CDKN2A Mutation Analysis, Protein Expression, and Deletion Mapping of Chromosome 9p in Conventional Clear-Cell Renal Carcinomas
2001; Elsevier BV; Volume: 158; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64001-1
ISSN1525-2191
AutoresPeter Schraml, Kirsten Struckmann, Rudolf Bednar, Wenting Fu, Thomas Gasser, Kim Wilber, Juha Kononen, Guido Sauter, Michael J. Mihatsch, Holger Moch,
Tópico(s)Cancer Genomics and Diagnostics
ResumoInactivation of tumor suppressor genes on chromosome 9p is considered a critical event in renal cell carcinoma pathogenesis. Alterations of CDKN2A on 9p21 have been reported in renal cancer cell lines, but their relevance for primary renal carcinomas is unclear. Loss of heterozygosity (LOH) was analyzed by using four polymorphic microsatellites at D9S970 (9p12-9p13), D9S171 (9p13), D9S1748 (9p21), and D9S156 (9p21) in 113 primary conventional clear-cell renal cell carcinomas (CRCCs). Allelic deletion was detected in 21 of 88 informative CRCCs (24%) with the highest rate of LOH being observed at D9S171 on 9p13 (20%). Chromosome 9p LOH was associated with short tumor-specific survival in stage pT3 RCC (P = 0.01). Fluorescence in situ hybridization analysis of 54 CRCCs revealed no homozygous CDKN2A deletions indicating that this mechanism of CDKN2A inactivation is rare in CRCC. Sequencing of 113 CRCCs showed that 13 tumors (12%) had a 24-bp deletion abrogating codons 4 through 11 of CDKN2A. Immunohistochemical CDKN2A expression was absent in normal renal tissue and was only detected in six of 382 CRCCs (1.5%) on a renal tumor microarray. These data suggest that CDKN2A alterations are present in a small subset of CRCCs and a second, yet unknown tumor suppressor gene proximal to the CDKN2A locus, may play a role in CRCC development. Inactivation of tumor suppressor genes on chromosome 9p is considered a critical event in renal cell carcinoma pathogenesis. Alterations of CDKN2A on 9p21 have been reported in renal cancer cell lines, but their relevance for primary renal carcinomas is unclear. Loss of heterozygosity (LOH) was analyzed by using four polymorphic microsatellites at D9S970 (9p12-9p13), D9S171 (9p13), D9S1748 (9p21), and D9S156 (9p21) in 113 primary conventional clear-cell renal cell carcinomas (CRCCs). Allelic deletion was detected in 21 of 88 informative CRCCs (24%) with the highest rate of LOH being observed at D9S171 on 9p13 (20%). Chromosome 9p LOH was associated with short tumor-specific survival in stage pT3 RCC (P = 0.01). Fluorescence in situ hybridization analysis of 54 CRCCs revealed no homozygous CDKN2A deletions indicating that this mechanism of CDKN2A inactivation is rare in CRCC. Sequencing of 113 CRCCs showed that 13 tumors (12%) had a 24-bp deletion abrogating codons 4 through 11 of CDKN2A. Immunohistochemical CDKN2A expression was absent in normal renal tissue and was only detected in six of 382 CRCCs (1.5%) on a renal tumor microarray. These data suggest that CDKN2A alterations are present in a small subset of CRCCs and a second, yet unknown tumor suppressor gene proximal to the CDKN2A locus, may play a role in CRCC development. Chromosome 9p is a candidate to harbor an important tumor suppressor gene with relevance for renal carcinoma progression. 