ANTICANCER RESEARCH 27: 1565-1570 (2007)

Expression Analysis of Components in Brush Biopsies of Oral Lesions

OLIVER DRIEMEL1, HARTWIG KOSMEHL2, JULIA ROSENHAHN3, ALEXANDER BERNDT4, TORSTEN E. REICHERT1, LUCIANO ZARDI5 and REGINE DAHSE2

1Department of Oral and Maxillofacial Surgery, University of Regensburg, Regensburg; 2Institute of Pathology, HELIOS Clinics Erfurt GmbH, Erfurt; 3Institute of Human Genetics and Anthropology and 4Institute of Pathology, University of Jena, Jena, Germany; 5Istituto Giannina Gaslini, Genova, Italy

Abstract. Background: Oral brush biopsies have proved to be is a heterotrimeric molecule with an ·, ‚ and Á a promising new non-invasive methodology in the diagnosis of chain. Different laminin isoforms arising from an exchange oral lesions. The extracellular matrix (ECM) molecules Á2 chain of single chains have been found with tissue- and of laminin-5 (L5Á2), tenascin-c (Tn-C) and the development-specific functions (4). Laminin-5 consists of isoform containing EDB (EDB-fn) are involved in matrix the ·3, ‚3 and Á2 chains and represents the main of remodeling during malignant transformation in oral carcinoma. the epithelial adhesion complex (5, 6). Laminin-5 is Materials and Methods: Expression of L5Á2, Tn-C and EDB-fn frequently expressed at the invasion front of epithelial was analysed in brush biopsy-obtained cells of benign tumors. The isolated Á2 chain of laminin-5 (L5Á2), or its inflammatory or hyperproliferative lesions and primary oral proteolytic fragments, act as potent factors in migration and squamous cell carcinoma (OSCC) using the Roche LightCycler invasion (7, 8). 2.0 System. Results and Conclusion: Oral carcinoma are Cellular fibronectin (fn) is a large adhesive protein detectable with mRNA resynthesis of the ECM molecules L5Á2 mediating interaction between cells and their environment and Tn-C in oral brush biopsies. EDB-fn mRNA was not by binding to integrin receptors and components. detected – the stroma myofibroblasts are apparently a preferential It appears in different isoforms due to alternative mRNA source of EDB-fn and sampling by oral brush biopsy harvests splicing of the EDA, EDB, and IIICS regions, and epithelial cells and does not reach the cells which do express subsequent post-translational modifications. The complete EDB-fn. The performance of gene expression analysis in brush EDB region may be entirely included or omitted in the fn biopsies is limited by a high RNase activity in the oral cavity. molecule. The fibronectin isoform containing EDB (EDB- fn) is undetectable in healthy adult oral tissues, with the Extracellular matrix (ECM) components have fundamental exception of tissues undergoing physiological remodeling or functions in cell attachment, differentiation, proliferation during wound healing. By contrast, its expression in tumors and migration. During cancer progression, the ECM of the and fetal tissues is high. Furthermore, it was demonstrated tissue in which the tumor grows is extensively remodeled, that EDB-fn is a marker of angiogenesis and that both by degradation of preexisting ECM molecules and by endothelial cells invading tumor tissues migrate along ECM the neosynthesis of ECM components, which in many cases fibers containing EDB-fn (9). are not present in the ECM of normal tissues (1). Isoforms Tenascin-C (Tn-C) is an ECM that of the ECM molecules laminin, fibronectin and tenascin-c modulates the adhesion of cells (10). Tn-C is induced or are structural components of tumor invasion (2, 3). repressed in a cell type- and species-dependent fashion by multiple factors. In general, Tn-C is abundantly expressed during embryonic development, yet it is re-expressed within the adult organism during normal and pathological Correspondence to: Dr. Regine Dahse, HELIOS Klinikum Erfurt, tissue remodeling. In normal adult tissues, Tn-C Institute of Pathology, Nordhäuser Str. 74, D-99089 Erfurt, expression is induced during neovascularisation and wound Germany. Tel: +361 781 2770, Fax: +361 781 2760, e-mail: healing, and at tissue sites that are subject to [email protected] biomechanical forces. In pathologies, Tn-C expression is Key Words: Oral brush biopsy, extracellular matrix, Á2 chain of associated with cancer, wound healing and inflammatory laminin-5, tenascin-c, EDB-fibronectin, LightCycler, ECM. diseases (reviewed in (11)).

0250-7005/2007 $2.00+.40 1565 ANTICANCER RESEARCH 27: 1565-1570 (2007)

We chose the tumor invasion markers Tn-C, L5Á2 and Table I. Sample characteristics. EDB-fn to analyse their expression in inflammatory, benign Sample Gender Age Diagnosis proliferative and malignant oral lesions, and to study the number (years) diagnostic reliability of these markers in oral brush biopsies. Brush biopsies are an innovative non-invasive technique for the N1 f 31 normal oral mucosa (control) detection of oral precancerous stages and manifest carcinomas. N2 f 28 normal oral mucosa (control) Biopsy brushes allow for harvesting oral epithelial cells N3 m 30 normal oral mucosa (control) N4 f 46 normal oral mucosa (control) from deep cell layers in which dysplastic changes find their N5 f 46 normal oral mucosa (control) origin. Oral brush biopsy methodology has been B 1 f 73 fibroma demonstrated to be suitable for tumor detection and B 2 m 61 lichen ruber planus diagnosis in cooperation with molecular analysis like DNA B 3 m 38 hyperplasia cytometry (12, 13), immunocytology (14), TP53 mutation B 4 m 71 florid ulcus B 5 f 89 leukoplakia analysis (15) and FISH (16). Recently, we demonstrated B 6 m 49 leukoplakia laminin-5 immunocytochemistry as a new tool for identifying B 7 f 55 aphthous inflammation dysplastic cells in oral brush biopsies (17). Based on these B 8 f 45 lichen ruber planus promising immunocytological results, we hypothesized in this B 9 m 80 verrucous leukoplakia study that an quantitative mRNA detection of ECM B 10 f 34 leukoplakia molecules in cells from oral brush biopsies might contribute Sample Gender Age Histopathology TNM Grading to the characterization of a malignant phenotype. number (years) classification Herein we present the first real time-PCR-based study on oral brush biopsies. The expression of the extracellular T1 f 80 oral SCC PT4a pN2cM0 G2 matrix molecules Tn-C, L5Á2 and EDB-fn in oral lesions T2 m 69 verrucous pTis carcin. in situ and oral squamous cell carcinoma is discussed from a cell T3 m 60 oral SCC pT2pN0cM0 G2 biological and clinical diagnostic point of view. T4 m 56 oral SCC pT2pN2cM0 G2 T5 f 41 oral SCC pT1pN0cM0 G2 Materials and Methods N: normal controls; B: benign oral lesions; T: tumor; m: male; f: Sampling. Oral brush biopsies with a sterile nylon cytology brush female; SCC: squamous cell carcinoma. (Medscand Medical AB, Sweden) were performed in 50 patients with benign inflammatory or hyperproliferative lesions (n=22), and primary oral squamous cell carcinoma (n=28). Brush biopsies of the control of all 55 samples, 20 cDNA samples were selected for gene oral mucosa were taken from healthy, non-smoking individuals as expression analysis: five samples of normal oral mucosa (controls), controls (n=5). All