubate for 30 min at 37" C in a 5 % CO, incubator (Fig. 1). Agarose gel
Agarose gel was prepared as follows: 70mg of agarose (A-37, Indubiose, L'lndustrie Biologique FranCaise, 35, Quai du Moulin de Cage, 92 Gennevilliers, France) per plate were weighed, added to 6.5 ml of distilled deionized water, and boiled until the agarose was fully dissolved. As the agarose cooled, 0.65 ml of 10 x medium I99 without bicarbonate, 0.65 ml horse serum, and 0.0395 ml of 7.5 % sodium bicarbonate were added. Next, 6.2 ml of the mixture were poured into each Petri dish (Falcon Plastics No. 3002, Oxnard, Calif., USA) and allowed to gel, then 2.3 mm wells were cut in the agrose gel. Seven /'I aliquots of either the saline or the test antigen cell suspension were added to triplicate wells. The plates were placed in moist chambers in a 5 % CO, atmosphere, and allowed to incubate for 18 h. Measurement of migration inhibition
After 18 h the leukocytes had migrated into the agarose-plastic interface. The cells were heatfixed to the plates by floating them in water at 87" C for 2 min and the agarose was removed by inverting the plates in hot water. The plates were dried. The cell migration was usually concentric. The average diameter of migration was determined by vernier measurement of two diameters at right angles.The area of migration for each well was calculated by the formula A nrS.Triplicate values were averaged and a migration index MI
=
average area of migration cells i antigen average area of migration cellst saline
was then calculated.
Ed-
LEUKOCYTE MIGRATION IN LUNG CARCINOMA
3 M KCI
LUNG
MINCE
u
’--0 .. .,^, I M KLI
v
c3
-b
175,000 x G 60 min
DIALY Z E I
I
NORMAL ANTIGEN
1,500 x G
/ ’*
EQUALIZE
PROTEIN CONCENTRATION
hr
J
5 X Soline
t
t 50
1 Buffy
t
Coot Leukocyte 2.2 x 108ml
FIGURE 1
Technique used for extraction of lung-tumor antigen in human leukocyte migration in ugurose assay.
Data analysis
The average coefficient of variance (standard deviationlmean) for the migration areas of these samples was 0.14. Analysis of our most recent work after gaining experience with the assay shows a coefficient of variance of only 0.076.
Analyses of our data showed that migration indices in the 0.70 to 0.80 range were not always statistically significant whereas indices of 0.7 or below were always significant (pGO.05) when the mean areas of migration for the antigen-treated and the control leukocytes were compared by the Patient selection t-test for unequal variance. For this reason and All patients had histologically confirmed lung because there was a clear division of reactivity cancer. Three patients had adenocarcinoma; the with migration indices of most lung cancer other 19 had either squamous-cell carcinoma o r patients below this value and of most controls undifferentiated carcinoma of the lung. Since above, we selected 1.3) was found in seven patients. Enhancement appeared to be a less specific phenomenon than
inhibition since it occurred with equal frequency to extracts of normal lung (in two patients) and to non-pulmonary tumor antigens (in three patients). Only two patients were specifically enhanced by a lung-tumor antigen and not by the corresponding control lung antigen. The significance of enhancement of migration at present is not clear. Some investigators consider it as an artifact of testing in allogeneic systems (Cochran et al., 1974) whereas others consider it as evidence of weak sensitization (Soborg, 1968; Federlin et a/., 1971). In a small number of patients tested serially we noted a shift from inhibition to enhancement with progressive or recurrent disease. In one instance this was associated with the development of skin test anergy to tumor antigens. Confirmation of these results must await further serial testing. Efforts in our laboratory are presently directed toward further purification and characterization of these antigens.
INHIBITION DE LA MIGRATION DES LEUCOCYTES HUMAINS DANS L'AGAROSE PAR DES EXTRAITS DE CANCER PULMONAIRE SOLUBILISES AU KCL Les auteurs ont utilist le test de migration des leucocytes dans I'agarose, rtcemment mis au point par Clausen (Clausen, 1971), pour tester 22 cas de cancer pulmonaire contre des extraits solubles de cancer pulmonaire allogtnique et de poumon allogtnique normal. Dans 17 cas, I'inhibition a ttP nettement plus marqute avec un extrait tumoral au moins (indice de migration moyen ( I M ) = 0.58) qu'avec I'extrait de poumon normal correspondant ( I M moyen = 0.83). Les sujets DNCB-positifs et ntgatifs ont rtagi aux extraits tumoraux avec une frfquence tgale. Chez trois malades, on a observP une inhibition sptcifique avec un extrait de tumeur autologue ( I M moyen = 0.53), mais pas avec un extrait de poumon normal (IM moyen = 0.83). On n'a nott aucune inhibition significative dans 7 cas de cancer pulmonaire testts contre des extraits de tumeur non pulmonaire ( I M moyen = 1.1). Avec les extraits de cancer pulmonaire, I'inhibition n'a e'tk notable que chez 6 des 53 ttmoins - dont des malades atteints d'autres tumeurs, des cas d'emphystme et des non fumeurs du m6me dge. Ces constatations confirment l'hypothtse de la prtsence d'antigtnes associts a la tumeur dans les extraits de cancer pulmonaire humain solubilists au KCI. REFERENCES
ANDERSEN,V., BJERRUM,O., BENDIXEN,G., SCHRODT, T., and DISSING, I., Effect of autologous
mammary tumor extracts on human leukocyte migration in vitro. Int. J . Cancer, 5, 357-363 (1970). A ~ S . H., ~ sPITLER, ~ ~ L. E., , F ~ 0.L,, ~ ~
F ~ H. H., H~~~~ ~ leukocyte ~ migration ~ in ugarose using four antigens in correlation with skin reactivity. J. Immunol., 110, 1174-1179 (1973).
828
AUERBACH, O., STOUT,A. P., HAMMOND,E. C., and GARFINKEL, L., Multiple primary bronchial carcinoma. Cancer, 20, 699-705 (1967). BRUGAROLAS, A., TAKITA, H., and VINCE NT , R. G . , ~ Skin, test in bronchogenic carcinoma. 11. Its value in 5, 319-325 ~ differential ~ diagnosis.~ J. Surg. Oncul., ~ , BULL,D. M., LEIBACH, .I.R., WILLIAMS, M . A., and
LEUKOCYTE MIGRATION IN LUNG CARCINOMA
HELMS,R. A., Immunity to colon cancer assessed by antigen-induced inhibition of mixed mononuclear cell migration. Science, 181,957-959 (1973). CLAUSEN,J. E., Tuberculin-induced migration inhibition of human peripheral leukocytes in agarose medium. Acta allerg., 26, 56-80 (1971). COCHRAN,A, J., GRANT,R. M., SPILG, W. G., MACKIE,R. M., Ross, C. E., HOGLE,D. E., and RUSSELL,J. M., Sensitization to tumor-associated antigens in human breast carcinoma. Int. J. Cancer, 14, 19-25 (1974). COCHRAN, A. J., MACKIE,R. M., THOMAS,C. E., GRANT,R. M., CAMERON-MOWAT, D. E., and SPILG, W. G. S., Cellular immunity to breast cancer and malignant melanoma. Brit. J . Cancer, 28, Supplement I, 77-81 (1973). COCHRAN, A. J., SPILG,W. G. S., MACKIE,R. N., and THOMAS,C. E., Post-operative depression of tumor-directed cell-mediated immunity in patients with malignant disease. Brit. med. J., IV, 67 (1972). EILBER, F. R., and MORTON,D. L., Impaired immunologic reactivity and recurrence following cancer surgery. Cancer, 25, 362-367 (1970). FEDERLIN,K., MAINI,R. N., RUSSELL, D. S., and DUMONDE, D. C., A micromethod for peripheral leukocyte migration in tuberculin sensitivity. J . clin. Purh., 24, 533-536 (1971). HOLLINSHEAD, A. C., STEWART,T. H. M., and HERBERMAN, R. B., Delayed hypersensitivity reactions to soluble membrane antigen of human malignant lung cells. J . nut. Cancer Inst., 52, 327-337 (1974). KJAER,M., In vitro demonstration of cellular hypersensitivity to tumor antigens by means of the leukocyte migration technique in patients with renal carcinoma. Europ. J . Cancer, 10, 523-531 (1974). KRANT,M. J., MANSKOPT,G., BRANDRUP,C. S., and MADOFF,M. A., Immunological alterations in bronchogenic carcinoma. Cancer, 21,623 (1968). LEONARD, E. J., MELTZER,S. M., BORSOS,T., and RAPP, H. J., Properties of tumor-specific antigens
solubilized by hypertonic potassium chloride. Nat. Cancer Inst. Monogr., 35, 129-134 (1972).
