CA-549: Immunohistochemistry and Serum Levels in Breast Carcinoma and Other Neoplasms*

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 19, No. 6 Copyright 1989, Institute for Clinical Science, Inc. CA-549: Immunohistochemistry and Serum Levels in Breast Carcinoma and Other Neoplasms* MUHAMMAD S. SHURBAJI, M.D.,ti ROY A. BEVERIDGE, M.D., DANIEL W. CHAN, P h.d.,!* ROBERT C. ROCK, M.D.,t$ and FRANCIS P. KUHAJDA, M.D.f$ Departments of Pathology, f Laboratory Medicine,t and Oncology, The Johns Hopkins Hospital and University Baltimore, MD ABSTRACT CA-549 is a high m olecular w eight acidic glycoprotein found in the serum of breast cancer patients. D etection of CA-549 in serum using an immunoradiometric assay has [1] correlated with disease course in breast canqer patients and [2 ] aided in establishing the diagnosis of breast cancer in patients with metastatic disease. This study examines the expression of CA-549 using im m unohistochem istry in norm al breast, benign breast disease, breast carcinoma, and a variety of other carcinomas. In addition, in 29 patients both serum and tissue samples were available for correlation. CA-549 was constitutively expressed in normal breast tissue, but immunohistochem ical positivity was restricted either to the luminal aspect or entire cell m em brane. All patients with benign breast disease had normal levels of CA-549 despite immunohistochemical positivity. Nearly all (98 percent) of breast carcinomas showed reactivity for CA-549, w ith a m ajority (82 percent) of the cases showing cytoplasmic positivity. In patients with both serum and tissue studied, those with cytoplasmic staining of the breast carcinomas had mean serum level of 174 U per ml (range 1.9 to 785), compared to 37.3 U per ml (range 2.1 to ) in those with only m em brane or luminal staining of tum or (p = ). Immunoreactive CA-549 was found in many norm al epithelia and in other types of carcinomas. CA-549 is [1] constitutively expressed on the cell m em brane of norm al breast epithelium, [2 ] commonly present in the cytoplasm of breast carcinomas, and [3] found often in a variety of carcinomas. A,,,, Address reprint requests to: M. S. Shurbaji, Introduction M.D., e/o Francis P. Kuhajda, M.D., Departm ent of Pathology, Johns Hopkins Hospital, 600 N. Wolfe Ar- Ar\ i. i i i.,. CA-549 IS a high m olecular w eight St., Baltimore, m d acidic glycoprotein d e te c te d by a new /89/ $01.20 Institute for Clinical Science, Inc.

2 CA-549 IMMUNOHISTOCHEMISTRY 409 monoclonal antibody, BC4E549, raised by im m unizing m ice w ith a partially purified m em brane p rep a ra tio n from T417 hum an breast tum or grown in nude m ic e. 2 C A -549 w as a d a p te d to an immunoradiometric serum assay and is a new marker for breast cancer. 1-3 It was dem onstrated by us earlier that CA-549 seru m assay is a re la tiv e ly specific marker for breast cancer and that it was superior to carcinoem bryonic antigen (CEA) in m onitoring disease progression. 1 Elevated serum levels were found in a minority of patients with other epithelial malignancies, but they tended to be low er th an th e levels seen w ith patients w ith breast carcinom a. 1 3 In light of the relative specificity and sensitivity of CA-549 as a serum m arker for breast carcinoma, the potential utility of CA-549 has b een evaluated as an im m unohistochem ical m arker for m am m ary carcinom a in formalin-fixed, paraffin em bedded tissue. In addition, both immunohistochemical positivity and the pattern of positivity were correlated with serum levels of CA-549 w hen available. M aterials and M ethods C a s e S e l e c t io n Serum sam ples w ere obtained from patients with benign breast disease and m alignant neoplasm s of breast, colon, lung, prostate, ovary, and endometrium. These random ly selected patients had participated in a previous study to evaluate CA-549 serum levels. 1 O f the 301 patients in the previous study, 84 had surgical pathology material available for immunohistochemical study. Both serum levels and tissue immunoreactivity were correlated in this population which consisted of 26 cases of benign breast disease, and 29 breast, 16 colon, eight prostate, and five lung carcinomas. To study fu rth er the im m unohistochem istry of CA-549, a variety of m alignant epithelial tum ors w ith available paraffin blocks, were chosen from the surgical pathology files of The Johns Hopkins Hospital. M o n o c l o n a l A n t ib o d ie s BC4N154* is a m urine IgM monoclonal antibody produced in fusion using sp le n ic ly m p h o c y te s from a m ouse immunized with hum an milk fat globule m em b ran es. This antibody was covalently linked to polystyrene beads and used only in the im m unoradiom etric (IRMA) assay. 2 BC4E549* is a m urine IgG l monoclonal antibody derived from fusion using sp le n ic ly m p h o c y te s from a m ouse im m unized w ith purified m em branes from the T417 hum an breast tum or cell line grown in nude m ice.2 This antibody was used as the labelled antibody in the IRMA serum assay and as the prim ary antibody for the immunohistochem ical studies. S e r u m A ssay Serum analyses were perform ed using an immunoradiometric assay (IRMA) as previously d e scrib e d. 1 2 Any CA-549 level >11 Units per ml was considered elevated. 1 I m m u n o h is t o c h e m is t r y Im m unohistochem ical staining was ad ap ted to a C ode-o n im m unohistochemical stainer. t Briefly, 6 x sections w ere cut from routinely processed 1 0 percent buffered formalin-fixed and paraffin em bedded tissue blocks, deparaffinized, rehydrated in Tris saline buffer (50 mm Tris, 150 mm NaCl), and treated w ith th re e p e rc e n t H 20 2 in absolute m ethanol for five m in to block endogenous pero x id ase activity. Follow ing * Hybritech, Inc., San Diego, CA. t Fisher Scientific, Pittsburgh, PA.

3 4 1 0 SHURBAJI, BEVERIDGE, CHAN, ROCK, AND KUHAJDA pronase treatm ent^ 0.5 mg per ml for 30 min at 37 C, slides were rinsed with Tris buffer and blocked w ith non-im m une horse serum, 1:100. BC4E549 monoclonal antibody diluted 1:400 in Tris-saline was applied for 30 min at 37 C, followed by biotinylated horse anti-mouse, 1 :1 0 0 and preformed, prediluted avidin-peroxidase com plex 1. A m inoethylcarbazole (Biomeda) was utilized as the chrom o gen. Non-immune mouse serum served as a negative control. The counterstain was M ayer s hematoxylin. Results I m m u n o h is t o c h e m ic a l D e t e c t io n o f CA-549 C e llu la r P a tte r n s : C A -549 w as expressed to a varying degree by a wide variety of norm al epithelial tissues (table I) and epithelial neoplasm s (table II). W ithin individual cells two patterns of staining were identified: a luminal/membranous pattern or cytoplasmic pattern. W ith the luminal/membranous pattern, CA-549 immunoreactivity was found in either th e lum inal aspect of cells occupying a gland, or rimming the entire cell m em brane; th e re was a concom itant absence of cytoplasmic positivity. W ith the cytoplasmic pattern diffuse staining of the cytoplasm usually occurred in the m ajority of the cells. In both staining patterns, nuclei w ere always negative. For classification purposes, tissues that show ed any d e g re e of u n e q u iv o c a l im m unoreactivity w ere considered as positive. Expression of any appreciable d e g re e of cytoplasm ic reactivity resulted in classification as the cytoplasmic pattern. Non-neoplastic Tissue: Normal epithelium from a variety of organs, when posi- t Behring Diagnostic, Lajolla, CA. Vector Lab, Burlingame, CA. 1Biomeda, Stoughton, MA. T i s s u e TABLE I CA-549 Immunohistochemical Positivity in Selected Normal Tissues P o s i t i v e S t a i n i n g * (Percent) Breast 26/26 (10 0 ) Colon 13/28 ( 46) Lung 6/6 (10 0 ) Prostate 13/20 ( 65) Endometrium 5/5 (10 0 ) *A11 normal epithelia showed only luminal/membranous pattern of staining. tive, tended to show uniform staining intensity and exclusively the lum inal/ m em branous p attern of cellular positivity. In figure 1A is illustrated this pattern of uniform luminal/membranous staining in the normal breast. A similar staining p a tte rn was seen in benign b reast disease (figure IB). Neoplastic Tissue: In contrast to the relatively uniform staining of non-neoplastic tissue, tum ors show ed m arked variation in staining intensity reflecting tum or heterogeneity as seen with other tum or m arkers. 7,8 In breast carcinoma, im m u n o h is to c h e m ic a l sta in in g for CA-549 was seen in 57/58 (98 percent) of cases. In these cases the majority of the tum or cells showed cytoplasmic staining Tissue TABLE II CA-549 Immunohistochemical Positivity in Selected Carcinomas *Cyto. ilum./ Mem. Total + (Percent) Negative (Percent) Breast ( 98) 1 ( 2) Colon ( 86) 4 (14) Prostate ( 54) 18 (46) Ovary (100) 0 ( 0) Endometrium (100) 0 ( 0) Pancreas ( 63) 3 (37) Liver ( 8) 1 1 (92) Head & Neck ( 88) 1 (12 ) Kidney ( 85) 1 (15) Bladder (100) 0 ( 0) Salivary Gland (100) 0 ( 0) *Cytoplasmic Pattern. tluminal/membranous Pattern.

4 CA-549 im m u n o h is t o c h e m is t r y 411 F ig u r e 1. Im m u n o histochemical staining for CA-549 in normal breast (A) an d in b re a s t w ith fibrocystic d isease (B). N o te th e e x c lu s iv e luminal staining (A 140 X, B 225 X ). (figure 2). The cytoplasmic staining p at te rn p redom inated, occurring in 47/57 (82 percent) of cases. The incidence of CA-549 imm unohistochem ical positivity in the other carci nomas ranged from 8 to percent with cytoplasmic staining in 0 to 80 percent of the tum ors depending on their prim ary site (table II). C o rre la tio n o f C A -549 L evels a n d Im m unohistochem istry: All 26 patients w ith benign b reast disease had norm al se ru m levels of CA -549 and show ed exclusively the lum inal/m em brane stain ing of norm al breast structures or fibro cystic disease. O f the 29 cases of breast carcinom a w ith both serum levels and im m unoperoxidase staining, 28 (96 percent) showed positive im m unohistochem ical staining for CA /28 (64 percent) had cyto plasmic staining and 10/28 (36 percent) th e lu m in a l m e m b r a n o u s p a t t e r n. CA-549 serum levels w ere elevated in 23/29 (79 percent). The m ean serum level for the entire group was U p er ml (range 1.9 to 785.0). T he m ean serum level for the cases that showed the cytoplasmic stain ing p a tte rn on im m u n ohistochem istry was U p e r ml (S.D. = 263.6, ran g e 1.9 to 785.0), w hile th e m ean

5 412 SHURBAJI, BEVERIDGE, CHAN. ROCK, AND KUHAJDA F ig u r e 2. (A): Low p o w er view illu s tra tin g the difference in immunoh is to c h e m ic a l sta in in g p a t t e r n fo r C A b e tw e e n th e c e n tr a lly located normal breast duct w ith faint lum inal stain ing, and the surrounding infiltrating duct carcinoma (200 X ). (B): Im m unohisto ch em ical stain in g for CA-549 in a case of infil trating duct carcinoma of breast. Note the presence o f in te n s e cy to p lasm ic staining, and contrast to exclusive luminal staining seen in figure 1 (600 X). se ru m le v e l for th e c a se s w ith th e lum inal/m em branous p a tte rn of im m unohistochemical staining was 37.3 U per ml (S.D. = 29.8, range 2.1 to ). Although these results show that there is a te n d e n c y for h ig h e r se ru m lev e ls am ong the cases w ith th e cytoplasm ic pattern of staining as com pared to those w ith lum inal/m em branous p a tte rn, the difference was of b o rd erlin e statistical s ig n ific a n c e ( s t u d e n t s t - t e s t p = ). Additionally, serum levels and immunohistochemical staining data w ere avail able on 16 colon, eight prostate, and five lung carcinom as. 15/16 (94 percent) of the colon adenocarcinom as w ere positive for CA-549 in a lu m inal/m em branous pattern by imm unohistochem istry. Only two of th ese patients (13 percent) had m odest elevations of the serum levels (11.6 and 15.1 U p er ml). Six (75 percent) of eight prostate ade nocarcinoma specim ens had immunohis-

6 CA-549 IMMUNOHISTOCHEMISTRY tochemical staining for CA-549 with only one case showing a cytoplasmic pattern; 5/8 (63 p ercen t) had elevated serum levels w ith a m ean of 14.7 U p e r ml (range 11.5 to 29.4 U per ml). The highest serum level, 29.4 U per ml, was seen in the patient with cytoplasmic staining. All five (100 percent) of the lung carcinomas w ere im m unoreaetive with one case show ing a cytoplasm ic p a tte rn. N one of th ese p atien ts had elevated serum levels. Discussion T he d e v e lo p m e n t of non-invasive techniques to m onitor disease progression in breast cancer patients remains an im portant clinical goal. Serum levels of CA-549 have been shown to be both a sensitive and specific m arker for tum or progression in patients with breast carcin o m a. 1 In a d d itio n, m ark ed ly hig h serum levels of CA-549 w ere strongly s u g g e stiv e o f b r e a s t c a rc in o m a in patients with metastatic disease. 1,3 Our immunohistochemical studies sought to determ ine if CA-549 was a breast-specific m arker in tissue and if serum levels correlated w ith tissue reactivity. Correlation of im m unohistochem ical studies and serum levels have been perform ed for carcinoem bryonic antigen (CEA ), 4 alpha-fetoprotein (AFP), and h u m a n c h o r io n ic g o n a d o tr o p h in (H C G ). 6,9 D espite the subjectivity of assessing immunohistochemical staining, th ese studies found th at im m unohistochemical positivity for these substances correlated with serum levels and w ere helpful in both tum or classification and m onitoring of m etastatic disease. Differentiation of embryonal carcinoma, endoderm al sinus tumors, choriocarcinoma, and sem inom a was enhanced using tissue and serum correlation.9 Studies with CEA show ed that tum ors w ith strong CEA im m unohistochem ical positivity were more likely to have elevated serum levels. Thus, tissue staining could be used to p red ict which patients would b e n e fit from long te rm follow -up of serum CEA levels for assessment of m etastatic disease. 4 O ur results show that although serum CA-549 levels were elevated in only 1.5 percent of healthy w om en, 1 nearly all epithelial structures in the normal breast expressed CA-549. Similarly, in patients w ith b reast cancer, elevated CA-549 levels ranged from 63 percent in partial remission to 83 percent with disease progression (1); yet, 98 percent of breast cancers d e m o n strated im m unohistochem ical positivity for CA-549. H ow ever, despite the fact that normal breast structures reacted as frequently as carcinoma, it was the pattern of CA-549 reactivity which appeared to differ in the benign and malignant breast tissue and to correlate with the likelihood of elaboration into the serum. Normal breast and benign b reast disease show ed exclusively m embranous or luminal positivity with concomitant normal serum levels, w hereas the carcinom as show ed p re dom inantly cytoplasm ic positivity and elevated serum levels. The pattern of immunohistochemical reactivity seen in this study is similar to the observations of D em ers et al3 on the im m unohistoc h e m istry of CA -549, and to th o se rep o rted w ith other related glycoprote in s. 5,10 A m ong th e b re a s t c a n cer patients with both CA-549 immunohistochemistry and serum levels, those whose tum or biopsies had cytoplasmic reactivity tended to have higher serum levels than those with luminal or membranous positivity, although in this small population of patients there was not statistical significance (p = ). T hough m arkedly e le v a te d serum levels of CA-549 strongly suggested the diagnosis of metastatic breast carcinoma in a patient with m etastatic disease of unknown primary, 1 tissue immunoreactivity is not lim ited to the breast. A wide

7 4 1 4 SHURBAJI, BEVERIDGE, CHAN, ROCK, AND KUHAJDA variety of norm al epithelia and carcinomas express CA-549 epitopes (table II). As a serum test, CA-549 has been show n to b e h e lp fu l in m o n ito rin g patients with breast carcinoma and may be a helpful adjunct in establishing the diagnosis of breast carcinoma. T he diagnostic utility of CA-549 im m unohistochemistry, however, remains limited. It is not a breast specific marker and the high frequency of positivity in breast carcinoma makes it an unlikely candidate as a prognostic indicator. The mechanisms responsible for the cellular localization of CA-549 and its secretion into the serum are unknown. Further studies aimed at understanding the processing of the CA-549 antigen and its subcellular localization are warranted as they could provide clues to the biochem istry of secretory proteins and differentiation of hum an carcinomas. A cknowledgment The authors wish to thank Hybritech Inc. for supplying the antibody. Dr. Kuhajda is a recipient of an American Cancer Society Career Developm ent Award, which partially supported this effort. References 1. Be v e r id g e, R. A., C h a n, D. W., Br u z e k, D., D a m r o n, D., E t t in g e r, D. S., R o c k, R. C., Sh u b a ji, M. S., and Ku h a jd a, F. P.: A new biomarker in monitoring breast cancer: CA-549. J. Clin. Oncol. 6: , B ray, K. R., K o d a, J. E., and G a u r, P. K.: Serum levels and biochemical characteristics of cancer-associated antigen CA-549, a circulating breast cancer marker. Cancer Res. 47: , D e m e r s, L. M., H arvey, H. A., G l e n n, J. D., and G a u r, P. K.: CA-549, a new m arker for patients with advanced breast cancer. J. Clin. Lab. Anal. 2: , G o s l in, R., O 'B r i e n, M. J., St e e l e, G., M ayer, R., W il s o n, R., C o r s o n, J. M., an d ZAMCHECK, N.: C o rre la tio n o f plasm a CEA and CEA tissue staining in poorly d ifferen tiated colorectal cancer. Am. J. M ed. 71: , H a rris, H.: The Ca antigen: Structure, function and clinical application. Tumor Markers in Clinical Practice, Concepts and Applications. Daar, A. S., ed. Oxford, Blackwell Scientific Publications, 1982, pp Javadpo u r, N., M c I n t ir e, K. R., and W a l d - m a n n, T. A.: Human chorionic gonadotrophin (HCG) and alpha-fetoprotein (AFP) in sera and tumor cells of patients with testicular seminoma. Cancer 42: , K u h a jd a, F. P., Bo h n, H., and M e n d e l s o h n, G.: P regnancy-specific beta-1 glycoprotein (SP-1) in breast carcinoma. Cancer 54: , K u h a jd a, F. P., O f f u t t, L. E., and M e n d e l so h n, G.: The distribution of carcinoembryonic antigen in breast carcinoma. Cancer 52: , K u r m a n, R. J., Sc a r d in o, P. T., M c I n t ir e, K. R., W a ld m a n n, T. A., and Javadpour, N.: Cellular localization of alpha-fetoprotein and human chorionic gonadotrophin in germ cell tumors of the testis using an indirect immunoperoxidase technique. Cancer 40: , M e sa-t e ja d a, R., Pa la k o d ety, R. B., L e o n, J. A., and K h a t c h e r ia n, A. O.: Immunocytochemieal distribution of a breast carcinoma associated protein identified by monoclonal antibodies. Am. J. Pathol. 730: , 1988.