Tenascin Patterns of Expression in Duct Carcinoma In Situ of the Breast

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1 266 Annals o f Clinical & Laboratory Science, vol. 30, no. 3, 2000 Tenascin Patterns of Expression in Duct Carcinoma In Situ of the Breast Basem F. Iskaros, Cristina P. Sison, and Steven I. Hajdu Department of Pathology, North Shore University Hospital, Manhasset, New York Abstract. Immunohistochemical methods were used to study tenascin (TN) expression in duct carcinoma in situ (DCIS) of the breast of different histologic types. We evaluated 82 lumpectomy specimens of DCIS. There were 5 cases of comedo type, 19 cases of noncomedo type, and 58 cases of mixed comedo and noncomedo type. In 44 cases, the intraductal carcinomas were associated with infiltrating (invasive) duct carcinoma. TN expression was studied by immunohistochemical methods using monoclonal mouse anti-human tenascin (DAKO- TN2M636;1:50 dilution). Positivity was recorded on a scale of 0 to 2+ for presence of TN staining around tumor ducts and thickness of TN-stained fibrous bands. TN showed positive correlation between thick bands around comedo DCIS and thin bands around noncomedo DCIS. The TN score had statistically significant positive association with high nuclear grade (p 0.004), periductal inflammatory infiltrate of DCIS (p ), associated extensive central necrosis of DCIS (p ), and comedo DCIS (p ). TN expression in the stroma was positively associated with tumor size (p ), extensive central necrosis (p 0.02), comedo DCIS (p ), and associated invasive carcinoma (p 0.006). The TN score did not correlate with duct size, multicentric carcinoma, or associated microcalcification. These results demonstrate the different biological nature of DCIS comedo type and suggest its preinvasive potential. Keywords: tenascin, breast carcinoma, duct carcinoma in situ, extracellular matrix, adhesion glycoprotein Introduction Tenascin (TN) is a glycoprotein component of the extracellular matrix (ECM). It is generally expressed at sites of epithelial-mesenchymal interactions during fetal development, and at the epithelial tumor-stromal interface of various benign and malignant epithelial tumors [1,2]. Early investigations on experimentally induced rare breast tumors detected TN in the stroma of malignant tumors but not in that of benign tumors. Thus T N was regarded as a stromal marker of epithelial malignancy [3]. Later, TN was noted in the normal and hyperplastic adult breast as thin bands in the periductal stromal region. In fibroadenomas and carcinoma in situ, TN expression is increased, forming thick periductal bands. In invasive breast Address correspondence to Steven I. Hajdu, M.D., Department o f Pathology, North Shore University Hospital, 300 Community Drive, Manhasset, NY 11030, USA; tel: , fax: carcinoma, TN expression is markedly increased in the stroma around infiltrating tumor cells [4,5]. It is evident that pathologic hyperplasias of the breast are characterized not only by cellular alterations but also by demonstrable quantitative and qualitative changes of the ECM. Moreover, evidence has shown that ECM proteins may modify gene expression and may serve as vehicles for cytokine-mediated signals of intercellular communication [6]. Duct carcinoma in situ (DCIS) has become clinically important since the advent of routine, high quality mammography [7,8]. Older series recorded an incidence of 1 % to 5% of DCIS [9]. Recent studies have shown that approximately 15 to 25% of mammographically screened patients have this type of early breast carcinoma [10,11]. This increase in detection provides a great opportunity to identify patients who are at risk for invasive breast cancer. Knowledge of the biologic potential of such lesions has an important impact on patient management and treatment options. Lagios et al [12], in 1989, were /00/ $1.50; 2000 by the Association of Clinical Scientists, Inc.