patients were asked to brush their teeth and five tumor samples and ten samples representing benign oral rinse the oral cavity with an antiseptic prior to sampling. Cell lesions (Table I). harvesting was performed by rotating the brush under light pressure over the lesion. The brush tip was cut off into sterile Eppendorf Qualitative detection of EDB- fibronectin. For RT-PCR detection of tubes and stored at –80ÆC prior to RNA isolation. One additional EDB-fn (the fibronectin isoform containing EDB), the brush biopsy was taken per patient and cells were transfered to a oligonucleotide primer pair: B1: 5’-CGGCCTGGAGTACAA slide for standard HE staining and cytology. Informed consent to TGTCAGTGT-3’ and B2: 5’-CAGGTGACACGCATGGTGTCT participate in the study was obtained from the patients. GGA-3’ located in exons III-7 and -8 flanking the EDB region were used (18). RNA of an osteosarcoma cell line, which was previously RNA isolation and cDNA synthesis. Total RNA was extracted from found to express EDB-fn (19), was used as positive PCR control. epithelial cells using the RNeasy Micro Kit (QIAGEN GmbH, Thirty rounds of amplification were performed with the Germany). Cell disruption and lysate homogenisation were following cycling protocol: initial denaturation at 94ÆC for 5 min; performed using QIAshredder Spin Columns (QIAGEN). Total 25 cycles with denaturation at 94ÆC for 30 sec, annealing at 56ÆC RNA was eluted with 15 Ìl RNase-free water. Digestion of for 30 sec and extension at 72ÆC for 60 sec; final extension: 72ÆC contaminating DNA was performed with RNase-free DNase I for for 5 min. A volume of 10 Ìl of the PCR products was 30 min at 37ÆC. Reaction was stopped for 10 min at 65ÆC and 10 Ìl electrophoresed through a standard 2% agarose gel stained with resulting total RNA were used for subsequent first strand cDNA SYBR-Green I for visualization under UV light. synthesis. Total RNA was reverse transcribed using random hexamers and the Transcriptor First Strand cDNA Synthesis Kit LightCycler-based relative mRNA quantification of L5Á2 and Tn-C. (Roche, Mannheim, Germany). Relative quantification of L5Á2 and Tn-C mRNA was performed RNA integrity was assessed with a control PCR for the pyruvate using the LightCycler 2.0 System (Roche, Germany). A calibrator- dehydrogenase gene with an intron spanning primer pair (forward: normalized relative quantification method with PCR efficiency 5’-GGTATGGATGAGGAGCTGGA-3’; reverse: 5’-CTTCCAC correction was selected: i.e., the amount of a target gene (L5Á2; Tn- AGCCCTCGACTAA-3’). This approach yields amplification C) is analysed relative to the amount of a reference gene in the same products from DNA 224 bp) and cDNA (102 bp). After quality sample. As a reference, the housekeeping gene G3PDH

1566 Driemel et al: Expression Analysis in Oral Brush Biopsies

Figure 1. Expression of the Á2 chain of laminin-5 in normal oral mucosa (N1-N5), benign oral lesions (B1-B10) and oral carcinoma (T1-T5) as detected using LightCycler-based quantitative RT-PCR. The relative mRNA amounts of laminin-5 Á2 normalized to the housekeeping gene G3PDH are shown.

(glyceraldehyde-3-phosphate dehydrogenase) was chosen that is line), but was not found in normal oral mucosa samples, expressed approximately at constant levels in most cell types. As benign inflammatory or proliferative oral lesions, or in the calibrator sample, pooled RNA of squamous cell carcinoma tumor samples. (expressing L5Á2 and Tn-C) was analysed. In addition to the correction of sample quality differences, normalization to a calibrator corrected the PCR results for different detection sensitivities. L5Á2. The relative mRNA amount per sample was determined with LightCycler-based RT-PCR in comparison to a reference To optimise the real-time PCR conditions, the optimal MgCl2 concentration and annealing temperature were determined using gene and a calibrator DNA dilution series. For the calculation FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany) of data, only the Cp (crossing point) values for the and melting curve analysis. Finally, reactions were performed in a appropriate PCR fragment obtained by the LightCycler volume of 20 Ìl and a master mix of the following composition was software and verified by melting point analysis were used. The used: 2 Ìl LightCycler DNA Master SybrGreen I (Roche, Mannheim, Cp values are a function of the PCR amplification efficiency. Germany), 3 mM MgCl2, 0.5 ÌM primers (10 ÌM). A volume of 2 Ìl of cDNA template (samples or standard in appropriate dilution) was The mean value of three amplifications was used for the data added to the master mix in pre-cooled capillaries. calculation if standard deviation was <0.1. Final results are The following oligonucleotide primer pairs were used: G3PDH: expressed as the target/reference ratio of the sample divided 5’-AGCAATGCCTCCTGCACCACCAAC-3’ and 5’-CCGGAGGG by the target/reference ratio of the calibrator. L5Á2 was found GCCATCCACAGTCT-3’; L5Á2: 5’-AGGCTGTCCAACGAAA at very low expression levels in normal oral mucosa. The TGGG-3’ and 5’-GGAGCTGTGATCCGTAGACCA-3’ (20); highest expression was detected in tumor samples (T1, T4, T5) Tn-C: 5’-AAGAGGCTCACAATCTCACG-3’ and 5’-CTCAGACA and in hyperproliferative benign oral lesions (B1, B3). Benign CGGCTAAATCTC-3’ (21). The experimental protocol for the Lightcyler was as follows: Initial oral lesions in general expressed L5Á2 at a midrange level denaturation: 95ÆC for 900 sec; amplification: 45 cycles of 95ÆC for (Figure 1). 10 sec, 56ÆC (G3PDH); 61ÆC (Tn-C) or 62ÆC (L5Á2) for 15 sec, 72ÆC for 12 sec; melting curve analysis: 1 cycle of 95ÆC for 1 sec followed Tn-C. LightCycler analysis of Tn-C was performed as by 64ÆC for 120 sec and continual fluorescence acquisition. All described for laminin-5. Normal oral mucosa cells did not experiments were performed in three replicates. express Tn-C. Tn-C mRNA was detectable in all tumor Relative standard curves for the targets (L5Á2, Tn-C) and the samples. Individual tumor samples (T1, T5) had very high reference gene (G3PDH) were created using the calibrator cDNA and dilution series of 100-10-4. For each sample, the relative Tn-C expression levels, others (T2, T3, T4) expressed Tn-C amount of the target gene and the reference gene was determined. at amounts lower than the average detection level of benign All calculations were made using Microsoft Excel software. oral lesions. Tn-C mRNA was detectable in 60% of the benign oral lesions (Figure 2). Results Discussion EDB-fn. Detection of EDB-fn with qualitative RT-PCR revealed a 413 bp amplification product representing the Mucosal lesions of various types are frequently found in the fibronectin isoform containing EDB. Such a PCR product oral cavity and a limited proportion of these will potentially was detected in the positive control (osteosarcoma cell progress to malignant tumors. The detection of a

1567 ANTICANCER RESEARCH 27: 1565-1570 (2007)

Figure 2. Expression of tenascin-C in normal oral mucosa (N1-N5), benign oral lesions (B1-B10) and oral carcinoma (T1-T5). premalignant or malignant lesion when small is one of the immunocytochemical marking of atypical cells in the brush most important factors in improving cancer diagnosis and biopsy preparations improves cellular localisation and therapy. Surgical biopsy is the most accurate method in the diagnosis. The positive predictive value in identifying diagnosis of oral lesions, however, it is not applicable to large atypical cells with lamin-5 immunocytochemistry was 97% numbers of patients. Neither macroscopic nor microscopic (17). At the mRNA expression level analysed in the study evaluation can give reliable information concerning the risk presented herein, L5Á2 was detected in benign inflammatory for cancer development, which is why markers indicating and proliferative oral lesions and in the tumor samples. high risk for tumor progression would be of significant value. Results of both studies indicate that laminin-5 mRNA The ECM molecules L5Á2 and Tn-C are known as synthesis already occurs in inflammatory tissue and markers for matrix remodeling during malignant premalignant lesions, as well as in carcinoma cells, and that transformation in OSCC. We were able to demonstrate in posttranscriptional and/or posttranslational processes lead this study that both molecules were detectable at the mRNA to a cellular laminin-5 accumulation in the tumor cells expression level in brush biopsies of oral carcinoma. This (detectable at the protein level with immunocytochemistry). means that isolated tumor cells from brush biopsies Cellular accumulation of laminin-5 in tumor cells seems not represent the biological properties of a malignant tumor. to be homogenous. The variable accumulation of laminin-5 This result underlines the applicability of the brush biopsy subunits was shown in a previous study in human colorectal technique as a novel non-invasive method for the detection neoplasia: expression was continuous and gradient-like in of oral precancerous stages and manifest carcinomas. In normal mucosa, enhanced at the periphery of adenomas, contrast to the cervix uteri, the oral mucosa has no and discontinuous in places in carcinomas and metastases transformation zone with aberrant surface cell layers. (25). Posttranslational modifications in the tumor cells Atypical cells are found in deeper cell layers or are might influence the laminin-5 protein and lead to protein detectable at the surface only in later disease stages. Brush accumulation. To give an example, membrane-type matrix biopsies are able to overcome this diagnostic problem: cells metalloproteinase-1 (Mt1-MMP) was found as an enzyme can be obtained non-invasively from all epithelial layers of processing L5Á2 and thereby creating fragments which the oral mucosa and early lesions are detectable. increased tumor cell scattering and migration (26). It is known from previous studies of the head and neck Recently, an extracellular co-deposition of laminin-5 with region that advanced malignant transformation and invasion the ECM molecule Tn-C was found in oral squamous cell correlate stringently with the resynthesis of laminin-5 (2; carcinoma, and a functional interaction of both molecules 23). Laminin-5, consisting of alpha3-, beta3-, and gamma2- during malignant progression was hypothesized (2). In chains, represents the main protein of the anchoring contrast to laminin, Tn-C isoforms are generated by filaments which connect the with the alternative splicing. Two main isoforms are known, of which hemidesmosome of the cell. It is frequently expressed at the the large Tn-C isoform is mainly expressed during tumor invasion zone of epithelial tumors (22, 23). This protein has stroma remodeling (27). In oral squamous cell carcinoma, recently proved to be an invasion marker for epithelial cells large Tn-C mRNA was detected in tumor cells at the site of and intracellular accumulation of monomeric gamma2- tumor invasion and the protein was deposited in the tumor chains has been widely observed in invasive carcinoma cells stroma (28). We detected Tn-C mRNA in benign oral lesions (24). Recently, we were able to demonstrate in a laminin-5 and in carcinoma, but not in normal oral mucosa. The Tn-C immunocytochemistry study on brush biopsies from benign expression level (as well as the laminin-5 Á2 chain expression) oral lesions and oral SSC that the laminin-5 did not discriminate between benign oral lesions and

1568 Driemel et al: Expression Analysis in Oral Brush Biopsies carcinoma. It is known that in adult tissues, Tn-C re- Experimentally, our LightCycler-based gene expression expression is induced during neovascularisation and wound analysis was performed as a calibrator-normalized relative healing and at tissue sites that are subject to biomechanical quantification method with PCR efficiency correction. This forces. In pathologies, Tn-C expression is associated with experimental design ensures the correction of individual cancer, wound healing and inflammatory diseases (11). In all mRNA quantity and quality differences, and different these pathological conditions, Tn-C has similar basic cellular detection sensitivities. However, we should state problems effects: it promotes the migration of wounded epithelial cells in isolating intact total RNA from brush biopsies. to close an injury and increases the infiltration of carcinoma Successful cDNA synthesis could be found in only about cells into the surrounding tissue. In previous studies, an 50% of cases as detected using control PCR for a intense Tn-C immunoexpression associated with inflammation housekeeping gene. This problem is mainly due to the high was seen in odontogenic cysts (29, 30). During chronic RNase activity of bacteria in the oral cavity and was periodontal inflammation, Tn-C was reduced in chronically obviously not solved by as aseptic as possible sampling inflamed connective tissue, showing punctate staining at the conditions. Oral brush biopsies as a innovative technique basement membrane zone (31). should preferably be combined with immunocytological We conclude from our results concerning the Tn-C marker analysis or other detection methods at the more expression in benign hyperproliferative and inflammatory oral stable protein level. lesions and oral carcinoma that the Tn-C matrix formation is highly variable and correlates to the activity of cell proliferation Acknowledgements (local fibroblasts, vascular cells, epithelial cells or exuded/migrated inflammatory cells) and to the number of This study was supported by grants from the European Union FP6, inflammatory cells. The Tn-C expression in acute inflammation LSHC-CT-2003-5032, STROMA. This publication reflects only the may be caused by Tn-C-positive inflammatory cells like authors’ view. The European Commission is not liable for any use that may be made of the information contained herein. histiocytes (macrophages) or lymphocytes and by serum Tn-C which may be accumulated in the exudative process. References A third ECM molecule was analysed in our study: the fibronectin isoform containing EDB. EDB-fn is a well 1 Kaspar M, Zardi L and Neri D: Fibronectin as target for tumor studied marker of wound healing, angiogenesis and of therapy. Int J Cancer 118(6): 1331-1339, 2006. tumor formation (32, 33). Human monoclonal antibodies 2 Berndt A, Borsi L, Hyckel P and Kosmehl H: Fibrillary specific for EDB-fn have demonstrated an impressive tumor co-deposition of laminin-5 and large unspliced tenascin-C in the targeting performance in a variety of tumor-bearing animals invasive front of oral squamous cell carcinoma in vivo and in and in patients with cancer (34). To understand the vitro. J Cancer Res Clin Oncol 127: 286-292, 2001. mechanisms of tissue remodeling during cancer progression, 3 Pyke C, Romer J, Kallunki P, Lund LR, Ralfkiaer E, Dano K it is important to know the type of cells that actively express and Tryggvason K: The g2 chain of kalinin/laminin-5 is fibronectin isoforms. In studies on the expression of preferentially expressed in invading malignant cells in human cancers. Am J Pathol 145: 782-791, 1994. fibronectin in human breast tissues, it was demonstrated 4 Engbring JA and Kleinman HK: The basement membrane that the splicing pattern of fibronectin pre-mRNA is matrix in malignancy. J Pathol 200(4): 465-470, 2003. dependent on the cell type and histology and that stromal 5 Rousselle P, Lunstrum GP, Keene DR and Burgeson RE: cells expressing EDB-fn were rather rare (35, 36). In human Kalinin: an epithelium-specific basement membrane adhesion colon adenomas and carcinomas, fibronectin mRNA was molecule that is a component of anchoring filaments. J Cell found in 96% of the tumors – the signals were mainly Biol 114(3): 567-576, 1991. detected in myofibroblasts, labelled with alpha-smooth 6 Masunaga T, Shimizu H, Ishiko A, Tomita Y, Aberdam D, Ortonne JP and Nishikawa T: Localization of laminin-5 in the muscle actin, in the cancer stroma (37). epidermal basement membrane. J Histochem Cytochem 44(11): Our group was able to demonstrate in previous double- 1223-1230, 1996. staining experiments in oral carcinoma cells that the stroma 7 Hlubek F, Jung A, Kotzor N, Kirchner T and Brabletz T: myofibroblasts are a preferential source of EDB-fn (23). In the Expression of the invasion factor laminin gamma2 in colorectal present brush biopsy study, EDB-fn was not detected, neither in carcinomas is regulated by beta-catenin. Cancer Res 61: 8089- normal oral mucosa samples nor in benign inflammatory or 8093, 2001. proliferative oral lesions, or in the tumor samples. Apparently, 8 Koshikawa N, Giannelli G, Cirulli V, Miyazaki K and Quaranta sampling by oral brush biopsy harvests epithelial cells and does V: Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5. J Cell Biol 148: 615-624, 2000. not reach the cells which express EDB-fn. 9 Borsi L, Balza E, Carnemolla B, Sassi F, Castellani P, Berndt A, Oral carcinomas are identifiable by L5Á2 and Tn-C Kosmehl H, Biro A, Siri A, Orecchia P, Grassi J, Neri D and mRNA synthesis in oral brush biopsies. For diagnostic tests, Zardi L: Selective targeted delivery of TNFalpha to tumor individual quantification seems not to be feasible. blood vessels. Blood 102(13): 4384-4392, 2003.

