Characterization of quantitative chromosomal abnormalities in renal cell carcinomas by interphase four-color fluorescence in situ hybridization

Cancer Genetics and Cytogenetics 158 (2005) 110–118

Lead article

Characterization of quantitative chromosomal abnormalities in renal cell carcinomas by interphase four-color fluorescence in situ hybridization Aline Ossard Receveura, Je´roˆme Couturierb, Vincent Molinie´c, Annick Vieillefondd, Franc¸ois Desanglese, Marine Guillaud-Bataillea, Gise`le Danglota, Philippe Coullina, Alain Bernheima,* a

Laboratoire de Cytoge´nomique des cancers CNRS UMR 8125, Institut Gustave Roussy, 39 rue Camille Desmoulins 94805, Villejuif cedex 5, France b Service de Ge´ne´tique Oncologique, Institut Curie, Paris, France c Service d’Anatomie Pathologique, Hoˆpital Foch, Suresnes, France d Service d’Anatomie pathologique, Hoˆpital Cochin Paris, France e Laboratoire de Cytoge´ne´tique, Hoˆpital du Val de Graˆce, Paris, France Received 9 June 2004; received in revised form 9 August 2004; accepted 10 August 2004

Abstract

Renal cell carcinomas (RCC) in adults are histologically heterogeneous solid tumors with specific chromosomal abnormality patterns included in the World Health Organization (WHO) classification. To overcome some of the drawbacks of cytogenetic and comparative genomic hybridization (CGH) analyses, we designed a first-generation cytogenetic diagnostic test using four-color fluorescence in situ hybridization (FISH) on interphase nuclei. We selected 51 bacterial artificial chromosome and P1-derived artificial chromosome clones covering 17 chromosomal regions involved in the abnormalities of the adult RCC histologic subtypes. An initial set of probes allowed the identification of clear-cell RCC, papillary RCC, and other RCC on a single slide. A second test allowed the detection of additional chromosomal abnormalities or aberrations specific to chromophobic RCC and oncocytomas. We tested 25 cases of RCC, and the results were in agreement with those of cytogenetic techniques and/or CGH methods. The techniques appeared to be very sensitive, because small tumoral cell clones that were undetected by other cytogenetic methods were identified with this method. It was concluded that the multicolor FISH test was specific and sensitive, easy to perform, and could be part of the investigation process in RCC. 쑖 2005 Elsevier Inc. All rights reserved.

1. Introduction Renal cell carcinomas (RCC) in adults are one of the best examples of tumor classification based on chromosomal aberrations [1]. RCC represent 2–4% of all human neoplasms, and their incidence has increased by 2% annually in industrial countries. They affect men more frequently than women, with peak onset in the 60s, and are rare among children and young adults. The classification of kidney cancers established in Heidelberg in 1997 [2] defines four histologic subtypes. Clear-cell renal cell carcinomas (ccRCC), papillary renal cell carcinomas (pRCC), and chromophobic carcinomas (chrRCC) are the malignant subtypes that account for approximately 80, 10, and 5% of cases, respectively, while oncocytomas are essentially benign tumors that

* Corresponding author. Tel.: ⫹331-42-11-54-15; fax: ⫹331-42-1152-60. E-mail address: [email protected] (A. Bernheim). 0165-4608/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2004.08.019

represent 5% of cases. This classification includes cytogenetic criteria because these RCC exhibit the following recurrent chromosomal abnormalities [3]: (1) ccRCC are characterized by the deletion of the short arm of chromosome 3 [4,5] together with a frequent trisomy 5q via a derivative translocation der(5)t(3;5) and 5q31, 9p21, and 14q22 as additional aberrant regions [6,7]; (2) papillary renal cell tumors (pRCT), including adenomas, do not have del(3p) but they exhibit polysomies of 7q and 17q; and (3) Y-chromosome loss in male patients is common in pRCC but can also be seen in ccRCC [8,9]. Carcinomas show additional trisomies of chromosomes 12, 16, and 20 [10,11]. The other subtypes of renal tumors are characterized by loss of chromosomes 1 and 14, or translocations involving 11q23 in oncocytomas, and loss of various chromosomes, particularly, 1, 2, 6, 10, 13, 17, and 21, in chrRCC [12–14]. Various drawbacks, such as poorly proliferating cell cultures, high chromosomal condensation of mitoses, and frequent complex rearrangements hamper conventional cytogenetics in RCC. Comparative genomic hybridization

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(CGH), a recent technique that detects quantitative chromosomal imbalances in tumoral DNA and does not require several cell cultures, has been used [15]. CGH, however, cannot detect balanced translocations, minor clones (⬍50%), or small deletions (5–10 megabases) [16]. Fluorescence in situ hybridization (FISH) with probes specific for the regions involved in the quantitative abnormalities offers an alternative. A four-color FISH test for the detection of specific abnormalities in different adult RCC using selected probes was developed. This test made possible the differential diagnosis between the ccRCC and pRCC subtypes on the one hand, and between the chrRCC and the oncocytoma subtypes on the other hand. Using this first-generation test, the diagnosis was made rapidly using only two or three slides per patient.

2. Materials and methods 2.1. Patients A total of 25 patients with sporadic RCC were studied. The sex ratio was 2.12 (17 men/8 women). All patients were adults, with an average age of 65.08 years (range 33–89 years). All patients underwent surgical treatment at the time of the diagnosis, which removed tumors ranging from 1.1– 13 cm in size. The tumor grades, pathologic stages, and tumor types were classified according to tumor node metastasis (TNM) classification and the World Health Organization (WHO) grading system [1]. The four classic histologic subtypes were present: 11 tumors were ccRCC, 6 were pRCC, 4 were oncocytoma, 2 were chrRCC, 1 could not be classified, and the diagnosis was dubious in the last one. Frozen samples from 10 of these patients were available for CGH on chromosomes. 2.2. Methods Tumor samples were prepared using cytogenetic techniques after short-term culture (one passage). The slides were prepared to obtain a high density of nuclei in the spread. 2.3. Selection of specific bacterial artificial chromosomes (BAC) and P1-derived artificial chromosomes (PAC) clones Regions involved in the four main RCC were selected, as reported in the literature. We selected several BAC or PAC for each of these regions to enhance the size and intensity of the spots generated after FISH. Within these regions, three specific genes have been claimed to be involved in RCC. On 3p25, the von Hippel-Lindau gene is distal to the breakpoints in the deletion of 3p and is involved particularly in hereditary tumors [17] but also in sporadic types [18]. On 7q31, the MET oncogene is inside the smallest overrepresented region in papillary RCC [19,20] and has been found mutated in familial and sporadic papillary RCC [21].