2 Tenascin expression and breast carcinoma 267 the first to suggest a relationship between histological features and the risk of local recurrence in patients with DCIS treated with less than mastectomy [12]. It was demonstrated that comedo DCIS represents a high nuclear grade subtype that carries an increased risk of both recurrent DCIS and progression to invasive mammary carcinoma [13,14]. By exclusion, the term noncomedo DCIS has come to refer to all other forms of DCIS. To understand the changes of the ECM proteins in DCIS, we used immunohistochemical methods with archival tissue sections of formalin-fixed, paraffin-embedded surgical materials. In this study, we demonstrated different patterns of expression of the ECM protein, TN, in comedo and noncomedo DCIS, and correlated the patterns of TN expression with the biologic behavior of DCIS. Materials and M ethods The pathology records of 82 patients with DCIS were retrieved from the files of North Shore University Hospital. Hematoxylin- and eosin-stained slides from all included cases were reviewed by two of the authors. These cases reflected a spectrum of DCIS including the pure comedo type. We randomly included DCIS cases associated with infiltrating (invasive) carcinoma of the breast. The biopsy specimens were categorized histopathologically, as shown in Table 1, by tumor variables including (1) histologic pattern of DCIS, (2) Table 1. Tumor characteristics in 82 cases of duct carcinoma in situ of the breast Parameter or characteristic Mean value % or no. of cases Tumor diameter (mm, mean ± SD) 13.3 ± 7.7 Duct diameter (mm, mean ± SD) 2.2 ± 1.4 Pure comedo type 5 6% Mixed type 58 70% Noncomedo type 19 23% Associated invasive carcinoma 44 54% Associated extensive central necrosis 50 61% Associated periductal inflammation 48 58% Associated microcalcification 62 76% Positive margin of excision 46 56% Multicentric tumors 33 40% extensive central necrosis (estimated as >50% of tumor duct lumen), (3) tumor size, (4) unicentric versus multicentric lesion, (5) presence of microcalcification, (6) associated invasive carcinoma, (7) positive margins of excision, and (8) associated inflammation. TN expression was detected by immunohistochemical methods as previously reported [15]. For immunostaining, all blocks were cut into 5- im sections and placed on lysine-coated glass slides. The sections were deparaffinized in xylene and rehydrated with a graded ethanol series (100% to 70%), then rinsed in water, incubated in 3% hydrogen peroxide for 20 min, washed in tap water, and pretreated with pro tease (1:100) in phosphate-buffered saline (PBS) for 30 min at room temperature. The slides were rinsed in tap water, distilled water, and PBS. The monoclonal antihuman mouse tenascin antibody (DAKO, Carpinteria, CA.,TN2M 636;l/50 dilution) was added and incubated for 4r h in a humidified chamber. Slides were washed in PBS and biotinylated antimouse antibody was added for 15 min. After an additional PBS wash, the slides were treated with 3,3- diaminobenzidine (DAKO, Carpinteria, CA) for 5 min. Slides were counterstained with hematoxylin, dehydrated, and mounted with glass coverslips. TN positivity in the periductal region was graded from 0 to 2+ as follows: 0, no staining; 1 +, stained periductal thin band (<4 im by ocular micrometer); 2+, stained periductal thick band (>3 (Jm). T N positivity in surrounding stroma was recorded as 0 when absent and 1 + when present. The relationships between TN score and the abovementioned histopathologic tumor variables were analyzed using Fishers exact test. P-values, based on a two-tailed test, were regarded as significant at less than Pairwise comparisons by Fishers exact test with Bonferroni s correction were carried out as needed. Results Expression of TN in 82 cases of DCIS of the breast was studied by immunohistochemistry and graded on a scale from 0 to 2+. Expression oftn was found as a thin band around hyperplastic ducts and DCIS of the breast, noncomedo type (Fig. 1). In DCIS, comedo type, TN expression was seen as thick bands around the tumor ducts (Fig. 2). In tumors associated with

3 268 Annals o f Clinical & Laboratory Science Fig. 1. Tenascin expression showing periductal staining form ing th in bands around duct carcinoma in situ, noncomedo type (xloo). Fig. 2. Tenascin expression showing periductal staining forming thick bands around duct carcinoma in situ, comedo type (xloo). m icroinvasion or frankly invasive carcinoma, T N expression was found in the tumor stroma surrounding the invading tumor cells. The p-values obtained by Fisher s exact test for each o f the tumor-associated variables are given in Table 2. There was significant association between the three levels o f nuclear grade and T N bands around tumor ducts. In the nuclear grade 3 group, 67% (26/39) had thick T N bands around the ducts and 33% (14/42) had thin T N bands. In the nuclear grade 2 group, 33% (13/39) had thick T N bands and 60% (25/42) had thin T N bands. In the nuclear grade 1 group, none had thick T N bands and 7% (3/42) had thin T N bands around tumor ducts. A higher nuclear grade was associated with thick T N bands around tumor d u cts. P airw ise c o m p a r iso n s sh o w ed that the proportion o f tumors with thick T N bands differed significantly between grades 2 and 3 (p <0.012). There was significant association between com edo type DCIS and thickness o f the T N bands around tumor ducts. Am ong the pure comedo type o f tumor, there were 10% (4/39) with thick T N bands and 2% (1/42) with thin bands. Am ong the mixed type o f tumor, there were 85% (33/39) with thick T N bands and 60% (25/42) with th in T N bands. A m ong other types o f tumors, there were 5% (2/39) with thick T N bands and 38% (16/42) with th in T N bands. Pairwise comparisons showed that the proportion o f tumors with thick bands was significantly different between the pure comedo and others (p<0.008) and between mixed type and others (p<0.0008). There was no difference in the proportion o f tumors with thick T N bands around tumor ducts between pure comedo and mixed type (p 0.39).

