ANTICANCER RESEARCH 27: 1565-1570 (2007) Expression Analysis of Extracellular Matrix 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 Laminin 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 fibronectin 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 protein 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 collagen 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 glycoprotein 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.