McCoy, J. L., JEROME,L. F., DEAN,J. H., CANNON, G. B., ALFORD,T. C., DOERING, T., and HEBERMAN, R. B., Inhibition of leukocyte migration by tumor-associated antigens in soluble extracts of human breast carcinoma. J. nut. Cancer Inst., 53, 11-17 (1974). REISFELD, R. A., PELLEGRINO,M. A., and KAHAN,B. D., Salt extraction of soluble HLA antigens. Science, 172, 1134-1136 (1971). SBBERG, M., In vifro migration of human autogenous and allogeneicleukocytes in cellular hypersensitivity. Acra med. scand., 184, 135-139 (1968).
S ~ B E RM., G , and BENDIXEN, G., Human lymphocyte migration as a parameter of hypersensitivity. Acra med. scand., 181, 247-256 (1967). SPITLER, L. E., Cellular immunity to melanoma antigens in malignant melanoma. Clin. Res., 21 (2), 654 (1974). SPITLER, L. E., LITTOY, F., SAGEBIEL,R. W., BLEVIS,M. S., and EPSTEIN,W. L., Malignant melanoma, cellular immunity to melanoma antigens. In preparation. TAKITA, H., and BRUGAROLAS, A., Skin tests in bronchogenic carcinoma. I. Correlation of the immunologic status and the extent of disease. J. Surg. Oncol., 5, 315-318 (1973). WELLS,S. A., BURDICK,J. F., CHRISTIANSEN, C., KETCHAM, A. S.,and ADKINS,P. C., Demonstration of tumor-associated delayed cutaneous hypersensitivity reactions in patients with lung cancer and in patients with carcinoma of the cervix. Nut. Cancer Inst. Monogr., 37, 197-203 (1973). WILSON,R. E., ALEXANDER, P., ROSENBERG, S. A., and SIMMONS, R. L., Horizons in tumor immunology. Arch. Surg., 109, 17-29 (1974). WOLBERG,W. H., Inhibition of leukocyte migration by human tumors; effect on patient survival. Arch. Surg., 109, 211-214 (1974).
829
INHIBITION OF HUMAN LEUKOCYTE MIGRATION IN AGAROSE BY KCl EXTRACTS OF CARCINOMA OF THE LUNG
A. W. BODDIE,JR., E. Carmack HOLMES, Jack A. ROTHand Donald L. MORTON Department of Surgery, School of Medicine, The Center for the Health Sciences, Los Angeles, Calif. 90027, USA; and the Veterans Hospital, Sepulreda, Calif. 91343, USA
The leukocyte migration in agarose assay recently developed by Clausen (Clausen, 1971), was used to test 22 lung cancer patients against soluble extracts of allogeneic lung cancer and allogeneic normal lung. Seventeen were inhibited to a significantly greater degree by at least one tumor extract (average migration index ( M I = 0.58) than by the corresponding normal lung extract (average M I = 0.83). DNCB-positive and D NCB-negative patients reacted with equal frequency to tumor extracts. Three patients tested against their autologous tumor and normal lung extracts were specifically inhibited by the tumor extract (average M I = 0.53) but not by the normal lung extract (average M I = 0.83). None of seven lung cancer patients tested against non-pulmonary tumor extracts was significantly inhibited (average M I = 1.1). Only 6/53 controls including patients with other tumors, patients with emphysema and age-matched non-smokers showed significant inhibition against any of the lung cancer extracts. These findings strongly suggest the presence of tumor-associated antigens in KCI-solubilized extracts of human lung cancer.
In recent years, several investigators have used the leukocyte migration inhibition assay of Serberg and Bendixen (1967) to detect tumorassociated antigens in a variety of human neoplasms including malignant melanoma and carcinoma of the breast, colon and kidney (Cochran et al., 1972, 1973; Andersen et al., 1970; McCoy et al., 1974; Bull et al., 1973; Kjaer, 1974). Efforts to further identify and purify human tumor-associated antigens have been hindered to some extent by the fact that the assay is a technically difficult one to perform and secondly by the fact that, with one exception (McCoy, 1974), most of these studies used insoluble membrane extracts which are not amenable to further purification. The leukocyte migration in agarose technique described by Clausen in 1971 has simplified the
assay (Clausen, 1971 ; Serberg and Bendixen, 1967) and has been shown to correlate well with delayed cutaneous hypersensitivity to a variety of common skin test antigens (Astor et al., 1973). Preliminary work with melanoma antigens has demonstrated the usefulness of the assay in the study of human tumor antigens (Spitler, 1974). In this study we have demonstrated cell-mediated immunity to soluble lung cancer antigens in lung cancer patients using the leukocyte migration in agarose technique. MATERIAL AND METHODS
Preparation of antigens
Soluble antigens were prepared according to the technique o f Reisfeld (Reisfeld et al., 1971; Leonard et al., 1972). Fresh tumors and control
Received : December 16, 1974.
823
BODDIE ET AL.
tissues obtained sterile from surgery or from necropsy were weighed and finely minced with a scalpel. The total number of viable cells in each specimen was determined by counting the number of cells which excluded trypan blue in a singlecell suspension of a weighed aliquot. Minced tissue was suspended in cold 3 M KCI at PH 7.4 with 1-3 x lo7 viable cells/ml and mixed for 16 to 24 h on a turning rotor at 4" C. The mixtures were centrifuged at 175,000 x g for 60 min at 4" C and the resulting supernatant was dialyzed against 200vol PBS with three changes of medium in 24 h. The dialysate was then centrifuged at 1,500x g for 15 min (in some of the later antigen preparations the dialysate was centrifuged at 136,000 x g for 15 min). The final supernatant was millipore-filtered, and its protein concentration was determined by the Biuret method. This material was used in the subsequent assays. Soluble antigen preparations with protein concentrations of 5.0 mg/ml were the most satisfactory. When mixed with leukocyte suspension in a I :10 ratio this gives a final antigen concentration of 454 yglml. More dilute preparations (227 ,ug/ml) were less reactive. More concentrated preparations sometimes reacted nonspecifically though some antigen tested in final concentrations up to 900 pg/ml showed no evidence of cellular toxicity. Soluble bacterial antigen contamination was excluded by culture of the tissue specimens and by testing the final soluble antigen preparations against 35 different antisera to common bacterial antigens in Ouchterlony gel diffusion. Aliquots of the antigen preparations were stored in liquid nitrogen at -70°C. Antigen activity did not appear to be affected by freeze-thawing. Leukocyte immobilization
Leukocyte migration in agarose was performed according to the technique of Clausen (1971). Thirty to 50 ml of blood were drawn from each patient and control into a syringe containing 3 to 5 m l of 1,000IU/ml phenol-free heparin (Bio-Heparin, Renalab, Division of Ries Biological, Los Angeles, Calif., USA). Three to 5 ml of 6 % Dextran (type 200-C mw 240,000, Sigma, St. Louis, Mo., USA) were then added and mixed thoroughly. The syringes were inverted for 45 to 60 min at 37" C to allow the red cells to sediment. The leukocyte-rich plasma was drawn off, diluted 2:l with warm Hanks' solution,
824
washed and centrifuged at 287 r g four times. The cells were counted. The percentage of white cells was determined by Wright's staining and the cells suspended to a final concentration of 2 . 2 ~ 1 0viable ~ WBCs per ml in medium 199 (Gibco, Grand Island, N.Y., USA) with 10% horse serum, 66 IU penicillin and 66 yglrnl streptomycin and 1.25 g NaNCO,/L (adjusted to PH 7.4 in a 5 % CO, atmosphere). Five /cl of test antigen, the normal tissue antigen and a saline control were added to separate test tubes, mixed with 50pI of cell suspension and allowed to incubate for 30 min at 37" C in a 5 % CO, incubator (Fig. 1). Agarose gel
Agarose gel was prepared as follows: 70mg of agarose (A-37, Indubiose, L'lndustrie Biologique FranCaise, 35, Quai du Moulin de Cage, 92 Gennevilliers, France) per plate were weighed, added to 6.5 ml of distilled deionized water, and boiled until the agarose was fully dissolved. As the agarose cooled, 0.65 ml of 10 x medium I99 without bicarbonate, 0.65 ml horse serum, and 0.0395 ml of 7.5 % sodium bicarbonate were added. Next, 6.2 ml of the mixture were poured into each Petri dish (Falcon Plastics No. 3002, Oxnard, Calif., USA) and allowed to gel, then 2.3 mm wells were cut in the agrose gel. Seven /'I aliquots of either the saline or the test antigen cell suspension were added to triplicate wells. The plates were placed in moist chambers in a 5 % CO, atmosphere, and allowed to incubate for 18 h. Measurement of migration inhibition
After 18 h the leukocytes had migrated into the agarose-plastic interface. The cells were heatfixed to the plates by floating them in water at 87" C for 2 min and the agarose was removed by inverting the plates in hot water. The plates were dried. The cell migration was usually concentric. The average diameter of migration was determined by vernier measurement of two diameters at right angles.The area of migration for each well was calculated by the formula A nrS.Triplicate values were averaged and a migration index MI
=
average area of migration cells i antigen average area of migration cellst saline
was then calculated.
Ed-
LEUKOCYTE MIGRATION IN LUNG CARCINOMA
3 M KCI
LUNG
MINCE
u
’--0 .. .,^, I M KLI
v
c3
-b
175,000 x G 60 min
DIALY Z E I
I
NORMAL ANTIGEN
1,500 x G
/ ’*
EQUALIZE
PROTEIN CONCENTRATION
hr
J
5 X Soline
t
t 50
1 Buffy
t
Coot Leukocyte 2.2 x 108ml
FIGURE 1
Technique used for extraction of lung-tumor antigen in human leukocyte migration in ugurose assay.
Data analysis
The average coefficient of variance (standard deviationlmean) for the migration areas of these samples was 0.14. Analysis of our most recent work after gaining experience with the assay shows a coefficient of variance of only 0.076.
Analyses of our data showed that migration indices in the 0.70 to 0.80 range were not always statistically significant whereas indices of 0.7 or below were always significant (pGO.05) when the mean areas of migration for the antigen-treated and the control leukocytes were compared by the Patient selection t-test for unequal variance. For this reason and All patients had histologically confirmed lung because there was a clear division of reactivity cancer. Three patients had adenocarcinoma; the with migration indices of most lung cancer other 19 had either squamous-cell carcinoma o r patients below this value and of most controls undifferentiated carcinoma of the lung. Since above, we selected 1.3) was found in seven patients. Enhancement appeared to be a less specific phenomenon than
inhibition since it occurred with equal frequency to extracts of normal lung (in two patients) and to non-pulmonary tumor antigens (in three patients). Only two patients were specifically enhanced by a lung-tumor antigen and not by the corresponding control lung antigen. The significance of enhancement of migration at present is not clear. Some investigators consider it as an artifact of testing in allogeneic systems (Cochran et al., 1974) whereas others consider it as evidence of weak sensitization (Soborg, 1968; Federlin et a/., 1971). In a small number of patients tested serially we noted a shift from inhibition to enhancement with progressive or recurrent disease. In one instance this was associated with the development of skin test anergy to tumor antigens. Confirmation of these results must await further serial testing. Efforts in our laboratory are presently directed toward further purification and characterization of these antigens.
INHIBITION DE LA MIGRATION DES LEUCOCYTES HUMAINS DANS L'AGAROSE PAR DES EXTRAITS DE CANCER PULMONAIRE SOLUBILISES AU KCL Les auteurs ont utilist le test de migration des leucocytes dans I'agarose, rtcemment mis au point par Clausen (Clausen, 1971), pour tester 22 cas de cancer pulmonaire contre des extraits solubles de cancer pulmonaire allogtnique et de poumon allogtnique normal. Dans 17 cas, I'inhibition a ttP nettement plus marqute avec un extrait tumoral au moins (indice de migration moyen ( I M ) = 0.58) qu'avec I'extrait de poumon normal correspondant ( I M moyen = 0.83). Les sujets DNCB-positifs et ntgatifs ont rtagi aux extraits tumoraux avec une frfquence tgale. Chez trois malades, on a observP une inhibition sptcifique avec un extrait de tumeur autologue ( I M moyen = 0.53), mais pas avec un extrait de poumon normal (IM moyen = 0.83). On n'a nott aucune inhibition significative dans 7 cas de cancer pulmonaire testts contre des extraits de tumeur non pulmonaire ( I M moyen = 1.1). Avec les extraits de cancer pulmonaire, I'inhibition n'a e'tk notable que chez 6 des 53 ttmoins - dont des malades atteints d'autres tumeurs, des cas d'emphystme et des non fumeurs du m6me dge. Ces constatations confirment l'hypothtse de la prtsence d'antigtnes associts a la tumeur dans les extraits de cancer pulmonaire humain solubilists au KCI. REFERENCES
ANDERSEN,V., BJERRUM,O., BENDIXEN,G., SCHRODT, T., and DISSING, I., Effect of autologous
mammary tumor extracts on human leukocyte migration in vitro. Int. J . Cancer, 5, 357-363 (1970). A ~ S . H., ~ sPITLER, ~ ~ L. E., , F ~ 0.L,, ~ ~
F ~ H. H., H~~~~ ~ leukocyte ~ migration ~ in ugarose using four antigens in correlation with skin reactivity. J. Immunol., 110, 1174-1179 (1973).
828
AUERBACH, O., STOUT,A. P., HAMMOND,E. C., and GARFINKEL, L., Multiple primary bronchial carcinoma. Cancer, 20, 699-705 (1967). BRUGAROLAS, A., TAKITA, H., and VINCE NT , R. G . , ~ Skin, test in bronchogenic carcinoma. 11. Its value in 5, 319-325 ~ differential ~ diagnosis.~ J. Surg. Oncul., ~ , BULL,D. M., LEIBACH, .I.R., WILLIAMS, M . A., and
LEUKOCYTE MIGRATION IN LUNG CARCINOMA
HELMS,R. A., Immunity to colon cancer assessed by antigen-induced inhibition of mixed mononuclear cell migration. Science, 181,957-959 (1973). CLAUSEN,J. E., Tuberculin-induced migration inhibition of human peripheral leukocytes in agarose medium. Acta allerg., 26, 56-80 (1971). COCHRAN,A, J., GRANT,R. M., SPILG, W. G., MACKIE,R. M., Ross, C. E., HOGLE,D. E., and RUSSELL,J. M., Sensitization to tumor-associated antigens in human breast carcinoma. Int. J. Cancer, 14, 19-25 (1974). COCHRAN, A. J., MACKIE,R. M., THOMAS,C. E., GRANT,R. M., CAMERON-MOWAT, D. E., and SPILG, W. G. S., Cellular immunity to breast cancer and malignant melanoma. Brit. J . Cancer, 28, Supplement I, 77-81 (1973). COCHRAN, A. J., SPILG,W. G. S., MACKIE,R. N., and THOMAS,C. E., Post-operative depression of tumor-directed cell-mediated immunity in patients with malignant disease. Brit. med. J., IV, 67 (1972). EILBER, F. R., and MORTON,D. L., Impaired immunologic reactivity and recurrence following cancer surgery. Cancer, 25, 362-367 (1970). FEDERLIN,K., MAINI,R. N., RUSSELL, D. S., and DUMONDE, D. C., A micromethod for peripheral leukocyte migration in tuberculin sensitivity. J . clin. Purh., 24, 533-536 (1971). HOLLINSHEAD, A. C., STEWART,T. H. M., and HERBERMAN, R. B., Delayed hypersensitivity reactions to soluble membrane antigen of human malignant lung cells. J . nut. Cancer Inst., 52, 327-337 (1974). KJAER,M., In vitro demonstration of cellular hypersensitivity to tumor antigens by means of the leukocyte migration technique in patients with renal carcinoma. Europ. J . Cancer, 10, 523-531 (1974). KRANT,M. J., MANSKOPT,G., BRANDRUP,C. S., and MADOFF,M. A., Immunological alterations in bronchogenic carcinoma. Cancer, 21,623 (1968). LEONARD, E. J., MELTZER,S. M., BORSOS,T., and RAPP, H. J., Properties of tumor-specific antigens
solubilized by hypertonic potassium chloride. Nat. Cancer Inst. Monogr., 35, 129-134 (1972).
McCoy, J. L., JEROME,L. F., DEAN,J. H., CANNON, G. B., ALFORD,T. C., DOERING, T., and HEBERMAN, R. B., Inhibition of leukocyte migration by tumor-associated antigens in soluble extracts of human breast carcinoma. J. nut. Cancer Inst., 53, 11-17 (1974). REISFELD, R. A., PELLEGRINO,M. A., and KAHAN,B. D., Salt extraction of soluble HLA antigens. Science, 172, 1134-1136 (1971). SBBERG, M., In vifro migration of human autogenous and allogeneicleukocytes in cellular hypersensitivity. Acra med. scand., 184, 135-139 (1968).
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