ABERRANTLY EXPRESSED CYTOKERATIN 1, ATUMOR-ASSOCIATED AUTOANTIGEN IN PAPILLARY THYROID CARCINOMA Steven D. LUCAS 1 *, Bo EK 2 , Lars RASK 2 , Jonas RASTAD 1 , Go ¨ ran ÅKERSTRO ¨ M 1 and Claes JUHLIN 1 1 Department of Surgery, Uppsala University Hospital, Uppsala, Sweden 2 Uppsala Genetic Center, Department of Cell Research, Swedish University of Agricultural Sciences, Uppsala, Sweden Papillary thyroid carcinoma (PT C) is a somewhat puzzling disease, combining a propensity to metastasize with an indolent clinical course. T he often pronounced T cell– dominated inflammatory infiltrate seen in PTC tumors has prompted us to search for signs of a tumor-induced immune response. In previous studies, we have demonstrated large tumor-specific depositsof IgG and complement in PT C tissue and isolated a putative target antigen. This investigation examines the presence of autoantibodies to cytokeratin 1, a high m.w. cytokeratin normally expressed only in suprabasal keratinocytes, in the serum and tumor tissue of PT C patients. Using immunoprecipitation and W estern blot, cytokeratin 1–reactive autoantibodies were demonstrated in 80% of the PTC sera tested, and tumor-derived antibodies were shown to precipitate cytokeratin 1. Using immunohistochemistry, cytokeratins1 and 10 were found in a large proportion of PT C tumors (39/44) but were absent from normal thyrocytes of most PTC-bearing glands. Our results indicate that this protein is expressed aberrantly in neoplastic cells and is immunogenic in this context. Int. J. Cancer 73:171–177, 1997.  1997 Wiley-Liss, Inc. Exploiting the immune system’s ability to recognize and destroy neoplastic cells has come into the limelight of cancer research. Several systems have been devised to selectively activate the immune system through immunization with tumor antigens, spe- cific introduction of cytokine genes such as IL2 by gene transfer and in vitro stimulation of autologous tumor infiltrating lympho- cytes (TILs) (Aebersold et al., 1991; Pardoll, 1993). Some of these techniques have reached the clinical setting, with promising results (Aebersold et al., 1991; Rosenberg et al., 1988). Such attempts to stimulate tumor immunity derive from basic concepts of foreign antigen immunity applied to induced cancers in animal models or human diseases, such as malignant melanoma, breast cancer and renal cancer, chosen on the basis of their high prevalence, lethal behavior and relative immunogenicity. However, a model of inherent tumor immunity capable of controlling or eradicating manifest disease, without intervening stimulation of the immune system, has been lacking. Papillary thyroid carcinoma (PTC) is a relatively uncommon disease (annual incidence: approx. 8/100,000), afflicting patients at any age. Despite the high incidence of local lymph node metastases in PTC (50–90%), overall 10-year survival following surgical resection is over 90%, approaching 100% in patients under 50 years of age (Samaan et al., 1992). In light of the intense inflammatory reaction commonly seen in PTC-inflicted glands, this remarkable behavior, combining high metastatic potential with relative clinical indolence, has prompted hypotheses of immuno- logic control of tumor growth (Hirabayashi and Lindsay, 1965; Kamma et al., 1988). In a previous study, we demonstrated tumor-specific cellular deposits of IgG and complement factors in over 80% of examined PTC cases, suggesting activation of tumor-induced immunity (Lucas et al., 1996b). Initial immunoprecipitation experiments aimed at isolating the responsible antigens have generated several proteins, one of which has been identified as a 35 kDa fragment of cytokeratin 1 (Lucas et al., 1996a). The present study has revealed the presence of cytokeratins 1 and 10, normally present exclusively in suprabasal keratinocytes, in primary PTC tumors and their metastases. More importantly, autoantibodies to cytokeratin 1 have been found in the serum and tumor tissue of patients with PTC and appear to be involved in the collapse of the cytoskeleton. Our findings suggest that tumor-specific immunity indeed may occur in PTC. MATERIAL AND METHODS Tissues and patients We have studied the tissues of thyroidectomy specimens imme- diately frozen in liquid nitrogen or fixed in neutral buffered formalin. Normal abdominal skin also was obtained at surgery and handled in the same manner. Routine histopathological diagnosis was made on hematoxylin/eosin-stained sections according to the WHO classification of thyroid tumors. For immunoprecipitation and Western blot studies, 6 PTC tumors and 1 normal thyroid were examined. Immunohistochemistry was performed on tissues com- prising PTC (n = 44), follicular thyroid carcinoma (FTC, n = 5), anaplastic thyroid carcinoma (ATC, n = 2), medullary thyroid carcinoma (MTC, n = 5), atoxic follicular thyroid adenoma (FTA, n = 6), multi-nodular atoxic goiter (MG, n = 8), Graves’ disease (GD, n = 8) and Hashimoto’s thyroiditis (HT, n = 3). In PTC, adjacent or contralateral non-neoplastic thyroid tissue (35 cases) and lymph node metastases (8 cases) also were obtained. Patient characteristics are presented in Table I. Primary PTC tumors ranged from 0.8 to 6.5 cm in diameter (average 2.6 cm), 4 of which represented clinically occult cancers (less than 1.0 cm). Eighteen of the tumors were encapsulated completely, 18 were multi-focal and 8 demonstrated peri-glandular invasion. Two cases were recurrences in patients who previously had undergone hemi- or total thyroidectomy. Inflammatory infil- trates were present in all PTC specimens but varied in extent and distribution; 14 tumors exhibited advanced chronic inflammation with germinal centers, and the remaining 30 had diffuse inflamma- tory infiltrates. Most of the PTC specimens also contained fibrosis, which in 17 cases represented much of the tumor mass and surrounding thyroid tissue. Preparation of cytoskeletal proteins Cytoskeletal proteins were prepared from frozen tissue as follows: approx. 0.5 g of tissue was thawed, minced with a scalpel and transferred to a 1.5 ml Eppendorf tube. The tissue was homogenized with a hand-held rotating blade homogenizer in 1 ml of lysis buffer (10 mM Tris-HCl, 140 mM NaCl, 5 mM EDTA, 1% Triton X-100, 5 mM dithiothreitol [DTT], pH 7.6), with 20 strokes at low speed. The homogenate was incubated for 1 hr at 4°C with frequent vortexing, then centrifuged at 10,000 g for 10 min at 4°C. The supernatant was drawn off and stored at -70°C for later isolation of tumor immune complexes (see below). The pellet was resuspended in 2 ml high salt buffer (10 mM Tris-HCl, 140 mM NaCl, 1.5 M KCl, 0.5%Triton X-100, 5 mM EDTA, 5 mM DTT, Contract grant sponsor: Medical Research Council of Sweden; Contract grant sponsor: Swedish Cancer Society. *Correspondence to: Endocrine Surgery Research Unit, Department of Surgery, Uppsala University Hospital, S-751 85 Uppsala, Sweden. Fax: 46-18-553601. e-mail: Steven.Lucas@kirurgi.uu.se Received 4 November 1996; Revised 3 June 1997 Int. J. Cancer: 73, 171–177 (1997)  1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer