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 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

547

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

HUMAN PATHOLOGY

TABLE 1. Extent of Staining*

Volume 27, No. 6 (June 1996)

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.)

548

549

HUMAN PATHOLOGY

Volume 27, No. 6 (June 1996)

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

550

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.

REFERENCES 1. Koprowski H, Herlyn M, Steplewski Z, et al: Specific antigen in s e r u m of patients with colon carcinoma. Science 212:53-55, 1981

GLYCOPROTEIN A-80 IN PROSTATIC CARCINOMA AND PIN (Gould et al) 2. Klug TL, Bast RC Jr, NiloffJM, et al: Monoclonal antibody immunoradiometric assay for antigenic determinant (CA 125) associated with human epithelial ovarian carcinomas. Cancer Res 44:10481053, 1984 3. HolmgrenJ, Lindholm L, Persson B, et al: Detection by monoclonal antibody of carbohydrate antigen CA 50 in serum of patients with carcinoma. Br MedJ 288:1479-1482, 1984 4. Metzgar RS, Rodriguez N, Finn OJ, et al: Detection of a pancreatic cancer-associated antigen (DU-PAN-2 antigen) in serum and ascites of patients with adenocarcinoma. Proc Natl Acad Sci U S A 81:5242-5246, 1984 5. Herlyn M, Sears HF, Verrill H, et al: Increased sensitivity in detecting tumor-associated antigens in sera of patients with colorectal carcinoma. J Immunol Methods 75:15-21, 1985 6. Chia D, Terasaki PI, Suyama N, et al: Use of monoclonal antibodies to sialylated Lewis×, and siaiylated Lewis~ for serological tests of cancer. Cancer Res 45:435-437, 1985 7. Papsidero LD, Nemoto T, Croghan GA, et al: Expression of ductal carcinoma antigen in breast cancer sera as defined using monoclonal antibody F36/22. Cancer Res 44:4653-4657, 1984 8. Nuti M, Teramoto YA, Mariani~Constantini NR, et al: A monoclonal antibody (B72.3) defined patterns of distribution of a novel tumor-associated antigen in human mammary carcinoma cell populations. Intl J Cancer 29:539-545, 1982 9. Miotti S, Aguanno S, Canevari S, et al: Biochemical analysis of human ovarian cancer-associated antigens defined by murine monoclonal antibodies. Cancer Res 45:826-832, 1985 10. Chung YS, Ho JJ, Kim YS, et al: The detection of human pancreatic cancer-associated antigen in the serum of patients. Cancer 60:1636-1643, 1987 11. Radosevich JA, Ma Y, Lee I, et al: Monoclonal antibody 443A6 as a probe for a novel antigen found in human carcinomas with glandular differentiation. Cancer Res 45:5808-5813, 1985 12. Lee I, Radosevich JA, Ma Y, et al: Immunohistochemical analysis of human pulmonary carcinomas using monoclonal antibody 44-3A6. Cancer Res 45:5813-5817, 1985 13. Lee I, RadosevichJA, Chejfec G, et al: Malignant mesotheliomas: Improved differential diagnosis from lung adenocarcinoma using monoclonal antibody 44-3A6. Am J Pathol 123:49%507, 1986 14. Piehl MR, Gould VE, Warren WH, et al: Immunohistochemical identification of exocrine and neuroendocrine subsets of large cell lung carcinomas. Pathol Res Pract 183:675-682, 1988 15. Rittenhouse HG, Manderino GL, Hass GM: Mucin-type glycoproteins as tumor markers. Lab Med 16:556-560, 1985 16. Gould VE, Shin SS, Manderino GL, et al: Selective expression of a novel mucin-type glycoprotein in human tumors: Immunohistochemical demonstration with Mab A-80. Hum Patho119:623-627, 1988 17. Jansson DS, Gould VE, Gooch GT, et al: Immunohistochemical analysis of colon carcinomas applying exocrine and neuroendocrine markers. APMIS 96:1129-1139, 1988 18. Shin SS, Gould VE, Gould JE, et al: Expression of a new mucin-type glycoprotein in select epithelial dysplasias and neoplasms detected immunocytochemicallywith Mab A-80. APMIS 97:1053-1067, 1989 19. Koukoulis GK, Jao W, Gooch GT, et al: Immunohistochemical evaluation of neoplastic and non-neoplastic breast diseases with Mab A-80. Pathol Res Pract 186:439-449, 1990 20. Chejfec G, Candel A, Jansson DS, et al: Immunohistochemical features of giant cell carcinoma of the lung: Patterns of expression of cytokeratins, vimentin and the mucinous glycoprotein recognized by Mab A-80. Uhrastruct Pathol 15:131-138, 1991 21. Eriksson ET, Schimmelpenning H, Rutquist LE, et al: Immunohistochemical expression of the mucin-type glycoprotein A-80 and prognosis in human breast cancer. Br J Cancer 67:1418-1422, 1993 22. Gould VE, Tomanova R, Monson R, et al: Immunophenotypic subsets of large cell pulmonary carcinomas. Lab Invest 70:150A, 1994 (abstr) 23. Kastendieck H: Correlations between atypical primary hyperplasia and carcinoma of the prostate. Pathol Res Pract 169:366-387, 1980 24. Helpap B: The biological significance of atypical hyperplasia of the prostate. Virchows Arch Pathol Anat 387:307-317, 1980 25. McNeal JE, Bostwick DG: Intraductal dysplasia: A premalignant lesion of the prostate. Hum Pathol 17:64-71, 1986 26. Oyasu R, Bahson RR, Nowles K, et al: Cytological atypia in

551

the prostate: Frequency, distribution and possible relevance to carcinoma. J Urol 135:959-962, 1986 27. Bostwick DG, Brawer MK: Prostatic intra-epithelial hyperplasia and early invasion in prostatic cancer. Cancer 59:788-794, 1987 28. McNealJE: The significance of duct-acinar dysplasia in prostatic carcinogenesis. Prostate 13:91-102, 1988 29. Quinn BD, Cho KR, EpsteinJI: Relationship of severe dysplasia to stage B adenocarcinoma of the prostate. Cancer 65:2328-2337, 1990 30. McNealJE, Villers A, Redwine EA, et al: Microcarcinoma in the prostate: Its association with duct-acinar dysplasia. Hum Pathol 22:644-652, 1991 31. Brawer MK: Prostatic intraepithelial neoplasia: A premalignant lesion. Hum Pathol 23:242-248, 1992 32. Bostwick DG, Amin MB, Dundore P, et al: Architectural patterns of highgrade prostatic intraepithelial neoplasia. HUM PATHOL 24:298-301, 1993 33. Bostwick DG, Srigley J, Grignon D, et al: Atypical adenomatous hyperplasia of prostate: Morphologic criteria for its distinction from well differentiated carcinoma. HUM PATHOL 24:819-832, 1993 34. Helpap BGT, Bostwick DG, Montironi R: The significance of atypical adenomatous hyperplasia and prostatic intraepithelial neoplasia for the development of prostate carcinoma. Virchows Arch A Pathol Anat 426:425-434, 1995 35. Brawer MK, Peehl DM, Stmney TA, et al: Keratin immunoreactivity in the benign and neoplastic human prostate. Cancer Res 45:3663-3667, 1985 36. Nagle RB, Brawer MK, KittelsonJ, et al: Phenotypic relationships of prostatic intraepithelial neoplasia to invasive prostatic carcinoma. A m J Pathol 138:119-128, 1991 37. O'Malley FP, Grignon DJ, Shum DT: Usefulness ofimmunoperoxidase staining with high molecular cytokeratin in the differential diagnosis of small-acinar lesions of the prostate gland. Virchows Arch A Pathol Anat 417:191-196, 1990 38. Montironi R, Magi Galuzzi C, Diamanti L, et al: Prostatic intraepithelial neoplasia: Qualitative and quantitative analyses of the blood capillary architecture on thin tissue sections. Pathol Res Pract 189:542-548, 1993 39. Hsu SM, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29:577-580, 1981 40. Moll R, Lee I, Gould VE, et al: Immunohistochemical analysis of Ewing's tumors: Patterns of expression of intermediate filament and desmosomal proteins indicate cell type heterogeneity and pluripotential differentiation Am J Pathol 127:288-304, 1987 41. Kim YD, Manderino GL, Gooch GT, et al: Isolation and characterization of the tumor-associated mucin-like glycoprotein defined by monoclonal antibody A-80. Cancer Ther Contr 1:277-291, 1991 42. Goldstein NS, Qian J, Bostwick DG: Mucin expression in atypical adenomatous hyperplasia of the prostate. HUM PATHOL 26:88%891, 1995 43. Schellhammer PF, Wright GL: Biomolecular and clinical characteristics of PSA and other candidate prostate tumor markers. Urol Clin North Am 20:597-606, 1993 44. Beard D, Haskell C: Carcinoembryonic antigen in breast cancer. Am J Med 80:241-145, 1986 45. Colcher D, Horan Hand P, Nuti M, et al: Differential binding to human mammary and nonmammary tumors of monoclonal antibodies reactive with carcinoembryonic antigen. Cancer Invest 1:12% 128, 1983 46. Coombes RC, Buckman R, Forrester JA, et al: In vitro and in vivo effects of a monoclonal antibody-toxin conjugate for use in autologous bone marrow transplantation for patients with breast cancer. Cancer Res 46:4217-4220, 1986 47. Cooper EH, Forbes MA, Hancock AK, et al: An evaluation of mucin like carcinoma associated antigen (MCA) in breast cancer. BrJ Cancer 59:79%800, 1989 48. Ellis IO, Hinton CP, MacNay J, et al: Immunocytochemical staining of breast carcinoma with a monoclonal antibody NCRC 11: A new prognostic indicator. Br Med J 290:881-883, 1985 49. Ellis IO, Robins RA, Elston CW, et al: A monoclonal antibody, NCRC 11, raised to human breast carcinoma. I. Production and immunohistological characterization. Histopathology 8:501-516, 1984

HUMAN PATHOLOGY

Volume 27, No. 6 (June 1996)

50. Foster C~, Dindsdale EA, Edwards PAW, et al: Monoclonal antibodies to the human mammary gland. II. Distribution of determinants in breast carcinomas. V'nrchowsArch A Path Anat 394:295-305, 1982 51. SchlomJ, Wunderlich D, Teramoto Y: Generation of human monoclonal antibodies reactive with human mammary carcinoma cells. Proc Natl Acad Sci U S A 77:6841-6845, 1980 52. Stacker SA, Thompson C, Riglar C, et al: A new breast antigen defined by a monoclonal antibody. J Nad Cancer Inst 75:801811, 1985 53. Thor A, Weeks MO, Schlom J: Monoclonal antibodies and breast cancer. Semin Oncol 13:393-401, 1986 54. ArklieJ, Taylor-PapadimitriouJ, Bodmer W, et al: Differentiation antigens expressed by epithelial cells in the lactating breast are also detectable in breast cancers. I n t J Cancer 28:23-29, 1981 55. Edwards DP, Grzyb KT, Dressler LG, et al: Monoclonal antibody identification and characterization of a Mr 43,000 membrane glycoprotein associated with human breast cancer. Cancer Res 46:1306-1317, 1986 56. Hilkens J, Buljs F, Hilgers J, et al: Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors. IntJ Cancer 34:197206, 1984 57. Teramoto YA, Mariani R, Wunderlich D, et al: The immunohistochemical reactivity of a human monoclonal antibody with tissue sections of human mammary tumors. Cancer 50:241-249, 1981 58. Tjandra~, McKenzie IFC: Murine monoclonal antibodies in breast cancer: An overview. BrJ Surg 75:1067-1077, 1988

552

59. Mesa-Tejada R, Palacodety LD, LeonJA, et al: Immunocytochemical distribution of a breast carcinoma associated glycoprotein identified by monoclonal antibodies. Am J Pathol 130:305314, 1988 60. Ohushi N, Page D, Merino MJ, et al: Expression of tumorassociated antigen (DF3) in atypical hyperplasias and in situ carcinomas of the human breast. J Nail Cancer Inst 79:109-117, 1987 61. Pinkus GS, Kurtin PJ: Epithelial membrane antigen: A diagnostic discriminant in surgical pathology. HUM PATHOL 16:929-940, 1985 62. Moss L, Greenwald D, Cullen B, et al: Cell to cell heterogeneity in the expression of carbohydrate-based epitopes of a mucin-type glycoprotein on the surface of human mammary carcinoma cells. J Cell Physiol (USA) 137:310-320, 1988 63. BurchellJ, Gendler S, Taylor-PapadimitriouJ, et al: Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin. Cancer Res 47:5476-5482, 1987 64. Staten ED, Lott S, Mnniz-Saavedra V, et al: Neuroendocrine carcinomas of the stomach: A clinicopathologic evaluation. Surgery 112:1039-1047, 1992 65. Hsu W, Deziel DJ, Gould VE, et al: Neuroendocrine differentiation and prognosis of extrahepatic biliary tract carcinomas. Surgery 110:604-611, 1991 66. Bostwick DG: High grade prostatic intraepithelial neoplasia: The most likely precursor of prostate cancer. Cancer 75:1823-1836, 1995