NOVEL MUTATION IN THE ATP-BINDING SITE OF THE MET ONCOGENE TYROSINE KINASE IN A HPRCC FAMILY Martina OLIVERO 1,3 , Guido V ALENTE 3 , Alberto BARDELLI 2,3 , Paola LONGATI 2,3 , Norma FERRERO 1,3 , Cecilia CRACCO 4 , Carlo T ERRONE 4 , Salvatore ROCCA-ROSSETTI 4 , Paolo M. COMOGLIO 2,3 and M. Flavia DI RENZO 1,3 * 1 Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment, Candiolo, Turin, Italy 2 Division of Molecular Oncology, Institute for Cancer Research and Treatment, Candiolo, Turin, Italy 3 Department of Biomedical Sciences, University of Turin Medical School, Turin, Italy 4 Department of Surgical and Medical Disciplines, Urological Clinic, University of Turin Medical School, Turin, Italy Germline mutations in the tyrosine-kinase domain of the M ET proto-oncogene were found in patients suffering from the hereditary predisposition to develop multiple papillary renal-cell carcinomas (hereditary PRCC, HPRCC). PRCCs are often multiple and bilateral even in patients without a family history. W e analyzed the germline of patients carrying multiple or single papillary tumors with and without family history. One patient had a familial cancer and carried a novel (V1110I) germline M ET mutation, located in M ET gene exon 16. This mis-sense mutation was found in affected members of this patient’s family. Interestingly, the V1110I mutation is located in the ATP-binding site of the MET kinase and is homologousto the V157I mutation that triggersthe sarcoma- genic potential of the v-erbB oncogene. T he V1110I mutated MET receptor is an active kinase and transforms NIH-3T3 fibroblasts in the in vitro assays. Patients without familiality did not show germline mutations in the MET kinase domain, showing that multiple and bilateral papillary kidney tumors develop in the absence of these mutations. In conclusion, we describe a new mutation in the M ET oncogene kinase domain, associated to H PRCC, affecting an amino-acid residue critical for kinase activation in different oncogenes. Int. J. Cancer 82:640–643, 1999.  1999 Wiley-Liss, Inc. Genetic studies of kidney tumors have led to a new and simpler classification of carcinomas derived from renal cells that still takes into account morphological information (Kovacs et al., 1997). Papillary renal-cell carcinoma (PRCC) is defined on the basis of papillary and/or tubular growth pattern, i.e., the presence of villous projections with a fibrous tissue axis, and absence of clear cells. The terms chromophilic, eosinophilic or basophilic, also used to describe PRCC in the past, are no longer widely accepted (Kovacs et al., 1997), because PRCC is constituted by cells with different amounts of cytoplasm and shows a variable affinity for routine staining. The subclassification in type-1 (with scanty cytoplasm) and type-2 PRCC (with abundant eosinophilic cytoplasm), which takes into account other morphological elements, is preferable (Delahunt and Eble, 1997). These tumors are morphologically distinct from clear-cell and chromophobe renal-cell carcinomas, and are genetically characterized by a constellation of chromosome changes including trisomy 7 and 17 (Kovacs, 1993). PRCCs account for approximately 10% of kidney tumors in surgical series. Zbar and Lerman (1998) recognized the inherited form of PRCC, characterized by the predisposition to develop multiple and bilateral PRCC, designated as hereditary PRCC (HPRCC). Mis-sense mutations in the kinase domain of the MET gene have been described in the majority of HPRCC families (reviewed in Zbar and Lerman, 1998). The MET proto-oncogene encodes the tyrosine-kinase receptor for hepatocyte growth factor/ scatter factor (HGF/SF), which stimulates biological programs leading to the invasive growth of epithelial cells. Renal tubular cells are well-known targets for HGF activity (reviewed in Goldberg and Rosen, 1993): HGF/SF has been implicated in branching tubulogenesis of the developing kidney (Woolf et al., 1995) and in regeneration after renal injury and nephrectomy (Nagaike et al., 1991). In addition, the MET receptor, barely detectable in the normal adult kidney, is over-expressed in a high percentage of renal-cell carcinomas (Natali et al., 1996). This suggests that MET-receptor expression is required for the develop- ment of these tumors. PRCCs have been frequently described as multiple and bilateral, often as a single macroscopic mass associated with many micro- scopic lesions (Kovacs, 1993). In general, the presence of multiple and bilateral renal neoplasms is considered a hallmark of inherited disease. Three of the MET gene germline mutations reported in HPRCCs have been found in patients carrying bilateral and multiple tumors without a family history of kidney cancer (re- viewed in Zbar and Lerman, 1998). Thus, the data reported so far suggest that patients with PRCC are at risk of carrying germline mutations of the MET-gene kinase domain. This prompted us to systematically examine the germline of patients with PRCC. MATERIAL AND METHODS Subjects Case reports of kidney cancers resected for cure in the last 5 years at the Medical School Urological Clinic were reviewed: 160 were diagnosed as papillary type, at least 60–70% of the tumor having a tubulo-papillary growth pattern. Familial data were available for 48 of these, which were re-examined by the patholo- gist; 10 cases proved to be bona fide PRCC, according to the Heidelberg classification (Kovacs et al., 1997), either type 1 or type 2, with a variable affinity for staining (Delahunt and Eble, 1997). During follow-up examination, 9 patients were informed and gave consent to molecular analysis. After identification of a HPRCC family, family members who gave informed consent were studied; some of them also consented to radiological examination with CT scan and ultrasonography. An asymptomatic HPRCC family mem- ber was considered unaffected when free of renal-cell tumors according to CT examination and ultrasonography. Mutation analysis In the search for germline mutations of the MET proto-oncogene, we scanned exons 4–8 (encoding the cysteine-rich extracellular region) and 15–20 (encoding the kinase domain) by direct sequenc- ing of PCR products in patients with bilateral and multiple PRCC. Exons 16–19 were analyzed both by direct sequencing and by SSCP in all the patients with PRCC (Table I). Exon 16 was sequenced in members of the family of patient 5 and in 80 normal Grant sponsors: Italian Association for Cancer Research; CNR Target Project on ‘‘Biotechnology.’’ G. Valente is now at Department of Medical Sciences, Amedeo Avogadro University, 28100 Novara, Italy. *Correspondence to: Laboratory of Cancer Genetics, I.R.C.C. Institute for Cancer Research and Treatment, SP 142, Km. 3.95, 10060 Candiolo (Torino), Italy. Fax: +39 011 9933225. E-mail: mfdirenzo@ircc.unito.it Received 22 January 1999; Revised 29 March 1999 Int. J. Cancer: 82, 640–643 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer