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Delayed cutaneous hypersensitivity reactions to tumor cell antigens and to nonspecific antigens
Delayed cutaneous hypersensitivity reactions to tumor cell antigens and to nonspecific antigens Prognostic significance in patients with lung cancer Samuel A. Wells, Jr., M.D. * (by invitation), James F. Burdick, M.D. * (by invitation), William L. Joseph, M.D. ** (by invitation), Christine L. Christiansen, B.S.* (by invitation), Walter G. Wolfe, M.D.*** (by invitation), and Paul C. Adkins, M.D., ** Bethesda, Md., Washington, D. C. and Durham, N. C.
A
decreased cutaneous reactivity to standard microbial antigens has been demonstrated in patients with Hodgkin's disease and in patients with carcinoma. In the latter group, however, anergy seems to be mostly confined to those patients who are generally debilitated.': ~ Eilber and Morton" found that patients with solid tumors could be sensitized topically to 2,4-dinitrochlorobenzene (DNCB) much less frequently than normal control subjects. These investigators also noted a positive correlation between cutaneous reactivity to DNCB and prognosis. Tumor-associated immune mechanisms are most frequently demonstrated by in vitro Read at the Fifty-third Annual Meeting of The American Association for Thoracic Surgery. Dallas, Texas, April 16, 17. and 18, 1973. "Tumor Immunology Section, Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. ""Department of Surgery, The George Washington University School of Medicine. Washington, D. C. """ Department of Surgery, Center, Durham. N. C.
Duke
University
Medical
techniques in which immune lymphocytes or serum antibody and tumor target cells are used. Some investigators have sought to demonstrate tumor immunity in vivo by evaluating the host's rejection of live tumor cells- 5 or by the elicitation of delayed cutaneous hypersensitivity reactions (DCHR) to tumor cell extracts. 6 , ; Regarding the latter technique, Oren and Herberman" have recently stressed the importance of using sterile tissue extracts of low protein concentration and of obtaining histologic confirmation of clinically positive skin test sites. In the present study we have evaluated 100 patients with lung disease. Seventy-five of these patients had cancer and 25 had benign lesions. Skin tests to common microbial antigens were conducted in all patients, and the sensitivity of each subject to DNCB was evaluated. In 18 patients, skin tests were done with membrane antigen extracts (MAE) of autologous or allogeneic tumor and control tissues. 557
55 8
The Journal of Thoracic and Cardiovoscular Surgery
Wells et al.
Subjects and methods Patients. One hundred patients, most of whom were from the Division of Thoracic and Cardiovascular Surgery at the George Washington University Hospital, were the subjects of this study. Sixty-seven patients had primary lung cancer. In 43 the diagnosis was squamous cell carcinoma, in 20 the diagnosis was adenocarcinoma, and in 4 the tumors were of other histologic types. In 8 patients the cancer was metastatic to the lung from other organs. The 25 control patients had been subjected to thoracotomy for cardiac or benign lung disease. The average age of the patients with malignant disease was 57.8 years, while the average age of those with nonmalignant disease was 39.5 years. All of the patients with malignant disease were ambulatory and none had lost more than 10 per cent of body weight since the onset of the present illness. No patients were subjected to skin tests sooner than 2 weeks after the operation, and none had received x-ray therapy or chemotherapy for 2 months prior to skin testing. Seventytwo of the 75 cancer patients were followed for 1 year from the time of skin testing. Test antigens. Standard microbial antigens. All patients were tested intradermally with 0.1 ml. each of three standard antigens: mumps antigen (Eli Lilly and Company, Indianapolis, Ind.), Candida (Dermatophytin 0 1: 100, Hollister-Stier Laboratories, Spokane, Wash.) , and streptokinase-streptodornase (Varidase, 40 units/ 10 units per milliliter, Lederle Laboratories Division, American Cyanamid Company, Pearl River, N. Y.). Tests were read at 48 hours by measurement of perpendicular diameters with vernier calipers. Induration greater than 5 mm. average diameter was considered positive. All patients were sensitized to DNCB by the technique described by Eilber and Morton" and modified by Catalona. D DNCB, 2,000 and 50 mcg., was applied topically at respective sites on the upper inner arm and the volar surface of the forearm. The sites were evaluated in 14 days. If a spontaneous flare of induration
and redness was present at both test sites the patient's reaction was called 4+; if only the 2,000 meg. site reacted, it was called 3+. If no reactivity occurred, a challenged dose of 50 meg. of DNCB was applied at a separate site on the forearm and evaluated at 48 hours. Marked redness and induration at the challenge site was called a 2+ reaction, and a weak reaction was designated 1+. When none of the test sites developed induration the patient was described as being DNCB negative. Preparation of the membrane extracts. Sterile lung tumors were obtained from the George Washington University Hospital operating room. A specimen of normal lung distant from the cancer, as well as 60 to 100 ml. of heparinized blood, were also obtained. Leukocytes were separated from whole blood in 2 per cent methyl cellulose as described by Burger."? MAE of tumor cells and control tissues were prepared by freeze-thawing and low ionic strength extraction, a method described by Davies" and Mann-" and modified by Oren and Herberman." A crude membrane pellet obtained by centrifuging the pooled extracts at 105,000 g for 1 hour was resuspended in saline and used for skin testing. Protein concentration of the MAE was measured by the method of Lowry and associates. '" All test MAE were adjusted to a protein concentration of 2.5 mg. per milliliter. Each test injection contained 0.1 ml. or 0.25 mg. of protein. Sterility of the final extracts was tested by culture on blood agar plates and in thioglycolate broth. Only sterile MAE were used. Antigen extracts for allogeneic testing were negative for the presence of hepatitis-associated antigen as tested by counterelectrophoresis.'' All MAE skin tests were read at 48 hours, and most positive skin test sites were biopsied with a 2 mm. skin drill, fixed in 10 per cent formalin, sectioned, and stained with hematoxylin and eosin.
Results All but 1 of the 25 patients with benign lung disease and all but 2 of the 75 patients
Volume 66 Number 4 October, 1973
Delayed cutaneous hypersensitivity reactions
with lung cancer reacted positively to at least one of the three standard microbial antigens. There was no decreased reactivity in the cancer patients either quantitatively or qualitatively. Such was not the case with DNCB sensitization, however, for cutaneous reactivity was clearly reduced in patients with malignant disease. Of the 25 control patients, 24 (96 per cent) were able to be sensitized to DNCB. On the other hand, in the lung cancer group, only 39 of 75 (52 per cent) were able to be sensitized, a highly significant (p < 0.005) difference. If one compares DNCB reactivity (36/59 = 69 per cent) in lung cancer patients with resectable disease to reactivity in control patients, the difference is significant (p < 0.01). Furthermore, if one compares the incidence of DNCB reactivity in the group with resectable cancer to that in patients with nonresectable cancer (3/16 = 19 per cent), the difference is again significant (p < 0.01). Also, if patients are grouped according to the histologic type of tumor, it is found (Table I) that the proportion of nonresponders is significantly greater among patients with squamous cell carcinoma (22/43) than among the patients with adenocarcinoma (5/20) (p < 0.01). Seventy-two of the 75 lung cancer patients were followed for 1 year, at which time the development of recurrent disease or death was correlated with initial DNCB reactivity. As can be seen in Table II, of the 36 cancer patients originally nonresponsive to DNCB, 29 (81 per cent) had died or had developed recurrent disease. Conversely, of the 36 cancer patients (39 were in this group initially but 3 were lost to follow-up) originally responsive to DNCB, 14 (39 per cent) had died or developed recurrent disease. The difference between the incidence of death or recurrence in these two groups is highly significant (p < 0.005). In Table III is shown the reactivity of lung cancer patients to control and tumor MAE. Ten of 18 patients developed DCHR to lung cancer MAE. Eight of these 10 cancer patients were tested to MAE prepared from
559
Table I. Reactivity to dinitrochlorobenzene (DNCB) _ _ _ _ _ _ _ _I"'-P_o_s_it_iv_e_1 Negative I Totals Squamous cell carcinoma Adenocarcinoma Totals
21 15
22
36
27
5
43
20 63
Legend: The table provides a comparison of responses to DNCB in patients with squamous cell carcinoma and in patients with adenocarcinoma. A total of 49 per cent of the patients with squamous cell carcinoma did not respond to DNCB. as compared to 25 per cent of those with adenocarcinoma (p < 0.01).
Table II. Reactivity
10
dinitrochlorobenzene
(DNCB) _ _ _ _ _ _ _1
Recurrence or death No recurrence Totals
Posith'e I Negative I Totals 14 22 36
29 7 36
43 29 72
Legend: The table provides a comparison at I year of the incidence of death and/or recurrence in responders and nonresponders to DNCB. The rate of recurrence or death in nonresponders was 81 per cent, as compared to 39 per cent in responders (p < 0.005).
autologous tumors, whereas the remammg 2 were tested with allogeneic MAE from tumors of their histologic type. Of the 10 patients demonstrating DCHR to tumor MAE, 4 also reacted positively to leukocyte MAE and 1 to MAE of normal lung. Positive reactions to autologous tumor MAE occurred in patients with squamous cell carcinoma as well as those with adenocarcinoma. Eight months after testing to MAE, 7 of the 8 patients who reacted to autologous MAE were alive with no evidence of recurrence, while only 3 of 8 patients with negative DCHR to tumor MAE had not developed a recurrent lesion. At 12 months, however, only 3 patients from each group were alive with no evidence of recurrence. Discussion
In the present study we found that patients with lung cancer did not have cutaneous anergy to standard microbial antigens. Our findings are in agreement with those of Solowey and Rapaport" and Lamb and coinvestigators.' Krant and associates"
The Journal of
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Wells et al.
Thoracic and Cardiovascular Surgery
Table III. Delayed skin reactivity to tumor and normal tissue membrane extracts in patients with lung cancer Patient C. S. R. D. 1. N. 1. H. C. H. M. S. J. S. E. K. 1. G. O. C.
V. V.
R. E. E. E. H. C. C.
E. F. T. Z. H. D.
B.
Normal I Leukocyte lung
ICommon antigens + + + + + + + + + + + + + + + + +
N.T. + +
N.T.
+
+
+
+ +*
-*
+ +*
+ + + + +*
+ -*
NT. -*
-*
-*
+ -*
Legend: N.T .• Not tested. + = Positive. - = Negative . • Denotes reactivity to allogeneic preparation.
in a previous study of lung cancer patients, found a decreased cutaneous reactivity to tuberculin. Many of their patients, however, were receiving x-ray therapy or chemotherapy or were in a terminal state at the time of skin testing, which perhaps accounts for the negative cutaneous reactivity. Krant also found a decreased reactivity to DNCB in the lung cancer patients studied. The lung cancer patients in our study reacted to standard microbial antigens, but reactivity to DNCB was markedly decreased; this was especially true in those patients with nonresectable disease. It was also found that initial reactivity to DNCB correlated well with recurrence and survival at 1 year. These findings are in agreement with those of Eilber and Morton," who first suggested a positive correlation between poor prognosis and negative DNCB response in patients with solid tumors. DNCB seems to be an especially valuable agent for assessing a patient's immunological responsiveness. Reactivity to common microbial antigens represents immunological recall to a prior sensitization but gives little
information regarding the body's competence to respond immunologically to a new immunogen. The elicitation of DCHR to autologous and allogeneic tumor MAE in 10 of 18 lung cancer patients supports the concept of host cellular immunity to tumor cell antigens. It was somewhat surprising that 5 of the 18 patients reacted to MAE of autologous or allogeneic leukocytes, because the test materials were bacteriologically sterile and the MAE contained a relatively low protein concentration. Oren and Herberman" found that 10 per cent of normal individuals had positive DCHR to autologous leukocyte MAE when the protein concentration was 0.33 mg. per 0.1 ml. or less. This reactivity could represent true autoimmunity or alternately the presence of delayed hypersensitivity mediators in the leukocyte membrane preparations. In the present study, we found positive DCHR to normal lung MAE in only 1 patient. The normal lung antigen had been added as an organ-specific control, and we had actually expected to see a greater incidence of reactivity. The fact that reactivity rarely occurred with MAE of autologous normal lung tissue is perhaps indicative of unique properties of the leukocyte preparation used. This could be related to the presence of soluble delayed hypersensitivity mediators attached to or associated with the lymphocyte MAE that were pelleted during the high-speed centrifugation; conversely, it could be related to substances released from polymorphonuclear leukocytes which were present at the beginning of the leukocyte extraction procedure. Although positive DCHR to tumor MAE seemed to correlate with a favorable prognosis at 8 months, such was not the case when patients were evaluated 1 year after testing. This by no means indicates that cutaneous reactivity to tumor MAE is not useful in evaluating tumor-associated immune reactions. Conversely, Fass,> Levinthal," and their colleagues have found that positive cutaneous reactivity to tumor MAE is associated with a favorable prognosis. Furthermore, Hersh'" has found that the assessment
Volume 66 Number 4
Delayed cutaneous hypersensitivity reactions
561
October, 1973
of a combination of in vivo and in vitro immunological parameters, performed serially, has been especially helpful in the clinical management of leukemia patients. We are currently beginning an immunotherapy study of patients with operable lung cancer in which we will use the immunological techniques discussed in this paper. Summary
Oncogenesis is favored by an environment of depressed immunity, but there are few studies in man which correlate both general immunological status and reactivity to tumor-associated antigens with the patient's clinical course. DCHR to microbial antigens (mumps, Candida, and streptokinase-streptodornase) and to a previously unencountered antigen, DNCB, were evaluated in 100 ambulatory patients, 75 with lung cancer and 25 with benign lung disease. Eighteen cancer patients were also tested with MAE of autologous or allogeneic tumor tissue. Twenty-four patients with benign lung disease had positive DCHR to both microbial antigens and DNCB. Of the 75 cancer patients, 73 developed DCHR to microbial antigens. Reactivity to DNCB was markedly depressed, with only 39 patients reacting; of 16 patients with nonresectable disease, only 3 reacted. Eight of 18 patients developed DCHR to autologous MAE of lung tumor. Seven of these patients were without recurrence at 8 months, but only 3 were alive without recurrence at 1 year. Three of 8 patients with negative DCHR to tumor MAE were alive without recurrent disease at 8 months and also at I year. These data demonstrated that in lung cancer patients a poor prognosis is associated with a depressed immune recognition of DNCB and perhaps a negative cutaneous reactivity to autologous tumor MAE. REFERENCES Lamb, D., Pilney, F., Kelly, W. D., and Good, R. A: A Comparative Study of the Incidence of Anergy in Patients With Carcinoma, Leukemia, Hodgkin's Disease and other Lymphomas, 1. ImmunoI. 89: 555, 1962.
2 Solowey, A. C., and Rapaport, FT.: Immunologic Responses in Cancer Patients, Surg. Gynecol, Obstet. 121: 756, 1965. 3 Eilber, F. R., and Morton, D. L.: Impaired Immunological Reactivity and Recurrence Following Cancer Surgery, Cancer 25: 362, 1970. 4 Arata, T., Ogawa, I., Tanaka, Y., and Hashimoto, K.: Transplantation of a Newly Established Human Cancer Cell Line, Gann 60: 649, 1969. 5 Southam, C. M.: Evidence for Cancer Specific Antigens in Man, Progr. Exp. Tumor Res. 9: 1, 1967. 6 Hughes, L. E., and Lytton, B.: Antigenic Properties of Human Tumors: Delayed Cutaneous Hypersensitivity Reactions, Br. Med. J. 1: 209, 1964. 7 Stewart, T. H. M.: The Presence of Delayed Hypersensitivity Reactions in Patients Toward Cellular Extracts of Their Malignant Tumors. I. The Role of Tissue Antigen, Non-specific Reactions of Nuclear Material and Bacterial Antigen as a Cause for This Phenomenon, Cancer 23: 1368, 1969. 8 Oren, M. E., and Herberrnan, R. B.: Delayed Cutaneous Hypersensitivity Reactions to Membrane Extracts of Human Tumor Cells, Clin, Exp. Immuno!. 9: 45, 1971. 9 Catalona, W. J., Taylor, P. T., Rabson, A. S., and Chretien, P. B.: A Method for Dinitrochlorobenzene Contact Sensitization: A Clinico-pathological Study, N. Eng!. J. Med. 286: 399, 1972. 10 Burger, D. R., and Vetto, R. M.: Improved Lymphocyte Serotyping Techniques for Organ Matching, J. Surg. Res. 10: 477, 1970. 11 Davies, D. A. L.: Mouse Histocompatibility Isoantigens Derived From Normal and From Tumor Cells, Immunology 11: 115, 1966. 12 Mann, D. L., Rogentine, G. N., Fahey, J. L., and Nathenson, S. G.: Solubilization of Human Leukocyte Membrane Isoantigens, Nature 217: 1180, 1968. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein Measurement With the Folin Phenol Reagent, J. BioI. Chern. 193: 265, 1951. 14 Alter, H. J., Holland, P. V., and Purcell, R. H.: Counterelectrophoresis for Detection of Hepatitis-Associated Antigen: Methodology and Comparison With Gel Diffusion and Complement Fixation, J. Lab. Clin. Med. 77: 1000, 1971. 15 Krant, M. J., Manskopf, G., Brandrup, C. S., and Madoff, M. A.: Immunological Alterations in Bronchogenic Cancer, Cancer 21: 623, 1968. 16 Fass, L., Herberman, R. B., and Ziegler, J.: Delayed Cutaneous Hypersensitivity Reactions to Autologous Extracts of Burkitt Lymphoma Cells, N. Eng!. J. Med. 232: 776, 1970.
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17 Levinthal, B. G., Halterman, R. H., Rosenberg, E. B., and Herberman, R. B.: Immune Reactivity of Leukemia Patients to Autologous Blast Celis, Cancer Res. 32: 1820, 1972. 18 Hersh, E. M., Whitecar, J. P., McCredie, K. B., et al.: Chemotherapy, Immunocompetence, Immunosuppression and Prognosis in Acute Leukemia, N. Engl. J. Med. 285: 1211, 1971.
Discussion WILLIAM S. BLAKEMORE Philadelphia, Pa.
I wish to discuss this presentation to emphasize my belief that there will be considerable future effort in this area of patient care. Our own investigation has been directed in an attempt to increase host resistance of the nonspecific immune therapy by the use of antigens from staphylococci. Since most adults have some immunity to Staphylococcus from previous exposure and since further exposure to these antigens appears to strengthen cellular hypersensitivity, we have selected these for study in patients with advanced malignancies. It would be of interest to know whether Dr.
The Journcl of Thoracic and Cardiovascular Surgery
Morton has information to determine if the lack of response to ONCB in some of his patients was due to the amount of unresectable tumor present, causing the patients to have a negative response to ONCB, or whether there was an underlying defect in the immune mechanism. DR. WELLS (Closing) In this particular instance, it is not known whether the patients were basically immunologically deficient and therefore developed lung cancer or whether the immunological deficiency developed as a result of the pulmonary malignancy. It is known that children with congenital immunodeficiency diseases, especially of the thymus-dependent lymphoid system, are especially prone to develop cancer. Furthermore, patients who have been iatrogenically immunosuppressed for organ allografting have an increased incidence of malignant disease. We did not follow this group of patients serially, evaluating cutaneous reactivity to ONCB or to tumor cell antigens. Perhaps if we had done so we might have been able to detect a change from ONCS positivity to ONCB negativity associated with the progression of malignant disease.
A
decreased cutaneous reactivity to standard microbial antigens has been demonstrated in patients with Hodgkin's disease and in patients with carcinoma. In the latter group, however, anergy seems to be mostly confined to those patients who are generally debilitated.': ~ Eilber and Morton" found that patients with solid tumors could be sensitized topically to 2,4-dinitrochlorobenzene (DNCB) much less frequently than normal control subjects. These investigators also noted a positive correlation between cutaneous reactivity to DNCB and prognosis. Tumor-associated immune mechanisms are most frequently demonstrated by in vitro Read at the Fifty-third Annual Meeting of The American Association for Thoracic Surgery. Dallas, Texas, April 16, 17. and 18, 1973. "Tumor Immunology Section, Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. ""Department of Surgery, The George Washington University School of Medicine. Washington, D. C. """ Department of Surgery, Center, Durham. N. C.
Duke
University
Medical
techniques in which immune lymphocytes or serum antibody and tumor target cells are used. Some investigators have sought to demonstrate tumor immunity in vivo by evaluating the host's rejection of live tumor cells- 5 or by the elicitation of delayed cutaneous hypersensitivity reactions (DCHR) to tumor cell extracts. 6 , ; Regarding the latter technique, Oren and Herberman" have recently stressed the importance of using sterile tissue extracts of low protein concentration and of obtaining histologic confirmation of clinically positive skin test sites. In the present study we have evaluated 100 patients with lung disease. Seventy-five of these patients had cancer and 25 had benign lesions. Skin tests to common microbial antigens were conducted in all patients, and the sensitivity of each subject to DNCB was evaluated. In 18 patients, skin tests were done with membrane antigen extracts (MAE) of autologous or allogeneic tumor and control tissues. 557
55 8
The Journal of Thoracic and Cardiovoscular Surgery
Wells et al.
Subjects and methods Patients. One hundred patients, most of whom were from the Division of Thoracic and Cardiovascular Surgery at the George Washington University Hospital, were the subjects of this study. Sixty-seven patients had primary lung cancer. In 43 the diagnosis was squamous cell carcinoma, in 20 the diagnosis was adenocarcinoma, and in 4 the tumors were of other histologic types. In 8 patients the cancer was metastatic to the lung from other organs. The 25 control patients had been subjected to thoracotomy for cardiac or benign lung disease. The average age of the patients with malignant disease was 57.8 years, while the average age of those with nonmalignant disease was 39.5 years. All of the patients with malignant disease were ambulatory and none had lost more than 10 per cent of body weight since the onset of the present illness. No patients were subjected to skin tests sooner than 2 weeks after the operation, and none had received x-ray therapy or chemotherapy for 2 months prior to skin testing. Seventytwo of the 75 cancer patients were followed for 1 year from the time of skin testing. Test antigens. Standard microbial antigens. All patients were tested intradermally with 0.1 ml. each of three standard antigens: mumps antigen (Eli Lilly and Company, Indianapolis, Ind.), Candida (Dermatophytin 0 1: 100, Hollister-Stier Laboratories, Spokane, Wash.) , and streptokinase-streptodornase (Varidase, 40 units/ 10 units per milliliter, Lederle Laboratories Division, American Cyanamid Company, Pearl River, N. Y.). Tests were read at 48 hours by measurement of perpendicular diameters with vernier calipers. Induration greater than 5 mm. average diameter was considered positive. All patients were sensitized to DNCB by the technique described by Eilber and Morton" and modified by Catalona. D DNCB, 2,000 and 50 mcg., was applied topically at respective sites on the upper inner arm and the volar surface of the forearm. The sites were evaluated in 14 days. If a spontaneous flare of induration
and redness was present at both test sites the patient's reaction was called 4+; if only the 2,000 meg. site reacted, it was called 3+. If no reactivity occurred, a challenged dose of 50 meg. of DNCB was applied at a separate site on the forearm and evaluated at 48 hours. Marked redness and induration at the challenge site was called a 2+ reaction, and a weak reaction was designated 1+. When none of the test sites developed induration the patient was described as being DNCB negative. Preparation of the membrane extracts. Sterile lung tumors were obtained from the George Washington University Hospital operating room. A specimen of normal lung distant from the cancer, as well as 60 to 100 ml. of heparinized blood, were also obtained. Leukocytes were separated from whole blood in 2 per cent methyl cellulose as described by Burger."? MAE of tumor cells and control tissues were prepared by freeze-thawing and low ionic strength extraction, a method described by Davies" and Mann-" and modified by Oren and Herberman." A crude membrane pellet obtained by centrifuging the pooled extracts at 105,000 g for 1 hour was resuspended in saline and used for skin testing. Protein concentration of the MAE was measured by the method of Lowry and associates. '" All test MAE were adjusted to a protein concentration of 2.5 mg. per milliliter. Each test injection contained 0.1 ml. or 0.25 mg. of protein. Sterility of the final extracts was tested by culture on blood agar plates and in thioglycolate broth. Only sterile MAE were used. Antigen extracts for allogeneic testing were negative for the presence of hepatitis-associated antigen as tested by counterelectrophoresis.'' All MAE skin tests were read at 48 hours, and most positive skin test sites were biopsied with a 2 mm. skin drill, fixed in 10 per cent formalin, sectioned, and stained with hematoxylin and eosin.
Results All but 1 of the 25 patients with benign lung disease and all but 2 of the 75 patients
Volume 66 Number 4 October, 1973
Delayed cutaneous hypersensitivity reactions
with lung cancer reacted positively to at least one of the three standard microbial antigens. There was no decreased reactivity in the cancer patients either quantitatively or qualitatively. Such was not the case with DNCB sensitization, however, for cutaneous reactivity was clearly reduced in patients with malignant disease. Of the 25 control patients, 24 (96 per cent) were able to be sensitized to DNCB. On the other hand, in the lung cancer group, only 39 of 75 (52 per cent) were able to be sensitized, a highly significant (p < 0.005) difference. If one compares DNCB reactivity (36/59 = 69 per cent) in lung cancer patients with resectable disease to reactivity in control patients, the difference is significant (p < 0.01). Furthermore, if one compares the incidence of DNCB reactivity in the group with resectable cancer to that in patients with nonresectable cancer (3/16 = 19 per cent), the difference is again significant (p < 0.01). Also, if patients are grouped according to the histologic type of tumor, it is found (Table I) that the proportion of nonresponders is significantly greater among patients with squamous cell carcinoma (22/43) than among the patients with adenocarcinoma (5/20) (p < 0.01). Seventy-two of the 75 lung cancer patients were followed for 1 year, at which time the development of recurrent disease or death was correlated with initial DNCB reactivity. As can be seen in Table II, of the 36 cancer patients originally nonresponsive to DNCB, 29 (81 per cent) had died or had developed recurrent disease. Conversely, of the 36 cancer patients (39 were in this group initially but 3 were lost to follow-up) originally responsive to DNCB, 14 (39 per cent) had died or developed recurrent disease. The difference between the incidence of death or recurrence in these two groups is highly significant (p < 0.005). In Table III is shown the reactivity of lung cancer patients to control and tumor MAE. Ten of 18 patients developed DCHR to lung cancer MAE. Eight of these 10 cancer patients were tested to MAE prepared from
559
Table I. Reactivity to dinitrochlorobenzene (DNCB) _ _ _ _ _ _ _ _I"'-P_o_s_it_iv_e_1 Negative I Totals Squamous cell carcinoma Adenocarcinoma Totals
21 15
22
36
27
5
43
20 63
Legend: The table provides a comparison of responses to DNCB in patients with squamous cell carcinoma and in patients with adenocarcinoma. A total of 49 per cent of the patients with squamous cell carcinoma did not respond to DNCB. as compared to 25 per cent of those with adenocarcinoma (p < 0.01).
Table II. Reactivity
10
dinitrochlorobenzene
(DNCB) _ _ _ _ _ _ _1
Recurrence or death No recurrence Totals
Posith'e I Negative I Totals 14 22 36
29 7 36
43 29 72
Legend: The table provides a comparison at I year of the incidence of death and/or recurrence in responders and nonresponders to DNCB. The rate of recurrence or death in nonresponders was 81 per cent, as compared to 39 per cent in responders (p < 0.005).
autologous tumors, whereas the remammg 2 were tested with allogeneic MAE from tumors of their histologic type. Of the 10 patients demonstrating DCHR to tumor MAE, 4 also reacted positively to leukocyte MAE and 1 to MAE of normal lung. Positive reactions to autologous tumor MAE occurred in patients with squamous cell carcinoma as well as those with adenocarcinoma. Eight months after testing to MAE, 7 of the 8 patients who reacted to autologous MAE were alive with no evidence of recurrence, while only 3 of 8 patients with negative DCHR to tumor MAE had not developed a recurrent lesion. At 12 months, however, only 3 patients from each group were alive with no evidence of recurrence. Discussion
In the present study we found that patients with lung cancer did not have cutaneous anergy to standard microbial antigens. Our findings are in agreement with those of Solowey and Rapaport" and Lamb and coinvestigators.' Krant and associates"
The Journal of
5 60
Wells et al.
Thoracic and Cardiovascular Surgery
Table III. Delayed skin reactivity to tumor and normal tissue membrane extracts in patients with lung cancer Patient C. S. R. D. 1. N. 1. H. C. H. M. S. J. S. E. K. 1. G. O. C.
V. V.
R. E. E. E. H. C. C.
E. F. T. Z. H. D.
B.
Normal I Leukocyte lung
ICommon antigens + + + + + + + + + + + + + + + + +
N.T. + +
N.T.
+
+
+
+ +*
-*
+ +*
+ + + + +*
+ -*
NT. -*
-*
-*
+ -*
Legend: N.T .• Not tested. + = Positive. - = Negative . • Denotes reactivity to allogeneic preparation.
in a previous study of lung cancer patients, found a decreased cutaneous reactivity to tuberculin. Many of their patients, however, were receiving x-ray therapy or chemotherapy or were in a terminal state at the time of skin testing, which perhaps accounts for the negative cutaneous reactivity. Krant also found a decreased reactivity to DNCB in the lung cancer patients studied. The lung cancer patients in our study reacted to standard microbial antigens, but reactivity to DNCB was markedly decreased; this was especially true in those patients with nonresectable disease. It was also found that initial reactivity to DNCB correlated well with recurrence and survival at 1 year. These findings are in agreement with those of Eilber and Morton," who first suggested a positive correlation between poor prognosis and negative DNCB response in patients with solid tumors. DNCB seems to be an especially valuable agent for assessing a patient's immunological responsiveness. Reactivity to common microbial antigens represents immunological recall to a prior sensitization but gives little
information regarding the body's competence to respond immunologically to a new immunogen. The elicitation of DCHR to autologous and allogeneic tumor MAE in 10 of 18 lung cancer patients supports the concept of host cellular immunity to tumor cell antigens. It was somewhat surprising that 5 of the 18 patients reacted to MAE of autologous or allogeneic leukocytes, because the test materials were bacteriologically sterile and the MAE contained a relatively low protein concentration. Oren and Herberman" found that 10 per cent of normal individuals had positive DCHR to autologous leukocyte MAE when the protein concentration was 0.33 mg. per 0.1 ml. or less. This reactivity could represent true autoimmunity or alternately the presence of delayed hypersensitivity mediators in the leukocyte membrane preparations. In the present study, we found positive DCHR to normal lung MAE in only 1 patient. The normal lung antigen had been added as an organ-specific control, and we had actually expected to see a greater incidence of reactivity. The fact that reactivity rarely occurred with MAE of autologous normal lung tissue is perhaps indicative of unique properties of the leukocyte preparation used. This could be related to the presence of soluble delayed hypersensitivity mediators attached to or associated with the lymphocyte MAE that were pelleted during the high-speed centrifugation; conversely, it could be related to substances released from polymorphonuclear leukocytes which were present at the beginning of the leukocyte extraction procedure. Although positive DCHR to tumor MAE seemed to correlate with a favorable prognosis at 8 months, such was not the case when patients were evaluated 1 year after testing. This by no means indicates that cutaneous reactivity to tumor MAE is not useful in evaluating tumor-associated immune reactions. Conversely, Fass,> Levinthal," and their colleagues have found that positive cutaneous reactivity to tumor MAE is associated with a favorable prognosis. Furthermore, Hersh'" has found that the assessment
Volume 66 Number 4
Delayed cutaneous hypersensitivity reactions
561
October, 1973
of a combination of in vivo and in vitro immunological parameters, performed serially, has been especially helpful in the clinical management of leukemia patients. We are currently beginning an immunotherapy study of patients with operable lung cancer in which we will use the immunological techniques discussed in this paper. Summary
Oncogenesis is favored by an environment of depressed immunity, but there are few studies in man which correlate both general immunological status and reactivity to tumor-associated antigens with the patient's clinical course. DCHR to microbial antigens (mumps, Candida, and streptokinase-streptodornase) and to a previously unencountered antigen, DNCB, were evaluated in 100 ambulatory patients, 75 with lung cancer and 25 with benign lung disease. Eighteen cancer patients were also tested with MAE of autologous or allogeneic tumor tissue. Twenty-four patients with benign lung disease had positive DCHR to both microbial antigens and DNCB. Of the 75 cancer patients, 73 developed DCHR to microbial antigens. Reactivity to DNCB was markedly depressed, with only 39 patients reacting; of 16 patients with nonresectable disease, only 3 reacted. Eight of 18 patients developed DCHR to autologous MAE of lung tumor. Seven of these patients were without recurrence at 8 months, but only 3 were alive without recurrence at 1 year. Three of 8 patients with negative DCHR to tumor MAE were alive without recurrent disease at 8 months and also at I year. These data demonstrated that in lung cancer patients a poor prognosis is associated with a depressed immune recognition of DNCB and perhaps a negative cutaneous reactivity to autologous tumor MAE. REFERENCES Lamb, D., Pilney, F., Kelly, W. D., and Good, R. A: A Comparative Study of the Incidence of Anergy in Patients With Carcinoma, Leukemia, Hodgkin's Disease and other Lymphomas, 1. ImmunoI. 89: 555, 1962.
2 Solowey, A. C., and Rapaport, FT.: Immunologic Responses in Cancer Patients, Surg. Gynecol, Obstet. 121: 756, 1965. 3 Eilber, F. R., and Morton, D. L.: Impaired Immunological Reactivity and Recurrence Following Cancer Surgery, Cancer 25: 362, 1970. 4 Arata, T., Ogawa, I., Tanaka, Y., and Hashimoto, K.: Transplantation of a Newly Established Human Cancer Cell Line, Gann 60: 649, 1969. 5 Southam, C. M.: Evidence for Cancer Specific Antigens in Man, Progr. Exp. Tumor Res. 9: 1, 1967. 6 Hughes, L. E., and Lytton, B.: Antigenic Properties of Human Tumors: Delayed Cutaneous Hypersensitivity Reactions, Br. Med. J. 1: 209, 1964. 7 Stewart, T. H. M.: The Presence of Delayed Hypersensitivity Reactions in Patients Toward Cellular Extracts of Their Malignant Tumors. I. The Role of Tissue Antigen, Non-specific Reactions of Nuclear Material and Bacterial Antigen as a Cause for This Phenomenon, Cancer 23: 1368, 1969. 8 Oren, M. E., and Herberrnan, R. B.: Delayed Cutaneous Hypersensitivity Reactions to Membrane Extracts of Human Tumor Cells, Clin, Exp. Immuno!. 9: 45, 1971. 9 Catalona, W. J., Taylor, P. T., Rabson, A. S., and Chretien, P. B.: A Method for Dinitrochlorobenzene Contact Sensitization: A Clinico-pathological Study, N. Eng!. J. Med. 286: 399, 1972. 10 Burger, D. R., and Vetto, R. M.: Improved Lymphocyte Serotyping Techniques for Organ Matching, J. Surg. Res. 10: 477, 1970. 11 Davies, D. A. L.: Mouse Histocompatibility Isoantigens Derived From Normal and From Tumor Cells, Immunology 11: 115, 1966. 12 Mann, D. L., Rogentine, G. N., Fahey, J. L., and Nathenson, S. G.: Solubilization of Human Leukocyte Membrane Isoantigens, Nature 217: 1180, 1968. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein Measurement With the Folin Phenol Reagent, J. BioI. Chern. 193: 265, 1951. 14 Alter, H. J., Holland, P. V., and Purcell, R. H.: Counterelectrophoresis for Detection of Hepatitis-Associated Antigen: Methodology and Comparison With Gel Diffusion and Complement Fixation, J. Lab. Clin. Med. 77: 1000, 1971. 15 Krant, M. J., Manskopf, G., Brandrup, C. S., and Madoff, M. A.: Immunological Alterations in Bronchogenic Cancer, Cancer 21: 623, 1968. 16 Fass, L., Herberman, R. B., and Ziegler, J.: Delayed Cutaneous Hypersensitivity Reactions to Autologous Extracts of Burkitt Lymphoma Cells, N. Eng!. J. Med. 232: 776, 1970.
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17 Levinthal, B. G., Halterman, R. H., Rosenberg, E. B., and Herberman, R. B.: Immune Reactivity of Leukemia Patients to Autologous Blast Celis, Cancer Res. 32: 1820, 1972. 18 Hersh, E. M., Whitecar, J. P., McCredie, K. B., et al.: Chemotherapy, Immunocompetence, Immunosuppression and Prognosis in Acute Leukemia, N. Engl. J. Med. 285: 1211, 1971.
Discussion WILLIAM S. BLAKEMORE Philadelphia, Pa.
I wish to discuss this presentation to emphasize my belief that there will be considerable future effort in this area of patient care. Our own investigation has been directed in an attempt to increase host resistance of the nonspecific immune therapy by the use of antigens from staphylococci. Since most adults have some immunity to Staphylococcus from previous exposure and since further exposure to these antigens appears to strengthen cellular hypersensitivity, we have selected these for study in patients with advanced malignancies. It would be of interest to know whether Dr.
The Journcl of Thoracic and Cardiovascular Surgery
Morton has information to determine if the lack of response to ONCB in some of his patients was due to the amount of unresectable tumor present, causing the patients to have a negative response to ONCB, or whether there was an underlying defect in the immune mechanism. DR. WELLS (Closing) In this particular instance, it is not known whether the patients were basically immunologically deficient and therefore developed lung cancer or whether the immunological deficiency developed as a result of the pulmonary malignancy. It is known that children with congenital immunodeficiency diseases, especially of the thymus-dependent lymphoid system, are especially prone to develop cancer. Furthermore, patients who have been iatrogenically immunosuppressed for organ allografting have an increased incidence of malignant disease. We did not follow this group of patients serially, evaluating cutaneous reactivity to ONCB or to tumor cell antigens. Perhaps if we had done so we might have been able to detect a change from ONCS positivity to ONCB negativity associated with the progression of malignant disease.