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Nonrandom changes of chromosome 10 in bladder cancer
Nonrandom
Changes of Chromosome 10 in
Bladder Cancer Detection by FISH to Interphase Nuclei Ming-Rang Wang, Bernard hrissel, Jacques Tailhdier, Jean-Louis Kbmhy, Yvette Fonck, And& Lautier, Moncef Benkhalifa, and Paul Malet
(FISH) to interphase nuclei has been a valuable method AR!!ZRACX Fluorescence in situ hybridization for examining the chromosome copies in tumor cells in clinical pmctice. ‘Ikveiva cases oftmnsitional cell carcinoma (‘ICC) of the bladder were investigated with a biotin-labeled repetitive DNA probe to detect numerical abermions of chromosome 10 in interphase nuclei. The cells containing one fluorescent signal were screened in two of seven non-invasive tumors and in fiur offive invasive tumors. ‘Ike patients presented two FISH spots of different sizes. More than two signals were seen in one invasive tumor. The findings suggest that partial or complete loss of a chromosome 10 is a nonrondom abermtion in bladder cancer.
INTRODUCI’ION
Slide Preparation
Karyotypes have been demonstrated to be an important prognostic index of human cancer. However, accurate karyotype analysis of solid tumors is frequently hampered by the small number of recognizable ma&phases andby a selective growth of normal cells in tissue culture. In situ hybridization in nonmitotic cells with chromosome-specific probes is a rapid and objective method for the detection of chromosome copies without the necessity to culture tumor cells or to prepare chromosome spreads. In bladder cancer, i&p), + 7, - 9, del(llp) and structural aberrations of chromosomes 1, 3, 6, 8, and 17 have been reported as nonrandom abnormalities [l] ,but little is known about the involvement of chromosome 10 in this disease. We have recently investigated 12 bladder ‘ICCs by fluorescence in situ hybridization to interphase nuclei with biotinylated repetitive DNA probe specific for chromosome 10, and observed nonrandom anomalies for this chromosome.
Tumor samples, obtained from tmnsum&eral resection, were first d&aggregated by scraping and then digasted with 0.2% type II collagenase (Sigma). Cells were collected by centrifugation and resuspended in a pr ewarmed (PC) bypotonic solution of sterile water/fetal calf serum (7~1,v/v). After incubation at 3pc for 1 hour, cells were &red in methanol/acetic acid (3:~ v/v) at rl”C. Single-cell suspensions were dropped on cool wet slides. Slides m allowed to air-dry overnight. They were subsequently washed with PBS and dehydrated via alcohol. The slides were stored at - 2o°C for FISH analysis within several months.
Fkom the Labomtoire d’Histologie-EmbryoIogieQtog4n4tique, Faculti de MBdecine de Clennont-Fermnd &4R. W., B. P., M. B., P. M.); Service d’llmlogie, CHLJde Clermont-Wrmnd u. T.), Labomtoire di4ndmnie hthohiaue. CHU de ClermonMkand m. K.); Labor&ire d&natomie Ad&g&e, Centre Jean &k&n, Cl&mon~Fermnd (Y F.); and Institut dXnut.omie Puthologique, ClermontFenund IA. L.1 Address &print requests
to: Ming-Bong d’H~bgie-Bmb~Iogieie-smbryolosieCytDg6n6tique. lkub Henri-Dunant B.P.38, 83001 Clermont-Fbrmnd
Wang, Labomtoire de Mbdecine, Place cedex, Fmnce.
Received January 5, 1993; accepted June 15, 1993. 8 Cancer Genet Cytogenet73:a-10 (1994) 0165-4608/94/$07.00
In situ IQ&rid&&ion The slides were treated witb RNase (XNI&ml) for 60 minutes at VCtorem~endogenousRNA. Fbrimp~men~ofDNA probe and antibody penetration, the slides were incubated with 5 &OO ml of pepsin in 0.01 N HCL for 10 minutes at 37°C. Hybridization was performed with a modification of the procedure described by Pi&e1 et al. [2]. Briefly, 20 ~1 of hybridization mixture containing 1 ng/pl biotin-labeled DNA pmbe (MM1, ONCOR), 0.5 crs/crl sheared salmon sperm DNA, 60% formamide, 10% dextmn sulfate in 2 x SCC was applied to each slide, ccnmred with a 22 x 22 mm coverslip, and sealed with rubber cement. Probe and target DNA were denatured simultaneously at 75°C for 8 minutes. Hybridization took place in a moist chamber at 37°C for 16 hours. ~dizationwashes were carried out in 85% formamide for 20 minutes and twice in 2 x SSC, pH 7 for 10 minutes each at 430C. Signals were detected with FTIC-conjugated avidin and amplified with biotinylated goat anti-avidin antibody followed by another leyer of avidin (avidin and anti-
1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0
9
Figure 1 In situ hybridization with the biotinylated probe D10Z1. a,b) One fluorescent signal in cases 6 and 8; c, d) three to five signals in case 10; e,f) two FISH signals of different sizes in cases 6 and 11 (arrows indicating the smaller ones).