9p losses were found in 24 to 36% of conventional clear-cell renal cell carcinomas (CRCCs) by microsatellite analyses or comparative genomic hybridization.1Cairns P Tokino K Eby Y Sidransky D Localization of tumor suppressor loci on chromosome 9 in primary human renal cell carcinomas.Cancer Res. 1995; 55: 224-227PubMed Google Scholar, 2Thrash-Bingham CA Greenberg RE Howard S Bruzel A Bremer M Goll A Salazar H Freed JJ Tartof KD Comprehensive allelotyping of human renal cell carcinomas using microsatellite DNA probes.Proc Natl Acad Sci USA. 1995; 92: 2854-2858Crossref PubMed Scopus (94) Google Scholar, 3Moch H Presti Jr, JC Sauter G Buchholz N Jordan P Mihatsch MJ Waldman FM Genetic aberrations detected by comparative genomic hybridization are associated with clinical outcome in renal cell carcinoma.Cancer Res. 1996; 56: 27-30PubMed Google Scholar, 4Kinoshita H Yamada H Ogawa O Kakehi Y Osaka M Nakamura E Mishina M Habuchi T Takahashi R Sugiyama T Contribution of chromosome 9p21-22 deletion to the progression of human renal cell carcinoma.Jpn J Cancer Res. 1995; 86: 795-799Crossref PubMed Scopus (33) Google Scholar A shorter recurrence-free survival was observed in CRCCs with 9p losses compared to tumors without 9p loss by comparative genomic hybridization.3Moch H Presti Jr, JC Sauter G Buchholz N Jordan P Mihatsch MJ Waldman FM Genetic aberrations detected by comparative genomic hybridization are associated with clinical outcome in renal cell carcinoma.Cancer Res. 1996; 56: 27-30PubMed Google Scholar 9p losses were also detected more frequently in renal carcinoma metastases as compared to their primaries, suggesting that alterations in this region contribute to tumor progression.5Bissig H Richter J Desper R Meier V Schraml P Schäffer A Sauter G Mihatsch M Moch H Evaluation of the clonal relationship between primary and metastatic renal cell carcinoma by comparative genomic hybridization.Am J Pathol. 1999; 155: 267-274Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Recent evidence has implicated CDKN2A located at chromosome 9p21 to be frequently aberrant in the germline of members of familial melanoma kindreds, but also in bladder and other solid tumors.6Foulkes WD Flanders TY Pollock PM Hayward NK The CDKN2A (p16) gene and human cancer.Mol Med. 1997; 3: 5-20Crossref PubMed Google Scholar CDKN2A encodes a 156-amino acid protein that exclusively associates with CDK4 and CDK6, inhibiting their complexation with D-type cyclins and the consequent phosphorylation of the retinoblastoma protein.7Lukas J Parry D Aagaard L Mann DJ Bartkova J Strauss M Peters G Bartek J Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16.Nature. 1995; 375: 503-506Crossref PubMed Scopus (864) Google Scholar This contributes to cell-cycle arrest. Inactivation of CDKN2A has been described as a consequence of homozygous deletion, rearrangement, hypermethylation, or point mutation.8Cairns P Tokino K Eby Y Sidransky D Homozygous deletions of 9p21 in primary human bladder tumors detected by comparative multiplex polymerase chain reaction.Cancer Res. 1994; 54: 1422-1424PubMed Google Scholar, 9Kamb A Liu Q Harshman K Tavtigian S Cordon-Cardo C Skolnick M Rates of p16 (MTS1) mutations in primary tumors with 9p loss.Science. 1994; 264: 416-417Crossref Google Scholar, 10Herman JG Merlo A Mao L Lapidus RG Issa JP Davidson NE Sidransky D Baylin SB Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers.Cancer Res. 1995; 55: 4525-4530PubMed Google Scholar The involvement of CDKN2A in human cancer is controversial. It has been shown that the rates of homozygous deletions or mutations is smaller in primary tumors than in cell lines.9Kamb A Liu Q Harshman K Tavtigian S Cordon-Cardo C Skolnick M Rates of p16 (MTS1) mutations in primary tumors with 9p loss.Science. 1994; 264: 416-417Crossref Google Scholar, 11Cairns P Mao L Merlo A Lee DJ Schwab D Eby Y Tokino K van der Riet P Blaugrund JE Sidransky D Rates of p16 (MTS1) mutations in primary tumors with 9p loss.Science. 1994; 265: 415-417Crossref PubMed Scopus (504) Google Scholar The role of CDKN2A in CRCC is uncertain.1Cairns P Tokino K Eby Y Sidransky D Localization of tumor suppressor loci on chromosome 9 in primary human renal cell carcinomas.Cancer Res. 1995; 55: 224-227PubMed Google Scholar, 12Cairns P Polascik TJ Eby Y Tokino K Califano J Merlo A Mao L Herath J Jenkins R Westra W Rutter JL Buckler A Gabrielson E Tockman M Cho KR Hedrick L Bova GS Isaacs W Koch W Schwab D Sidransky D Frequency of homozygous deletion at p16/CDKN2 in primary human tumours.Nat Genet. 1995; 11: 210-212Crossref PubMed Scopus (656) Google Scholar Homozygous deletions of CDKN2A have been detected in up to 56% of kidney cancer cell lines.13Kamb A Gruis NA Weaver-Feldhaus J Liu Q Harshman K Tavtigian SV Stockert E Day III, RS Johnson BE Skolnick MH A cell cycle regulator potentially involved in genesis of many tumor types.Science. 1994; 264: 436-440Crossref PubMed Scopus (2808) Google Scholar Hypermethylation of the promoter region of CDKN2A were reported in 23% of RCC cell lines.10Herman JG Merlo A Mao L Lapidus RG Issa JP Davidson NE Sidransky D Baylin SB Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers.Cancer Res. 1995; 55: 4525-4530PubMed Google Scholar To further determine the significance of CDKN2A alterations in primary CRCC we performed: 1) sequence analysis of CDKN2A in 113 CRCCs; 2) fluorescence in situ hybridization (FISH) to search for homozygous CDKN2A deletions; 3) immunohistochemical CDKN2A expression analysis using a renal tumor microarray containing 532 renal tumors; and 4) loss of heterozygosity (LOH) analysis using four polymorphic microsatellites on 9p. All renal tumors from the archive of the Institute for Pathology, University of Basel, were reviewed by one pathologist (HM). One hundred thirteen consecutive nephrectomy specimens from 1985 to 1994 with CRCC14Kovacs G Akhtar M Beckwith B Bugert B Cooper C Delahunt B Eble J Fleming S Ljungberg B Medeiros L Moch H Reuter V Ritz E Roos G Schmidt D Srigley J Störkel S Van den Berg E Zbar B The Heidelberg classification of renal cell tumours.J Pathol. 1997; 183: 131-133Crossref PubMed Scopus (1146) Google Scholar were selected for this study. Histological grading and tumor staging were done according to Thoenes and colleagues15Thoenes W Stoerkel S Rumpelt H Histopathology and classification of renal cell tumors (adenomas, oncocytomas, and carcinomas): the basic cytological and histopathological elements and their use for diagnostics.Pathol Res Pract. 1986; 181: 125-143Crossref PubMed Scopus (601) Google Scholar and International Union Against Cancer (UICC).16UICC Sobin L Wittekind C TNM Classification of Malignant Tumours. ed 5. Wiley-Liss, New York, Chichester, Weinheim, Brisbane, Singapore, Toronto1997Google Scholar There were 11 grade 1, 71 grade 2, and 31 grade 3 tumors. Forty tumors were stage pT1, eight were pT2, 64 were pT3, and one was pT4. An extensive tissue sampling to ensure perinephric fat infiltration may account for the relative high proportion of high-stage cases in this tumor set. The mean tumor diameter was 6.6 ± 3.0 cm. There was lymph node metastasis at nephrectomy in seven patients and evidence for hematogenous metastasis (pM1/cM1) in 19 patients. Overall and tumor-specific survival data were obtained by reviewing the hospital records, by direct communication with the attending physicians, and from the Cancer Registry of Basel. Patients were evaluated from the time of biopsy diagnosis to the last known follow-up. Overall survival was available from 100 patients and tumor-specific survival was available from 83 patients. The mean follow-up time was 48.9 months (median, 48 months; range, 2 to 131 months). Thirty-five patients had a 5-year follow-up. Forty-one patients died within 5 years after surgery. There was a significant association between short tumor-specific survival and presence of metastasis (P < 0.0001), pT stage (P < 0.001), and histological grade (P < 0.05). Formalin-fixed, paraffin-embedded tumor tissue from each of the 113 patients was selected on the basis of hematoxylin and eosin (H&E)-stained sections to ensure a minimum of 75% tumor cells in the samples. Twenty to 30 mg of normal and tumor tissue was scraped away from the paraffin blocks using a scalpel. Areas of normal tissue were defined using H&E tissue sections. Deparaffinizing of the tissues and DNA extraction were performed according to the QIAmp Tissue Kit protocol (Qiagen, Basel, Switzerland). Analysis of allelic deletions was performed using primers for microsatellites D9S970 (9p12-9p13), D9S171 (9p13), D9S1748 (9p21), and D9S156 (9p21). Primer sequences were obtained from the Genome Data Base. The locus of D9S1748 lies upstream of exon 1 of CDKN2A.17Mao L Merlo A Bedi G Shapiro GI Edwards CD Rollins BJ Sidransky D A novel p16INK4A transcript.Cancer Res. 1995; 55: 2995-2997PubMed Google Scholar Primers were labeled with T4-polynucleotide kinase (Catalys, Wallisellen, Switzerland) and γ32P-ATP (Amersham/Pharmacia, Zurich, Switzerland) for microsatellite analysis. Polymerase chain reaction (PCR) amplification was performed in a total of 15 μl containing 50 to 100 ng DNA, 1× Taq buffer (Qiagen), 200 μmol/L dNTPs, 3 pmol of each primer, 0.4 pmol32P-labeled primer, and 1 U Taq DNA polymerase (Qiagen). An initial denaturation step of 95°C for 3 minutes followed by 35 cycles each of 95°C for 30 seconds, 50°C to 55°C for 30 seconds, and 72°C for 1 minute with a final extension step of 72°C for 5 minutes comprised the PCR profile. Products were separated by electrophoresis in denaturing 6% polyacrylamide gels followed by autoradiography as previously described.18Schraml P Muller D Bednar R Gasser T Sauter G Mihatsch MJ Moch H Allelic loss at the D9S171 locus on chromosome 9p13 is associated with progression of papillary renal cell carcinoma.J Pathol. 2000; 190: 457-461Crossref PubMed Scopus (47) Google Scholar For informative cases, allelic loss was scored if the radiographic signal of one allele was >50% reduced in the tumor DNA as compared with the corresponding normal allele. FISH was performed as previously described.19Sauter G Moch H Carroll P Kerschmann R Mihatsch M Waldman F Chromosome-9 loss detected by fluorescence in situ hybridization in bladder cancer.Int J Cancer. 1995; 64: 99-103Crossref PubMed Scopus (50) Google Scholar One section from each tissue block was stained with H&E to ensure the presence of at least 90% tumor cells and nuclei of formalin-fixed tissue blocks were dissociated and dropped onto slides. Two-color FISH was performed using a 180-kb Spectrum Orange-labeled 9p21 probe (Vysis, Downer's Grove, IL), spanning the minimal homozygously deleted region that includes CDKN2A and excludes CDKN2B as described by Cairns and colleagues,1Cairns P Tokino K Eby Y Sidransky D Localization of tumor suppressor loci on chromosome 9 in primary human renal cell carcinomas.Cancer Res. 1995; 55: 224-227PubMed Google Scholar together with a corresponding Spectrum Green-labeled centromeric 9 α satellite probe (CEP 9, Vysis). Slide pretreatment, hybridization, and washing procedures were performed as previously described.19Sauter G Moch H Carroll P Kerschmann R Mihatsch M Waldman F Chromosome-9 loss detected by fluorescence in situ hybridization in bladder cancer.Int J Cancer. 1995; 64: 99-103Crossref PubMed Scopus (50) Google Scholar The hybridization mixture contained 3 μl of each of the probes and Cot-1 DNA (1 mg/ml). Slides were counterstained with 0.2 μmol/L 4,6-diamidino-2-phenylindole. At least 100 nuclei were selected for scoring using 4,6-diamidino-2-phenylindole staining. A loss of one CDKN2A allele was defined as presence of less 9p21 than centromere 9 signals in >40% of nuclei. A tumor was considered monosomic for chromosome 9 if >50% of the nuclei showed only one signal for (chromosome) 9p21 and CEP9. A homozygous deletion of 9p21 was considered, if >50% of the nuclei showed centromere 9 signals without 9p21 signals. Exon 1 and exon 2 of CDKN2A were amplified with two primer sets (Table 1), each resulting in two overlapping fragments. PCR samples for the first PCR contained ∼100 ng of genomic DNA, 10 pmol of each primer, 1× Taq buffer (Qiagen), 200 μmol/L of each nucleotide (dATP, dCTP, dGTP, dTTP) and 1 U Taq polymerase (Qiagen) in a 20 μl total volume. PCR conditions for the step-down PCR were: 95°C for 3 minutes; 10 cycles of 95°C for 40 seconds, 62.5°C to 64.6°C for 40 seconds, 72°C for 1 minute; 25 cycles of 95°C for 40 seconds, 55.5°C to 57°C for 30 seconds, 72°C for 1 minute; 72°C for 5 minutes. PCR conditions for exon 3 were: 95°C for 3 minutes; 35 cycles of 95°C for 40 seconds, 55°C for 30 seconds, 72°C for 1 minute; 72°C for 5 minutes.Table 1PCR Primers for CDKN2A Mutation AnalysisFirst PCR Exon 1Forward 5′-CGG AGA GGG GGA GAG CAG-3′Reverse 5′-GAC CGT AAC TAT TCG GTG CGT T-3′Forward 5′-CAG CAT GGA GCC TTC GGC TGA-3′Reverse 5′-GCG CTA CCT GAT TCC AAT TC-3′ Exon 2Forward 5′-GCT CTA CAC AAG CTT CCT TTC C-3′Reverse 5′-CAG CTC CTC AGC CAG GTC C-3′Forward 5′-CTG GAC ACG CTG GTG GTG CT-3′Reverse 5′-GGG CTG AAC TTT CTG TGC TGG-3′ Exon 3Forward 5′-GTG CCA CAC ATC TTT GAC CTC A-3′Reverse 5′-CGG TGA CTG ATG ATC TAA GT-3′Second PCR Exon 1Forward 5′-M13*Primers were tailed with M13 universal (−21) and M13 reverse (−29), respectively.-AGA GGG GGA GAG CAG GCA-3′Reverse 5′-CCA GCA GCG CCC GCA CCT C-3′Forward 5′-GAG CCT TCG GCT GAC TGG CTG-3′Reverse 5′-M13-CAA ACT TCG TCC TCC AGA GT-3′ Exon 2Forward 5′-M13-AAG CTT CCT TTC CGT CAT GCC-3′Reverse 5′-GCC AGG TCC ACG GGC AGA C-3′Forward 5′-M13-GAC ACG CTG GTG GTG CTG CA-3′Reverse 5′-TCT GAG CTT TGG AAG CTC TC-3′ Exon 3Forward 5′-GAC CTC AGG TTT CTA ACG CCT-3′Reverse 5′-CGG TGA CTG ATG ATC TAA GT-3′* Primers were tailed with M13 universal (−21) and M13 reverse (−29), respectively. Open table in a new tab The products of the first PCR were analyzed on a 1.2% agarose gel. One μl of the supernatant of excised, snap-frozen, and re-thawed bands were taken for the second PCR. The samples for the second PCR contained 10 pmol of each primer, 1× Taq buffer, 200 μmol/L of each nucleotide and 1 U of Taq polymerase (Qiagen). PCR conditions were: 95°C for 3 minutes, 30 cycles of 95°C for 30 seconds, 55°C to 65°C for 30 seconds, 72°C for 30 seconds; 72°C for 5 minutes. IRD800-labeled primers (MWG-Biotech, Ebersberg, Germany) were used to direct cycle-sequencing of the PCR products. Cycle sequencing was done according to the protocol of the manufacturer's instructions (MWG-Biotech). Sequence products were analyzed on a LICOR-DNA sequencer (Model 4000). Tumor DNA that showed a sequence change was subjected to a second PCR and sequence analysis. In those cases the matched normal DNA was also examined. To evaluate the prevalence of CDKN2A expression in renal tumors, a renal tumor microarray was used containing tumor specimens from 532 renal tumors and tissue from six normal kidneys. The construction of the tumor microarray was previously described.20Kononen J Bubendorf L Kallioniemi A Barlund M Schraml P Leighton S Torhorst J Mihatsch MJ Sauter G Kallioniemi OP Tissue microarrays for high-throughput molecular profiling of tumor specimens.Nat Med. 1998; 4: 844-847Crossref PubMed Scopus (3492) Google Scholar, 21Moch H Schraml P Bubendorf L Mirlacher M Kononen J Gasser T Mihatsch MJ Kallioniemi OP Sauter G High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma.Am J Pathol. 1999; 154: 981-986Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar There were 383 conventional (clear cell), 57 papillary, 23 chromophobe, three collecting duct RCCs, and 17 oncocytomas. The presence of tumor tissue on the arrayed samples was verified on one H&E-stained section. Sections (5-μm thick) were cut for immunohistochemistry. Standard indirect immunoperoxidase procedures were used for immunohistochemistry (ABC-Elite; Vector Laboratories, Burlingame, CA) on the tumor microarray as previously described.21Moch H Schraml P Bubendorf L Mirlacher M Kononen J Gasser T Mihatsch MJ Kallioniemi OP Sauter G High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma.Am J Pathol. 1999; 154: 981-986Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar A well-characterized antibody to CDKN2A7Lukas J Parry D Aagaard L Mann DJ Bartkova J Strauss M Peters G Bartek J Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16.Nature. 1995; 375: 503-506Crossref PubMed Scopus (864) Google Scholar was used for detection of CDKN2A expression (1:25, Ab-1; Oncogene Research Products, Cambridge, MA) after pronase pretreatment. Tumors were considered positive if an unequivocal nuclear positivity was seen in tumor cells. Cases of prostate and bladder carcinoma with nuclear CDKN2A expression served as external positive control. Contingency table analysis was used to analyze the relationship between allelic deletion, grade, stage, and presence of metastasis. Overall survival rates were plotted using the Kaplan-Meier method. Statistical differences between tumors with and without 9p deletions were determined with the log-rank test. The proportional hazard model was used to test for independent prognostic information. Eighty-eight normal/tumor pairs could be interpreted for LOH analysis on chromosome 9p. Twenty-one of 88 CRCCs (24%) showed allelic deletion with at least one microsatellite. Most deletions were detected at D9S171 on 9p13. Twelve of 59 informative tumors (20%) displayed allelic loss in this region. In contrast, only seven of 73 informative cases (10%) showed LOH at D9S1748 (9p21), eight of 68 (9%) demonstrated allelic deletion at D9S156 (9p21), and five of 68 (7%) displayed LOH at D9S970 (9p12-9p13). Allelic deletion at only one of the microsatellite loci examined were found in six tumors (10%) at D9S171, followed by three tumors at D9S1748 and D9S156 (4% each), respectively, and two tumors at D9S970 (Figure 1, A and B). LOH at 9p was not associated with tumor stage, histological grade, and metastasis (Table 2). Also, the separate analysis of the individual microsatellites did not reveal a relationship between LOH and morphological parameters. Tumor-specific survival data were available for 62 patients being informative for at least one 9p microsatellite. Fifteen of these patients showed LOH. Tumor-specific survival rates were only analyzed in stage pT3 RCCs, because there were too few tumor-related deaths in pT1/2 RCCs. In univariate survival analysis patient prognosis was associated with 9p deletion in stage pT3 RCCs. Tumor-specific 5-year survival was 58% for patients without 9p deletion (n = 30), whereas all patients with 9p deletions (n = 7) died of disease (P = 0.01, Figure 2). The analysis of pT3 CRCCs without metastasis at the time of nephrectomy also revealed a significant association between 9p deletions and poor prognosis (P = 0.02), although the number of tumors with deletions was small (n = 4) within this analysis. Cox proportional hazard analysis including the variables histological grade, presence of metastasis, and 9p deletion showed that LOH at 9p was not an independent predictor of poor prognosis in pT3 CRCCs (Table 3).Table 2LOH Analysis: 9p Deletion and Tumor Phenotype in Conventional (Clear-Cell) RCCNumber of tumors (n)9p LOH n (%)P valueGrade G193 (33) G25410 (19)0.87 G3258 (32)pT Stage pT1/24112 (29) pT3/4479 (19)0.27Metastasis pN0, cM07619 (25) pN1 or cM1122 (17)0.53 Open table in a new tab Table 3LOH Analysis: Proportional Hazard Analysis for pT3 Conventional (Clear-Cell) Renal CarcinomaVariableRelative riskP value9p-deletion1.60.5Histologic grade2.70.06Metastasis4.40.01 Open table in a new tab There was a 24-bp deletion within exon 1 in 13 of 113 tumors (12%) (Figure 3A). One patient had this deletion also in the normal DNA. This in-frame mutation results in a truncation of 8 amino acids of CDKN2A. Because of the special feature of the sequence in the start coding region of CDKN2A there were 25 possible variants of deletions leading to the same sequence alteration (Figure 3B). The 24-bp deletion was not associated with tumor stage or presence of metastases. Interestingly, the deletion was not detected in grade 1 RCCs, but in 9% of grade 2 and 22% of grade 3 tumors (Table 4). This trend did not reach significance (P = 0.07). There was no relationship between the 24-bp deletions and tumor-specific survival (P = 0.15).Table 4Sequence Analysis: 24-bp Deletion of Exon 1 and Tumor PhenotypeNumber of tumors (n)24-bp Deletion n (%)P valuepT Stage pT1/2486 (12%) pT3/4657 (11%)0.7Histologic grade G1110 G2706 (9%)0.07 G3327 (22%)Metastasis pN0, cM09010 (11%) pN1 or cM1233 (13%)0.8 Open table in a new tab A G to A transition was detected in codon 140 of exon 2 in one patient. The mutation causes an amino acid exchange from alanine to threonine. This mutation was also detected in the normal DNA of the patient and possibly represents a polymorphism. One hundred eight of 113 CRCCs (96%) showed a G to C transversion in the noncoding sequence of exon 3 that was also found in the matched normal DNA of each tumor. Eighty-nine of these tumors were homozygous and 19 were heterozygous for this transversion. There were only five tumors showing the wild-type sequence of the gene data base.22Serrano M Hannon GJ Beach D A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4.Nature. 1993; 366: 704-707Crossref PubMed Scopus (3340) Google Scholar Interestingly, the homozygous C/C genotype was associated with a low grade of differentiation (P < 0.05) (Table 5). To further evaluate the frequency of the C haplotype in the normal population, we analyzed 48 blood samples of healthy donors. The analysis revealed a C-allele frequency of 90% of the individuals tested.Table 5Sequence Analysis: Homozygous Transversion in Exon 3 of p16 and Tumor PhenotypeNumber of tumors*Five tumors were excluded from analysis: 1 tumor was not interpretable, 4 tumors showed the wild type of exon 3. (n)Homozygous transversion n (%)P valuepT-Stage pT1/24637 (77%) pT3/46252 (81%)0.6Histologic grade G1116 (55%) G26756 (84%)0.03 G33027 (90%)Metastasis pN0, cM08568 (80%) pN1 or cM12317 (91%)0.2* Five tumors were excluded from analysis: 1 tumor was not interpretable, 4 tumors showed the wild type of exon 3. Open table in a new tab High-quality hybridization signals for both centromeric and gene-specific probes were obtained in 54 tumors. Four of 54 CRCCs (7%) showed physical 9p21 deletions, but none of the tumors had a homozygous CDKN2A deletion according to our definition. There were two additional tumors with chromosome 9 monosomy. To determine the frequency and potential implications of CDKN2A expression in RCC, we analyzed a cohort of 532 renal tumors on a tumor microarray. We observed six CRCCs with nuclear CDKN2A expression. These tumors showed neither CDKN2A mutations nor D9S1748 deletions. Papillary (n = 57), chromophobe (n = 23), and collecting duct carcinomas (n = 3) as well as oncocytoma (n = 17) did not show CDKN2A expression. Prostate and bladder carcinomas served as external positive controls. In these controls, a strong nuclear staining was detectable. Representative tumors are shown in Figure 4, A and B. We demonstrated an association between LOH on chromosome 9p with short patient survival. Chromosome 9p deletions were detected in 24% of CRCCs using four highly polymorphic microsatellite markers. A slightly higher rate of chromosome 9p deletions was reported by Cairns and colleagues,1Cairns P Tokino K Eby Y Sidransky D Localization of tumor suppressor loci on chromosome 9 in primary human renal cell carcinomas.Cancer Res. 1995; 55: 224-227PubMed Google Scholar who detected deletions in 14 of 42 primary CRCCs (33%). Other groups reported 9p LOH in 16 to 33% of CRCCs.2Thrash-Bingham CA Greenberg RE Howard S Bruzel A Bremer M Goll A Salazar H Freed JJ Tartof KD Comprehensive allelotyping of human renal cell carcinomas using microsatellite DNA probes.Proc Natl Acad Sci USA. 1995; 92: 2854-2858Crossref PubMed Scopus (94) Google Scholar, 23Bugert P Kovacs G Molecular differential diagnosis of renal cell carcinomas by microsatellite analysis.Am J Pathol. 1996; 149: 2081-2088PubMed Google Scholar, 24Thrash-Bingham CA Salazar H Freed JJ Greenberg RE Tartof KD Genomic alterations and instabilities in renal cell carcinomas and their relationship to tumor pathology.Cancer Res. 1995; 55: 6189-6195PubMed Google Scholar, 25Schullerus D Herbers J Chudek J Kanamaru H Kovacs G Loss of heterozygosity at chromosomes 8p, 9p, and 14q is associated with stage and grade of non-papillary renal cell carcinomas.J Pathol. 1997; 183: 151-155Crossref PubMed Scopus (97) Google Scholar The significant difference in tumor-specific survival between tumors with and without 9p deletion suggests that a tumor suppressor gene on 9p is involved in tumor progression. In lung, bladder, head and neck cancer, and melanoma,12Cairns P Polascik TJ Eby Y Tokino K Califano J Merlo A Mao L Herath J Jenkins R Westra W Rutter JL Buckler A Gabrielson E Tockman M Cho KR Hedrick L Bova GS Isaacs W Koch W Schwab D Sidransky D Frequency of homozygous deletion at p16/CDKN2 in primary human tumours.Nat Genet. 1995; 11: 210-212Crossref PubMed Scopus (656) Google Scholar, 26Fountain JW Karayiorgou M Ernstoff MS Kirkwood JM Vlock DR Titus-Ernstoff L Bouchard B Vijayasaradhi S Houghton AN Lahti J Kidd VJ Housman DE Dracapoli NC Homozygous deletions within human chromosome band 9p21 in melanoma.Proc Natl Acad Sci USA. 1992; 89: 10557-10561Crossref PubMed Scopus (332) Google Scholar allele losses and homozygous deletions were most frequently observed at 9p21. Allelic imbalance has generally been considered to represent loss of genetic material, but the use of PCR-based techniques makes it difficult to differentiate allele loss from allele gain. Although it cannot be excluded that some of our tumors had gains we think that the vast majority of our tumors analyzed showed LOH. In approximately half of the cases signal reduction of one allele was near absolute in the tumors. Only four of 24 tumors (17%) with LOH displayed a 50% reduction of one allele. In addition, our FISH results coincide with previous cytogenetic data in that regional or entire gains of chromosome 9 are rare events in kidney tumors.27Mitelman F Catalog of Chromosome Aberrations in Cancer. ed 5. Wiley-Liss, New York, Chichester, Brisbane, Toronto, Singapore1994Google Scholar Hence, signal reduction of one allele very likely reflects loss ra
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