•
Therapeutic Radiology
Carcinoembryonic Antigen and Skin Test Reactivity in Tumor Radiotherapy 1 Manuel Vider, M.D., Rafiah Kashmiri, M.Sci., Bernard Moses, A.B., Davie Earlywine, L.P.N., William R. Meeker, M.D., Joella F. Utley, M.D., and Yosh Maruyama, M.D. Serial carcinoembryonic antigen (CEA) levels were obtained from 122 cancer patients. In a random selection, the levels in 67 of these patients were compared with clinical response to radiotherapy. Skin tests were also performed for histoplasmin, tuberculin and mumps. CEA levels, skin-delayed hypersensitivity reaction (DHR) and clinical tumor response were evaluated and correlated. Clinical response of tumors to radiotherapy was more often seen in patients with positive skin tests, but no correlation was observed between skin test reactivity and CEA response curves. INDEX TERMS:
Immunity. Neoplasms, therapy. Skin. Therapeutic radiology, immunity
Radiology 119:677-681, June 1976
•
• (CEA) was first isolated in 1965 from human colonic cancers and fetal colonic tissue (15). This antigen was originally thought to be entodermally derived, tumor-specific and diagnostic for colon carcinoma. A large number of publications have since described the clinical application of CEA in the study of cancer and the management of the cancer patient (5-8, 12, 13, 19,22,25,27,30,32,35,36,39). In this paper, we shall analyze our experience with the CEA test in a radiotherapeutic clinic, with a separate test of immunological reactivity, i.e., delayed skin hypersensitivity response (DHR) to standard bacterial, viral and mycotic antigens (PPD, histoplasmin and mumps) and correlate these with clinical tumor response in a randomly sampled patient subpopulation. ARCINOEMBRYONIC ANTIGEN
C
BACKGROUND ON DELAYED HYPERSENSITIVITY AND IMMUNE RESPONSE IN CANCER
In 1921, MacCarty and Blackford (23) reported that regional lymphatic grandular involvement and postoperative longevity in gastric carcinoma appeared to be related. MacCarty and Mahle (24) summarized 99 of 200 cases followed for eight years and reported that (a) individuals with no lymph gland imvolvement and showing histological differentiation lived 14 % longer than those with no gland involvement and poor differentiation; (b) patients with partial gland involvement and differentiated histology lived 20 % longer than those with poor differentiation; (c) those with complete gland replacement showed no increased longevity despite differentiation; and (d) differentiation was more frequently associated with lymph gland involvement. Partial lymph node involvement was observed in 42.6 % and extensive lymph node involvement was noted in 7 % of the cases. This suggests a role for lymph node function and survival in gastric cancer. In 1954, Black and colleagues (3)
also concluded that survival in gastric carcinoma was related to nuclear differentiation and lymphoid infiltration. In 1930, Witebsky (38) produced an antiserum to human gastric cancer in guinea pigs. He then used alcoholic extracts to absorb the antisera of normal gastric tissue and found that the antiserum was still capable of producing a precipitation reaction against the cancer extract. This suggested that there was a different antigen in gastric cancer not present in the corresponding normal tissue. Moore and Foote (26) in 1949 related the prognosis of medullary carcinoma of the breast to the histological association with lymphocyte infiltration and germinal center formation and further speculated that "this rather characteristic lymphoid infiltrate indicates some adjustment between tumor and host." Berg (4) and Black and co-workers (3) also drew attention to the importance of a lymphoplasmacytic cell infiltrate in breast carcinoma and showed that such patients had a better prognosis. Roberts and Jones (33) found reduced DHR to Varidase as breast cancer progressed. Hodgkin's disease patients who are normally tuberculin-positive have negative skin tests with exacerbations of their disease, which revert to positive again during remissions. This represents a reversible defect in delayed hypersensitivity reactions which is not specific for tuberculin antigen since such aberrations are also seen in mumps and fungal skin test antigens (34). Cutaneous anergy to microbial antigens has been reported in leukemia, carcinoma and Hodgkin's disease (9, 28). Lamb et al. (20) found that 53 % of patients with Hodgkin's disease were anergic early in the course of the disease. Likewise, anergy was more frequent in all subjects studied by Hughes and Lytton (18) although no correlation with the histology of the tumors was observed. Anergy has also been found with carcinoma
1 From the Departments of Radiation Medicine (M. V., D. E., J. F. U., Y. M.) and Surgery (R. K., W. R. M.), University of Kentucky, A. B. Chandler Medical Center, Lexington, Ky. Presented at the Sixtieth Scientific Assembly and Annual Meeting of the Radiological Society of North America, Chicago, 111., Dec. 1-6, 1974. Supported in part by Grants CA 05258 and CA 17786, National Cancer Institute. shan
677
678
MANUEL VIDER AND OTHERS
Table I:
Cod ing System for Patterns of Response to Treatment
June 1976
Table III: Skin Test Results (Mean Diameter of Positive Skin Reactions (em) for Tumors of Different Sites)
The laboratory CEA response was coded using the following scheme. The digit describes the measurable CEA response in the plasma: 1. Normal Range, detectable, but less than 2.5 nq/ml 2. Sustained elevation, greater than 2.5 nq/rnl " 3. Progressive rise, to levels greater than 2.5 nq/ml 4. Progressive decline, initial levels greater than 2.5 nq/ml,
The clinical response of the tumor to radiotherapy can also be described. This was done using the following digitized scheme: 1. No clinical evidence of disease 2. Widespread tumor or unresponsive tumor* 3. Tumor progression, patient deterioration 4. Tumor response or clinical improvement X. Uncertain outcome. * Based upon experience of an earlier study (36), category 2 was not found to be of any value and was therefore not used in th is study.
and generally becomes more evident as the tumor condition progresses. Solowey and Rapaport (29) utilized streptokinasestreptodornase and the antigens of purified protein derivatives and reported 27.5 % positive tests in a population of 91 cancer patients. Grace and Kondo studied the autoreactivity of patients to their own tumor extracts and passive transferred antibodies in sera (16). The skin and tumor response tests revealed a depression of homograft responses in patients with advanced cancer (1, 16). When taken from the same donor, behavior of homografted skin also paralleled that of the tumor. Stewart (31), considering the inefficiency of the defensive mechanism as shown by the OHR, hypothesized that antigens that compete or share the final common path of the cell-mediated DHR of the host against the tumor can produce regression of certain tumors. In studies of host reactivity to neoantigens, Eilber and Morton (11) reported anergy to dinitrochlorobenzene (ONCB) delayed hypersensitivity reaction following cancer surgery. Of the 110 cases with negative DHR preoperatively, 45 were found inoperable. On the other hand, of the 70 cases that showed positive OHR, 90 %
Table II:
CasesGrouped by Organs
Site Lung Head and neck Uterus Colon and rectum Lymphomas Breast Skin Upper G I Urinary tract Multiple myeloma Prostate and testicle Ovary Brain Pancreas Total
Number of Cases 32 15 13 13 11 12
Site Lung Head, neck and eye Uterus Colorectal Lymphomas Breast Skin Upper GI Urinary tract, prostate and testicle Ovary Brain
No. Patients
+No. Reactors/No. Tested
Diameter of Skin Reaction (cm)*
12 13
6/12 13/13
2.0 2.8
11 7 4 8 3 4 3
7/11 4/7 2/4 6/8 1/3 2/4 2/3
2.8 3.5 1.3 1.8 1.0 1.8 4.8
1 1
0/1 0/1
NR NR
N R = No nreactor. + At least 1 positive test (mean reaction of all positive tests) . * Positive reactors only.
were operable and these patients were subsequently free of disease six months postoperatively. In all of these investigations, there have been attempts to apply a variety of immunological tests and assays as monitors for tracing response to therapy and for predicting tumor behavior in the cancer patient. Few studies have been applied to CEA and its possible relevance to other immunological tests in radiotherapy. This study was directed to that question. MATERIALS AND METHODS
Patient Population
Serial CEA titers were measured in 122 patients randomly selected from the patient population of the Radiation Medicine Clinic. The patients are 90 % white, and entirely from the Appalachian mountain and central Kentucky region. Many different neoplasms were seen. (TABLE I.)
One hundred and twenty-two patients were examined in a study in the Radiation Oncology Clinic of the University of Kentucky Medical Center, Lexlnqton, Kentucky. The study was carried out in a double blind manner. The samples were taken and transferred to the laboratory, and stored until all samples of the same patient had been collected. The results were obtained only Table IV:
Relationship Between Positive Skin Tests* and the CEA Response Curve
Code No.
Pattern of CEA Response
4
1
3 5 3
3
Negative CEA «2.5 ng/ml) Progressively rising CEA level (> 2.5 nq/ml) Falling CEA levels
5
2
2 2 122
4
Frequency of CEA Response to Therapy
Total * Any skin reaction greater than 0.5 em.
Pel' Cent of Group
23
35
23
35
21
30
-
--
67
100
after a conclusion was reached regarding clinical response. The first sample was taken before radiotherapy was started, the second was taken at the midpoint of the radiotherapeutic plan, i.e., at 3,000 rads in a planned course of 6,000 rads or at 1,000 rads in a planned course of 2,000 rads. The third sample was taken at the end of treatment and the fourth two months after the completion of treatment.
Radioimmunoassay Procedure
Clinical Response to Rad iotherapy
Skin Test Antigens and Methods Skin tests for Histoplasma (mycotic antigen), tuberculin as PPD (mycobacterial antigen) and mumps (viral antigen) were performed routinely on all patients. These were read at 24 and 48 hours and scored by standard scoring methods which measure erythema and induration in centimeters of diameter.
2.
3.
Tuberculosis: The tuberculin purified protein derivative (PPO) (Mantoux), was produced by Connaught and distributed by Panray, Ormont Co., Englewood, N.J., bioequivalent to 5 U.S. units; 0.1 ml of the antigen (5 U.S. units) was injected intradermally. Mumps: The mumps skin test antigen, produced by Lilly (V-1059) was injected in a dose of 0.1 ml intradermally. Histoplasmosis: The histoplasmin skin test antigen produced by Parke-Davis was injected intradermally in the dose of 0.1 ml.
These agents to exposed. reactivity
three antigens represent the most common which the population of this region have been The population of Kentucky showed a higher than that of all the states surrounding it to tuTable VI:
Frequency of Clinical Response
Unknown
1
No clinical tumor Tumor progression Tumor response
3
-
Per Cent of Group
7 13
19
11
16
36
54
67
100
11
* Any skin reaction greater than 0.5 cm.
berculin antigen (PPO). Thirty per cent of metropolitan and 40 % of farm inhabitants have skin reactions greater than 4 mm in diameter (31). The percentage of PPO with 10 or more mm reactivity was 8 % for metropolitan, 7 % for non-farm population and 8 % for farm inhabitants. The incidence of reaction to PPD with 4 or more mm was 30 % for metropolitan inhabitants, 25 % for non-farming population and 40 % for farmers. Kentucky dwellers also showed the highest incidence of reactivity to histoplasmin of any state in the Ohio Valley where this fungal disease is endemic. About 70 % have reactions greater than 4 mm in diameter. Mumps, a viral antigen, has a high incidence in all populations surveyed ("'-190 % ) (20). Any patient with a positive reaction was considered a reactor whether to one or multiple antigens. Because of the battery selected, a positive exposure history often could not be obtained. RESULTS
Of the serial CEA levels sampled in 122 patients, 78 cancer patients (64 %) had CEA levels greater than 2.5 ng/ml. Fifty-seven patients or 73 % showed correlation between the tumor-therapy response and the CEA levels during this period (35, 36). Sixty-seven patients were randomly selected for further study by skin test reactivity, and complete data was available for analysis.
Tumor Types and Skin Test Reactivity TABLE II shows the types and numbers of tumors in the study of CEA titers and response patterns and
Mean Skin Reactivity* (in cm) Related to the Clinical Response and CEA Curve Response
\ (__--Clinical Response to Radiotherapy Mean Skin Test Reaction Category Pattern (diameter of Response of Response in cm) Clinical response Type 3 Clinical response Type 4
Pattern of Clinical Response
Totals
The method in use (36) was developed by Gold (15) and modified by Hansen et al. (17). Basically, with use of a standard radioimmunoassay of 1251-labeled monospecific goat anti-CEA antibody, a sample of patient plasma, anticoagulated with EDTA was assayed for CEA. Plasma levels were not considered positive unless greater than 2.5 nanograms/ml. TABLE I describes the method used for charting the CEA levels found in patient plasma, and the scoring system for patient-tumor response to radiotherapy.
Therapeutic Radiology
Table V: Relationship of Positive Skin Test* and Clinical Radiotherapy Response
4
1.
679
CARCINOEMBRYONIC ANTIGEN AND SKIN TEST REACTIVITY
Vol. 119
Nonresponders
1.1
Responders
2.7
* Positive ski n tests only.
(.------CEA Response to Radiotherapy-------.., Category Pattern Mean Skin of Response of Response Test Reaction CEA curve pattern Type 3 CEA curve pattern Type 4
Progressive rise
1.5cm
Declines with treatment
1.8 cm
MANUEL VIDER AND OTHERS
680
TABLE III gives the mean diameter of the positive skin test reactions in the patients sampled in each tumor category who reacted positively to the skin test antigen.
CEA Response Pattern and Skin Test Reactivity TABLE IV presents data on the positive skin test reactivity and the pattern of the CEA response observed: 23/67 or 35 % had negative CEA tests and a positive skin test; 23/67 or 35 % had progressively rising CEA levels and positive skin tests; 21/67 or 30 % had CEA levels which fell in response to therapy, and positive skin tests. This indicates that there is no constant pattern of plasma CEA progression or decline in relation to skin test reactivity.
Clinical Tumor Response and Skin Test Reactivity TABLE V shows the different patterns of tumor response to radiotherapy in skin test positive patients. In this table, the clinical response was classified as: Unknown 1 = No clinical tumor 3 Progressive tumor despite radiotherapy 4 Tumor response with radiotherapy.
= =
There was a significantly greater proportion of patients with positive skin tests who showed clinical response to radiotherapy. This was seen in 36/67 or 54 % of patients who showed a positive response by DHR skin tests. In general, there was a better response to treatment in the radiotherapy patients who had positive skin tests among those studied here.
Relationship of Positive DHR Response, CEA Response and Clinical Tumor Response TABLE VI further subdivides and analyzes DHR reactivity, CEA response patterns and clinical response pattern. In the present study, 31 % of those with positive CEA level had a clinical response to therapy and had at least one positive response to the battery of skin tests. In those who had a clinical response to therapy, the OHR test responders also had a greater mean skin test reaction measured in centimeters (2.7 ern) compared with nonresponders (Type 3) whose diameter was 1.1 cm. However, the responder type of CEA curve (Type 4) did not show a different degree of DHR response compared to nonresponder CEA curves (Type 3) when matched on the basis of the intensity of the skin test (1.8 cm vs. 1.5 em).