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The NOV oncogene at 8q24 is potentially involved [22] in RCC, and trisomy 8 may be observed in some pRCC [10]. BAC overpassing these genes were selected. Because candidate genes have not yet been characterized for the other chromosomal regions, we chose contiguous BAC for each of them. A total of 61 BAC and PAC (Table 1) were selected from the University of California Santa Cruz (UCSC) (http:// www.genome.ucsc.edu/goldenpath) or from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) online databases and were first validated by FISH on normal mitoses. These probes were associated in different hybridization mixes (Fig. 1). 2.4. DNA extraction DNA was extracted from BAC and PAC according to the “Maxi Preps” method (Nucleobond, Dwen, Germany) after culture in 2YT medium supplemented with chloramphenicol or kanamycin. DNA was then digested by the Tru1 enzyme (MBI Fermentas, Vilnius, Lithuania) and electrophoresed on a 0.8% agarose gel to evaluate the fragment sizes and the amount of DNA. 2.5. Probes and labeling Four fluorochromes were used for labeling, Alexa 488 (green), Alexa 594 (red), Alexa 633 (mauve), and Cy3 (orange). The Alexa fluorochromes came from Molecular Probes (Leiden, The Netherlands), and Cy3 was from Amersham Pharmacia (Buckinghamshire, England). The random-primed DNA labeling method with the polymerase Klenow fragment [Kf exo-] (MBI Fermentas) was used for the first three fluorochromes. To accomplish labeling with Cy3, another enzyme, Sequenase (USB Amersham, Buckinghamshire, England) was used, and the labeled DNA was purified on a column (Dyex Quiagen, Courtaboeuf, France) before hybridization. An indirect method was used for Alexa 633: BAC DNA was first labeled by nick translation with the biotin 16 dUTP (Enzo-Roche, Indianapolis, IN) nick translation kit (QBiogen, Illkirch, France), using 1 µg DNA and subsequently revealed using avidin-A633 (USB Amersham). Forty nanograms of labeled DNA was used for coprecipitation for each probe. 2.6. Hybridization Slides were denatured for 150 seconds in 70% formamide/ 2× standard saline citrate (SSC) at 75⬚C and dehydrated through a series of ethanol treatments (70, 85, and 100%). DNA probes were coprecipitated with 10 µg of human Cot 1 DNA (Invitrogen, Carlsbad, CA) and resuspended in a hybridization mixture (50% formamide, 10% dextran sulfate, and 2× SSC, pH7). Hybridization was then performed overnight in a moist chamber at 37⬚C. Post-hybridization washes were done in NP-40/2× SSC at 75⬚C for 2 minute 30 seconds, and 5 minutes in phosphate-buffered saline at

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Table 1 List of the 51 BAC and PAC selected Clone name

GenBank identification no.

Size (kb)

RP11-470E10 RP11-25C11 RP11-329A2 CTD-2027D14 CTD-2062A1 RP1-204O11 CTB-13N12 CTB-11K1 CTB-114A6 RP11-4K16 RP11-534K13 RP11-775B15 RP11-354P17 RP11-344A7 RP11-1P8 RP11-796E2 RP11-1036M20 RP11-864A19 RP-11-794A8 RP-11-643C12 RP-11-724F15 RP11-140H17 RP11-502K10 RP11-343C2 RP5-104C10 RP5-963K23 RP4-791K14 RP11-139C10 RP11-160O2 RP11-535I13 RP11-141D5 RP11-806H10 RP11-559K16 RP11-42O15 RP11-108P2 RP11-386O9 RP11-422A6 RP11-120J4 RP11-355H10 RP3-377H14 RP1-271M21 RP1-30I19 RP11-248M22 RP11-347I22 RP11-186N15 RP11-512C24 RP11-157E11 RP11-138N13 RP11-672A2 RP11-689N19 RP11-259H21

AC069276 AC027121 AC077690 AC010354 AC010378 AC034243 AC004416 AC006159 AC002542 AC009514 AC020603 AC021733 AL353732 AL137O22 AL512606 AC025164 AC016136 AC012083 AL359792 AL049778 AL133444 AC009032 AC009131 AC026464 AL162615 AL031685 AL035685 AC010723 AC016698 AC010153 AC032035 AC061992 AC025609 AL354872 AL512443 AL360297 AC008278 AC073399 AC010145 AL022723 AL031983 AL022727 AL512284 AL353586 AL512770 AL355390 AL158812 AL139375 AP001189 AP000752 AC068081

184 162 199.8 228.3 137.7 191 32.1 92.1 188 169 202.3 158.2 173.2 169 153 216.8 176.7 166 180 205.5 192.3 184.5 180.8 190 140 115.9 155 174 154.2 104.1 158 134 163 151.7 161.3 169.8 136.8 150 198.9 148.34 134.2 144.8 151.6 191.6 192.2 158.9 173.5 146.7 186.3 194.1 152.5

room temperature followed by, in some cases, indirect labeling. Slides were then counterstained with 4’,6-diamidino2-phenylindole in Vectashield antifading medium (Vector Laboratories, Burlingame, CA). The study was conducted double blindly to make us unaware of the cytogenetic and histologic results and, therefore, to not influence the FISH results.

Genes

GRM7 UBE202 CTNNA1/KIAA0416/SIL1 MET CAV1/2 ST7 MAL2 NOV IFNA 1/2/13/6 IFNA 16/10/7/4 BTG1 EEA1 CNIL/GMFB/CGR19/CDKN3 CDKN3 CYB5M/NFAT5/NQO1 SNTB2/HAS3 SNTB2/VPS4/LOC64146 B4GALT5 KIAA0757/ZNF KCNB1

BIRC5 SSI-3 TIMP2 CTH PTGER3/ZNF265 DDX1 MYCN HLA-G/HLA-F GABRR1/UBD OR2JH2 LOC57118 LOC57118 FLJ10578

GL002/GARP FLJ11238 FLJ11238

Localization (Mb) UCSC 9.7 9.58 9.36 145.4 140 145.6 116.6 116.4 119.4 120 121.1 120.3 24.2 23.7 24.5 95.2 95.9 95.7 53 52.8 53.2 72.2 71.7 72 48.1 48.2 47.7 15.5

79.7 80 80 81.4 82 82.2 16.3 16.5 16.6 33.3 31.1 32.6 13.1 13.4 12.8 75.9 76.2 76.4 87.6 88 87.9

Locus 3p25.3 3p25.3 3p25.3 5q31.2 5q31.2 5q31.2 7q31.2 7q31.2 7q31.2 8q24.12 8q24.12 8q24.12 9p21.3 9p21.3 9p22 12q22 12q22 12q22 14q22.2 14q22.2 14q22.2 16q22.1 16q22.1 16q22.1 20q13.13 20q13.13 20q13.13 Yq11.22 Yq111.22 Yq11.22 17q25.3 17q25.3 17q25.3 1p31.1 1p31.1 1p31.1 2p24.3 2p24.3 2p24.3 6p22.1 6p22.1 6p22.1 10p13 10p13 10p13 13q22.2 13q22.2 13q22.2 11q13.5 11q13.5 11q13.5

2.7. CGH Tumor DNA (400 ng) was labeled with fluorochrome Alexa 488 (green) and reference DNA (200 ng) with Alexa 594 (red). Mixtures of tumor and reference DNA were coprecipitated with 20 µg of human Cot1 DNA, resuspended in 8.4 µL of hybridization mixture, and then co-hybridized on normal lymphocyte metaphases.

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Fig. 1. Description of different tests.

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At least 15 adequate mitoses were captured for each case. The ratio of tumor DNA to normal DNA along each chromosome was analyzed by CGH Quips Karyotyper software (Vysis, Downers Grove, IL). A loss of copy number was recorded when the ratio was below 0.75, and a gain was noted when the ratio was above 1.25.

3. Results From the data in the literature and our own experience with RCC, 17 chromosomal regions were investigated (Fig. 2). The localization of each probe was verified by FISH on normal metaphases. Of 61 probes, 51 were consistent with their in-silico mapping: 8 hybridized on two different chromosomes, and 2 were at another location (data not shown). The 16% value of inconsistencies is constantly found in such screening of BAC libraries (personal results). Among various mechanisms, it can be due to a possible contamination during the transfer of the BAC from one well to the other, a chimerism of the clone, or a duplication within the genome. Three BAC were pooled for the 17 regions to enhance the size and intensity of the spots, allowing for nearly 100% detection on interphasic cells. Each probe was labeled with one of the four fluorochromes, which allowed each region in a particular mix to be uniquely identified by a primary color.

The 25 tumors were investigated using two sets of probes (i.e., two slides) and sometimes three when chrRCCs were suspected. Up to four different fluorochrome-labeled probes were clearly visible on each slide. The percentage of nuclei exhibiting “monosomic” or “polysomic” signals varied from 5 to 100%. The results of the interphase tests are summarized in Table 2. A total of 21/25 results were concurred with the preestablished histological diagnosis. The four remaining cases will be described in detail later. 3.1. ccRCC and orientation test Loss of 3p25 was identified in 11 tumors, and the histologic features corresponded to the ccRCC subtype. It was in accordance with conventional cytogenetic analyses in 10/ 11 cases, showing a monosomy 3 (T4, T6, T7, T11), a del(3p) (T2, T9, T10), or an imbalanced 3p translocation (T1, T3, and T5). In T8, a 3p deletion was found in 60% of nuclei with interphase FISH while the karyotype was determined to be normal. An obvious interpretation was that normal stromal cell metaphases had been karyotyped while the 3p⫺ tumor cells had been detected by FISH (Fig. 3A). 3.2. ccRCC and additional abnormalities A gain of 5q (T1–T7 and T10) was observed in eight of nine tumors, four of which displayed a deletion of 14q (T3,

Fig. 2. Representation of the 17 regions. Colors used are the same as the fluorochromes.

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Table 2 Summary of findings by various cytogenomic techniques Tumors

Histology

Karyotype

Orientation TEST

Additional TEST

CGH

Subtype deduced

T1

ccRCC

12% del(3p)

100% ⫹5

NT

ccRcc

T2

ccRCC

32% del(3p)

30% ⫹5

NT

ccRcc

T3

ccRCC

52.8% del(3p)

29.2%(⫹5)/12.5%(⫺14)

NT

ccRcc

T4

ccRCC

43.8% del/(3p)

36.8% (⫹5)

NT

ccRcc

T5

ccRCC

50.6% del/(3p)

56.4% (⫹5, ⫺14)

(⫺3p,⫹5q,⫺14)

ccRcc

T6 T7 T8 T9 T10 T11

ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC

41.7% del(3p) 15% del(3p) 60% del(3p) 50% del(3p) 72% del(3p) 100% del(3p)

49%(⫹5)/22%(⫺14) 42%(⫹5)/22%(⫺14) Normal NT 46.34%(⫹5) NT

Unclassifiable

Normal

Normal(1,11,14)

NT NT NT NT (⫺3p) (⫺3p,⫹5q,⫺6q, ⫺9,⫺13,⫺18) Normal

ccRcc ccRcc ccRcc ccRcc ccRcc ccRcc

T12

46,XX,add(3)(p12),⫹7, ⫺8,⫺13, ⴚ14,⫺16,⫹3mar[12] 46, XY,del(3)(p13)[8]/46,XY, add(2)(q37),del(3)(p13)[4] 44,X,⫺X,der(3;8)(p?10;q?10), ⫹7,⫺8, ⴚ14[9]/45,X, idem,ⴙ5[3] 47, XY,ⴚ3,ⴙ5,⫹7,der(17)t(3;17) (q11;qter),add(21)(p11)[12] 45,XY,der(3)t(3;5)(p12;q22), ⴚ14[12] 74,ⴚ3, inc 45, X,⫺X,ⴚ3,⫺6,ⴚ14,⫹3mar[12] 46,XY[12] 46,XY,del(3p)[12] 47,XY,del(3p),⫹mar[12] 44,XY,⫺3,⫺9,der(10)t(10;13), ⫺13⫹mar[12] 46,XY,ⴚ3,⫹mar[12]

T13 T14

pRCC pRCC

48,XY,ⴙ7,ⴙ17[12] 48, X,⫺Y,ⴙ7,ⴙ7,ⴙ17[12]

10%(⫹12×2) Normal

(⫹7,⫹17) NT

T15

pRCC

29%(⫹8×2,⫹12×2,⫹20×2)

NT

pRCC

T16

pRCC

NT

(⫹3q,⫹7, ⫹12,⫹17) Normal

pRCC

Imprecise

96.6% (⫹7×2, ⫹17×2) 22%(⫹7)/2% (⫹7,⫹17)

43.3%(⫹12×2,⫹20×2)

T17

pRCC

T18

pRCC

81,XXY,⫺Y,⫺1,⫺3,ⴙ7,ⴙ7,⫺9, ⫺9,⫺10,⫺11,⫺14,⫺15,⫺19, der(19)t(3;19)(q11;p13),⫺21, ⫺22,⫺22[9] 49,XX,ⴙ7,ⴙ7,add(11)(p15),⫹12, ⫺14,⫹17,add(19)(p13)[12] 58,XX,⫹X,⫹2,⫹5,t(5;15),ⴙ7,ⴙ7, ⫹8,⫺11,⫹13,⫹16,ⴙ17,⫹19, ⴙ20,⫹2mar[6] 47,X,⫺Y,ⴙ7,⫹16[12]

68% (⫹7,⫹17) 91.3% (⫹7×2, ⫹17×2,⫺Ya) 90.3% (⫹7×2, ⫹17×2,⫺Ya)

chrRCC or oncocytoma pRCC pRCT

Normal

NT

pRCT

T19 T20

pRCC chrRCC

49,XX,ⴙ7,ⴙ17,ⴙ20[12] 46,XY[12]

23% (⫹7, ⫹/⫺Ya) 80% (⫹7,⫹17) Normal

70% (⫹20) Normal

NT Normal

T21 T22 T23

chrRcc Oncocytoma Oncocytoma

Normal Normal Normal

(⫺6) 6.4%(⫹11)/38.7%(⫹1) 12%(⫹11)/44%(⫹1)

NT Normal Normal

T24

Oncocytoma

Hypotetraploid 46,XY[12] 46,XY,der(15)t(?8;15)(?q22; q26)[12]b 45,X,⫺Y[12]

pRCC chrRCC or oncocytoma chrRCC Oncocytoma Oncocytoma

NT

NT

?

T25

Oncocytoma

Nonclonal abnormalities

20% (⫹7, ⫹/⫺Ya) 20% (⫹7)

16%(⫹20)

NT

?

Boldface type indicates items that are specific chromosomal abnormalities. Abbreviation: NT, not tested. a Loss of chromosome Y is not observed in all nuclei. b The der(15)t(8?;15) appeared to be a der(15)t(11;15) from a FISH labeled mitosis with 11q13 probe.

T5, T6, and T7); T8 had no additional abnormality. No deletion of 9p was detected in any tumor (Fig. 3C). 3.3. pRCT and the orientation test Polysomies 7q and 17q were discovered in 9 cases (T13–T19, T24, and T25). None of them had a loss of 3p. These results were mostly in agreement with cytogenetic analysis, if the karyotypes were not too complex. All the cases with an association of polysomies 7 and 17 corresponded to the pRCC histologic subtype, with the exception

of case T17. Although it had chromosomal characteristics of pRCC, among others, including a ⫹5, its histology was dubious. T18 was a pRCC with a single trisomy 7; however, a trisomy 16 was observed in the karyotype but FISH did not confirm it (Fig. 3B). 3.4. pRCT and additional abnormalities Additional specific chromosomal abnormalities were investigated in seven patients. Two of them did not show any chromosomal abnormalities, in accordance with cytogenetics (T14 and T18). Five cases (T13, T15, T16, T19, and

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Fig. 3. (A and B) Results of test 1. (A) Nuclei with only one green spot correspond to ccRCC. (B) Polysomy 7 and 17 and loss of Y diagnose pRCC nuclei. (C and D) Results of additional abnormalities. (C) Loss of 14q and gain of 5q in a ccRCC nucleus. (D) Gain of 12q and 20q in a pRCC nucleus. The few spots visible are doublets that explain the presence of two small spots in one area.

T25) had gains of chromosomes 8q, 12q, 16q, or/and 20 (Fig. 3D). These rearrangements were partially detected on the karyotype. 3.5. Rare tumors and the test Four cases had histologic features of oncocytoma. T24 and T25 had a trisomy 7 in 20% of the cells, with a partial correlation with the karyotype (loss of Y in T24). T22 and T23 did not show abnormalities in the first test; however, a trisomy 1p31 was detected in both cases, and a trisomy 11q13 was detected in some cells (Table 2). The two chrRCC subtypes were not efficiently classified by this method. T12 was an undifferentiated tumor without abnormality detected by FISH and a normal CGH, while the karyotype showed a monosomy 3 and a marker. In the few mitoses available, an abnormal submedian chromosome was labeled by the 3p25 probe, suggesting a structural rearrangement with no evidence of deletion of 3p25. The normality of both FISH tests of T12 could not allow us to orientate the histologic diagnosis. In fact, the histology of T12 looked like a renal cell carcinoma, but with an unusual appearance; it was then unclassifyable.

3.6. CGH Aberrations were revealed in 5 out of 10 investigated cases: 3 ccRCC (T5, T10, and T11) and 2 pRCC (T13, T16). The five other tumors were normal (T12, T17, T20, T22, and T23). Only aberrations for which the number of clones exceeded 50% (i.e., the primary chromosomal aberrations) were evidenced by this technique. When additional abnormalities were present with a low rate of abnormal clones (⬍ 50%), they remained undetected. 4. Discussion Renal cell carcinomas, particularly ccRCC, are heterogeneous, with a mixture of stromal and tumoral cells. The rate of pathologic nuclei counted varied between 5 and 100%, demonstrating this genomic heterogeneity of this renal cell carcinoma on a cell-by-cell basis. The comparison of FISH results with those of other cytogenetic techniques showed that sensitivity and specificity of the detection of specific chromosomal abnormalities was often better with the fourcolor FISH test. The efficiency of CGH for detection of

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quantitative changes in RCC was disappointing because of the dilution of tumor cells by normal cells. Sometimes, a positive selection of abnormal mitoses has been observed after cell culture, an important step in the procedure of cytogenetic diagnosis, as in T1 or T23. The various chromosomal probes used to assign each case of the four major renal cell tumor subtypes have different specificities: 3p25 seems to be highly discriminant, either by its deletion in each ccRCC or its integrity in the other subtypes. Few deletions of 3p outside 3p25 have been described, such as FHIT in 3p14. It could be useful to also detect those losses. The additional detection test for chromosomal abnormalities for ccRCC seems adequate, although the inclusion of another probe of 5q could be useful to secure the diagnosis. Duplication of 5q, which derived from t(3;5) or a gain of chromosome 5, has been claimed to be a good prognostic indicator by Gunawan et al. [23] in a series of 118 patients. Deletions of 9p21 and 14q22~qter have been found in ccRCC, and both these aberrations signal a poor prognosis [24,25]. In our study, the four patients who had a gain of 5q without 14q loss had either small or lowgrade tumors, concurrent with a good prognosis. In contrast, the four patients who had a deletion of 14q22 in addition to the 5q gain presented with a large tumor diameter and loco-regional extension, which concurs with the likelihood of a dismal outcome. Over-representation of both chromosomes 7 and 17 was a strong indicator of papillary tumors. The presence of additional gains, mainly of chromosomes 12, 16, and 20, gave a clear indication of the cancerous nature of the tumor, thus a pRCC. According to Junker et al., their presence could be significant for determining the prognosis [26]. The precise signification of a single trisomy 7 remains to be determined. This isolated chromosomal gain is not specific to malignancy and it can be observed even in normal tissue [27]. Trisomy 7 could, however, be an interesting factor in the difficult discrimination between the oncocytomas and chrRCC. It is present in several oncocytomas while it is absent in chrRCC, which have a very high rate of loss of heterozygosity at several chromosomes. Nagy et al. [28] have demonstrated this through the use of microsatellite allelotyping. The four-color FISH diagnostic testing mainly differentiates between ccRCC and pRCC with a high degree of confidence while revealing important information to help differentiate between oncocytomas and chrRCC. It also constitutes a basis for making chromosomal prognosis factors more available to clinicians via a decision tree, which remains to be established.

Acknowledgments We thank Lorna Saint-Ange for her excellent editorial assistance and Didier Fauvet for his technical skills.

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