Assessment of Cellular Immune Response to Cancer of the Breast

ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 9, No. 6 Copyright 1979, Institute for Clinical Science, Inc. Assessment of Cellular Immune Response to Cancer of the Breast RONALD B. HERBERM AN, M.D. Laboratory o f Immunodiagnosis National Cancer Institute, Bethesda, MD 20014 ABSTRACT Cellular immune competence and cell-m ediated immunity to tumor antigens have been studied in patients with breast cancer. Some patients have b een shown to have depressed lym phoproliferative responses to phytohem agglutinin and in mixed leukocyte culture. In some cases, this depression appeared attributable to suppressor cells. Many patients with breast cancer had a cellular immunity to extracts of autologous or allogeneic tumors, as detected by lymphoproliferation and leukocyte m igration inhibition assays. In addition, some breast cancer patients reacted to antigens associated with murine mammary tumor virus. Some of the tests for cellular immunity have revealed correlations with clinical course and, therefore, may be of use in the m anagem ent of patients with breast cancer. Introduction A ssessm ent of cellu lar im m unity in breast cancer patients is of considerable interest in two different respects. First, such studies should help to provide a b etter understanding of the biology of breast cancer, particularly in regard to the possible role of the immune response in resistance against tum or growth. Im portant questions in this regard are: (a) is the presence of breast cancer associated with depressed cellular im m une com petence and (b) do breast cancers have tumorassociated antigens w hich can induce cell-m ediated im m unity in the autochthonous host and w hich m ight be involved in re sista n c e ag ain st tum or growth? Second, tests of cellular immunity might have practical clinical value for im m unodiagnosis or m anagem ent of breast cancer. Some tests might be useful 4 6 7 in the detection of breast cancer by screening of general populations or groups of women at high risk of developing breast cancer. They might also aid in the differen tial diagnosis of m alignant versus b e nign breast diseases. In patients w ith known breast cancer, cellular immunity assays m ight help to determ ine the stage of disease and assess prognosis, thereby helping to identify patients w ith poor prognosis w ho sh o u ld receiv e chem o therapy or other treatm ent after surgery. F urtherm ore, after m astectom y, serial testing of patients might aid in the early d e te c tio n o f re c u rre n t or m etastatic disease. D epressed Cellular Im m une Com petence In vitro assays of cell-m ed iated im m une reactivity have been used to look for 0091-7370/1100-0467 $01.20 Institute for Clinical Science, Inc.

468 HERBERMAN decreased immune com petence in cancer p a tie n ts. D e c re a se d p ro life ratio n of lymphocytes in response to m itogens has been extensively studied, b u t clear depression has been largely restricted to patie n ts w ith ad v a n ced or in o p e ra b le cancer.24 It should be noted, however, that significant depression has been seen even with patients with stage I or II disease,24, and using the relative proliferation index to quantitate better the response relative to norm al donors, fo und d e p re sse d lym phoproliferative responses to phytohem agglutinin and in mixed leukocyte cultures (MLC) were found by u s10 in about one-third of breast cancer patients. In a recent analysis of data from tests performed within four months after mastectomy, we observed some correlation b e tw een depressed responses in MLC and clinical course. However, the trend has been in a paradoxical direction, with stage I and II patients with depressed MLC responses having a prolonged disease-free interval compared to patients with normal MLC reactivity.* Further studies are needed to understand the basis for this association, but there have been indications that some patients with depressed responses do not have an intrinsic defect in their lymphocytes but rather have suppressor cells w hich can inhibit lym phoproliferative responses.13 In addition to disease-related im m unodepression, th erap y can also cause depressed imm unity. We have observed persistent depressed proliferative responses in breast cancer patients who received postoperative radiotherapy. Cellular Immunity to Tumor-Associated Antigens M any tum or-associated antigens or oth er antigens can e lic it im m une responses in the tum or-bearing patient. As * Cannon, G. B., Dean, J. D., and Herberman, R. B.: manuscript in preparation. sessm ent of such responses, particularly cell-m ediated immune responses, have b e e n ex te n siv ely stu d ie d in sev eral laboratories. It has been important to determ ine w hether or not some of the tests can identify tumor-associated antigens in breast cancer and w hether or not the existence of cellular immunity to the tumors is related to clinical status. Antigens can often be recognized by the host w hen present in very small amounts, and recognition and response could occur prior to release of the antigens into the circulation. It might, therefore, be expected that im m unological reactions w ould be d e tected while tumors were still small and localized, and that assays for such reactions might be more sensitive markers for breast cancer than the circulating tumor markers. D etection of cellular immune reactions in breast cancer patients might also serve as a means for identifying and isolating new breast-cancer-associated antigens, which could then be used for raising heterologous antibodies and setting up radioimmunoassays to measure antigen levels in the circulation. There have been several studies with breastcancer patients which have dem onstrated d elayed cutaneous h y p ersen sitivity reactions to tumor extracts. After some patients were found to have skin reactions to crude membrane extracts of autologous and allogeneic tumors,1soluble skin reactive antigens were then prepared from the crude extracts by sonication and separated by Sephadex G-200 chrom atography. Sephadex fractions of extracts of normal breast tissue from cancer patients and normal breast tissue from patients with fibrocystic disease, as well as those from breast cancer, gave reactivity in some patients with breast cancer. These data suggested that some patients had im m une reactiv ity against organassociated antigens. On further separation by gradient polyacrylam ide gel electrophoresis,12 two adjacent gel regions gave positive reactions.

ASSESSM ENT OF RESPONSE TO BREAST CANCER 469 In prelim inary tests for skin reactivity, one fraction (region 2b) appeared to contain a breast-cancer-associated antigen and the other (region 2a) a breast-tissueasso c ia te d an tig e n. In th e se in itia l studies, region 2a gave positive reactions in patients with localized breast cancer and not in patients w ith dissem inated cancer, w hereas the other region gave positive results in m ost breast cancer patients. In more recen t tests w ith larger num bers of patients and with further splitting of gel regions, region 2a from both a large primary tumor and from MCF-7, a cell line derived from breast cancer, again gave a high incidence of positive reactions with good specificity in patients with localized breast cancer.22 The region 2b 1-3 from MCF-7 also showed good specificity and reacted only in patients with localized disease. The ability to separate breasttumor-associated skin reactive antigens from an established cell line is very encouraging, since this should be a source for large, standardized batches of antigen for extensive clinical testing. Efforts are also u n d e r way to prepare specific antibodies against the isolated skin reactive antigens, which may be used to set up radioim m unoassays for these antigens. A num ber of investigators have studied the proliferative response of lymphocytes from breast cancer patients to tumor cells or to tumor extracts. It is difficult to estimate the clinical utility of most of the data. Since the num bers of patients in most studies w ere small, no clinical correlations were made, and allogeneic extracts w ere usually em ployed. The use o f allogeneic materials has been found to present the p o ten tial for resp o n se to norm al alloantigens,11 and it is difficult to distinguish this from reactivity to tumorassociated antigens. Such problem s in interpretation can be elim inated by the use o f autologous tum or cells or extracts. However, this would obviously restrict the usefulness of this test to the study of known cancer patients, after surgical removal of tumor. Dean et al9 studied 34 patients with breast cancer and observed significant proliferative responses in 12 patients to either intact autologous tum or cells or crude extracts of autologous tumors. The reactivity appeared to be directed against tumor-associated antigens, since normal breast tissue of reactive patients did not stim ulate their lymphocytes. W hen the data from the lym phoproliferative responses to tumor antigens and in MLC were analyzed in regard to subsequent clinical course, it was found th at th e combined use of both tests provided a significant discrim ination b e tw e en patients developing recurrent disease and those rem aining disease free.* Patients who had responses in MLC in the normal range and were unreactive to tumor antigens had a poor prognosis, with recurrence of disease in the majority. In contrast, m ost p atien ts who w ere depressed in MLC or who were normal in M LC and reactive to tum or antigens rem ained free of disease. Although the basis for this correlation is unclear, such testing may prove very useful in substaging breast cancer patients and identifying patients w ith poor prognosis who m ight benefit from further therapy after m astectomy. The leukocyte m igration inhibition assay, which is considered a close in vitro correlate of delayed cutaneous hypersensitivity, has been widely used to study cell-m ediated immunity of breast cancer patients. Anderson et al2 first showed that the m igration o f leukocytes of some patients with breast cancer was inhibited on exposure to autologous tumor extracts and not to normal tissues. D ecreased reactivity was seen in patients who w ere free of evident disease after mastectomy, but this was difficult to evaluate because the p a * Cannon, G. B., Dean, J. H., and Herberman, R. B., manuscript in preparation.

470 HERBERMAN tients also received radiotherapy. Segall et al21 confirm ed the reactivity of the majority of breast cancer patients to autologous tum or extracts. They looked for cross-reactivity to other breast cancers and obtained only one positive result. M ost su b seq u en t studies, how ever, have found good reactivity of b reast cancer patients to allogeneic tum or extracts.8,14,20 McCoy e ta l 19 also found reactivity of the majority of breast cancer patients against extracts of MCF-7, a cell line derived from breast cancer. T he occurrence of reactivity of breast cancer patients against common antigens in allogeneic as well as autologous extracts derived from tumors suggests the potential usefulness of this procedure for initial diagnosis. H ow ever, although only a small proportion of normal women have shown reactivity in these studies, patients with benign breast diseases have displayed a h ig h er frequency of reactivity. ^S"14-19"20 Patients whose breast biopsies showed some form of breast disease had reactivity sim ilar to that of breast cancer patients, whereas women whose biopsies showed no evidence of disease failed to react.7 These results, and the reactivity of some cancer patients against extracts of benign breast lesions, indicate that patients with neoplastic and benign breast diseases may become sensitized to breast tissue antigens. Sim ilar to the findings described previously for delayed cutaneous hypersensitivity reactions, Kadish e t al4 reported preliminary evidence for the separation of breast-cancer-associated antigens from b reast tissu e a n tig en s. A highm olecular-w eight fraction elicited reactions in 50 percent of breast cancer patients and in only 5 percent of controls. In contrast, a low-molecular-weight fraction elicited reactivity in patients with benign and m alignant b reast diseases. This finding suggests that two different antigens could be used for clinical testing, one that m ight be useful to screen for patients with breast diseases and the other that might h e lp to d iscrim inate b rea st cancer patients from patients with benign breast diseases. It was of interest that the reactivity of wom en with benign diseases was strongly influenced by surgery, w ith a drop in reactivity within one m onth after surgery; in contrast, the reactivity o f breast cancer patients rem ained high during this period.7 A nother potential application of this assay m ight be to assess the prognosis of breast cancer patients. Black et al3 have exam ined this issue most extensively, using cryostat sections of tumor tissues as source of antigens. T he frequency of reactivity in their study was related to stage, w ith 90 percent of patients with in situ cancer reacting and only about one third of stage II patients reacting against autologous tumor sections. The relationship betw een reactivity and disease status at the tim e of testing has not been w ell defined in most studies, but recent analysis of data in our laboratory16 indicates that reactivity tended to decrease one to six m onths prior to clinical recurrence of disease. Black et a l4 m ade the intriguing observation that many patients with breast cancer reacted in leukocyte m igration inhibition assays with mouse milk from a hig h m am m ary tum or virus (MTV) 4- strain and usually not with milk from a low MTV strain. A higher incidence of reactivity was noted in patients with other favorable prognostic indicators,6 and reactivity to the virus-containing milk correlated rather well w ith reactivity of patients to autologous or allogeneic breast cancer m aterials.5 Zachrau et al25 eluted a protein from the breast tissues that appeared to have a m olecular w eight similar to that of the gp52 of MTV. This protein itself, how ever, was not shown to elicit migration inhibition or to be antigenically related to gp52. McCoy et al15 confirmed that many breast cancer patients can react in leuko

ASSESSMENT O F RESPONSE TO BREAST CANCER 47] cyte m igration in h ibition w ith MTV + mouse milk, and also with purified MTV and gp52. In contrast, very few normal donors or patients w ith other types of cancer have reacted to these m aterials. The significance o f these reactions rem ains to be d e term in ed, since som e breast cancer patients have also reacted in m igration inhibition to gp69/71 of m urine leukem ia virus, w hich does not crossreact serologically w ith gp52. The cellm ediated immune response m ight be recognising a common determ inant on these two viral glycoproteins, or these results m ight simply reflect a hyperreactivity of breast cancer patients to glycoproteins of various types. Regardless of the nature of the antigenic specificities recognized, however, m easurem ent of reactivity to these viral reagents may be quite useful for m onitoring of breast cancer patients. Although considerable information has been obtained with the usual direct capillary tube leukocyte migration inhibition assay, this procedure has several disadvantages. It is technically difficult, requires large volumes of blood, is not quantitative and does not perm it analysis of the mechanism of the reaction. McCoy and oth ers17,18,23 in our laboratory have m odified the leukocyte m igration assay in several ways to overcome most of these problems. It has been possible to perform indirect assays using human granulocytes from normal donors as indicator cells, for m easurem ent of lymphokine (leukocyte inhibitory factor) production in supernatants from mononuclear cells of patients incubated w ith tum or extracts or viral antigens. The migration phase of the assay has been performed by an agarose microdroplet m ethod, which reduces the num ber of granulocytes and supernatants required an d sim p lifies th e perform ance and analysis of the test. By incubating the mononuclear cells with antigen in conical microfuge tubes,16 high titers (1:100 to 1:1000) of lymphokine have been generated from small num bers of cells. These im provem ents in the sensitivity and technical ease of the leukocyte m igration inhibition assay make it more likely that it will eventually come into wide-scale use in clinical laboratories. Conclusions Studies of cellular immunity in breast cancer patients have provided some im portant inform ation regarding the function of the im m une system in breast cancer. It seem s reasonably clear that some breast cancer patients have depressed cellular im m une com petence. There is also substantial evidence for cell-m ediated immunity to breast cancer antigens and to antigens related in some way to m ouse m am m ary tum or virus. However, the specificity of the immune reactions needs to be clarified. Patients w ith benign diseases of the breast, as well as w ith carcinom a of the breast, have shown considerable reactivity in some of these assays and it seems likely that breast tissue antigens or o th er antigens are being recognized in addition to tumorassociated antigens. In regard to the practical value of these tests, the need to perform lymphoproliferation assays with autologous tumor materials and the occurrence of reactivity by patients with benign disease in leukocyte migration inhibition would appear to p reclu d e any use of th e se tests for detection or diagnosis of breast cancer. However, some of these techniques may provide assistance in the m anagem ent of patients with breast cancer. O ur data indicate that the m easurem ent of lymphoproliferative responses in M LC and to autologous tumor antigens may help to id en tify p a tie n ts w ith a high risk of developing recurrence of disease after mastectomy. In addition, serial m onitoring with leukocte m igration inhibition assay may provide early indication of recurrent disease. F u rth e r studies of the

472 HERBERMAN relationship betw een immune reactivity and clin ical course may also provide insight into some interesting and im portant biological factors in regard to host defense against progressive growth of breast cancer. References 1. A l f o r d, C., H o l l i n s h e a d, A. C., and H e r b e r m a n, R. B.: Delayed cutaneous hypersensitivity reactions to extracts of malignant and normal human breast cells. Ann. Surg. 178:20-24, 1973. 2. An d e r s e n, V., Bj e r r u m, O., B e n d ix e n, G., Sc h io d t, T., and DlSSING, I.: Effect of autologous mammary tumor extracts on human leukocyte migration in vitro. Int. J. Cancer 5:357-363, 1970. 3. B l a c k, M. M., L e is, H. P., S h o r e, B., and Z a c h r a u, R. E.: Cellular hypersensitivity to breast cancer: Assessm ent by a leukocyte migration procedure. Cancer33:952-958,1974. 4. B l a c k, M. M., M o o r e, D. H., S h o r e, B., Z a c h r a u, R. E., and L e i s, H. P., J r.: Effect of murine milk samples and human breast tissues on human leukocyte migration indices. Cancer Res. 34:1054-1060, 1974. 5. B l a c k, M. M., Z a c h r a u, R. E., Sh o r e, B., a n d L e i s, H. P., J r.: B io lo g ical c o n s id e ra tio n s o f tu m o r-sp e c ific a n d v iru s-a ss o c ia te d a n tig e n s of h u m a n b re a s t can c e r. C a n c e r R es. 36:769-774, 1976. 6. B l a c k, M. M., Z a c h r a u, R. E., S h o r e, B., M o o r e, D. H., and L e i s, H. P., J r.: Prognostically favorable immunogens of human breast cancer tissue: antigenic similarity to murine mammary tumor virus. Cancer 35:121-128, 1975. 7. C a n n o n, G. B., M cc o y, J. L., Je r o m e, L. J., Re d d ic k, R., A l f o r d, C., T e n l e y, V., and H e r ber m an, R. B.: Immunological relationship betw een breast carcinoma and benign breast disease as detected by the leukocyte migration inhibition assay. J. Nat. Cancer Inst. 61.1 1 8 1-1 1 8 6, 1978. 8. C o c h r a n, A. 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ASSESSM ENT OF RESPONSE TO BREAST CANCER 473 21. Se g a l l, A. W e i l e r, O., G e n i n, ]., L a c o u r, J., and L a c o u r, F.: In vitro study of cellular im m unity against autochthonous human cancer. Int. J. Cancer 9:417-425, 1972. 22. W e e s e, J. H e r b e r m a n, R. B., H o l l in s h e a d, R. B., C a n n o n, G., Ke e l s, M., and O l d s h a m, R. K.: Specificity of delayed cutaneous hypersensitivity reactions to extracts of human tumor cells. J. Nat. Cancer Inst. 60:255-263, 1978. 23. W e e s e, J. L., M cc o y, J. L., D e a n, J. H., O r - t a l d o, J. R., B u r k, K. R., and H e r b e r m a n, R. B.: Brief communication: Technical modifications of the human agarose microdroplet leukocyte migration inhibition assay. J. Immunol. Methods 24:363-370, 1978. 24. W h it t a k e r, M. G., and C l a r k, C. G.-. D e pressed lymphocyte function in carcinoma of the breast. Brit. J. Surg. 58:717-720, 1971. 25. Z a c h r a u, R. E., B l a c k, M. M., D io n, A. S., S h o r e, B., I sa c, M., An d r a d e, A. M and W i l l ia m s, C. J.: Prognostically significant protein components of human breast cancer tissues. Cancer Res. 36:3143-3146, 1976. STATEMENT OF OWNERSHIP, MANAGEMENT AND CIRCULATION (Act of October 23, 1962; Section 4369, Title 39, United States Code) Date of Filing September 20, 1979 Title of Publication ANNALS OF CLINICAL AND LABORATORY SCIENCE Frequency of Issue Bimonthly Location of Known Office of Publication 230 N. Broad St., Philadelphia, PA. 19102 Location of the Headquarters or General Business Offices of the Publisher Same as above Publisher Institute for Clinical Sciences, Inc. Editor F. William Sunderman, M.D., Ph.D. Managing Editor Same as above Owner Institute for Clinical Sciences, Inc. Known Bondholders, Mortgagees, and Other Security Holders Owing or Holding 1 Percent or More of Total Amount of Bonds, Mortgages or Other Securities None Average No. Copies Each Issue During Preceding 12 Months Actual Number of Copies of Single Issue Published Nearest to Filing Date A. Total No. Copies Printed (Net Press Run) 1750 1700 B. Paid Circulation 1. Sales Through Dealers and Carriers, Street Vendors and Counter Sales None None 2. Mail Subscriptions 1542 1536 C. Total Paid Circulation 1542 1536 D. Free Distribution by Mail, Carrier or Other Means, Samples, Complimentary, and Other Free Copies None None E. Total Distribution (Sumof CandD) 1542 1536 F. Copies not Distributed 1. Office Use, Left-over, Unaccounted Spoiled After Printing 208 164 2. Returns from News Agents None None G. Total (Sumof EandF shouldequal net press runshownina) 1750 1700 I certify that the statements made by me are correct and complete F. WILLIAM SUNDERMAN, M.D., Ph.D.