cyclin-d1 and Head-and-Neck-Neoplasms

To evaluate the role of cancer stem cell biomarkers in diagnosis and prognosis of OSCC patients.. The search strategy was entered into PubMed NLM, EBSCO CINAHL, EBSCO Dentistry & Oral Sciences Source, Wiley Cochrane Library, and Scopus. The full text eligible studies (n=7) were assessed for their quality using the JBI Critical Appraisal Checklist to evaluates the methodological quality of the studies based on possibility of bias in its design, conduct, and analysis. Selected studies were further analysed based on different parameters such as publication year, sample size, and outcomes.. A total of 432 studies were identified through the search strategy. A total of 306 records were removed before screening either because of duplication or marked ineligible by the automation tools. The screened records were 126 out of which 104 were removed as they were not conducted on OSCC. Twenty-two reports were sought for retrieval, however, we could not find the full text of 3 studies and12 studies were excluded because the biomarkers were not associated with cancer stem cells. The most common cancer stem cell biomarkers associated with OSCC were MCT1,VEGF-A, GD15, HIF1 α, Ki67, Hsp 70, Cyclin D1, and CD44.. Various stem cell biomarkers have been found to have diagnostic and prognostic role in oral squamous cell carcinoma such as Cyclin D1, VEGF-A, GD15, and CD44. They can be used to predict the overall survival rate, local progression-free survival rate, and distant metastasis-free survival rate in Head and Neck cancer patients.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Neoplastic Stem Cells; Prognosis; Squamous Cell Carcinoma of Head and Neck; Vascular Endothelial Growth Factor A

To evaluate published evidence on the predictive value of CCND1 amplification/cyclin D1 overexpression as malignant transformation risk markers in potentially malignant disorders (PMDs) of the head and neck.. We searched PubMed, Embase, Web of Science, and Scopus for studies published before June 2018. We conducted a meta-analysis to quantify the impact of CCND1/cyclin D1 amplification/overexpression on malignant transformation of head and neck PMDs.. CCND1/cyclin D1 amplification/overexpression is important to predict the malignant transformation risk of head and neck PMDs, especially oral PMDs.

Topics: Cell Transformation, Neoplastic; Cyclin D1; Gene Amplification; Head and Neck Neoplasms; Humans; Predictive Value of Tests

The correlation between cyclin D1 overexpression and the clinical outcome of head and neck cancer is not defined. The aim of this meta-analysis was to evaluate the prognostic value of cyclin D1 in patients with head and neck cancer. A search thorough Ovid MEDLINE was performed to enroll all eligible articles. Twenty-two studies comprising a total of 1,929 patients with different head and neck cancers were included. Cyclin D1 overexpression was significantly associated with lymph node metastasis [OR 2.25; 95 % confidence interval (CI) 1.76-2.87] and worse disease-free survival (OR 3.06; 95 % CI 2.42-3.87]. Subgroup analysis revealed that cyclin D1 overexpression correlated significantly with nodal metastasis for laryngeal cancer (OR 2.26; 95 % CI 1.61-3.16) and was a significant poor predictor for nasopharyngeal cancer (OR 4.44; 95 % CI 1.89-10.42). Our meta-analysis suggests that cyclin D1 overexpression could represent an important prognostic indicator for patients with head and neck cancer.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Disease-Free Survival; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Genetic Markers; Global Health; Head and Neck Neoplasms; Humans; Prognosis; Squamous Cell Carcinoma of Head and Neck; Survival Rate

Biomarkers are increasingly being used in many cancers to select patients for oncological treatment paradigms based on their inherent genetic properties. However, in head and neck cancers, there are no personalised therapies available outside the context of a clinical trial. A number of studies suggest there are intrinsic tumour properties of head and neck cancers that affect their response to chemotherapeutic agents. This paper aimed to review their evidence base.. A narrative review was conducted following a search of the PubMed database.. The review identified a number of biomarkers predicting response to chemotherapy in head and neck cancers. The paper discusses these in detail, and explores where future research could be directed in order to deliver personalised therapies for patients with head and neck cancers.

Topics: Antineoplastic Agents; bcl-X Protein; Biomarkers, Tumor; Carcinoma, Squamous Cell; Clinical Trials as Topic; Cyclin D1; ErbB Receptors; Evidence-Based Medicine; Gene Expression Regulation; Glutathione Transferase; Head and Neck Neoplasms; Humans; Mutation; Polymorphism, Genetic; Predictive Value of Tests; Prognosis; Randomized Controlled Trials as Topic; Sensitivity and Specificity; Tubulin; Tumor Suppressor Protein p53

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy that continues to be difficult to treat and cure. In many organ systems and tumor types, there have been significant advances in the understanding of the molecular basis for tumorigenesis, disease progression and genetic implications for therapeutics. Although tumorigenesis pathways and the molecular etiologies of HNSCC have been extensively studied, there are still very few diagnostic clinical applications used in practice today. This review discusses current clinically applicable molecular markers, including viral detection of Epstein-Barr virus and human papillomavirus, and molecular targets that are used in diagnosis and management of HNSCC. The common oncogenes EGFR, RAS, CCND1, BRAF, and PIK3CA and tumor suppressor genes p53, CDKN2A and NOTCH are discussed for their associations with HNSCC. Discussion of markers with potential future applications is also included, with a focus on molecular alterations associated with targeted therapy resistance.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Epstein-Barr Virus Infections; Gene Expression Regulation, Neoplastic; Genes, erbB-1; Genes, p16; Genes, p53; Genes, ras; Head and Neck Neoplasms; Humans; Papillomavirus Infections; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins B-raf; Receptors, Notch; Squamous Cell Carcinoma of Head and Neck

Whether cyclin D1 (CCND1) gene variants increase susceptibility to head and neck cancer (HNC) is undetermined. Therefore, we performed the present meta-analysis to systematically assess any possible association between CCND1 variants (G870A and G1722C) and HNC risk. Seventeen studies for CCND1 G870A and three studies for CCND1 G1722C were included. Overall, CCND1 polymorphisms (G870A and G1722C) had no association with increased HNC risk (p>0.05). In the subgroup analysis by smoking status, significantly increased HNC risk was found among smokers under allele contrast, homozygous comparison and recessive models (p<0.05), smoking carriers of A allele and AA genotype appearing at elevated risk. In conclusion, while there was overall a lack of any association between CCND1 polymorphisms (G870A and G1722C) and HNC risk, smokers carrying the A allele and AA genotype of the CCND1 G870A polymorphism may be susceptible to HNC development.

