PROGNOSTIC SIGNIFICANCE OF THE CA 2+ BINDING PROTEIN S100A2 IN LARYNGEAL SQUAMOUS-CELL CARCINOMA Libero LAURIOLA 1 * , Fabrizio MICHETTI 2 , Nicola MAGGIANO 1 , Jacopo GALLI 3 , Gabriella CADONI 3 , Beat W. SCHA ¨ FER 4 , Claus W. HEIZMANN 4 and Franco O. RANELLETTI 5 1 Institute of Pathology, Universita ` Cattolica del S. Cuore, Rome, Italy 2 Institute of Anatomy, Universita ` Cattolica del S. Cuore, Rome, Italy 3 Institute of Otolaryngology, Universita ` Cattolica del S. Cuore, Rome, Italy 4 Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zurich, Zurich, Switzerland 5 Institute of Histology, Universita ` Cattolica del S. Cuore, Rome, Italy We investigated by immunocytochemistry the expression of the Ca 2 binding protein S100A2 in 62 cases of laryngeal squamous-cell carcinoma (SCC). S100A2 was detected in 18/19 (95%) low-grade tumors and in 22/43 (51%) high-grade tumors, which were partially keratinizing. The remaining 21/43 (49%) high-grade tumors were non-keratinizing, ana- plastic tumors and clearly S100A2-negative. In normal laryn- geal squamous epithelium and in laryngeal SCC, S100A2 expression was strictly associated with that of cytokeratins 14 (P  0.0002) and 17 (P  0.0021), suggesting an associa- tion of S100A2 expression and cell commitment to squa- mous differentiation. A correlation was found between S100A2 tumor positivity and longer relapse-free (P  0.0005) and overall (P  0.0095) survival. Int. J. Cancer (Pred. Oncol.) 89:345–349, 2000. © 2000 Wiley-Liss, Inc. The relationship between prognosis and histological grading of tumors has been extensively investigated. In laryngeal squamous- cell carcinoma (SCC), conflicting results have been reported be- tween survival and tumor cell differentiation (Wiernik et al., 1991). Moreover, multivariate analyses have shown that differen- tiation is an independent prognostic factor, but this issue is still disputed (Wiernik et al., 1991). Thus, the identification of factors related to tumor cell differentiation may be useful for a better understanding of the relationship between histological grading and tumor behavior. The S100 family of calcium-binding proteins has been impli- cated in pleiotropic cellular events, with specific functions for each of the family members, such as regulation of proliferation, differ- entiation, motility, and extracellular signal transduction as well as apoptosis and cancer metastasis (Scha ¨fer and Heizmann, 1996; Heizmann and Cox, 1998). Interestingly, genes coding for S100 proteins are clustered with genes encoding structural proteins required for epidermal cornification (Scha ¨fer et al., 1995; Mischke et al., 1996). S100A2, a member of the S100 protein family, is consistently expressed in the skin. In particular, S100A2 protein appears to be expressed specifically in the basal–parabasal layer of normal skin and in well-differentiated skin tumors (Shrestha et al., 1998). The vast majority of laryngeal cancers are of the squamous-cell type with a variable degree of differentiation ranging between well-differentiated, keratinizing, and anaplastic, non-keratinizing tumors (Wiernik et al., 1991). Preliminary immunohistochemical experiments showed that both normal squamous laryngeal epithe- lium and laryngeal SCC express S100A2 protein. Since S100A2 protein expression appears to be positively associated with squa- mous-cell differentiation (Shrestha et al., 1998), we studied the clinical significance of S100A2 expression in laryngeal SCC. MATERIAL AND METHODS Patients Our study included 62 untreated consecutive primary laryngeal SCC patients admitted to the Department of Otolaryngology of the Catholic University, Rome. Histological grading and TNM classi- fication were performed on conventional paraffin sections accord- ing to the recommendations of the International Union Against Cancer (Hermanek et al., 1987). Accordingly, tumors were graded as well- (G1), moderately (G2), or poorly (G3–G4) differentiated. Histopathological grading of laryngeal SCC based only on the presence or absence of keratin was made as recommended by Wiernik et al. (1991). The former and the latter were placed into the keratinizing and the non-keratinizing, anaplastic groups, re- spectively. The anaplastic group included cellular features com- patible with squamous cells, but keratin was not recognized within the tumor cells or as aggregates among the neoplastic cells. At our institution, all primary laryngeal cancer patients receive standard therapeutic management: therapeutic surgical treatment (curative surgery) of the primary tumor (T) related to the lesion extension, therapeutic neck node dissection when there is lymph node in- volvement at clinical presentation (N+) according to the “wait- and-see” policy under strict follow-up conditions, post-surgical radiotherapy for locally advanced tumors (T4), and neck lymph node metastasis with extranodal spread according to the following treatment protocol: 180 cGy a day for 5 days in a week for a total of 70 Gy. All patients in this study were treated according to this standard procedure. Thirty-eight patients underwent total laryngec- tomy, 17 supraglottic laryngectomy, 2 hemilaryngectomy, and 5 cordectomy. At surgery, 8 patients with clinically positive neck nodes underwent a therapeutic neck dissection. The median fol- low-up period was 44 months (range 2–90 months). Immunohistochemical analysis Tumor tissues obtained at surgery and 2 autopsy specimens of normal larynx were fixed in formalin and paraffin-embedded ac- cording to standard procedures. Tissue sections were treated with 0.3% H 2 O 2 in methanol for 10 min to block endogenous peroxi- dase activity. For the immunolocalization of S100A2, sections were incubated with normal rabbit serum for 15 min, then with rabbit anti-serum against S100A2, diluted 1:200, for 1 hr. The specificity of the rabbit anti-serum against human recombinant S100A2 was assessed by Western blotting, as described elsewhere (Ilg et al., 1996). For cytokeratin (CK) immunolocalization, con- secutive sections were incubated for 1 hr with anti-CK14 (clone LL002), anti-CK17 (clone E3), and anti-CK19 (clone b170) mono- clonal antibodies (MAbs; Novocastra, Newcastle-upon-Tyne, UK) at 1:100 dilution. Indirect immunostaining was achieved using the ABC (Vector, Burlingame, CA) technique. Endogenous biotin was saturated by a biotin blocking kit (Vector). The peroxidase was developed with a DAB substrate kit (Vector). Negative controls were performed using normal rabbit or mouse serum, omitting the primary antibodies. All immunostained sections were evaluated by Grant sponsor: Swiss National Science Foundation; Grant number: 31- 50510.97; Grant sponsor: MURST. Ministero per L’Universita ` e La Ricerca Scientifica e Tecnologı `ca. *Correspondence to: Libero Lauriola, Istituto di Anatomia Patologica, Universit` a Cattolica del S. Cuore, Largo F. Vito 1, 00168 Roma, Italy. Fax: +0103963051157. E-mail: ibiap@rm.unicatt.it Received 25 November 1999; Revised 28 February 2000 Int. J. Cancer (Pred. Oncol.): 89, 345–349 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer