Tenascin (TN) is a large glycoprotein of the extracellular

Transcription

1 Tenascin Expression in Intraepithelial Neoplasia and Invasive of the Uterine Cervix Basem F. Iskaros, MD; Leopold G. Koss, MD Tenascin (TN) is a large glycoprotein of the extracellular matrix that is transiently expressed in various tissues during embryogenesis and growth. It is also expressed in wound healing and tumor stroma. 3 Tenascin may have a role in cell adhesion, migration, and promotion of growth during fetal development and oncogenesis. 6 The stroma and its extracellular matrix located in and around invasive tumors may show a desmoplastic reaction that differs morphologically from that of the normal stroma of the same organ. There is evidence that this desmoplastic reaction is related to tumor invasion and is the result of a derangement of cell-cell adhesion. Because TN is an important component of the desmoplastic reaction, its expression may be an index of the invasive potential of a malignant tumor. 7 9 As an example, in lesions of the female breast, TN forms thin periductal bands in ductal hyperplasia, within fibroadenomas, and around ductal carcinomas in situ. In invasive breast carcinomas, however, TN expression is markedly increased in the stroma surrounding infiltrating tumor cells. 7 Recently, we correlated the patterns of TN expression in Accepted for publication February 3,. From the Department of Pathology, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY. Presented in part at the annual meeting of the US and Canadian Academy of Pathology, Boston, Mass, March, 99. Reprints: Leopold G. Koss, MD, Department of Pathology, Montefiore Medical Center, East th St, Bronx, NY 67. Objective. To determine whether the expression of the matrix protein tenascin (TN) is of diagnostic or prognostic value in cervical intraepithelial neoplasia (CIN). Design. Tenascin expression was evaluated in 7 formalin-fixed, paraffin-embedded biopsy and surgical specimens of the uterine cervix. Specimens included lowgrade squamous neoplastic lesions (CIN I), 3 high-grade squamous neoplastic lesions (CIN II and CIN III), microinvasive carcinomas, and invasive squamous carcinomas. Five normal cervices and examples of cervicitis were used as controls. Expression of TN was studied by immunohistochemistry with a monoclonal mouse anti-human tenascin antibody. Tenascin expression in the basement membrane and in the stroma was arbitrarily graded as normal or slightly, moderately, or markedly increased. Results. In the normal cervix, TN formed a thin band along the basement membrane of the squamous epithelium, except for the transformation zone, where the bands splintered and delicate TN fibers were present in the adjacent stroma. In cervicitis, TN bands were splintered in the basement membrane and the protein was weakly expressed in the stroma infiltrated by inflammatory cells. In the CIN lesions, regardless of grade, the TN bands in the basement membrane were slightly ( cases) or moderately ( cases) increased. In CIN lesions with chronic stromal inflammation, a slight increase in stromal staining was observed, similar to the findings in cervicitis. In microinvasive and frankly invasive squamous cell carcinomas, TN expression was markedly increased in the basement membrane and in the stroma surrounding the invasive nests of cancer cells. Conclusion. Tenascin expression may be of value in the assessment of early stromal invasion in cancer of the uterine cervix. Tenascin expression is of no value in distinguishing various grades of CIN and, therefore, is not a predictor of future behavior. (Arch Pathol Lab Med. ;: 6) invasive breast and colon carcinomas with prognosis.,9 In invasive colon cancer, an increase in TN expression in the stroma correlated positively with patient survival, but there was no significant correlation in patients with invasive mammary carcinoma. In the uterine cervix, TN expression was reported to be increased in precancerous lesions associated with human papillomavirus and was thought to be an expression of epithelial growth and stromal remodeling. In a more recent study, the increase in TN expression in cervical lesions was attributed to an inflammatory component associated with cervical intraepithelial neoplasia (CIN) lesions. Because of these conflicting results, we undertook an immunohistochemical study to investigate whether the differences in TN expression in various grades of squamous intraepithelial lesions of the uterine cervix may be of value in assessing the degree of abnormality and, hence, their future behavior. MATERIALS AND METHODS The pathology records and paraffin blocks of biopsies and surgical specimens for 7 archival cases of inflammatory and neoplastic lesions of the uterine cervix were retrieved. There were examples of CIN, of which were low-grade squamous neoplastic lesions (CIN I) and 3 were high-grade squamous neoplastic lesions (CIN II and CIN III). There were also microinvasive carcinomas, as defined by the International Federation of Gynecologists and Obstetricians (FIGO) criteria, and invasive squamous cell carcinomas. Five biopsy cases of cervicitis and cases of normal cervix from hysterectomies performed for unre- Arch Pathol Lab Med Vol, September Tenascin Expression in Cervical Neoplasia Iskaros & Koss

2 Figure. A, Tenascin (TN) expression in normal uterine cervix. A thin band of TN is seen at the level of the basement lamina of the squamous epithelium. In the adjacent transformation zone, TN fibrils are present in the stroma. B, Cervicitis. Tenascin forms a somewhat thickened band in the area of the basement lamina and is present in the underlying stroma in the form of thin filaments (original magnification [A and B]). Figure. Low-grade squamous intraepithelial lesion of the uterine cervix. A, Hematoxylin-eosin stained section. B, Tenascin (TN) expression in the form of a somewhat thickened ( ) and splintered band in the area of the basement lamina. There is no TN expression in the underlying stroma (original magnification [A and B]). Figure 3. High-grade squamous intraepithelial lesion of the uterine cervix. A, Hematoxylin-eosin stained section. B, Tenascin (TN) expression in the form of a thickened and splintered band in the area of the basement lamina ( ). A narrow band of fine TN fibers is present in the underlying stroma. In other areas of this and other similar lesions, there was no TN expression in the stroma (original magnification [A and B]). Arch Pathol Lab Med Vol, September Tenascin Expression in Cervical Neoplasia Iskaros & Koss 3

3 Figure. High-grade squamous intraepithelial lesion with focal extension into the stroma (pseudoinvasion). Around the tumor dip, tenascin forms a well-defined band, similar to the band in the area of the epithelial basement lamina ( ) (original magnification ). lated reasons were added as normal controls. All blocks were cut into - m sections and placed on lysine-coated glass slides. The following protocol was followed: the sections were deparaffinized in xylene and rehydrated with a graded ethanol series (% to 7%), followed by a rinse in water, incubation in 3% hydrogen peroxide for minutes, a wash in tap water, and pretreatment with protease (:) in phosphate-buffered saline (PBS) for 3 minutes at room temperature. The slides were rinsed sequentially in tap water, distilled water, and PBS. The monoclonal anti-human mouse TN antibody (Dako Corporation, Carpinteria, Calif; TNM636; : dilution) was added, and slides were incubated for hours in a humidified chamber. Slides were washed in PBS and a biotinylated anti-mouse antibody (Dako) was added for minutes, followed by a rinse in PBS, and streptavidin reagent for minutes. After an additional PBS wash, the slides were treated with 3,3-diaminobenzidine (Dako) for minutes. Slides were counterstained with hematoxylin, dehydrated, and mounted with glass coverslips. Two sets of controls were included in each run. Sections of an adenomatous polyp of colon were used as a positive control. Sections of an invasive carcinoma of the uterine cervix were used as a negative reagent control by omitting exposure to TN antibody. Immunohistochemical staining patterns and intensity of TN expression in different cervical lesions were graded from to 3 as follows: indicated a thin basement membrane band ( m by ocular micrometer);, Table. Tenascin (TN) Expression and Cases Associated With Stromal Inflammation* Diagnosis LGSIL HGSIL No. of cases TN score 3 TN in stroma None Slight Marked 6 Microinvasive Frank Invasive * LGSIL indicates low-grade squamous intraepithelial lesion; HGSIL, high-grade squamous intraepithelial lesion; and TN score, expression of tenascin in the basement membrane. somewhat thickened basement membrane ( 3 m); and 3, thick basement membrane band ( 3 m). Tenascin positivity in the stroma was recorded as either absent, slight (limited to thin filaments), or marked (forming broad bands). RESULTS In the normal cervix, TN formed a thin, delicate, continuous band within the basement membrane of the squamous epithelium. In the transformation zone, TN expression in the form of thin bands was also evident in the adjacent stroma (Figure, A). In cervicitis, the TN bands were thickened in the basement membrane, and the protein was also present in the adjacent stroma in the form of thin filaments (Figure, B). In the squamous intraepithelial lesions of various grades, the TN bands were either slightly ( ) or moderately ( ) thickened in the basement membrane (Figures and 3; Tables and ). There were some differences in the level of thickening among various segments of the lesions, and the data represent the dominant pattern. An inflammatory infiltrate consisting mainly of lymphocytes in adjacent stroma was present in 3 cases of CIN ( cases of low grade and cases of high grade), case of microinvasive carcinoma, and cases of frank invasive carcinoma (Tables and ). Slight stromal staining in the form of thin bands was observed in of the 3 CIN cases, regardless of grade, with Figure. Tenascin expression around plugs of microinvasive (A) and fully invasive cervical carcinomas. Broad bands and thick fibers surround the plugs of invasive tumor (B) (original magnification [A and B]). Arch Pathol Lab Med Vol, September Tenascin Expression in Cervical Neoplasia Iskaros & Koss

4 Table. Tenascin (TN) Expression and Cases Without Stromal Inflammation* Diagnosis LGSIL HGSIL No. of cases TN score 3 TN in stroma None Slight Marked 3 Microinvasive Frank Invasive * LGSIL indicates low-grade squamous intraepithelial lesion; HGSIL, high-grade squamous intraepithelial lesion; and TN score, expression of tenascin in basement membrane. inflammatory stromal reaction. The stromal staining pattern was similar to that observed in cervicitis. Around the stromal dips of the thickened CIN, the TN bands were limited to the basement membrane (Figure ). By contrast, in microinvasive and frankly invasive carcinoma of the cervix, the TN expression was markedly increased in the stroma and around the invasive nests of tumor, even in the absence of inflammation (Figure ; Tables and ). COMMENT Histologic and colposcopic studies have documented that in about 9% of cases the initial neoplastic events in the epithelium of the cervix take place in the area of squamous epithelium adjacent to the columnar epithelium, known as the transformation zone. It is assumed that only a relatively small proportion of squamous intraepithelial lesions will progress to invasive cancer, with the probability of progression increasing for high-grade lesions. The biologic mechanisms leading to invasions are unknown, but it may be assumed that in some of these lesions a genetic rearrangement in cancer cells or in adhesion molecules may occur, conferring on the cancer cells the ability to break through the basement membrane. 3 The relationship of the precancerous lesions of the uterine cervix with human papillomavirus of various types has led to the suggestion that the presence of human papillomavirus of high-risk type (such as types 6 or ) is possibly predictive of tumor progression. Still, even in these patients, there is no certitude whatsoever of the natural course of the disease. Furthermore, the determination of viral type and follow-up of patients are complicated and costly. Therefore, a simple laboratory method based on evaluation of biopsy material that would predict the behavior of CIN would be of significant clinical value. This was the background of the TN study reported in this article. It has been shown that expression of TN is increased in some malignant tumors when compared with that of normal adult tissues.,9 It was proposed that TN plays an important role in stromal alterations associated with malignant tumors of the breast, prostate, and colon. 7,6,7 Still, the role of TN in tumor stroma is controversial. While some reports suggest that the TN protein paves the way for tumor cells to invade and metastasize, other reports suggest that increased TN expression in the tumor stroma acts as a boundary to contain the tumor cells and to prevent further progression and metastases. Regardless of this controversy, the TN expression may be increased at sites of active tissue remodeling. It is of interest that the epithelial cells, whether benign or malignant, may be the source of TN., In this study, we have shown that TN fibrils are present in the stroma of the normal transformation zone, in cervicitis, and in CIN lesions with inflammatory infiltrate of the stroma. The last observation suggests that inflammatory cells may induce TN synthesis and release by fibroblasts, probably by releasing cytokines and growth factors, such as transforming growth factor-, which has been shown to stimulate fibroblasts to synthesize TN. 3, In squamous intraepithelial lesions of different grades, the TN expression was either normal or slightly or moderately increased in the basement membrane (Tables and ). The degree of basement membrane thickening was somewhat variable from one segment of the lesion to another. These segmental differences may account for the controversy in the published data., Our results are similar to those previously reported by Pöllänen et al, who concluded that TN expression cannot be used as a marker of CIN progression. We agree with this conclusion. On the other hand, in microinvasive and frankly invasive carcinoma, TN expression was markedly increased in the form of fibers and broad bands in the stroma surrounding invading nests of cancer cells, regardless of the presence or absence of an inflammatory infiltrate. Thus, TN staining may be helpful in separating microinvasive carcinoma from pseudoinvasion of invaginating papillae of abnormal squamous epithelium (compare Figure with Figure ). References. Chiquet-Ehrismann R, Hagios C, Schenk S. The complexity in regulating the expression of tenascin. BioEssays. 99;7: Erickson HP. Tenascin-c, tenascin-r and tenascin-x: a family of talented proteins in search of functions. Curr Opin Cell Biol. 993;: Koukoulis GK, Gould VE, Bhattacharyya A, et al. Tenascin in normal, reactive, hyperplastic and neoplastic tissue: biologic and pathologic implication. Hum Pathol. 99;: Mackie E, Tucker RP, Halfter W, Chiquet-Ehrismann R. 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Enhanced tenascin expression in cervical and vulvar koilocytotic lesions. Am J Pathol. 99;: Pöllänen R, Soini Y, Vuopala S, Laara E, Lehto V-P. Tenascin in human papillomavirus associated lesions of the uterine cervix. J Clin Pathol. 996;9: 3.. Koss LG, Gompel C. Introduction to Gynecologic Cytopathology With Histologic and Clinical Correlations. Baltimore, Md: Williams & Wilkins; 999: Koss LG. Epidermoid carcinoma of the uterine cervix and related precancerous lesions. In: Koss LG, ed. Diagnostic Cytology and Its Histopathologic Bases. th ed. Philadelphia, Pa: JB Lippincott; 99:37.. Ho GYF, Bierman MD, Beardsley L, et al. Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med. 99;33:3.. Koss LG. Human papillomavirus: passenger, driver or both? [editorial]. Hum Pathol. 99;9: Bigner DD, Humphery PA. Tenascin expression in prostatic hyperplasia, intraepithelial neoplasia, and carcinoma. Hum Pathol. 993;:9 99. 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5 7. Sakai T, Kawakatsu H, Hirota N, et al. Specific expression of tenascin in human colonic neoplasms. Br J Cancer. 993;67: 6.. Jahkola T, Toivonen T, Smitten KV, Blomqvist C, Virtanen I. Expression of tenascin in invasion border of early breast cancer correlates with higher risk of distant metastasis. Int J Cancer. 996;69: Shoji T, Kaamiya T, Tsubura A, et al. Tenascin staining positivity and the survival of patients with invasive breast carcinoma. J Surg Res. 993;: Sugawara I, Hirakoshi J, Masunaga A, et al. Reduced tenascin expression in colonic carcinoma with lymphogenous metastasis. Invasion Metastasis. 99; : Ishihara A, Yoshida T, Tamaki H, Sakakura T. Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance. Clin Cancer Res. 99;:3.. Lightner VA, Marks JR, McCachren SS. Epithelial cells are an important source of tenascin in normal and malignant human breast tissues. Exp Cell Res. 99;: Chiquet-Ehrismann R, Kalla P, Pearson CA. Participation of tenascin and transforming growth factor beta in reciprocal epithelial-mesenchymal interaction of MCF7 cells and fibroblasts. Cancer Res. 99;9:3 3.. Sakai T, Kawakatsu H, Ohta M, Saito M. Tenascin induction in tenascin nonproducing carcinoma cell lines in vivo and by TGF-beta in vitro. J Cell Physiol. 99;9: Arch Pathol Lab Med Vol, September Tenascin Expression in Cervical Neoplasia Iskaros & Koss