4 Tenascin expression and breast carcinoma 269 Table 2. Tenascin (TH) expression around tumor ducts and in the tumor stroma, in relation to variables associated with duct carcinoma in situ of the breast Variable or characteristic TN around tumor ducts TN in tumor stroma thick band (total N=39) thin band (total N=42) P present absent (total N=31) (total N=51) P Tumor diameter (mm, mean ± SD) ± ± ± ± Nuclear grade (%, N) grade 1 grade 2 grade 3 0% (0) 33.3% (13) 66.7% (26) 7.1% (3) 59.5% (25) 33.3% (14) % (0) 45.2% (14) 54.8% (17) 7.8% (4) 47.1% (24) 45.1% (23) 0.31 Comedo type (%, N) pure comedo mixed others 10.3% (4) 84.6% (33) 5.1% (2) 2.4% (1) 59.5% (25) 38.1% (16) % (1) 93.5% (29) 3.2% (1) 7.8% (4) 56.9% (29) 35.3% (18) Associated invasive carcinoma % positive (N) 56.4% (22) 52.4% (22) % (23) 41.2% (21) Associated extensive central % positive (N) necrosis 82.1% (32) 42.9% (18) % (24) 51.0% (26) 0.02 Associated inflammation % positive (N) 79.5% (31) 40.5% (17) % (21) 52.9% (27) 0.25 Associated microcalcification % positive (N) 74.4% (29) 76.2% (32) % (22) 78.4% (40) 0.60 Margin of excision % positive (N) 58.1% (18) 54.9% (28) 0.82 Multicentric tumors % multicentric (N) 38.5% (15) 42.9% (18) % (11) 43.1% (22) 0.64 Tumor diameter % large (> 20 mm) (N) 25.6% (10) 19.0% (8) % (15) 5.9% (3) There was significant association between inflammation and thickness of thetn band around the ducts. Among the inflamed tumors, 80% (31/39) had thick TN bands around the tumor ducts and 41% (17/42) had thin TN bands around the tumor ducts. Among the non-inflamed tumors, 21% (8/39) had thick TN bands around the tumor ducts and 60% (25/42) had thin TN bands. There was significant association between extensive central necrosis and the thickness oft N bands around tumor ducts. Among tumors with extensive central necrosis, 82% (32/39) had thick TN bands around tumor ducts, versus 43% (18/42) with thin bands. Among tumors negative for extensive necrosis, only 18% (7/39) had thick TN bands, as opposed to 57% (24/42) with thin TN bands.

5 270 Annals o f Clinical & Laboratory Science There were no significant associations between the thickness of the TN bands around tumor ducts and microcalcification, associated invasion, positive margins of exclusion, multicentricity, or tumor size. There was significant association between the presence of associated invasion and expression of TN in the stroma. Among tumors with associated invasion, 74% (23/31) showed TN expression in the stroma and 41% (21/51) did not. Among those without associated invasion, 26% (8/31) showed TN expression in the stroma while 59% (30/51) did not. There was significant association between comedo type DCIS and the expression of TN in the stroma. Among the pure comedo type of tumors, there were only 3% (1/31) positive fortn in the stroma and 8% (4/51) negative for TN in the stroma. Among the mixed type of tumors, there were 94% (29/31) positive fort N in the stroma and 57% (29/51) negative for TN in the stroma. Among the other types of tumors, there were 3% (1/31) positive fort N and 35% (18/ 51) negative fort N in the stroma. Pairwise comparisons showed that presence of TN in the stroma was significantly different between the mixed type and others (p ). There was no significant difference in the proportion of tumors with TN in the stroma between the pure comedo and mixed types (p 0.357) or between pure comedo and other types (p 0.380). There was significant association between extensive central necrosis and the expression oftn in the stroma. Among those tumors associated with extensive central necrosis, 77% (24/31) were positive for TN in the stroma and 51% (26/51) were negative fortn in the stroma. Among those tumors without extensive necrosis, 23% (7/31) were positive for T N in the stroma while 49% (25/51) were negative. Large tumors (ie, tumors with diameter >20 mm) tended to express TN in the stroma more often than small tumors (ie, tumors with diameter <20 mm). There were no significant associations between the presence of TN in the tumor stroma and the three levels of nuclear grade, inflammation, microcalcification, positive margin of excision, or multicentricity. Discussion DCIS has traditionally been viewed as one entity, but recent studies suggest that, like invasive carcinoma, DCIS represents a heterogeneous group of lesions [16,17]. In the late 1980s, several groups began to suggest that there were biologic differences between fully developed comedo DCIS and other DCIS variants. Some authors have presented data to show that comedo DCIS is more likely to be associated with occult invasion and multicentricity [13,14]. Also, it has been proposed that noncomedo DCIS with necrosis is an intermediate lesion between noncomedo and comedo DCIS [14,18]. In our study, 71% of all randomly selected DCIS cases were mixed comedo and noncomedo DCIS. This observation demonstrates the known heterogeneity of this disease and also suggests progression of noncom edo DCIS to the more aggressive comedo type. In the normal human breast, TN expression, as visualized by immunohistochemistry, is reported to be a constant ECM component throughout life, although there is markedly increased expression in carcinogenesis and malignant growth. Derangements of cell-cell adhesion, together with a change of ECM protein turnover, are critical events in tumor progression and are involved in invasion. The role of TN in tumor invasion and metastasis is poorly understood. The relationship between TN expression and prognosis in malignant tumors is controversial [ 19-21]. TN synthesis is induced in the stroma as a result of interaction with the neoplastic epithelium. Transforming growth factor beta is a regulatory protein that is reported to stimulate the production oftn [22,23]. Recently, we reported the patterns o ft N expression and their correlation with established prognostic factors of invasive breast carcinoma [15]. In this study, we demonstrated different patterns oftn expression in comedo and noncomedo DCIS. Also, we found direct correlation between TN expression in the stroma of DCIS and tumor size, extensive central necrosis, comedo DCIS, and DCIS associated with invasion. These findings demonstrate the biologic difference between comedo and non-comedo DCIS, and support previous studies that showed high incidence of invasion associated with comedo DCIS [13,14]. They also support the known biologic difference of comedo DCIS and its preinvasive potential. Although there are reports of association of multicentric lesions with comedo DCIS and invasive carcinoma [15,4,25], our results did not show correlation between TN expression and m ulticentricity. T he im plications o f the

6 Tenascin expression and breast carcinoma 271 multicentricity of DCIS are unclear. Presence of extensive central necrosis in noncomedo DCIS was found to have a pattern oftn expression around tumor ducts and in the tumor stroma similar to that in the comedo type DCIS (Table 2). This observation strongly supports the existence of variants, such as an aggressive form of noncomedo type or an intermediate lesion between noncomedo and comedo DCIS. A cknow ledgem ents. We thank Ms. Eleanore Boss and Ms. Lisa Moskowitz for skillful technical assistance, and Ms. Mei L. Wu for excellent secretarial assistance. References 1. Erickson HP, Bourdon MA. Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Ann Rev Cell Biol 1998;5: Erickson HP, Lightner VA. Hexabrachion protein (tenascin, cytotactin, brachionectin) in connective tissues, embryonic brain and tumors. Adv Cell Biol 1988;2: Mackie EJ, Chiquet-Ehrisman R, Pearson CA, et al. Tenascin is a stromal marker for epithelial malignancy in the mammary gland. Proc N atl Acad Sci USA 1987;84: HoweedyAA, Virtanen I, LaitinenL, etal. Differential distribution of tenascin in the normal, hyperplastic and neoplastic breast. Lab Invest 1990;63: Gould VE, Koukoulis GK, Virtanen I. Extra-cellular matrix proteins and their receptors in the normal, hyperplastic and neoplastic breast. Cell Diff Dev 1990;32: Nathan C, Sporn M. Cytokines in context. J Cell Biol 1991;113: Lagios MD. Human breast precancer: cancer status. Cancer Surv 1993;2: Margolin FR, Lagios MD. Mammographic detection of early breast cancer: ten years experience in a community hospital. West J Med 1986;144: Rosner D, Bedwani RN, Vana J, et al. Non-invasive breast carcinoma: results of a national survey of the American College of Surgeons. Ann Surg 1980; 192: Schnitt SJ, Silen N, Sadowsky NL, et al. Ductal carcinoma in situ (intraductal carcinoma) of the breast. N Engl J Med 1988;318: Schwartz GF, Feig SA, Patchefsky AS. Significance and staging of nonpalpable carcinomas of the breast. Surg Gynecol Obstet 1988;166: Lagios M D, M argolin FR, W estdahl PR, et al. Mammographically detected duct carcinoma in situ. Cancer 1989;63: PageDL, Steel CM, Dixon JM. ABC of breast diseases. Carcinoma in situ and patients at high risk of breast cancer. Brit Med J 1995;310: Silverstein MJ, Cohlan BG, Gierson ED, et al. Duct carcinoma in situ: 227 cases without microinvasion. Eur J Cancer 1992;28: Iskaros BF, Hu X, Sparano JA, Fineberg SA. Tenascin pattern of expression and established prognostic factors in invasive breast carcinoma. J Surg Oncol 1998;68: Patchefsky AS, Schwartz FG, Finkelstein SD, et al. Heterogeneity of intraductal carcinoma of the breast. Cancer 1989;63: Lennington WJ, Jensen RA, Dalton LW, et al. Ductal carcinoma in situ of the breast. Heterogeneity of individual lesions. Cancer 1994;73: Scott MA, Lagios M D, Axelsson K, et al. Duct carcinoma in situ of the breast: reproducibility of histological subtype analysis. Hum Pathol 1997;28: Moch H, Torhost J, Durmuller U, et al. Comparative analysis of the expression of tenascin and established prognostic factors in human breast cancer. Pathol Res Pract 1993;189: Shoji T, Kamiya T, Tsubura A, et al. Tenascin staining positivity and the survival of patients with invasive breast carcinoma. J Surg Res 1993;55: Iskaros BF, Tanaka KE, H u X, et al. Morphologic pattern of tenascin as a diagnostic biomarker in colon cancer. J Surg Oncol 1997;64: Walker RA, Dearing SJ, Gallacher B. Relationship of transforming growth factor beta 1 to extracellular matrix and stromal infiltrates in invasive breast carcinoma. Brit J Cancer 1994;69: C hiquet-e hrism an R, Kalla P, Pearson CA. Participation of tenascin and transforming growth factor beta in reciprocal epithelial-mesenchymal interactions of MCF7 cells and fibroblasts. Cancer Res 1994;49: Lagios MD, Westdahl PR, Margolin FR, et al. Duct carcinom a in situ. R elationship o f extent of noninvasive disease to the frequency of occult invasion, multicentricity, lymph node metastasis, and short-term treatment failure. Cancer 1982;50: Boyages J, Delaney G, Taylor R. Predictors of local recurrence after treatment of ductal carcinoma in situ: a meta-analysis. Cancer 1999;85: