Immunolocalization of glycoprotein A-80 in prostatic carcinoma and prostatic intraepithelial neoplasia

Immunolocalization of glycoprotein A-80 in prostatic carcinoma and prostatic intraepithelial neoplasia

Immunolocalization of Glycoprotein A-80 in Prostatic Carcinoma and Prostatic Intraepithelial Neoplasia VICTOR E. GOULD, MD, VERA DOLJANSKAIA, BS, GARY...

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Immunolocalization of Glycoprotein A-80 in Prostatic Carcinoma and Prostatic Intraepithelial Neoplasia VICTOR E. GOULD, MD, VERA DOLJANSKAIA, BS, GARY GOOCH, PhD, AND DAVID G. BOSTWICK, MD A-80 is a mucin-like glycoprotein associated with exocrine differentiation that shows little or no expression in normal exocrine cells and typical adenomas, but is upregulated in dysplasia and adenocarcinoma of certain organs. Its expression has not been systematically examined in prostatic adenocarcinoma and its putative precursor, prostatic intraepithelial neoplasia (PIN). The authors applied a mouse monoclonal antibody against A-80 in paraffin-embedded sections from 103 cases of prostatic carcinoma, 26 cases of nodular hyperplasia, 7 autopsy samples from normal young adult prostates, and 12 fetal prostates. All but one cancer reacted, although expression was heterogeneous; 75 of 103 stained extensively ( > 3 + on a 0 to 5+ scale) and strongly. Staining extent and inteusity were independent of tumor grade, and tended to be strong even when focal. Seventy-seven of 84 foci (92%) of high-grade PIN and 38 of 52 loci (73%) of low-grade PIN stained for A-80; reactions were most extensive and intense in high grade PIN. Only 5 of 26 cases (19%) of hyperplasia reacted, and this consisted of weak to moderate staining in sporadic

cells; the remainder were negative. Normal adult prostatic epithelium did not express A-80 except for weak and inconsistent staining in loci of inflammation and infarction; atrophic glands were negative. Fetal prostate showed focally strong reactivity. These results indicate that A-80 is selectively expressed in most cases of intraepithelial neoplasia and prostate cancer, but is usually absent in benign and hyperplastic epithelium. The upregulation of glycoprotein A-80 in PIN and adenocarcinoma parallels observations in other organs, such as the breast and colon, suggesting that this is a significant oncodevelopmental molecule with potential clinical applications. HUM PATHOL 547-552. Copyright © 1996 by W.B. Saunders Company Key words: prostate, prostatic carcinoma, prostatic intraepithelial neoplasia, mucins, glycoproteins, A-80, immunohistochemistry, prognosis. Abbremai~om: PIN, prostatic intraepithelial neoplasia; Mab, monodonal antibodies; IgM, immunoglobulin M, CEA, carcinoembryoulc antigen; PSA, prostate~pecilic antigen.

Several m u c i n type glycoproteins a n d related proteins have b e e n isolated f r o m h u m a n carcinomas a n d cell lines in an effort to develop organ- a n d t u m o r specific diagnostic markers. M o n o c l o n a l antibodies (Mab) have b e e n raised against s o m e o f these i n c l u d i n g CA 19-1, 1 CA 125, 2 CA 50, 3 Du-Pan-2, 4 C O - 5 1 . 4 / C A 199, 5 CSLEX 1, 6 F 3 6 / 2 2 , 7 B72.3, s MoV2, 9 Span-l, l° 443A6,11-14 a n d CEA, 15'16 a n d A-80.16'17 Most o f these antibodies recognize c a r c i n o m a in m o r e t h a n o n e site, a n d are often expressed in reactive, hyperplastic, fetal, and, rarely, in n o r m a l epithelia. Yet some, such as Mab A80, offer significant p r o m i s e as investigative, diagnostic, a n d p r o g n o s t i c t o o l s ) 8-22 Prostatic c a r c i n o m a is the m o s t f r e q u e n t visceral c a n c e r in m e n a n d a leading cause o f c a n c e r death. R e c e n t work indicates that prostatic intraepithelial neoplasia (PIN) is the m o s t likely p r e c u r s o r o f prostatic carcinoma, b u t m o s t o f this research has c e n t e r e d o n the histological a n d cytological features o f P I N a n d o n correlations with c a r c i n o m a a n d s e r u m prostate specific antigen (PSA) c o n c e n t r a t i o n . 23-'~3 I m m u n o h i s t o c h e m i cal studies o f PIN have f o c u s e d p r e d o m i n a n t l y o n the continuity o f the basal cell layer as d e f i n e d by cytokeratin reactivity with antibodies that recognize diverse polypeptides. 27,~5-3s Early studies with Mab A-80 s h o w e d frequently strong immunoreactivity in some cases o f prostatic car-

