Oncogene (2000) 19, 754 ± 761 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell carcinomas: increased copy number and overexpression of the MET proto-oncogene Liubov Glukhova1, Christian Lavialle2, Didier Fauvet1, Ilse Chudoba3, GiseÁ le Danglot1, Eric Angevin4, Alain Bernheim1 and Anne-FrancËoise Goguel*,1 1Laboratoire de CytogeÂneÂtique et GeÂneÂtique Oncologiques, UMR 1599, PR2, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif cedex, France; 2Laboratoire de GeÂneÂtique Oncologique, Unite Mixte de Recherche 1599 du Centre National de la Recherche Scienti®que, Institut Gustave Roussy, 94805 Villejuif cedex, France; 3Institut fur Humangenetik und Anthropologie, D-07740 Jena, Germany; 4Unite d'ImmunotheÂrapie, Institut Gustave Roussy, 94805 Villejuif cedex, France Molecular cytogenetic analysis of several sporadic throughout tumour progression (Kovacs, 1993b). papillary renal cell carcinomas and of their xenografts Indeed, the most common non papillary subtype, clear in immunode®cient mice had previously allowed us to cell renal carcinoma, is characterized by deletions in delimit a minimal overrepresented region of chromo- chromosome arm 3p, whereas the 3p region is intact in some 7 shared by all of them to band 7q31. We have papillary RCC. In addition, the latter exhibits an re®ned the location of the overlapping region to the increase in chromosome number typically including junction of the subbands 7q31.2 and 7q31.3 by reverse chromosomes 7 and 17. These anomalies are also painting with two dierently labelled probes prepared found in early renal lesions such as adenomas, from the small chromosome 7 derivatives microdissected suggesting that they could be involved in tumour from the cells of two distinct tumours. This small region initiation. The evolution of papillary tumours is was shown to contain the MET proto-oncogene, present characterized by additional polysomy of chromo- at three to four copies per cell as determined by somes 8, 12, 16, and 20 (Kovacs et al., 1991; Kovacs, Southern blot analysis. The increased copy number of 1993b). the MET gene was found to be associated with its Trisomy 7 is the most frequent cytogenetic abnorm- overexpression at the mRNA level. However, no change ality in early as well as advanced stages of papillary in MET copy number or expression level was observed in RCCs, suggesting that a gene (or genes) located on the cells from two xenografted tumours serially chromosome 7 plays a role in the pathogenesis of these transplanted into immunode®cient mice, as compared to tumours. One approach to pinpoint such a gene those from the corresponding initial tumours. Our results consists in seeking tumours that have retained only a indicate that expression of the MET proto-oncogene limited region of chromosome 7 as the sole over- above a critical threshold is required for the maintenance represented genetic material from this chromosome. of the tumorigenic phenotype of at least some papillary Reduction of the size of the chromosomal region renal cell carcinomas, but does not further increase bearing a candidate gene is more likely to occur in during tumour progression. Oncogene (2000) 19, 754 ± advanced tumours as a consequence of chromosome 761. rearrangements during tumour progression. We have previously reported the molecular cytogenetic charac- Keywords: chromosome 7 polysomy; 7q31 chromoso- terization of four sporadic metastatic papillary RCCs mal region; papillary renal cell carcinoma; MET and of their counterparts xenografted in severe receptor tyrosine kinase combined immunode®cient (SCID) mice. Among them, two tumours and their corresponding xenografts were found to harbour one to four small derivative Introduction chromosomes (hereafter designated `minichromo- somes'). In both cases, all the minichromosomes were The papillary subtype of renal cell carcinoma (RCC) composed of small fragments derived solely from accounts for about 10% of all kidney tumours. In chromosome 7. Moreover, the der(7) minichromo- general, the diagnosis of papillary RCC is based on somes from both tumours appeared to only have in cytological and growth pattern characteristics (Weiss et common a small region located within band 7q31 al., 1995; Amin et al., 1997). However, renal tumours (Glukhova et al., 1998). exhibit a heterogeneous morphology and their histolo- In the present work, we have re®ned the location of gical features can change during tumour progression the unique region of overlap between the two types of (Kovacs, 1993a; Lager et al., 1995; Wilhelm et al., minichromosomes to the junction of bands 7q31.2 and 1995). Molecular cytogenetic analysis allows the 7q31.3 and shown that it contains the MET proto- distinction of papillary RCC from other RCCs based oncogene. Tumour cell DNA and RNA were analysed on speci®c chromosomal anomalies that persist by Southern and Northern blotting to assess the MET gene copy number and mRNA accumulation level, respectively. In order to determine whether these two *Correspondence: A-F Goguel parameters might evolve during tumour progression, a Received 1 June 1999; revised 15 October 1999; accepted comparison was made between initial tumours and 8 December 1999 their xenografted counterparts serially transplanted MET gene in minichromosomes from two papillary RCCs L Glukhova et al 755 into SCID mice, the latter exhibiting a higher nuclear grade as de®ned by Fuhrman et al. (1982). Results Characterization of the 7q31 region carried on minichromosomes We have previously reported that the karyotype of the cells from the three tumours RCC-1, RCC-43 and RCC-47 (nuclear grade 1 ± 2) and from the correspond- ing xenografted tumours RCC-1 P6, RCC-43 P5 and RCC-47 P3 (grade 3 ± 4), serially transplanted into SCID mice for six, ®ve or three passages, respectively, exhibited a total or partial polysomy of chromosome 7 (Glukhova et al., 1998; see Materials and methods for tumour nomenclature). RCC-1 and RCC-1 P6 ex- hibited one or two t(7;2;7) chromosomes per cell and this derivative chromosome was shown to contain a duplicated 7q21-qter region. RCC-43 and RCC-47, as well as RCC-43 P5 and RCC-47 P3, had additional material from chromosome 7 associated with mini- chromosomes. Molecular cytogenetic analysis indicated that a chromosomal region centred on band 7q31 was shared by the der(7) minichromosomes from both tumours (Glukhova et al., 1998). To con®rm these observations, reverse painting on Figure 1 Detection of an overlapping 7q31 subregion common chromosomes from normal cells was carried out by to the minichromosomes of RCC-43 P5 and RCC-47 P3. Reverse painting was carried out by simultaneous hybridization to normal simultaneous hybridization with two probes prepared cell metaphases of two probes generated from the microdissected from the microdissected minichromosomes of the two minichromosomes of the xenografted tumours RCC-43 P5 and xenografted tumours RCC-43 P5 and RCC-47 P3 and RCC-47 P3 and stained with two dierent ¯uorochromes: stained with rhodamine and ¯uorescein, respectively rhodamine and ¯uorescein, respectively. Photographs of the same (see Materials and methods). Fluorescence microscopic normal chromosome 7, taken with dierent UV-light ®lters, are shown. From top to bottom: 4',6-diamidino-2-phenylindole examination showed the speci®c regional staining of (DAPI) counterstaining; double-staining with the two minichro- only chromosome 7 previously observed with each mosome-speci®c probes showing the region of overlap at the individual probe (Glukhova et al., 1998), but disclosed, border of bands 7q31.2 and 7q31.3 (yellow signal); single-staining in addition, the presence of a small overlapping area with the RCC-43 P5 minichromosome-speci®c probe (red signals); single-staining with the RCC-47 P3 minichromosome-speci®c (yellow signal) at the junction of one of the RCC-43 probe (green signals); diagram of chromosome 7 showing the P5-speci®c red signals (7q31.2 ± q31.3) and of one of location of the overlapping region the RCC-47 P3-speci®c green signals (7q31.1 ± q31.3) (Figure 1). is not present in the minichromosomes from RCC-43 Refined mapping of the overlapping 7q31 subregion and RCC-47 P3. associated with the minichromosomes With the aim of mapping more precisely the region of Southern blot determination of the MET gene copy overlap, ¯uorescent in situ hybridization (FISH) number in RCC tumour cells analyses were carried out, using as probes two nonchimeric and nonoverlapping yeast arti®cial chro- In view of the presence of MET sequences in YAC mosomes (YACs), 746H5 and 880B6 that target the 746H5 (see Materials and methods), we chose to 7q31.2 ± q31.3 and 7q31.3 ± q32.1 regions, respectively. measure directly the copy number of the MET gene As a control, they were ®rst hybridized to the RCC-1 by Southern blot analysis of the DNA extracted from tumour cells. The expected number of ¯uorescent the cells of both the RCC-43 and RCC-47 tumours and signals per metaphase were observed with each of of the RCC47 P3 xenografted tumour, using two them: one twin-spot on each of the two normal dierent probes. (Cells from the RCC-43 P5 xeno- chromosomes 7 and two twin-spots on each t(7;2;7) grafted tumour could not be studied because of their derivative chromosome (data not shown). For both inability to grow into mass culture). In order to RCC-43 and RCC-47 P3, 746H5-speci®c spots were validate the results obtained, DNA from the cells of found, besides the two normal chromosomes 7, on all the RCC-1 tumour and of the RCC-1 P6 xenografted of the minichromosomes. The number of associated tumour (which harbour a number of MET-bearing signals indicated that each minichromosome contrib- chromosome 7 long arms that can be precisely uted about two copies of the 746H5-hybridizing counted) was analysed in parallel, together with DNA region per cell (Figure 2).