1569 ANTICANCER RESEARCH 27: 1565-1570 (2007)

10 Deryugina EI and Bourdon MA: Tenascin mediates human 25 Sordat I, Rousselle P, Chaubert P, Petermann O, Aberdam D, glioma cell migration and modulates cell migration on Bosman FT and Sordat B: Tumor cell budding and laminin-5 fibronectin. J Cell Sci 109: 643-652, 1996. expression in colorectal carcinoma can be modulated by the 11 Chiquet-Ehrismann R and Chiquet M: Tenascins: regulation tissue micro-environment. Int J Cancer 88(5): 708-717, 2000. and putative functions during pathological stress. J Pathol 4: 26 Koshikawa N, Minegishi T, Sharabi A, Quaranta V and Seiki M: 488-499, 2003. Membrane-type matrix metalloproteinase-1 (MT1-MMP) is a 12 Remmerbach TW, Weidenbach H, Hemprich A and Boecking processing enzyme for human laminin gamma 2 chain. J Biol A: Earliest detection of oral cancer using non-invasive brush Chem 280(1): 88-93, 2005. biopsy including DNA-image cytometry: report on four cases. 27 Borsi L, Carnemolla B, Nicolo G, Spina B, Tanara G and Zardi L: Anal Cell Pathol 25: 159-166, 2003. Expression of different tenascin isoforms in normal, hyperplastic 13 Remmerbach TW, Mathes SN, Weidenbach H, Hemprich A and neoplastic human breast tissues. Int J Cancer 52(5): 688-692, and Boecking A: Noninvasive brush biopsy as an innovative tool 1992. for early detection of oral carcinomas. Mund Kiefer 28 Hindermann W, Berndt A, Borsi L, Luo X, Hyckel P, Gesichtschir 8: 229-236, 2004. Katenkamp D and Kosmehl H: Synthesis and protein 14 Ogden GR, Cowpe JG, Chisholm DM and Lane EB: DNA distribution of the unspliced large tenascin-C isoform in oral and analysis of oral exfoliative cytology in the squamous cell carcinoma. J Pathol 189(4): 475-480, 1999. detection of oral cancer. Eur J Cancer B Oral Oncol 30 B: 29 Amorim RF, Godoy GP, Galvao HC, Souza LB and Freitas RA: 405-408, 1994. Immunohistochemical assessment of extracellular matrix 15 Scheifele C, Schlechte H, Bethke G and Reichart PA: components in syndrome and non-syndrome odontogenic Detection of TP53-mutations in brush biopsies from oral keratocysts. Oral Dis 10: 265-270, 2004. leukoplakias. Mund Kiefer Gesichtschir 6: 410-414, 2002. 30 de Oliveira MD, de Miranda JL, de Amorim RF, de Souza LB 16 Veltman JA, Hopman AH, Bot FJ, Ramaekers FC and Manni JJ: and de Almeida FR: Tenascin and fibronectin expression in Detection of chromosomal aberrations in cytologic brush odontogenic cysts. J Oral Pathol Med 33: 354-359, 2004. specimens from head and neck squamous cell carcinoma. 31 Haapasalmi K, Makela M, Oksala O, Heino J, Yamada KM, Cancer 81(5): 309-314, 1997. Uitto VJ and Larjava H: Expression of epithelial adhesion 17 Driemel O, Dahse R, Hakim SG, Tsioutsias T, Pistner H, and integrins in chronic inflammation. Am J Pathol Reichert TE and Kosmehl H: Laminin-5 immunocytochemistry: 147: 193-206, 1995. a new tool for identifying dysplastic cells in oral brush biopsies. 32 Liu H, Chen B, Zardi L and Ramos DM: Soluble fibronectin Cytopathology (OnlineEarly Articles doi:10.1111/j.1365-2303. promotes migration of oral squamous-cell carcinoma cells. Int J 2006.00401.x), 2006. Cancer 78(2): 261-267, 1998. 18 Tavian D, De Petro G, Colombi M, Portolani N, Giulini SM, 33 Berndt A, Kosmehl H, Borsi L, Luo XM, Zardi L and Katenkamp Gardella R and Barlati S: RT-PCR detection of fibronectin D: Evidence of ED-B fibronectin synthesis in human tissues by EDA+ and EDB+ mRNA isoforms: molecular markers for non-radioactive RNA in situ hybridization. Investigations on hepatocellular carcinoma. Int J Cancer 56: 820-825, 1994. carcinoma (oral squamous cell and breast carcinoma), chronic 19 Kilian O, Dahse R, Alt V, Zardi L, Rosenhahn J, Exner U, inflammation (rheumatoid synovitis) and fibromatosis (Morbus Battmann A, Schnettler R and Kosmehl H: Expression of Dupuytren). Histochem Cell Biol 109: 249-255, 1998. EDA+ and EDB+ fibronectin splice variants in . Bone 34 Ebbinghaus C, Ronca R, Kaspar M, Grabulovski D, Berndt A, 35(6): 1334-1345, 2004. Kosmehl H, Zardi L and Neri D: Engineered vascular-targeting 20 Akutsu N, Amano S and Nishiyama T: Quantitative analysis of antibody-interferon-gamma fusion protein for cancer therapy. laminin 5 gene expression in human keratinocytes. Exp Int J Cancer 116(2): 304-313, 2005. Dermatol 14(5): 329-335, 2005. 35 Matsumoto E, Yoshida T, Kawarada Y and Sakakura T: 21 Katenkamp K, Berndt A, Hindermann W, Wunderlich H, Expression of fibronectin isoforms in human breast tissue: Haas KM, Borsi L, Zardi L and Kosmehl H: mRNA production of extra domain A+/extra domain B+ by cancer expression and protein distribution of the unspliced tenascin- cells and extra domain A+ by stromal cells. Jpn J Cancer Res C isoform in prostatic adenocarcinoma. J Pathol 203(3): 771- 90(3): 320-325, 1999. 779, 2004. 36 Yoshida T, Matsumoto E, Hanamura N, Kalembeyi I, Katsuta K, 22 Pyke C, Salo S, Ralfkiaer E, Romer J, Dano K and Ishihara A and Sakakura T: Co-expression of tenascin and Tryggvason K: Laminin-5 is a marker of invading cancer cells fibronectin in epithelial and stromal cells of benign lesions and in some human carcinomas and is coexpressed with the ductal carcinomas in the human breast. J Pathol 182(4): 421- receptor for urokinase plasminogen activator in budding 428, 1997. cancer cells in colon adenocarcinomas. Cancer Res 55(18): 37 Hanamura N, Yoshida T, Matsumoto E, Kawarada Y and 4132-4139, 1995. Sakakura T: Expression of fibronectin and tenascin-C mRNA by 23 Kosmehl H, Berndt A, Strassburger S, Borsi L, Rousselle P, myofibroblasts, vascular cells and epithelial cells in human colon Mandel U, Hyckel P, Zardi L and Katenkamp D: adenomas and carcinomas. Int J Cancer 73(1): 10-15, 1997. Distribution of laminin and fibronectin isoforms in oral mucosa and oral squamous cell carcinoma. Br J Cancer 81(6): 1071-1079, 1999. Received January 8, 2007 24 Katayama M and Sekiguchi K: Laminin-5 in epithelial tumour Revised March 12, 2007 invasion. J Mol Histol 35(3): 277-286, 2004. Accepted March 21, 2007

1570