DISCUSSION
In this series, 78 of our 122 patients (64 %) showed detectable plasma levels of CEA and 57 (73 %) showed
June 1976
correlation between the response curves traced by the CEA and clinical response. Clinical response of tumors to radiotherapy was more often seen in patients with positive skin tests, but there was no correlation observed between skin test reactivity and the CEA response curves. Local therapy and destruction of the tumor by irradiation could result in the release of tumor-associated antigens. One would then expect that immediately following irradiation, monitors of serum CEA level in tumor-bearing patients would change with radiotherapy and response to therapy. An initially nondetectable titer may indicate a small or localized, or a non-CEA-producing tumor. A positive titer will usually indicate an advanced localized tumor or more widespread tumor Local radiotherapy may increase, decrease, or not affect such a tumor. A decrease in CEA titers with treatment may reflect radioresponsiveness of the tumor and its destruction. No change would indicate CEA-producing sites outside the treatment field or radioresistance of the tumor under irradiation. Progressively rising titers would indicate an increasing number of CEA-producing sites outside the treatment field or radioresistance. Our data indicate that such correlations are observed with the level of plasma CEA (35, 36) in patients undergoing radiation therapy. Skin test reactivity has also been advocated as a test for immune reactivity. A positive reaction indicates that a more favorable tumor response can be expected. Such observations were confirmed in these studies. Lundy et al. (21) showed correlation between ONCB reactivity for recurrence and survival in localized disease of the head and neck. In general, the results of these studies again show that no single test was completely accurate in predicting tumor response. We found a lack of correlation between the skin tests and CEA response which is consistent with the hypothesis that these two tests monitor separate mechanisms of immunological response in cancer patients. Thus, the responsiveness by the CEA curve seems to be unrelated to the mechanism that causes delayed hypersensitivity (OHR) reactions to the histoplasmin, PPO and mumps skin tests. This can be explained, assuming that the CEA response curves and OHR are separate parameters and may not involve the same mechanisms that relate to the Clinical response of the patient to tumor therapy. We observed a lack of correlation which is sometimes seen when many different tests or diagnostic procedures are combined looking for improvements of the accuracy of the collective tests. The objective in the planning and carrying out of this work was to clarify and extend the interpretation and application of currently available immunological tests to the monitoring and management of patients undergoing radiotherapy. We conclude that clinical response of tumors to radiotherapy was more often seen in patients with positive skin tests, but there was no correlation observed between skin test reactivity and the CEA re-
Vol. 119
CARCINOEMBRYONIC ANTIGEN AND SKIN TEST REACTIVITY
sponse curves. However, it was apparent that there is a need for further development of more precise batteries of immunological tests to assess the immunological response of the cancer patient to treatment than those reported here. Yosh Maruyama, M.D. Department of Radiation Medicine University of Kentucky A. B. Chandler Medical Center Lexington, Ky. 40506
REFERENCES 1. Anthony HM, Templeman GH, Madsen KE, et al: The prognostic significance of DHR skin tests in patients with carcinoma of bronchus. Cancer 34: 1901-1906, Dec 1974 2. Black MM, Opler SR, Speer FD: Microscopic structure of gastric carcinomas and their regional lymph nodes in relation to survival. Surg Gynec Obstet 98:725-734, Jun 1954 3. Black MM, Speer Fp, Opler SR: Structural representations of tumor-host relationships in mammary carcinoma. Biologic and prognostic significance. Am J Clin Pathol 26:250-265, Mar 1956 4. Berg JW: Inflammation and prognosis in breast cancer. A search for host resistance. Cancer 12:714-720, Jul 1959 5. Chu TM, Reynoso G, Hansen HJ: Demonstration of carcinoembryonic antigen in normal human plasma (letter). Nature 238: 152-153,21 Jul1972 6. Chu TM, Nemoto T: Evaluation of carcinoembryonic antigen in human mammary carcinoma. J Nat Cancer Inst 51:11191122, Oct 1973 7. Concannon JP, Dalbow MH, Frich JC Jr: Carcinoembryonic antigen (CEA) plasma levels in untreated cancer patients and patients with metastatic disease. Radiology 108: 191-193, Jul 1973 8. Costanza ME, Das S, Nathanson L, et al: Carcinoembryonic antigen. Report of a screening study. Cancer 33:583-590, Feb 1974 9. Dubin IN: The poverty of the immunological mechanisms in patients with Hodgkin's disease. Ann Intern Med 27:898-913, Dec 1947 10. Edwards LB, Acquaviva FA, Livesay VT, et al: An atlas of sensitivity to tuberculin, PPD-B and histoplasmin in the United States. Am Rev Resp Dis 99:1-132, Apr 1969 11. Eilber FR, Morton DL: Impaired immunological reactivity and recurrence following cancer surgery. Cancer 25:3Q2-367, Feb 1970 12. Elias EG, Holyoke ED, Chu TM: Carcinoembryonic antigen (CEA) in feces and plasma of normal subjects and patients with colorectal carcinoma. Dis Colon Rectum 17:38-41, Jan-Feb 1974 13. Freedman SO: Carcinoembryonic antigen: current clinical applications. J Allergy Clin Immunol 50:348-357, Dec 1972 14. Gold P: Tumor-specific antigen in GI cancer. Hospital Practice 7:79-88, Feb 1972 15. Gold P, Freedman SO: Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J Exp Med 121:439-462, 1 Mar 1965 16. Grace JT Jr, Kondo T: Investigations of host resistance in cancer patients. Ann Surg 148:633-641, Oct 1958 17. Hansen HJ, Lance KP, Krupey J: Demonstration of an ionsensitive antigenic site on carcinoembryonic antigen using zirconyl phosphate gel (abst). Clin Res 19:143, Jan 1971 18. Hughes LE, Lytton B: Antigenic properties of human tumours: delayed cutaneous hypersensitivity reactions. Br Med J 1: 209-212, 25 Jan 1964
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Therapeutic Radiology
19. Khoo SK, MacKay EV: Carcinoembryonic antigen in cancer of the female reproductive system: sequential levels and effects of treatment. Aust NZ J Obstet Gynecol 13: 1-17, Feb 1973 20. Lamb 0, Pilney F, Kelly WD, et al: A comparative study of the incidence of anergy in patients with carcinoma, leukemia, Hodgkin's disease and other lymphomas. J Immunol 89:555-558, Oct 1962 21. Lundy J, Wanebo H, Pinsky C, et al: Delayed hypersensitivity reactions in patients with squamous cell cancer of the head and neck. Am J Surg 128:530-533, Oct 1974 22. MacSween JM, Warner NL, Mackay IR: The detection of carcinoembryonic antigen in whole serum from patients with malignant and nonmalignant disease. Clin Immunol Immunopathol 1: 330-345, Apr 1973 23. MacCarty WC, Blackford JM: Involvement of regional lymphatic glands in carcinoma of the stomach. Ann Surg 74:811-843, 1921 24. MacCarty WC, Mahle AE: Relation of differentiation and lymphocytic infiltration to postoperative longevity in gastric carcinoma. J Lab elin Med 6:473-480, Jun 1921 25. Meeker WR, Kashmiri R, Hunter L, et al: Clinical evaluation of carcinoembryonic antigen test. Arch Surg 107:266-274, Aug 1973 26. Moore OS Jr, Foote FW Jr: The relative favorable prognosis of medullary carcinoma of the breast. Cancer 2:635-642, Jul 1949 27. Ona FV, Zamcheck N, Dhar P, et al: Carcinoembryonic antigen (CEA) in the diagnosis of pancreatic cancer. Cancer 31:324327, Feb 1973 28. Parker F Jr, Jackson H Jr, Fitzhugh G, et al: Studies of diseases of the lymphoid and myeloid tissues. IV. Skin reactions to human and avian tuberculin. J Immunol 22:277-282, Apr 1932 29. Solowey AC, Rapaport FT: Immunologic responses in cancer patients. Surg Gynec Obstet 121:756-760, Oct 1965 30. Sorokin JJ, Sugarbaker PH, Zamcheck N, et al: Serial carcinoembryonic antigen assays. Use in detection of cancer recurrence. JAMA 228:49-53, 1 Apr 1974 31. Stewart THM: The presence of delayed hypersensitivity reactions in patients toward cellular extracts of their cellular tumors. 2. A correlation between the histologic picture of lymphocyte infiltration of the tumor stroma, the presence of such a reaction, and a discussion of the significance of this phenomenon. Cancer 23: 1380-1387, Jun 1969 32. Ravry M, Moertel CG, Schutt AJ, et al: Usefulness of serial serum carcinoembryonic antigen (CEA) determinations during anticancer therapy or long-term follow-up of gastrointestinal carcinoma. Cancer 34: 1230- 1234, Oct 1974 33. Roberts MM, Jones-Williams W: The delayed hypersensitivity reaction in breast cancer. Br J Surg 61:549-552, Jul 1974 34. Vidae CE: Hodgkin's disease and its management. Bull Roswit Park Memoriallnst 1:105, 1956 35. Vider M, Kashmlri R, Hunter L, et al: Carcinoembryonic antigen (CEA) monitoring in the management of radiotherapy patients. Oncology 30:257-272, 1974 36. Vider M, Kashmiri R, Meeker WR, et al: Carcinoembryonic antigen (CEA) monitoring in the management of radiotherapeutic and chemotherapeutic patients. Am J Roentgenol 124:630-635, Aug 1975 37. Vincent RG, Chu TM: Carcinoembryonic antigens in patients with carcinoma of the lung. J Thorac Cardiovasc Surg 66: 320-328, Aug 1973 38. Witebsky E, Rose NR, Shulman S: Studies of normal and malignant tissue antigens. Cancer Res 16:831-841, Oct 1956 39. Zamcheck N, Moore TL, Dhar P, et al: Carcinoembryonic antigen in benign and malignant diseases of the digestive tract. Natl Cancer Inst Monogr 35:433-439, Dec 1972
Therapeutic Radiology
Carcinoembryonic Antigen and Skin Test Reactivity in Tumor Radiotherapy 1 Manuel Vider, M.D., Rafiah Kashmiri, M.Sci., Bernard Moses, A.B., Davie Earlywine, L.P.N., William R. Meeker, M.D., Joella F. Utley, M.D., and Yosh Maruyama, M.D. Serial carcinoembryonic antigen (CEA) levels were obtained from 122 cancer patients. In a random selection, the levels in 67 of these patients were compared with clinical response to radiotherapy. Skin tests were also performed for histoplasmin, tuberculin and mumps. CEA levels, skin-delayed hypersensitivity reaction (DHR) and clinical tumor response were evaluated and correlated. Clinical response of tumors to radiotherapy was more often seen in patients with positive skin tests, but no correlation was observed between skin test reactivity and CEA response curves. INDEX TERMS:
Immunity. Neoplasms, therapy. Skin. Therapeutic radiology, immunity
Radiology 119:677-681, June 1976
•
• (CEA) was first isolated in 1965 from human colonic cancers and fetal colonic tissue (15). This antigen was originally thought to be entodermally derived, tumor-specific and diagnostic for colon carcinoma. A large number of publications have since described the clinical application of CEA in the study of cancer and the management of the cancer patient (5-8, 12, 13, 19,22,25,27,30,32,35,36,39). In this paper, we shall analyze our experience with the CEA test in a radiotherapeutic clinic, with a separate test of immunological reactivity, i.e., delayed skin hypersensitivity response (DHR) to standard bacterial, viral and mycotic antigens (PPD, histoplasmin and mumps) and correlate these with clinical tumor response in a randomly sampled patient subpopulation. ARCINOEMBRYONIC ANTIGEN
C
BACKGROUND ON DELAYED HYPERSENSITIVITY AND IMMUNE RESPONSE IN CANCER
In 1921, MacCarty and Blackford (23) reported that regional lymphatic grandular involvement and postoperative longevity in gastric carcinoma appeared to be related. MacCarty and Mahle (24) summarized 99 of 200 cases followed for eight years and reported that (a) individuals with no lymph gland imvolvement and showing histological differentiation lived 14 % longer than those with no gland involvement and poor differentiation; (b) patients with partial gland involvement and differentiated histology lived 20 % longer than those with poor differentiation; (c) those with complete gland replacement showed no increased longevity despite differentiation; and (d) differentiation was more frequently associated with lymph gland involvement. Partial lymph node involvement was observed in 42.6 % and extensive lymph node involvement was noted in 7 % of the cases. This suggests a role for lymph node function and survival in gastric cancer. In 1954, Black and colleagues (3)
also concluded that survival in gastric carcinoma was related to nuclear differentiation and lymphoid infiltration. In 1930, Witebsky (38) produced an antiserum to human gastric cancer in guinea pigs. He then used alcoholic extracts to absorb the antisera of normal gastric tissue and found that the antiserum was still capable of producing a precipitation reaction against the cancer extract. This suggested that there was a different antigen in gastric cancer not present in the corresponding normal tissue. Moore and Foote (26) in 1949 related the prognosis of medullary carcinoma of the breast to the histological association with lymphocyte infiltration and germinal center formation and further speculated that "this rather characteristic lymphoid infiltrate indicates some adjustment between tumor and host." Berg (4) and Black and co-workers (3) also drew attention to the importance of a lymphoplasmacytic cell infiltrate in breast carcinoma and showed that such patients had a better prognosis. Roberts and Jones (33) found reduced DHR to Varidase as breast cancer progressed. Hodgkin's disease patients who are normally tuberculin-positive have negative skin tests with exacerbations of their disease, which revert to positive again during remissions. This represents a reversible defect in delayed hypersensitivity reactions which is not specific for tuberculin antigen since such aberrations are also seen in mumps and fungal skin test antigens (34). Cutaneous anergy to microbial antigens has been reported in leukemia, carcinoma and Hodgkin's disease (9, 28). Lamb et al. (20) found that 53 % of patients with Hodgkin's disease were anergic early in the course of the disease. Likewise, anergy was more frequent in all subjects studied by Hughes and Lytton (18) although no correlation with the histology of the tumors was observed. Anergy has also been found with carcinoma
1 From the Departments of Radiation Medicine (M. V., D. E., J. F. U., Y. M.) and Surgery (R. K., W. R. M.), University of Kentucky, A. B. Chandler Medical Center, Lexington, Ky. Presented at the Sixtieth Scientific Assembly and Annual Meeting of the Radiological Society of North America, Chicago, 111., Dec. 1-6, 1974. Supported in part by Grants CA 05258 and CA 17786, National Cancer Institute. shan
677
678
MANUEL VIDER AND OTHERS
Table I:
Cod ing System for Patterns of Response to Treatment
June 1976
Table III: Skin Test Results (Mean Diameter of Positive Skin Reactions (em) for Tumors of Different Sites)
The laboratory CEA response was coded using the following scheme. The digit describes the measurable CEA response in the plasma: 1. Normal Range, detectable, but less than 2.5 nq/ml 2. Sustained elevation, greater than 2.5 nq/rnl " 3. Progressive rise, to levels greater than 2.5 nq/ml 4. Progressive decline, initial levels greater than 2.5 nq/ml,
The clinical response of the tumor to radiotherapy can also be described. This was done using the following digitized scheme: 1. No clinical evidence of disease 2. Widespread tumor or unresponsive tumor* 3. Tumor progression, patient deterioration 4. Tumor response or clinical improvement X. Uncertain outcome. * Based upon experience of an earlier study (36), category 2 was not found to be of any value and was therefore not used in th is study.
and generally becomes more evident as the tumor condition progresses. Solowey and Rapaport (29) utilized streptokinasestreptodornase and the antigens of purified protein derivatives and reported 27.5 % positive tests in a population of 91 cancer patients. Grace and Kondo studied the autoreactivity of patients to their own tumor extracts and passive transferred antibodies in sera (16). The skin and tumor response tests revealed a depression of homograft responses in patients with advanced cancer (1, 16). When taken from the same donor, behavior of homografted skin also paralleled that of the tumor. Stewart (31), considering the inefficiency of the defensive mechanism as shown by the OHR, hypothesized that antigens that compete or share the final common path of the cell-mediated DHR of the host against the tumor can produce regression of certain tumors. In studies of host reactivity to neoantigens, Eilber and Morton (11) reported anergy to dinitrochlorobenzene (ONCB) delayed hypersensitivity reaction following cancer surgery. Of the 110 cases with negative DHR preoperatively, 45 were found inoperable. On the other hand, of the 70 cases that showed positive OHR, 90 %
Table II:
CasesGrouped by Organs
Site Lung Head and neck Uterus Colon and rectum Lymphomas Breast Skin Upper G I Urinary tract Multiple myeloma Prostate and testicle Ovary Brain Pancreas Total
Number of Cases 32 15 13 13 11 12
Site Lung Head, neck and eye Uterus Colorectal Lymphomas Breast Skin Upper GI Urinary tract, prostate and testicle Ovary Brain
No. Patients
+No. Reactors/No. Tested
Diameter of Skin Reaction (cm)*
12 13
6/12 13/13
2.0 2.8
11 7 4 8 3 4 3
7/11 4/7 2/4 6/8 1/3 2/4 2/3
2.8 3.5 1.3 1.8 1.0 1.8 4.8
1 1
0/1 0/1
NR NR
N R = No nreactor. + At least 1 positive test (mean reaction of all positive tests) . * Positive reactors only.
were operable and these patients were subsequently free of disease six months postoperatively. In all of these investigations, there have been attempts to apply a variety of immunological tests and assays as monitors for tracing response to therapy and for predicting tumor behavior in the cancer patient. Few studies have been applied to CEA and its possible relevance to other immunological tests in radiotherapy. This study was directed to that question. MATERIALS AND METHODS
Patient Population
Serial CEA titers were measured in 122 patients randomly selected from the patient population of the Radiation Medicine Clinic. The patients are 90 % white, and entirely from the Appalachian mountain and central Kentucky region. Many different neoplasms were seen. (TABLE I.)
One hundred and twenty-two patients were examined in a study in the Radiation Oncology Clinic of the University of Kentucky Medical Center, Lexlnqton, Kentucky. The study was carried out in a double blind manner. The samples were taken and transferred to the laboratory, and stored until all samples of the same patient had been collected. The results were obtained only Table IV:
Relationship Between Positive Skin Tests* and the CEA Response Curve
Code No.
Pattern of CEA Response
4
1
3 5 3
3
Negative CEA «2.5 ng/ml) Progressively rising CEA level (> 2.5 nq/ml) Falling CEA levels
5
2
2 2 122
4
Frequency of CEA Response to Therapy
Total * Any skin reaction greater than 0.5 em.
Pel' Cent of Group
23
35
23
35
21
30
-
--
67
100
after a conclusion was reached regarding clinical response. The first sample was taken before radiotherapy was started, the second was taken at the midpoint of the radiotherapeutic plan, i.e., at 3,000 rads in a planned course of 6,000 rads or at 1,000 rads in a planned course of 2,000 rads. The third sample was taken at the end of treatment and the fourth two months after the completion of treatment.
Radioimmunoassay Procedure
Clinical Response to Rad iotherapy
Skin Test Antigens and Methods Skin tests for Histoplasma (mycotic antigen), tuberculin as PPD (mycobacterial antigen) and mumps (viral antigen) were performed routinely on all patients. These were read at 24 and 48 hours and scored by standard scoring methods which measure erythema and induration in centimeters of diameter.
2.
3.
Tuberculosis: The tuberculin purified protein derivative (PPO) (Mantoux), was produced by Connaught and distributed by Panray, Ormont Co., Englewood, N.J., bioequivalent to 5 U.S. units; 0.1 ml of the antigen (5 U.S. units) was injected intradermally. Mumps: The mumps skin test antigen, produced by Lilly (V-1059) was injected in a dose of 0.1 ml intradermally. Histoplasmosis: The histoplasmin skin test antigen produced by Parke-Davis was injected intradermally in the dose of 0.1 ml.
These agents to exposed. reactivity
three antigens represent the most common which the population of this region have been The population of Kentucky showed a higher than that of all the states surrounding it to tuTable VI:
Frequency of Clinical Response
Unknown
1
No clinical tumor Tumor progression Tumor response
3
-
Per Cent of Group
7 13
19
11
16
36
54
67
100
11
* Any skin reaction greater than 0.5 cm.
berculin antigen (PPO). Thirty per cent of metropolitan and 40 % of farm inhabitants have skin reactions greater than 4 mm in diameter (31). The percentage of PPO with 10 or more mm reactivity was 8 % for metropolitan, 7 % for non-farm population and 8 % for farm inhabitants. The incidence of reaction to PPD with 4 or more mm was 30 % for metropolitan inhabitants, 25 % for non-farming population and 40 % for farmers. Kentucky dwellers also showed the highest incidence of reactivity to histoplasmin of any state in the Ohio Valley where this fungal disease is endemic. About 70 % have reactions greater than 4 mm in diameter. Mumps, a viral antigen, has a high incidence in all populations surveyed ("'-190 % ) (20). Any patient with a positive reaction was considered a reactor whether to one or multiple antigens. Because of the battery selected, a positive exposure history often could not be obtained. RESULTS
Of the serial CEA levels sampled in 122 patients, 78 cancer patients (64 %) had CEA levels greater than 2.5 ng/ml. Fifty-seven patients or 73 % showed correlation between the tumor-therapy response and the CEA levels during this period (35, 36). Sixty-seven patients were randomly selected for further study by skin test reactivity, and complete data was available for analysis.
Tumor Types and Skin Test Reactivity TABLE II shows the types and numbers of tumors in the study of CEA titers and response patterns and
Mean Skin Reactivity* (in cm) Related to the Clinical Response and CEA Curve Response
\ (__--Clinical Response to Radiotherapy Mean Skin Test Reaction Category Pattern (diameter of Response of Response in cm) Clinical response Type 3 Clinical response Type 4
Pattern of Clinical Response
Totals
The method in use (36) was developed by Gold (15) and modified by Hansen et al. (17). Basically, with use of a standard radioimmunoassay of 1251-labeled monospecific goat anti-CEA antibody, a sample of patient plasma, anticoagulated with EDTA was assayed for CEA. Plasma levels were not considered positive unless greater than 2.5 nanograms/ml. TABLE I describes the method used for charting the CEA levels found in patient plasma, and the scoring system for patient-tumor response to radiotherapy.
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Table V: Relationship of Positive Skin Test* and Clinical Radiotherapy Response
4
1.
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CARCINOEMBRYONIC ANTIGEN AND SKIN TEST REACTIVITY
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Nonresponders
1.1
Responders
2.7
* Positive ski n tests only.
(.------CEA Response to Radiotherapy-------.., Category Pattern Mean Skin of Response of Response Test Reaction CEA curve pattern Type 3 CEA curve pattern Type 4
Progressive rise
1.5cm
Declines with treatment
1.8 cm
MANUEL VIDER AND OTHERS
680
TABLE III gives the mean diameter of the positive skin test reactions in the patients sampled in each tumor category who reacted positively to the skin test antigen.
CEA Response Pattern and Skin Test Reactivity TABLE IV presents data on the positive skin test reactivity and the pattern of the CEA response observed: 23/67 or 35 % had negative CEA tests and a positive skin test; 23/67 or 35 % had progressively rising CEA levels and positive skin tests; 21/67 or 30 % had CEA levels which fell in response to therapy, and positive skin tests. This indicates that there is no constant pattern of plasma CEA progression or decline in relation to skin test reactivity.
Clinical Tumor Response and Skin Test Reactivity TABLE V shows the different patterns of tumor response to radiotherapy in skin test positive patients. In this table, the clinical response was classified as: Unknown 1 = No clinical tumor 3 Progressive tumor despite radiotherapy 4 Tumor response with radiotherapy.
= =
There was a significantly greater proportion of patients with positive skin tests who showed clinical response to radiotherapy. This was seen in 36/67 or 54 % of patients who showed a positive response by DHR skin tests. In general, there was a better response to treatment in the radiotherapy patients who had positive skin tests among those studied here.
Relationship of Positive DHR Response, CEA Response and Clinical Tumor Response TABLE VI further subdivides and analyzes DHR reactivity, CEA response patterns and clinical response pattern. In the present study, 31 % of those with positive CEA level had a clinical response to therapy and had at least one positive response to the battery of skin tests. In those who had a clinical response to therapy, the OHR test responders also had a greater mean skin test reaction measured in centimeters (2.7 ern) compared with nonresponders (Type 3) whose diameter was 1.1 cm. However, the responder type of CEA curve (Type 4) did not show a different degree of DHR response compared to nonresponder CEA curves (Type 3) when matched on the basis of the intensity of the skin test (1.8 cm vs. 1.5 em).
DISCUSSION
In this series, 78 of our 122 patients (64 %) showed detectable plasma levels of CEA and 57 (73 %) showed
June 1976
correlation between the response curves traced by the CEA and clinical response. Clinical response of tumors to radiotherapy was more often seen in patients with positive skin tests, but there was no correlation observed between skin test reactivity and the CEA response curves. Local therapy and destruction of the tumor by irradiation could result in the release of tumor-associated antigens. One would then expect that immediately following irradiation, monitors of serum CEA level in tumor-bearing patients would change with radiotherapy and response to therapy. An initially nondetectable titer may indicate a small or localized, or a non-CEA-producing tumor. A positive titer will usually indicate an advanced localized tumor or more widespread tumor Local radiotherapy may increase, decrease, or not affect such a tumor. A decrease in CEA titers with treatment may reflect radioresponsiveness of the tumor and its destruction. No change would indicate CEA-producing sites outside the treatment field or radioresistance of the tumor under irradiation. Progressively rising titers would indicate an increasing number of CEA-producing sites outside the treatment field or radioresistance. Our data indicate that such correlations are observed with the level of plasma CEA (35, 36) in patients undergoing radiation therapy. Skin test reactivity has also been advocated as a test for immune reactivity. A positive reaction indicates that a more favorable tumor response can be expected. Such observations were confirmed in these studies. Lundy et al. (21) showed correlation between ONCB reactivity for recurrence and survival in localized disease of the head and neck. In general, the results of these studies again show that no single test was completely accurate in predicting tumor response. We found a lack of correlation between the skin tests and CEA response which is consistent with the hypothesis that these two tests monitor separate mechanisms of immunological response in cancer patients. Thus, the responsiveness by the CEA curve seems to be unrelated to the mechanism that causes delayed hypersensitivity (OHR) reactions to the histoplasmin, PPO and mumps skin tests. This can be explained, assuming that the CEA response curves and OHR are separate parameters and may not involve the same mechanisms that relate to the Clinical response of the patient to tumor therapy. We observed a lack of correlation which is sometimes seen when many different tests or diagnostic procedures are combined looking for improvements of the accuracy of the collective tests. The objective in the planning and carrying out of this work was to clarify and extend the interpretation and application of currently available immunological tests to the monitoring and management of patients undergoing radiotherapy. We conclude that clinical response of tumors to radiotherapy was more often seen in patients with positive skin tests, but there was no correlation observed between skin test reactivity and the CEA re-
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sponse curves. However, it was apparent that there is a need for further development of more precise batteries of immunological tests to assess the immunological response of the cancer patient to treatment than those reported here. Yosh Maruyama, M.D. Department of Radiation Medicine University of Kentucky A. B. Chandler Medical Center Lexington, Ky. 40506
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