M, R. W a n g et al.
10
avidin, Vector, in a final concentration of 25 ~tg/ml in 4 x SCC, pH Z6, with 15% dry milk, 0.2% Tween 20). Nuclei were allowed to counterstain in 1 ~ / m i propidium iodide {PI), 1 ~lg/mi diAmtdinophenylindole {DAPI) in Tris-HCL: glycerol (1:9, v/v) with 0.2% di-azobicyclo-{2,2,2)-octane {DABCO) as an antifadin~ agent. The evaluation of in situ hybridization was performed according to the criteria described by Hopmen at al. [3]. RESULTS The histopathologic grade and clinical stage of the tumors studied, as well as chromosomal aberrations as detected by FISH with the probe DIOZ1, are summarized in Table I and in Figure 1. Clonal abnormalities for c h r o m o s o m e 10 were detected in six of 12 TCCs. In seven non-invasive tumors, two cases presented evident aberrations. Case 5 showed 4 3 % of cells with one FISH signal. In case 6, two abnormal cell populations were seen, one contAinin~ a fluorescent signal, and an-
other containing two spots of different sizes (the size of the larger one was the same as that in the normal diploid cells). Clonal changes were screened in four of five invasive TCCs. Case 11 was similar to case 6, and had two aberrant clones. Cases 8 and 12 both presented a hybridization signal in the abnormal cells. In case 10, one FISH spot was detected in 34% of cells and more than two signals were found in 11% of cells.
DISCUSSION Hopman at al. [3] were the first to use FISH on the interphase nuclei to study transitional cell carcinomas of the urinary bladder. As a powerful tool, this technique allows objective detection of the chromosome changes, especially numerical aberrations in the tumor cells, and avoidance of certain problems that have hampered cytogenetic analysis of solid tumors. However, FISH information is generated only for those chromosome segments to which the probes hybridize. When hybridization is performed with the chromosomespecific DNA probes that recognize repetitive sequences in the centromeric regions, a centromeric sequence present in, for instance, a marker chromosome, or a dicentric chromosome, can cause an extra signal. In contrast, a deletion involving (part of) the centromere can lead to a reduction in the number of detectable FISH spots in interphase nuclei [4]. Therefore, one must be careful with the interpretation of interphase cytogenetic results. There have been few published reports of transitional cell carcinomas of the bladder containing aberrant chromosome 10, and all the findings were obtained by conventional karyotype analysis [5-8]. We examined 12 TCCs by in situ hybridization to interphase nuclei and found a relatively high incidence of tumors with chromosome 10 aberrations. In these cases with abnormal clones, the percentages of cells conraining only one fluorescent signal were at least 20%, which suggested a probable monosomy. In cases 6 and 11, two spots of different sizes were detected. This denotes a structural aberration involving the centromeric region, either a deletion or a duplication, or a monosomy plus a marker containing a
Table 1
Sl|mmAl'~of basic
clinical information and FISH results of 12 TCCs
Aberrations for chromosome 10 as detected Case Stage Grade by HSH {percentage of nuclei) 1 2 3 4 5 6 7 8
Ta Ta Ta Ta Ta Ta Ta T1
G1 G1 G2 G2 G2 G2 G3 G1
None a None None None Mono (44%} Mono (45%} del? {49%} None Mono (21%)
9 10
T2 T2
G3 G3
II 12
T3a T3
G2 G3
None Mono (34%) tri (5%) tetra {5%) hyper {1%) Mono (46%) del? (49%) Mono (91%)
a None, non-clona]aberration for chromosome10 was detected. Abbreviations: mono, monnsomy;tri, trisomy;tetra, four FISHspots per nucleus; hyper, morethan fourFISHspots per nucleus; del?,uncertain deletion. part of the sequence to which the probe hybridizes. A review of the published reports of aberrations involving chromosome 10 suggests that our patients may contain a daletion. In case 10, in addition to a monosomy clone, cells with three, four, and five fluorescent spots were detected, which indicates that there was a cytogenetic heterogeneity in this tumor. In summary, our results suggest: 1) partial or complete loss of a chromosome 10 can be a nonrandom aberration in bladder cancer [as they were found in both invasive and noninvasive carcinomas], and 2) some bladder tumors are heterogeneous. REFERENCES 1. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia. 2nd Ed. Elsevier Science Publishing Co., New York, pp. 805-813. 2. PinkelD, Stmume T, GrayJW (1986):Cytngeneticanalysis using quantitative, high-sensitivity fluorescence hybridization. Proc Natl Acad Sci USA 63:2934-2938. 3. Hopman AHN, Ramaekers FCS, Raap AK, BeckJLM, Devilee P, van der Ploeg M, VooijsGP (1966):In situ hybridization as a tool to study numerics] chromosome aberrations in solid bladder tumors. Histochemistry 69:307-316. 4. Hopman AHN, Moesker O, Smeets AWGB,Pauwels RPE, Vooijs GP, Ramaekers FCS {1991):Numerical chromosome 1, 7, 9 and 11 aberrations in bladder cancer detected by in situ hybridization. Cancer Res 51:644-651. 5. Gibes Z, Pmut GR Jr.,Connolly JG, PontesJE, Sandberg A A (1984}: Non-random chromosomal changes in transitionalcellcarcinoma of the bladder. Cancer Res 44:1257-1264. 6. Atkin NB, Baker M C (1965):Cytogenetic study often carcinoma of the bladder: Involvement of chromosomes 1 and 11. Cancer Genet Cytngenet 15:253-268. 7. BergerCS, Sandberg AA, Todd IAD, Pannington RD, Haddad FS, Hecht BK, Hecht F {1986):Chromosomes in kidney, ureter,and bladder cancer. Cancer Genet Cytogenet 23:1-24. 8. Smeets W, Pauwels R, Laamk~rs L, Debruyne F,Geraedts J {1987): Chromosomal analysisofbladder cancer: HI. Non-random alterations. Cancer Genet Cytogenet 29:29-41.
Changes of Chromosome 10 in
Bladder Cancer Detection by FISH to Interphase Nuclei Ming-Rang Wang, Bernard hrissel, Jacques Tailhdier, Jean-Louis Kbmhy, Yvette Fonck, And& Lautier, Moncef Benkhalifa, and Paul Malet
(FISH) to interphase nuclei has been a valuable method AR!!ZRACX Fluorescence in situ hybridization for examining the chromosome copies in tumor cells in clinical pmctice. ‘Ikveiva cases oftmnsitional cell carcinoma (‘ICC) of the bladder were investigated with a biotin-labeled repetitive DNA probe to detect numerical abermions of chromosome 10 in interphase nuclei. The cells containing one fluorescent signal were screened in two of seven non-invasive tumors and in fiur offive invasive tumors. ‘Ike patients presented two FISH spots of different sizes. More than two signals were seen in one invasive tumor. The findings suggest that partial or complete loss of a chromosome 10 is a nonrondom abermtion in bladder cancer.
INTRODUCI’ION
Slide Preparation
Karyotypes have been demonstrated to be an important prognostic index of human cancer. However, accurate karyotype analysis of solid tumors is frequently hampered by the small number of recognizable ma&phases andby a selective growth of normal cells in tissue culture. In situ hybridization in nonmitotic cells with chromosome-specific probes is a rapid and objective method for the detection of chromosome copies without the necessity to culture tumor cells or to prepare chromosome spreads. In bladder cancer, i&p), + 7, - 9, del(llp) and structural aberrations of chromosomes 1, 3, 6, 8, and 17 have been reported as nonrandom abnormalities [l] ,but little is known about the involvement of chromosome 10 in this disease. We have recently investigated 12 bladder ‘ICCs by fluorescence in situ hybridization to interphase nuclei with biotinylated repetitive DNA probe specific for chromosome 10, and observed nonrandom anomalies for this chromosome.
Tumor samples, obtained from tmnsum&eral resection, were first d&aggregated by scraping and then digasted with 0.2% type II collagenase (Sigma). Cells were collected by centrifugation and resuspended in a pr ewarmed (PC) bypotonic solution of sterile water/fetal calf serum (7~1,v/v). After incubation at 3pc for 1 hour, cells were &red in methanol/acetic acid (3:~ v/v) at rl”C. Single-cell suspensions were dropped on cool wet slides. Slides m allowed to air-dry overnight. They were subsequently washed with PBS and dehydrated via alcohol. The slides were stored at - 2o°C for FISH analysis within several months.
Fkom the Labomtoire d’Histologie-EmbryoIogieQtog4n4tique, Faculti de MBdecine de Clennont-Fermnd &4R. W., B. P., M. B., P. M.); Service d’llmlogie, CHLJde Clermont-Wrmnd u. T.), Labomtoire di4ndmnie hthohiaue. CHU de ClermonMkand m. K.); Labor&ire d&natomie Ad&g&e, Centre Jean &k&n, Cl&mon~Fermnd (Y F.); and Institut dXnut.omie Puthologique, ClermontFenund IA. L.1 Address &print requests
to: Ming-Bong d’H~bgie-Bmb~Iogieie-smbryolosieCytDg6n6tique. lkub Henri-Dunant B.P.38, 83001 Clermont-Fbrmnd
Wang, Labomtoire de Mbdecine, Place cedex, Fmnce.
Received January 5, 1993; accepted June 15, 1993. 8 Cancer Genet Cytogenet73:a-10 (1994) 0165-4608/94/$07.00
In situ IQ&rid&&ion The slides were treated witb RNase (XNI&ml) for 60 minutes at VCtorem~endogenousRNA. Fbrimp~men~ofDNA probe and antibody penetration, the slides were incubated with 5 &OO ml of pepsin in 0.01 N HCL for 10 minutes at 37°C. Hybridization was performed with a modification of the procedure described by Pi&e1 et al. [2]. Briefly, 20 ~1 of hybridization mixture containing 1 ng/pl biotin-labeled DNA pmbe (MM1, ONCOR), 0.5 crs/crl sheared salmon sperm DNA, 60% formamide, 10% dextmn sulfate in 2 x SCC was applied to each slide, ccnmred with a 22 x 22 mm coverslip, and sealed with rubber cement. Probe and target DNA were denatured simultaneously at 75°C for 8 minutes. Hybridization took place in a moist chamber at 37°C for 16 hours. ~dizationwashes were carried out in 85% formamide for 20 minutes and twice in 2 x SSC, pH 7 for 10 minutes each at 430C. Signals were detected with FTIC-conjugated avidin and amplified with biotinylated goat anti-avidin antibody followed by another leyer of avidin (avidin and anti-
1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0
9
Figure 1 In situ hybridization with the biotinylated probe D10Z1. a,b) One fluorescent signal in cases 6 and 8; c, d) three to five signals in case 10; e,f) two FISH signals of different sizes in cases 6 and 11 (arrows indicating the smaller ones).
M, R. W a n g et al.
10
avidin, Vector, in a final concentration of 25 ~tg/ml in 4 x SCC, pH Z6, with 15% dry milk, 0.2% Tween 20). Nuclei were allowed to counterstain in 1 ~ / m i propidium iodide {PI), 1 ~lg/mi diAmtdinophenylindole {DAPI) in Tris-HCL: glycerol (1:9, v/v) with 0.2% di-azobicyclo-{2,2,2)-octane {DABCO) as an antifadin~ agent. The evaluation of in situ hybridization was performed according to the criteria described by Hopmen at al. [3]. RESULTS The histopathologic grade and clinical stage of the tumors studied, as well as chromosomal aberrations as detected by FISH with the probe DIOZ1, are summarized in Table I and in Figure 1. Clonal abnormalities for c h r o m o s o m e 10 were detected in six of 12 TCCs. In seven non-invasive tumors, two cases presented evident aberrations. Case 5 showed 4 3 % of cells with one FISH signal. In case 6, two abnormal cell populations were seen, one contAinin~ a fluorescent signal, and an-
other containing two spots of different sizes (the size of the larger one was the same as that in the normal diploid cells). Clonal changes were screened in four of five invasive TCCs. Case 11 was similar to case 6, and had two aberrant clones. Cases 8 and 12 both presented a hybridization signal in the abnormal cells. In case 10, one FISH spot was detected in 34% of cells and more than two signals were found in 11% of cells.
DISCUSSION Hopman at al. [3] were the first to use FISH on the interphase nuclei to study transitional cell carcinomas of the urinary bladder. As a powerful tool, this technique allows objective detection of the chromosome changes, especially numerical aberrations in the tumor cells, and avoidance of certain problems that have hampered cytogenetic analysis of solid tumors. However, FISH information is generated only for those chromosome segments to which the probes hybridize. When hybridization is performed with the chromosomespecific DNA probes that recognize repetitive sequences in the centromeric regions, a centromeric sequence present in, for instance, a marker chromosome, or a dicentric chromosome, can cause an extra signal. In contrast, a deletion involving (part of) the centromere can lead to a reduction in the number of detectable FISH spots in interphase nuclei [4]. Therefore, one must be careful with the interpretation of interphase cytogenetic results. There have been few published reports of transitional cell carcinomas of the bladder containing aberrant chromosome 10, and all the findings were obtained by conventional karyotype analysis [5-8]. We examined 12 TCCs by in situ hybridization to interphase nuclei and found a relatively high incidence of tumors with chromosome 10 aberrations. In these cases with abnormal clones, the percentages of cells conraining only one fluorescent signal were at least 20%, which suggested a probable monosomy. In cases 6 and 11, two spots of different sizes were detected. This denotes a structural aberration involving the centromeric region, either a deletion or a duplication, or a monosomy plus a marker containing a
Table 1
Sl|mmAl'~of basic
clinical information and FISH results of 12 TCCs
Aberrations for chromosome 10 as detected Case Stage Grade by HSH {percentage of nuclei) 1 2 3 4 5 6 7 8
Ta Ta Ta Ta Ta Ta Ta T1
G1 G1 G2 G2 G2 G2 G3 G1
None a None None None Mono (44%} Mono (45%} del? {49%} None Mono (21%)
9 10
T2 T2
G3 G3
II 12
T3a T3
G2 G3
None Mono (34%) tri (5%) tetra {5%) hyper {1%) Mono (46%) del? (49%) Mono (91%)
a None, non-clona]aberration for chromosome10 was detected. Abbreviations: mono, monnsomy;tri, trisomy;tetra, four FISHspots per nucleus; hyper, morethan fourFISHspots per nucleus; del?,uncertain deletion. part of the sequence to which the probe hybridizes. A review of the published reports of aberrations involving chromosome 10 suggests that our patients may contain a daletion. In case 10, in addition to a monosomy clone, cells with three, four, and five fluorescent spots were detected, which indicates that there was a cytogenetic heterogeneity in this tumor. In summary, our results suggest: 1) partial or complete loss of a chromosome 10 can be a nonrandom aberration in bladder cancer [as they were found in both invasive and noninvasive carcinomas], and 2) some bladder tumors are heterogeneous. REFERENCES 1. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia. 2nd Ed. Elsevier Science Publishing Co., New York, pp. 805-813. 2. PinkelD, Stmume T, GrayJW (1986):Cytngeneticanalysis using quantitative, high-sensitivity fluorescence hybridization. Proc Natl Acad Sci USA 63:2934-2938. 3. Hopman AHN, Ramaekers FCS, Raap AK, BeckJLM, Devilee P, van der Ploeg M, VooijsGP (1966):In situ hybridization as a tool to study numerics] chromosome aberrations in solid bladder tumors. Histochemistry 69:307-316. 4. Hopman AHN, Moesker O, Smeets AWGB,Pauwels RPE, Vooijs GP, Ramaekers FCS {1991):Numerical chromosome 1, 7, 9 and 11 aberrations in bladder cancer detected by in situ hybridization. Cancer Res 51:644-651. 5. Gibes Z, Pmut GR Jr.,Connolly JG, PontesJE, Sandberg A A (1984}: Non-random chromosomal changes in transitionalcellcarcinoma of the bladder. Cancer Res 44:1257-1264. 6. Atkin NB, Baker M C (1965):Cytogenetic study often carcinoma of the bladder: Involvement of chromosomes 1 and 11. Cancer Genet Cytngenet 15:253-268. 7. BergerCS, Sandberg AA, Todd IAD, Pannington RD, Haddad FS, Hecht BK, Hecht F {1986):Chromosomes in kidney, ureter,and bladder cancer. Cancer Genet Cytogenet 23:1-24. 8. Smeets W, Pauwels R, Laamk~rs L, Debruyne F,Geraedts J {1987): Chromosomal analysisofbladder cancer: HI. Non-random alterations. Cancer Genet Cytogenet 29:29-41.