STAGING OF HEAD AND NECK SQUAMOUS CELL CARCINOMA USING THE MET ONCOGENE PRODUCT AS MARKER OF TUMOR CELLS IN LYMPH NODE METASTASES Giorgio CORTESINA 1 * , Tiziana MARTONE 1 , Emanuela GALEAZZI 1 , Martina OLIVERO 2,3 , Antonella DE STEFANI 1 , Mario BUSSI 1 , Guido VALENTE 4 , Paolo M. COMOGLIO 2,3 and M. Flavia DI RENZO 2,3 1 Department of Clinical Physiopathology, University of Torino School of Medicine, Torino, Italy 2 Department of Biomedical Sciences and Oncology, University of Torino School of Medicine, Torino, Italy 3 Institute for Cancer Research and Treatment, Candiolo, Torino, Italy 4 Department of Medical Sciences, Amedeo Avogadro University, Novara, Italy In head and neck squamous cell carcinomas (HNSCC), metastasis to cervical lymph nodes is a major determinant of patient outcome. To detect metastases, we used the MET oncogene as marker, which encodes the receptor for hepa- tocyte growth factor/scatter factor, mediating epithelial cell motility and invasiveness. The MET gene is expressed in epithelia and over-expressed in carcinomas of specific histo- types, but not in lymphatic tissue. A total of 151 lymph nodes from 20 squamous cell carcinomas were studied with both in-depth histology and end-point and real-time quantitative RT-PCR. MET-encoded sequences were found in 61 of 151 nodes (40%), of which 24 (16%) were found metastatic by in-depth histopathology. Parallel routine histopathologic analysis of 654 lymph nodes from the same cases identified 36 metastases (5%). Real-time quantitative RT-PCR was used to measure MET gene-specific mRNA in normal tissues, pri- mary tumors and lymphatic metastases and showed a 2– 8- fold increased expression in tumor cells which metastasize. RT-PCR for 3 cytokeratins expressed in HNSCC (K4, K10 and K13) proved to be less sensitive in detecting occult lym- phatic metastases. Western blot analysis demonstrated the presence of the full-size MET receptor in primary tumors and lymph node metastases; immunohistochemistry showed re- ceptor localization in tumor cells. Altogether, these data demonstrate that the MET gene product is a valuable marker with which to detect occult tumor cells in lymph nodes, thanks to its high expression in metastatic cells. After RT- PCR analysis we were able to attribute a more advanced stage to 10 out of 20 HNSCC cases, including 5 cases classi- fied as tumor-free after routine histopathology. Int. J. Cancer (Pred. Oncol.) 89:286 –292, 2000. © 2000 Wiley-Liss, Inc. Squamous cell carcinoma is the most frequent epithelial tumor occurring in the head and neck. These tumors can arise throughout the lining of the upper aero-digestive tract and tend to metastasize to regional lymph nodes, rather than to spread hematogenously. It is widely accepted that the presence of lymph node metastases is the most important prognostic factor in head and neck squamous cell carcinoma (HNSCC). Thus, the assessment of cervical nodes is an important step in both pre-operative and post-operative management of HNSCC patients and neck dissection is considered both a staging and a curative procedure (for a review, see Snow and Bartelink, 1995). Detection of metastases is routinely per- formed by histopathological analysis and the frequency of early metastatic relapse indicates that a number of disseminated tumor cells are undetectable by current methods. It is therefore manda- tory to find both a method and a marker to detect and to diagnose microscopic and occult metastases. Recently, polymerase chain reaction (PCR)-based techniques have been applied in an attempt to facilitate detection of micrometastases in lymph nodes and, as a marker, the expression of either tissue-specific and/or tumor-spe- cific molecules has been proposed. Oncogene abnormalities may be considered rational cancer markers, as either mutation or increase in expression can be directly correlated with transformation. Over-expression of ty- rosine kinase oncogenes has been reported in many carcinomas (for a review, see Kolibaba and Druker, 1997) and can help show low numbers of transformed cells within a normal context. The present report uses MET gene-specific RNA as a marker. MET is an oncogene expressed in epithelium, over-expressed in several epithelium-derived tumors and not expressed in lymphatic and in other mesoderma-derived tissues (Di Renzo et al., 1991). In addi- tion, MET is one of the oncogenes in the growth factor receptor family that is related to tumor cell invasiveness and metastasis. MET encodes a transmembrane tyrosine kinase identified as the receptor for a polypeptide known as hepatocyte growth factor (HGF) or scatter factor (SF). The MET oncogene is activated in a human gastric carcinoma cell line where it is amplified and over- expressed (Giordano et al., 1989). We have also shown that this gene, expressed in several normal human epithelial tissues (Di Renzo et al., 1991), is often over-expressed in carcinomas of specific histotypes (Di Renzo et al., 1995a,b). Recently, germline and somatic missense mutations in the MET proto-oncogene have been identified in papillary renal cell carcinomas (Schmidt et al., 1997; Olivero et al., 1999), confirming the link between the MET gene and cancer. This report investigates the expression of the MET oncogene in primary HNSCC and cervical lymph nodes and we have compared MET with epithelial-specific marker. Expres- sion levels were measured with a quantitative RT-PCR method and shown to be higher in metastases than in primary tumors. MATERIAL AND METHODS Patients, tissue samples, and cell lines The tissues were obtained from the Pathology Department after surgery of informed patients. Samples were obtained from curative resections of head and neck squamous cell carcinomas of patients not previously treated with radio- or chemotherapy. Twenty cases of HNSCC were selected: for each grading and pathological stag- ing were determined. Details of patients are shown in Table I. Samples of primary tumors, normal mucosa, and cervical lymph nodes of the same patients were snap-frozen and stored in liquid nitrogen. Of the 805 lymph nodes resected by the surgeon from these 20 patients, 151 were sampled (at least 1 of each level from each patient) during lymphadenectomy. Each of these 151 lymph nodes was divided in 2 parts and snap-frozen for parallel in-depth histopathology and molecular analyses. In-depth pathology con- sisted of the analysis of at least 3 sections at different levels with Grant sponsor: Italian Association for Cancer Research; Grant sponsor: CNR Target Project on Biotechnology; Grant sponsor: Giovanni Ar- menise-Harvard Foundation for Advanced Scientific Research. *Correspondence to: University of Torino School of Medicine, Institute for Cancer Research (I.R.C.C.), Laboratory of Cancer Genetics, SP 142, Km 3.95, 10060 Candiolo (Torino), Italy. Fax: +39 011 993.3524. E-mail: mfdirenzo@ircc.unito.it Received 26 October 1999; Revised 9 February 2000 Int. J. Cancer (Pred. Oncol.): 89, 286 –292 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer