Cyclin D1 Polymorphism and Expression in Patients with Squamous Cell Carcinoma of the Head and Neck
2001; Elsevier BV; Volume: 159; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63038-6
ISSN1525-2191
AutoresSarah Holley, Gary Parkes, Christoph Matthias, Ulrike Bockmühl, V. Jähnke, Korinna Leder, Richard C. Strange, Anthony A. Fryer, Paul R. Hoban,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoWe have previously reported that the cyclin D1 (CCND1) GG870 genotype was associated with poorly differentiated tumors and reduced disease-free interval in patients with squamous cell carcinoma of the head and neck (SCCHN). We have now examined the association of this and a secondCCND1 polymorphism with gene expression and outcome in SCCHN patients. Analysis of a CCND1 G/C1722polymorphism revealed that CCND1 CC1722genotype was associated with poorly differentiated tumors [P = 0.005; odds ratio (OR), 5.7; 95% CI, 1.7 to 19.2), and reduced disease-free interval (P = 0.003; Hazard Ratio (HR), 7.3; 95% CI, 1.1 to 27.2.) independently from the influence ofCCND1 GG870 genotype. Patients whose tumors were negative for cyclin D1 were associated with reduced disease-free interval (P = 0.028; HR, 4.1; 95% CI, 1.4 to 14.2). Although G/C1722 genotypes were not associated with expression, we found a significant trend between reduced expression of cyclin D1 in patients with theCCND1 GG870 genotype (P= 0.04). Splicing of CCND1 mRNA in head and neck tissues was modulated by CCND1 A/G870alleles, thus CCND1 transcript a was spliced equally from CCND1 A870 and G870alleles, whereas CCND1 transcript b was spliced mainly from the CCND1 A870 allele. Our analysis has also identified differences in cyclin D1 genotype and protein expression and the pathogenesis of SCCHN in males and females. Thus, CCND1 CC1722 genotype was more common in female patients (P = 0.019; OR, 3.3; 95% CI, 1.3 to 10) and cyclin D1 expression was more frequent (chi-square1, 3.96; P= 0.046) and at higher levels (P = 0.004) in tumors from female patients. In summary, our data show that the twoCCND1 polymorphic sites are independently associated with tumor biology and clinical outcome. CCND1 A/G870 alleles affect gene expression in head and neck tissues. We also provide preliminary evidence that the molecular genetics of SCCHN development may be influenced by patient gender. We have previously reported that the cyclin D1 (CCND1) GG870 genotype was associated with poorly differentiated tumors and reduced disease-free interval in patients with squamous cell carcinoma of the head and neck (SCCHN). We have now examined the association of this and a secondCCND1 polymorphism with gene expression and outcome in SCCHN patients. Analysis of a CCND1 G/C1722polymorphism revealed that CCND1 CC1722genotype was associated with poorly differentiated tumors [P = 0.005; odds ratio (OR), 5.7; 95% CI, 1.7 to 19.2), and reduced disease-free interval (P = 0.003; Hazard Ratio (HR), 7.3; 95% CI, 1.1 to 27.2.) independently from the influence ofCCND1 GG870 genotype. Patients whose tumors were negative for cyclin D1 were associated with reduced disease-free interval (P = 0.028; HR, 4.1; 95% CI, 1.4 to 14.2). Although G/C1722 genotypes were not associated with expression, we found a significant trend between reduced expression of cyclin D1 in patients with theCCND1 GG870 genotype (P= 0.04). Splicing of CCND1 mRNA in head and neck tissues was modulated by CCND1 A/G870alleles, thus CCND1 transcript a was spliced equally from CCND1 A870 and G870alleles, whereas CCND1 transcript b was spliced mainly from the CCND1 A870 allele. Our analysis has also identified differences in cyclin D1 genotype and protein expression and the pathogenesis of SCCHN in males and females. Thus, CCND1 CC1722 genotype was more common in female patients (P = 0.019; OR, 3.3; 95% CI, 1.3 to 10) and cyclin D1 expression was more frequent (chi-square1, 3.96; P= 0.046) and at higher levels (P = 0.004) in tumors from female patients. In summary, our data show that the twoCCND1 polymorphic sites are independently associated with tumor biology and clinical outcome. CCND1 A/G870 alleles affect gene expression in head and neck tissues. We also provide preliminary evidence that the molecular genetics of SCCHN development may be influenced by patient gender. Squamous cell carcinoma of the head and neck (SCCHN) comprise ∼5% of newly diagnosed malignancies in Northern Europe and the United States. Annually, more than 500,000 new cases are registered worldwide and the incidence of the disease is increasing.1Johnson NW Ranasinghe AW Warnakulasuriya KAAS Potentially malignant lesions and conditions of the mouth and oropharynx: natural history—cellular and molecular markers of risk.Eur J Cancer Prev. 1993; 2: 31-51Crossref PubMed Google Scholar Survival rates for the disease are poor, clinical outcome can vary among patients with tumors from the same site, with comparable tumor stage, nodal status, and histological grade.2Muir C Weiland L Upper aerodigestive tract cancers.Cancer. 1995; 75: 147-153Crossref PubMed Scopus (219) Google Scholar, 3Janot F Klijanienko J Russo A Marnet JP de Braud F El-Naggar AK Pignon JP Luboinski B Cvitkovic E Prognostic value of clinicopathological parameters in head and neck squamous cell carcinoma: a prospective analysis.Br J Cancer. 1996; 73: 531-538Crossref PubMed Scopus (97) Google Scholar, 4Magnano M Bussi M de Stefani A Milan F Lerda W Ferrero V Gervasio F Ragona R Gabriele P Valenta G Cortesina G Prognostic factors for head and neck tumour recurrence.Acta Otolaryngol. 1995; 115: 833-838Crossref PubMed Scopus (21) Google Scholar Chronic consumption of tobacco and alcohol are recognized risk factors although it is unclear which traits determine tumor behavior and therefore prognosis. Studies have demonstrated elevated levels of cell proliferation in a high proportion of SCCHN tumors, and proliferation rates have been related to patient survival, and used in patient treatment strategies.5Kotelnikov VM Coon J Haleem A Taylor S Hutchinson J Panje W Caldarelli D Preisler HD Cell kinetics of head and neck cancers.Clin Cancer Res. 1995; 1: 527-537PubMed Google Scholar, 6Corvo R Giaretti W Sanguineti G Geido E Orecchia R Guenzi M Margarino G Bacigalupo A Garaventa G Barbieri M Vitale V In vivo cell kinetics in head and neck squamous cell carcinomas predicts local control and helps guide radiotherapy regimen.J Clin Oncol. 1995; 48: 1843-1850Google Scholar, 7Tomasino RM Daniele E Bazan V Morello V Tralongo V Nuara R Nagar C Salvato M Ingria F Restivo S Dardanoni G Vecchione A Russo A Prognostic significance of cell kinetics in laryngeal squamous cell carcinomas: clinical pathological associations.Cancer Res. 1995; 55: 6103-6108PubMed Google Scholar Thus, genes that encode regulators of cell proliferation may prove useful in establishing patient prognosis or as targets in therapy regimens. The cyclin D1 gene (CCND1) encodes cyclin D1 protein, which is expressed in response to mitogenic signals promoting transition through the restriction point in the G1 phase of the cell cycle.8Sherr CJ Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4927) Google Scholar Increased expression of cyclin D1 has been associated with increased cell proliferation.9Quelle DE Ashmun RA Shurtleff SA Kato JY Bar-Sagi D Roussel MF Sherr CJ Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts.Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (978) Google Scholar, 10Musgrove EA Lee CSL Buckley MF Sutherland RL Cyclin D1 induction in breast cancer cells shorten G1 and is sufficient for cells arrested in G1 to complete the cell cycle.Proc Natl Acad Sci USA. 1994; 91: 8022-8026Crossref PubMed Scopus (337) Google Scholar CCND1 amplification leading to deregulated CCND1 expression is common in tumors from patients with SCCHN.11Callender T El Naggar AK Lee MS Frankenthaler R Luna MA Batsakis JG PRAD-1 (CCND1)/cyclin D1 oncogene amplification in head and neck squamous cell carcinoma.Cancer. 1994; 74: 152-158Crossref PubMed Scopus (230) Google Scholar, 12Jares P Fernadez PL Campo E Nadal A Bosch F Aiza G Nayach I Trassera J Cardesa A PRAD-1/cyclin D1 gene amplification correlates with messenger mRNA overexpression and tumour progression in human laryngeal carcinomas.Cancer Res. 1994; 54: 4813-4817PubMed Google Scholar Cyclin D1 protein overexpression has been shown to correlate with reduced 5-year and overall survival in SCCHN patients.13Michalides R van Veelen N Hart A Loftus B Wientjens E Balm A Overexpression of cyclin D1 correlates with recurrence in a group of forty seven operable squamous cell carcinomas of the head and neck.Cancer Res. 1995; 55: 975-978PubMed Google Scholar Other studies have shown that cyclin D1 protein overexpression is associated with poor prognosis in primary hypopharyngeal, laryngeal, esophageal, and oral squamous cell carcinomas.14Masuda M Hirakawa N Nakashima T Kuratomi Y Komiyama S Cyclin D1 overexpression in primary hypolaryngeal carcinomas.Cancer. 1995; 78: 390-395Crossref Scopus (101) Google Scholar, 15Pignatora L Pruneri G Carboni N Capaccio P Cesana BM Neri A Buffa R Clinical relevance of cyclin D1 protein overexpression in laryngeal squamous cell carcinoma.J Clin Oncol. 1998; 16: 3069-3077PubMed Google Scholar, 16Ishikawa T Furihata M Ohtsuki Y Murakami H Inoue A Ogoshi S Cyclin D1 overexpression related to retinoblastoma protein expression as a prognostic marker in human oesophageal squamous cell carcinoma.Br J Cancer. 1998; 77: 92-97Crossref PubMed Scopus (62) Google Scholar Furthermore cyclin D1 antisense experiments have demonstrated that the gene may be a potential target for therapeutic intervention in SCCHN.17Nakashima T Clayman GL Antisense inhibition of cyclin D1 in human head and neck squamous cell carcinoma.Arch Otolaryngol Head Neck Surgery. 2000; 126: 957-961Crossref PubMed Scopus (42) Google Scholar CCND1 is polymorphic with a common A/G substitution at nucleotide 870 in the conserved splice donor region of exon 4 of the gene.18Betticher DC Thatcher N Altermatt HJ Hoban P Ryder WDJ Heighway J Alternate splicing produces a novel cyclin D1 transcript.Oncogene. 1995; 11: 1005-1011PubMed Google Scholar CCND1 alleles have been shown to be associated with splicing of CCND1 mRNA in both normal and tumorous lung tissue. In heterozygotes, transcript a is spliced equally from CCND1 G870 and A870 alleles, whereas transcript b is spliced mainly from the CCND1 A870allele.18Betticher DC Thatcher N Altermatt HJ Hoban P Ryder WDJ Heighway J Alternate splicing produces a novel cyclin D1 transcript.Oncogene. 1995; 11: 1005-1011PubMed Google Scholar In non-small cell lung cancer patients,CCND1 AA870 genotype is associated with poor prognosis.18Betticher DC Thatcher N Altermatt HJ Hoban P Ryder WDJ Heighway J Alternate splicing produces a novel cyclin D1 transcript.Oncogene. 1995; 11: 1005-1011PubMed Google Scholar In contrast we have recently demonstrated in SCCHN that the CCND1 GG870 genotype was associated with poorly differentiated tumors and independently from tumor differentiation, with reduced patient disease-free interval.19Matthias C Branigan K Jahnke V Leder K Haas J Heighway J Jones PW Strange RC Fryer AA Hoban P Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.Clin Cancer Res. 1998; 4: 2411-2418PubMed Google Scholar, 20Matthias C Jahnke V Jones PW Hoban PR Alldersea J Worrall S Anthony A Fryer AA Strange RC Cyclin D1, glutathione S-transferase and cytochrome P450 genotypes and outcome in patients with upper aerodigestive tract cancers: assessment of the importance of individual genes using multivariate analysis.Cancer Epidemiol Biomarkers. 1999; 8: 815-823PubMed Google Scholar A second common G/C polymorphism at nucleotide 1722 within CCND1 3′UTR has also been described (G/C1722 sequence information is available at the NCBI SNP database at http://www.ncbi.nlm.nih.gov/SNP), although the influence of this polymorphism on tumorigenesis has not been examined.21Heighway J Hae III polymorphism with 3′ untranslated region of PRAD 1.Nucleic Acids Res. 1991; 19: 5451Crossref PubMed Scopus (13) Google Scholar In this study we have further investigated the role of CCND1 allelism in SCCHN. We have examined the relationship of theCCND1 G/C1722 polymorphism with A/G870 alleles and clinical outcome. In addition we have further studied the influence of CCND1 alleles on mRNA splicing and protein expression in head and neck tissue. SCCHN patients (n = 294) were studied from our original cohort of 384 patients previously described.19Matthias C Branigan K Jahnke V Leder K Haas J Heighway J Jones PW Strange RC Fryer AA Hoban P Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.Clin Cancer Res. 1998; 4: 2411-2418PubMed Google Scholar, 20Matthias C Jahnke V Jones PW Hoban PR Alldersea J Worrall S Anthony A Fryer AA Strange RC Cyclin D1, glutathione S-transferase and cytochrome P450 genotypes and outcome in patients with upper aerodigestive tract cancers: assessment of the importance of individual genes using multivariate analysis.Cancer Epidemiol Biomarkers. 1999; 8: 815-823PubMed Google Scholar These patients were selected because of the availability of DNA and do not represent a clinical subgroup. The clinical pathological characteristics are similar to those of the total cohort.19Matthias C Branigan K Jahnke V Leder K Haas J Heighway J Jones PW Strange RC Fryer AA Hoban P Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.Clin Cancer Res. 1998; 4: 2411-2418PubMed Google Scholar Briefly, the case group comprised German Caucasians suffering a single histologically confirmed oral cavity pharyngeal or laryngeal squamous cell carcinoma. Patients were recruited at first presentation or during follow-up between 1994 and 1996. Malignancies were staged using the TMN classification system.22Phermanek P Sobin LH Union Internationale Contre le Cancer. TNM Classification of Malignant Tumours. ed 4. UICC, Springer-Verlag, Berlin1987Crossref Google Scholar All tumors were squamous cell carcinoma and were histologically graded as: well (G0–1), moderately (G2), and poorly (G3) differentiated. Margins of the resected specimen were examined by a histopathologist and judged for tumor-free margins using the international R0-R2 system; R0, microscopically proven free tumor margins; R1, microscopic infiltration and macroscopically free margin; R2, macroscopic tumor infiltration of the margin. Where details were available we examined the association of cyclin D1 genotype and protein expression with factors known to influence clinical outcome: tumor site (n = 268), tumor size (T1 to T4) (n = 258), histological differentiation (G0/1 to G3) (n = 168) and the presence of nodes at time of surgery (n = 166). We also examined the association of genotypes and protein expression with tumor recurrence (defined as disease-free interval) in 151 patients. The study to identify associations with disease-free interval was performed only in patients in which a R0 resection could be achieved during initial treatment. Patients suffering a tumor re-growth during the first 6 months after initial treatment and those suffering extra-capsular tumor spread in any of the resected lymph nodes were excluded to avoid misjudged recurrences because of residual tumor growth. Lymph node involvement was determined before surgery using ultrasound, computed tomography, and nuclear magnetic resonance imaging and later histologically proven in the neck dissection specimen. Peripheral blood samples were collected in ethylenediaminetetraacetic acid. Tumor and histologically normal head and neck tissues (salivary gland or muscle) were collected at time of surgery and snap-frozen in liquid nitrogen and stored at −70°C before use. DNA was extracted from frozen tissue and blood using a phenol-chloroform method.23Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989Google Scholar mRNA was isolated from frozen normal and tumor tissues using the MicrofastTrack RNA isolation kit (Invitrogen, Groningen, The Netherlands). CCND1 G/C1722 genotypes were identified in DNA isolated from peripheral blood using a restriction fragment length polymorphism-PCR based assay.24Betticher D Heighway J Haselton PS Altermatt MJ Ryder WDJ Cerny T Thatcher N Prognostic significance of CCND1 (cyclin D1) overexpression in primary resected non-small cell lung cancer.Br J Cancer. 1996; 73: 294-300Crossref PubMed Scopus (223) Google Scholar Briefly reactions were performed in 25 μl containing 1× Taq polymerase buffer (Promega, Southampton, UK), 100 μmol/L dNTPs, 0.25 μg of each primer, 2% dimethyl sulfoxide, 0.25 U of Taq polymerase (Promega, UK) and 0.1 μg of DNA. Reactions were performed on an automated thermal cycler with an initial denaturation of 94°C (2 minutes) and cycled 34 times with annealing temperature of 57°C (1 minute), an extension at 72°C (1 minute), and denaturation of 94°C (1 minute). PCR products were digested with HaeIII following the manufacturer’s guidelines (New England Biolabs, Hitchin, Hertfordshire, UK). Alleles were resolved on 3% agarose gels stained with ethidium bromide. mRNA extracted from snap-frozen tissues was reverse-transcribed to cDNA using a superscript preamplification system (Gibco-BRL Life Technologies, Paisley, UK). Analysis of CCND1 transcriptsa and b was performed using a nested PCR strategy.18Betticher DC Thatcher N Altermatt HJ Hoban P Ryder WDJ Heighway J Alternate splicing produces a novel cyclin D1 transcript.Oncogene. 1995; 11: 1005-1011PubMed Google Scholar Reactions containing 2 μl of cDNA were performed in 100-μl volumes containing 1× PCR buffer (Promega), 100 μmol/L dNTPs, 0.5 μg each primer, 2 U of Taq polymerase (Promega). Primary reactions were cycled 32 times, following which 1 μl of PCR product was nested for 22 cycles. PCR conditions were initial denaturation temperature 94°C (2 minutes), annealing 55°C (1 minute), elongation 74°C (1 minute), and denaturation 94°C (1 minute). PCR products were digested with the restriction enzymeScrFI (New England Biolabs) and alleles visualized on 8% acrylamide gels and silver stained. Paraffin-embedded tumor material was available in 135 cases from the cohort of 294 patients. Immunohistochemistry was performed using a Shandon Sequenza (Shandon Scientific Limited, Cheshire, UK) as described.25Dhar KK Branigan K Parkes J Howells REJ Hand P Musgrove C Strange RC Fryer AA Redman CWE Hoban PR Expression and subcellular localisation of cyclin D1 protein in ovarian epithelial tumour cells.Br J Cancer. 1999; 81: 1174-1181Crossref PubMed Scopus (60) Google Scholar A mouse monoclonal cyclin D1 antibody (DCS-6; Novocastra, Newcastle-upon-Tyne, UK) was used. A cyclin D1-positive breast tumor provided positive control material. Negative controls had no primary antibody applied and included a section of normal head and neck tissue. Slides were graded as negative (0 to 10 to 50% cells stained), moderate (>50 to 75% cells stained), and strong (>75% cells stained). Statistical analysis was performed using Stata, version 5 (Stata Corporation, College Station, TX). Pearson chi-square tests were used to identify linkage between CCND1 genotypes and associations of genotypes with gender. Association of genotypes and protein expression with clinicopathological parameters were analyzed and corrected for imbalances in age and gender using logistic regression. Associations between cyclin D1 protein and genotype and cyclin D1 protein expression with gender were analyzed using the Armitage trend test. Cox’s proportional hazard regression model was used in the analysis of the effects of cyclin D1 staining and genotypes on disease-free interval. Kaplan-Meier curves were generated for graphical representation of associations with disease-free interval. A probability level of 5% was considered statistically significant. The frequencies of CCND1 G/C1722genotypes in 294 patients with SCCHN are shown (Table 1). Allele frequencies conformed to Hardy Weinberg equilibrium. Furthermore significant linkage disequilibrium was demonstrated between G/C1722 and A/G870 alleles, thus 32 of 34 (94%) individuals with the CCND1 CC1722 genotype were also GG870 (Table 2). The distribution of CCND1G/C1722 genotypes was significantly different between female and male patients with the CCND1CC1722 genotype being more common in female patients (Table 1). This association remained significant after correction for smoking and alcohol consumption (P = 0.036; OR, 3.7). CCND1A/G870 genotypes were not associated with patient gender.19Matthias C Branigan K Jahnke V Leder K Haas J Heighway J Jones PW Strange RC Fryer AA Hoban P Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.Clin Cancer Res. 1998; 4: 2411-2418PubMed Google Scholar, 20Matthias C Jahnke V Jones PW Hoban PR Alldersea J Worrall S Anthony A Fryer AA Strange RC Cyclin D1, glutathione S-transferase and cytochrome P450 genotypes and outcome in patients with upper aerodigestive tract cancers: assessment of the importance of individual genes using multivariate analysis.Cancer Epidemiol Biomarkers. 1999; 8: 815-823PubMed Google ScholarTable 1Distribution of CCND1 G/C1722 Genotypes and Tumor Protein Expression in SSCHN PatientsCCND1 G/C1722 genotypeCyclin D1 protein expressionGG (%)GC (%)CC (%)Positive*Positive protein expression was defined as >10% tumor cells expressing cyclin D1 and was more frequent in tumors from female patients (chi-square21 = 3.96;P = 0.046). (%)Negative (%)Total cases117 (39.8)141 (48.0)36 (12.2)112 (83.0)23 (17.0) Male106 (41.7)121 (47.6)27 (10.6)87 (79.8)22 (20.2) Female†Using logistic regression analysis corrected for imbalances in age, with CCND1 GG1722 as a reference, the CCND1 CC1722 genotype was more common in female patients P = 0.025; OR, 3.3; 95% CI, 1.2 to 9.51.11 (27.5)20 (50.0)9 (22.5)25 (96.1)1 (3.9)Tumor site Laryngeal SCC81 (41.5)90 (46.2)24 (12.3)56 (82.4)12 (17.6) Pharyngeal SCC16 (32.7)26 (53.1)7 (14.3)27 (81.8)6 (18.2) Oral Cavity SCC9 (37.5)12 (50.0)3 (12.5)13 (81.2)3 (18.8) Multiple sites11 (42.3)13 (50.0)2 (7.7)16 (88.9)2 (11.1)T-factor T136 (38.7)51 (54.8)6 (6.5)22 (73.3)8 (26.7) T227 (42.2)29 (45.3)8 (12.5)25 (92.6)2 (7.4) T321 (42.0)21 (42.0)8 (16.0)14 (100)0 (0.0) T419 (37.3)25 (49.0)7 (13.7)31 (75.6)10 (24.4)Histological differentiation‡Using logistic regression correcting for age and gender in the model CCND1 CC1722 was associated with poor tumor differentiation (G0/1, G2 versus G3), P = 0.005; OR, 5.7; 95% CI, 1.7 to 19.2. Well5 (55.6)4 (44.4)0 (0.00)5 (71.4)2 (28.6) Moderate47 (40.2)58 (49.6)12 (10.3)45 (76.3)14 (23.7) Poor11 (26.2)21 (50.0)10 (23.8)24 (80.0)6 (20.0)Lymph nodes Negative38 (40.0)49 (51.6)8 (8.4)43 (79.6)11 (20.4) Positive23 (32.4)34 (47.9)14 (19.7)30 (76.9)9 (23.1)* Positive protein expression was defined as >10% tumor cells expressing cyclin D1 and was more frequent in tumors from female patients (chi-square21 = 3.96;P = 0.046).† Using logistic regression analysis corrected for imbalances in age, with CCND1 GG1722 as a reference, the CCND1 CC1722 genotype was more common in female patients P = 0.025; OR, 3.3; 95% CI, 1.2 to 9.51.‡ Using logistic regression correcting for age and gender in the model CCND1 CC1722 was associated with poor tumor differentiation (G0/1, G2 versus G3), P = 0.005; OR, 5.7; 95% CI, 1.7 to 19.2. Open table in a new tab Table 2Association of CCND1 A/G870 and G/C1722 Genotypes in SCCHN PatientsCCND1 G/C1722 genotypesCCND1 A/G870 genotypesAA (%)AG (%)GG (%)GG (%)54 (47.4)49 (43.0)11 (9.6)G/C (%)11 (8.1)87 (64.0)38 (27.9)CC (%)0 (0.0)2 (5.9)32 (94.1)Significant linkage disequilibrium was demonstrated betweenCCND1 G/C1722 and A/G870 alleles (P < 0.001; chi-square4 = 136.3). Open table in a new tab Significant linkage disequilibrium was demonstrated betweenCCND1 G/C1722 and A/G870 alleles (P < 0.001; chi-square4 = 136.3). We next examined the data for associations between the CCND1G/C1722 genotypes and factors that influence outcome in the total group. There was no association between patient genotype and age at tumor presentation (data not shown) and no association was found between CCND1 genotypes and tumor stage and tumor site (Table 1). However, we found using logistic regression with correction for age and gender, CCND1CC1722 genotypes were associated with poorly compared to well and moderately differentiated tumors (Table 1). As before, we found that CCND1 GG870genotype was associated with poorly differentiated tumors19Matthias C Branigan K Jahnke V Leder K Haas J Heighway J Jones PW Strange RC Fryer AA Hoban P Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.Clin Cancer Res. 1998; 4: 2411-2418PubMed Google Scholar (P = 0.016; OR, 2.2; 95% CI, 1.15 to 4.21), however on including both CCND1 genotypes in a model with age and gender we found CCND1CC1722 remained significantly associated with tumor differentiation (P = 0.036; OR, 4.91; 95% CI, 1.1 to 21.8), whereas CCND1 GG870was no longer significantly associated (P = 0.76; OR, 1.17; 95% CI, 0.43 to 3.23). CCND1CC1722 genotype was more common in patients with tumor nodes, although this did not achieve statistical significance (P = 0.075; OR, 2.8; 95% CI, 0.9 to 8.9). We further examined the association of CCND1G/C1722 genotypes with differentiation and tumor nodes in the cases with laryngeal tumors. CCND1CC1722 genotypes were associated with poorly differentiated tumors (P = 0.006; OR, 9.2; 95% CI, 1.9 to 44.9) and the presence of tumor nodes in laryngeal tumors (P = 0.046; OR, 4.3; 95% CI, 1.0 to 18.1). Cyclin D1 expression was detected in 112 of 135 (83%) of tumors examined (Table 1). The proportion of tumor cells expressing cyclin D1 was variable between different tumors. Thus expression was low in 35 of 135 (25.9%) cases, moderate in 56 of 135 (41.4%) cases, and high in 21 of 135 (15.5%) cases. Cyclin D1 protein expression was not associated with patient age at presentation neither did we find an association with tumor stage, differentiation, or the presence of tumor nodes (Table 1). Analysis of cyclin D1 expression with patient gender revealed that tumor expression of cyclin D1 was high in both sexes (Table 1). However, significantly more tumors from females expressed cyclin D1 compared with those from males (Table 1). Furthermore, a significant trend was observed between an increase in the proportion of cells expressing cyclin D1 in the tumors from female patients, compared with those from males (Table 3).Table 3Association of Cyclin D1 Expression in Tumors with CCND1 A/G870 Genotypes and Patient Gender in SCCHNCyclin D1 protein*Levels of cyclin D1 expression are defined as the proportion of tumor cells expressing protein and are, negative ( 75%).Patient genderCCND1 genotypeFemale†Female patients are associated with high levels of cyclin D1 expression (Armitage test for trend P = 0.004).MaleCCND1 AG870/AA870CCND1 GG870‡CCND1 GG870 genotype is associated with low expression of cyclin D1 protein (Armitage test for trend, P = 0.049).Negative1 (4.3)22 (95.7)14 (63.6)8 (36.4)Low5 (14.3)30 (85.7)22 (66.7)11 (33.3)Moderate12 (21.4)44 (78.6)42 (79.2)11 (20.8)High8 (38.1)13 (61.9)17 (85.0)3 (15.0)Figures in parenthesis are percent.* Levels of cyclin D1 expression are defined as the proportion of tumor cells expressing protein and are, negative ( 75%).† Female patients are associated with high levels of cyclin D1 expression (Armitage test for trend P = 0.004).‡ CCND1 GG870 genotype is associated with low expression of cyclin D1 protein (Armitage test for trend, P = 0.049). Open table in a new tab Figures in parenthesis are percent. We next examined CCND1 genotype and protein expression data for associations with patient disease-free interval. The Kaplan-Meier plot demonstrated that CCND1 CC1722genotype was associated with reduced time to tumor recurrence (Figure 1A). Using Cox’s proportional hazards model to correct for imbalances in patient age and gender, withCCND1 GG1722 genotype as reference,CCND1 CC1722 was significantly associated with an increased proportion of patients having tumor recurrence after 2 years (Table 4). On including tumor differentiation in the model this association was reduced (P = 0.042; HR, 3.76; 95% CI, 1.05 to 13.47). As previously reported CCND1 GG870genotype was associated with reduced disease-free interval19Matthias C Branigan K Jahnke V Leder K Haas J Heighway J Jones PW Strange RC Fryer AA Hoban P Polymorphism within the cyclin D1 gene is associated with prognosis in patients with squamous cell carcinoma of the head and neck.Clin Cancer Res. 1998; 4: 2411-2418PubMed Google Scholar (Table 4). Including both genotypes in the model, although not significant CCND1GG870 (P = 0.12; HR, 2.0; 95% CI, 0.83 to 4.85) and CCND1CC1722 (P = 0.102; HR, 3.2; 95% CI, 0.8 to 12.9) were both associated with an increase in the proportion of patients suffering a tumor recurrence after 2 years. Patients whose tumors did not express cyclin D1 were associated with reduced disease-free interval (Figure 1B). Accordingly, the tumor recurrence after 2 years was significantly higher in patients in this group (Table 4). Furthermore absence of tumor cyclin D1 protein expression seemed to be associated with disease-free interval in patients with laryngeal tumors (P = 0.047; HR, 3.7; 95% CI, 1.01 to 5.89).Table 4Association of CCND1 Genotypes, Tumor Cyclin D1 Protein Expression, and Prognostic Indicators with Tumor Recurrence in SCCHNPrognostic indicatorPHR95% CIProportion of patients with recurrenceCCND1 G/C1722*P values were obtained using Cox's regression co
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