cinoma, whereas n o d u l a r hyperplasia a n d n o r m a l adult epithelium were negative, or rarely a n d only focally reactive. 16'1s In this study, the a u t h o r s show that A-80 is expressed in m o s t cases o f prostatic a d e n o c a r c i n o m a irrespective o f grade. Notably, A-80 immunoreactivity was also n o t e d in m o s t foci o f PIN; staining was strongest a n d m o s t extensive in high-grade PIN. This wellcharacterized o n c o d e v e l o p m e n t a l m a r k e r provides m o lecular s u p p o r t for the p r e m a l i g n a n t character o f P I N a n d reinforces its clinicopathologic significance.

From the Rush Medical College, Chicago, IL; Abbott Laboratories, North Chicago, IL; and Mayo Clinic, Rochester, MN. Accepted for publication November 29, 1995. Address correspondence and reprint requests to Victor E. Gould, MD, Pathology Department, Rush Medical College, 1653 W Congress Pkwy, Chicago, IL 60612. Copyright © 1996 by W.B. Saunders Company 0046-8177/96/2706-001855.00/0

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MATERIALS AND METHODS One hundred twenty-nine specimens representing a broad spectrum of prostatic lesions, obtained by radical prostatectomy or transurethral resection, were selected from the surgical pathology files of Presbyterian-St. Luke's Medical Center. These samples included 103 cancers and 26 nodular hyperplasias. PINs31-33were identified in 88 cases of carcinoma and 16 cases of hyperplasia; there were 52 foci of low-grade and 84 of high-grade PIN, respectively. Basal cell hyperplasia was observed in 20 cancer cases and 10 hyperplasia cases. Five normal adult prostate samples and 12 fetal specimens (courtesy of Dr N.S. Gould, Michael Reese Medical Center, Chicago IL) derived from autopsy files were included for comparison. All tissue samples were fixed in formalin, conventionally processed, and embedded in paraffin. Hematoxylin-eosinstained sections were examined, and up to three blocks per case were selected for immunostaining. The immunohistochemical protocol has been published) 6'17Briefly, 5-/~m-thick sections were cut; slides were set in a slide warmer at 60°C for 1 hour, deparaffinized in xylene, and rehydrated in graded alcohols. No enzymatic or microwave pretreatment was applied before exposure to the antibody. The sections were incubated with Mab A-80 in a humid chamber overnight at 4°C

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TABLE 1. Extent of Staining*

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TABLE 2.

Glycoprotein A-80 Immunoreactivity in 103 Prostate Cancers

0

No. of eases 1/103

1+

2+

10/103 22/103

Comparison of A-80 Immunoreactivity in the Benign a n d M a l i g n a n t Prostate

3+

4+

5+

38/103

21/103

11/103

* Extent defined as percentage of stained cells: O = negative, 1+ = <1%, 2+ = 1% to 5%, 3+ = 6% to 50%, 4+ = 50% to 95%, 5+ = >95%. 4o

at a concentration of the immunoglobulin of 100 # g / m L . Subsequent steps were performed with the avidin-biofin-peroxidase method as outlined by Hsu et a139 using commercial reagents (Vector Laboratories, Burlingame, CA). Sites of antigen-antibody binding were visualized with 3,3'-diaminobenzidine (Aldrich Chemical, Danvers, MA). Finally, the sections were briefly counterstained with Mayer's hematoxylin. Wellcharacterized samples of colon and breast carcinomas served as positive controls; sections immunostained with an irrelevant mouse immunoglobulin served as negative controls. The extent of A-80 immunostaining was measured by the percentage of reactive tumor cells and was graded as negative (0) or 1 + to 5+ positive.4° Reactions in nonmalignant lesions were not quantified; instead, the presence or absence of unequivocal immunoreactivity was noted. The intensity of the reactions was rated as weak, moderate, or strong; in cases of heterogeneous intensity, the rating was based on the predominant pattern. Mab A-80 (Abbott Laboratories, North Chicago, IL) is a mouse IgM, raised against a mucinous glycoprotein derived from the human colon carcinoma cell line LS-174T. Data on the generation of this antibody, and on the isolation and characterization of the pertinent glycoprotein have been published.16,1s.41

RESULTS T h e f i n d i n g s a r e o u t l i n e d in T a b l e s 1 a n d 2. O f 103 c a r c i n o m a s , 102 (99%) d i s p l a y e d A-80 i m m u n o r e activity, a n d m o s t s h o w e d r e a c t i o n s t h a t were r a t e d as 3 + , 4 + , o r 5 + , with literally n o b a c k g r o u n d staining; t h e e x t e n t a n d i n t e n s i t y o f t h e r e a c t i o n s varied, b u t n e i t h e r a p p e a r e d to c o r r e l a t e with t u m o r g r a d e . I n welld i f f e r e n t i a t e d c a n c e r , r i c h c y t o p l a s m i c s t a i n i n g was conc e n t r a t e d at t h e a p i c a l b o r d e r ; i f i n t r a l u m i n a l s e c r e t i o n s

Tissue/Diagnosis

No. of Cases

Immunoreactivity (No. of Positive Cases) (%)

Fetal prostate Normal adult prostate Nodular hyperplasia Basal cell hyperplasia

12 7 26 30

12 0 5 4

Low grade PIN High grade PIN

52 84

38 (73) 77 (92)

(100) (0) (19) (13)

Intensity* 1-2 -l-2Jl 1-2 2-3

Adenocarcinoma, all cases

Gleason 5 Gleason 6 Gleason 7 Gleason 8 Gleason 9 Gleason 10

103 8 30 27 22 12 4

102 7 30 27 22 12 4

(99) (87) (100) (100) (100) (100) (100)

2-3 2-3 2-3 2-3 2-3 2-3 2-3

* 1 = weak, 2 : moderate, and 3 = strong. t Reactions involved scattered cells.

o r crystalloids w e r e p r e s e n t , t h e y o f t e n r e a c t e d s t r o n g l y (Fig 1). I n p o o r l y d i f f e r e n t i a t e d c a n c e r with solid clusters a n d rows o f t u m o r cells, r e a c t i o n s were diffusely cytoplasmic, with s o m e a c c e n t u a t i o n at t h e cell b o r d e r s (Figs 2 a n d 3). P o o r l y d e f i n e d acini a n d i n f i l t r a t i n g i n d i v i d u a l cells s h o w e d c y t o p l a s m i c A-80 e x p r e s s i o n ; mucin-producing areas stained convincingly whereas c l e a r " h y p e r n e p h r o i d " l o c i r e a c t e d unevenly. I n a r e a s with n o r m a l o r h y p e r p l a s t i c acini a d j a c e n t to carcinoma, the strong staining of the latter contrasted notably with t h e negativity o f t h e f o r m e r (Figs 2 a n d 3). C a r c i n o m a - i n v a d i n g s e m i n a l vesicles d i s p l a y e d s t r o n g but uneven tan-brown cytoplasmic and luminal immun o s t a i n i n g , w h i c h s t o o d in c o n t r a s t with t h e coarsely g r a n u l a r , yellow c y t o p l a s m i c c e r o i d b o d i e s in t h e vesicular e p i t h e l i u m (Fig 4). C a n c e r cells i n v a d i n g p e r i n e u r a l spaces s h o w e d c o n s i s t e n t l y i n t e n s e i m m u n o r e a c t i v i t y (Fig 5). T h e single A-80-negative c a r c i n o m a was small, well d i f f e r e n t i a t e d , n o t a s s o c i a t e d with PIN, a n d was n o n r e a c t i v e with a b a t t e r y o f n e u r o e n d o c r i n e m a r k e r s . Seventy-seven o f 84 cases (92%) o f h i g h g r a d e P I N a n d 38 o f 52 (73%) o f low-grade P I N s h o w e d v a r i a b l e

FIGURE 1. Carcinoma comprised of well-defined glands strongly and diffusely reactive for A-80; the staining is pafficularly intense in the tumor cell apices and in some intraluminal aggregates (arrows). (Original magnification ×480.) FIGURE 2. Carcinoma comprised predominantly of solid clear cell clusters strongly and extensively reactive for A-80; note unstained normal ducts (arrows). (Original magnification ×240.) FIGURE 3. Carcinoma consisting of mostly solid rows of cells; note diffuse and focally strong A-80 reactions contrasting with nonreactive hyperplastic glands. (Original magnification >(160.) FIGURE 4. Carcinoma invading seminal vesicle; compare focally strong but uneven A-80 reactions in neoplastic glands with the barely perceptible yellow granularity of the vesicular epithelium (arrows). (Original magnification x240.) FIGURE 5. Carcinoma invading periprostatic tissue; note strong A-80 staining predominantly in perineural neoplastic glands. (Original magnification x 160.) FIGURE6. PIN;note two adjacent ducts one of which is strongly and extensively reactive for A-80 while an adjacent hyperplastic gland remains unstained. (Original magnification ×480.) FIGURE 7. Hyperplastic glands adjacent to carcinoma (not in the field); A-80 staining is noted in sporadic, individual cells (arrows), while most cells are negative as are the corpora amylacea. (Original magnification × 160.) FIGURE 8. Normal adult prostate; no convincing A-80 reaction is noted. (Original magnification ×160.)

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but unequivocal A-80 immunoreactivity; high-grade PIN stained more intensely and often more extensively than low-grade PIN. Individual ducts and groups of ducts showing PIN were irregularly immunoreactive, with some papillary projections staining more than others; the strongest reactions were seen in the luminal cell borders, but cytoplasmic staining and some "spilling" into lumina were also noted (Fig 6). Notably, some hyperplastic glands without PIN showed weak, focal reactions when in the vicinity of or engulfed by ducts with PIN (Fig 7). The acini of " p u r e " nodular hyperplasia not associated with PIN or carcinoma were virtually always A80-negative including those with papillary projections; intraluminal secretions and corpora amylacea were also negative (Fig 7). Rarely, weak staining was noted in the cytoplasm or apex of isolated cells. Sporadic cells showing stronger staining were present in topographic association with intensely immunoreactive PIN (Fig 7) or with carcinoma. Foci of chronic inflammation and basal cell hyperplasia showed rare and weak focal staining, whereas foci of infarction were invariably negative as were atrophic appearing glands. Normal adult acini were consistently A-80-negative (Fig 8). In second-to third trimester fetal prostates, scattered ductal cells showed moderate staining, whereas in samples from less mature prostates, primitive ducts lined by transitional type epithelium showed distinct and frequently extensive apical staining.

DISCUSSION T h e r e are data regarding the presence of histochemically defined mucins in prostatic hyperplasia and carcinoma 42; however, information on the expression of well characterized, oncodevelopmental type glycoproteins in prostatic adenocarcinoma and PIN is 1imited. 43 In this study, the authors used a Mab directed against the mucin type glycoprotein A-80, and found expression in most foci of PIN and almost all prostate cancers regardless of glandular differentiation. Only weak and sporadic reactions were noted in nodular hyperplasia, and usually in samples with adjacent foci of PIN or adenocarcinoma. Numerous antisera and antibodies that recognize epitopes present in a heterogeneous "family" of heavy molecular weight mucinous glycoproteins, which may include carcinoembryonic antigen (CEA), have been applied to immunohistochemical studies of various carcinomas including those primary in the breast, lung, and colon. 16-19'44-48 Although comparisons are hindered by differences among tumors, methods, terminology, and the diversity of the probes themselves, several comm o n denominators have emerged: (a) immunoreactions tend to be uneven49-53; (b) metastatic carcinomas tend to react as the corresponding primariesS°'s4-s7; and, (c) reactions are generally detected in carcinomas of several sites, and in some nonneoplastic counterpart cells and tissues. 5s'58 The identification of A-80 in 99% of prostate cancers places it among the highest of the high levels of

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expression of the various glycoprotein markers used in the phenotypic analysis of carcinomas. 8"~1,19,45,50,55,57,5961 Although the expression o f A-80 was usually strong and extensive, some cases showed more limited reactions. This pattern, as suggested previously, is frequent with mucin-like glycoproteins and may reflect heterogeneity in their production and their variable or incomplete glycosylation.62 Alternatively, heavy glycosylation may result in the steric blocking of antibody binding, thereb Y maskin ~ g the e P ito P e and dimmin g immunostaining. 6" Yet the findings with Mab A-80 in prostatic carcinoma are similar to those in carcinomas of the breast, 19 colon, a7 lung, 22 stomach, 64 and extrahepatic biliary tree, 65 indicating consistent, albeit selective, ~8'19 upregulation of this glycoprotein with carcinogenesis. The presence of A-80 immunoreactivity in mucinous foci of prostatic adenocarcinoma parallels observations in "colloid" carcinomas of the colon 17 and breast. 19 This finding suggests that Mab A-80 can recognize its pertinent epitope in a setting that differs considerably from and probably lacks the three-dimensional organization characteristic of the cell m e m b r a n e as previously outlined. 19 If this epitope remained stable in fluids, A-80 assays in aspirated material from the tumors or in serum may prove clinically useful. The clinicopathologic relevance of PIN as a putative precursor of prostatic carcinoma has been well established; significantly, the risk factor relationship is particularly important for high-grade P I N . 23"34'66 The authors have now f o u n d that h i g h g r a d e PIN was almost always intensely reactive with Mab A-80, whereas lowgrade PIN was less reactive. This contrasted with the acini of nodular hyperplasia, which were not A-80 reactive. These observations parallel findings in the breast in which the atypical and proliferative variants of fibrocystic disease associated with an increased incidence of carcinoma were A-80 reactive, whereas the typical co u nter p arts and fibroadeno mas did not react. ~619 r ' " The consistent A-80 reactions in h i g h g r a d e dysplasias of the breast and prostate are also reminiscent of earlier observations in high-grade glandular dysplasia associated with Barrett's esophagus. 18 Notably, some bland-appearing hyperplastic prostatic glands were A-80 reactive, but mostly when temporally and topographically associated with high-grade PIN or adenocarcinoma; these observations are also analogous to earlier findings in the breast. 19 The "perfect," organ-specific transformation marker remains elusive; nevertheless, these results show that A-80 is a strong and consistent immunohistochemical probe for most prostatic carcinomas and high-grade PIN. The epitope recognized by Mab A-80 is well preserved in most conventionally prepared tissue and cytological samples. 18 These findings indicate that this probe may be clinically useful for the diagnosis and characterization of prostatic carcinoma and the dysplasias that may precede it.

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