Topics: Cyclin D1; Female; Genes, bcl-1; Genetic Predisposition to Disease; Head and Neck Neoplasms; Humans; Male; Polymorphism, Single Nucleotide; Smoking

CCND1 plays a critical role in cell cycle control and may contribute to head and neck cancer. We performed a meta-analysis of eleven case-control studies that examined the association between CCND1 G870A polymorphism and head and neck cancer risk. Overall, no significant association of this polymorphism with head and neck cancer was found (for AA vs. GG: OR=0.96, 95% CI=0.59-1.58, P<0.01 for heterogeneity; for GA vs. GG: OR=1.00, 95% CI=0.74-1.35, P<0.01 for heterogeneity; for the dominant model GA/AA vs. GG: OR=0.98, 95% CI=0.69-1.39, P<0.01 for heterogeneity; for the recessive model AA vs. GG/GA: OR=0.94, 95% CI=0.66-1.33, P<0.01 for heterogeneity). In subgroup analysis by ethnicity, we also did not find any significant association in European and Asians populations. All the results were not materially altered in any genetic model after the studies which did not fulfill Hardy-Weinberg equilibrium were excluded. In conclusion, our meta-analysis strongly suggested that the CCND1 G870A polymorphism is not associated with head and neck cancer risk.

Topics: Case-Control Studies; Cyclin D1; Genetic Predisposition to Disease; Genotype; Head and Neck Neoplasms; Humans; Polymorphism, Single Nucleotide; Risk Factors

Head and neck squamous cell carcinoma is the sixth most common type of neoplasm worldwide, but its prognosis has not improved significantly in recent years. Therefore, efforts need to be intensified to gain a better understanding of this disease and develop novel treatment strategies. Inhibition of cytidine 59-diphosphate 1,2-diacyl-sn-glycerol: inositol transferase by inostamycin, an antibiotic isolated from Streptomyces sp. MH816-AF15, induces G1 cell cycle arrest accompanied by a decrease in cyclin D1 and phosphorylated RB protein levels, along with suppression of in vitro invasive ability through reduced production of matrix metalloproteinases (MMP-2 and MMP-9) and cell motility in head and neck cancer cell lines. Furthermore, inostamycin abrogated the stimulatory effect of VEGF (vascular endothelial growth factor) on growth and migration activities of endothelial cells by targeting extracellular signal-regulated kinase-cyclin D1 and p38 pathways, respectively. Because inostamycin has both antiproliferative and anti-invasive abilities, inhibition of phosphatidylinositol synthesis could be a potent therapeutic strategy for head and neck cancer as the 'cancer dormant therapy', i.e. a therapeutic concept to prolong 'time to treatment failure' or 'time to progression'.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cyclin D1; Furans; G1 Phase; Head and Neck Neoplasms; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Phosphatidylinositols; Vascular Endothelial Growth Factor A

Cancer is the result of sequential genetic changes over time that transform a cell into a malignant and ultimately invasive entity. The insight that cancerous cells arise from a series of mutations in oncogenes and tumor suppressors, commonly known as multistep carcinogenesis, has been conceptually elaborated and proven in the last 20 years. Although knowledge about late steps of cancerogenesis and disease progression has greatly advanced, the initial molecular events remain largely unknown. Basic research in Drosophila has started the quest to find early markers that detect initial clonal expansion of precancerous cells. These efforts were spurred by novel findings demonstrating that certain mutations transform cells into super-competitors that expand at the expense of the surrounding epithelial cells without inducing histological changes. This mechanism, discovered as super competition in the fly, might also lie at the heart of a clinical observation termed 'field cancerization'. This review aims to bring together current understanding from basic research on cell competition and clinical studies that have analyzed field characteristics to highlight parallels and possible connections.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Drosophila; Genes, Tumor Suppressor; Genetic Markers; Head and Neck Neoplasms; Humans; Loss of Heterozygosity; Models, Biological; Neoplasms; Oncogenes; Precancerous Conditions

Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma with a poor prognosis that may be confused with less aggressive diseases, such as small lymphocytic lymphoma and follicular lymphoma. In many cases immunophenotyping, particularly analysis of reactivity for CD5 and CD10, is an important adjunct to morphology that usually distinguishes MCL from follicular lymphoma; the former is CD5(+)/CD10(-), whereas follicular lymphoma is the reverse. We report a case of MCL, initially diagnosed as follicular lymphoma, that at presentation expressed neither CD5 nor CD10. At relapse, it was still CD5(-), but CD10 was now detected. Studies for a t(11;14) translocation and CYCLIN D1 protein expression, however, permitted a revised diagnosis of MCL. An MCL with this immunophenotype and classical morphology has not been previously reported.

Topics: Aged; CD5 Antigens; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Diagnosis, Differential; Female; Head and Neck Neoplasms; Humans; Immunophenotyping; Lymphoma, Mantle-Cell; Neprilysin; Translocation, Genetic

Head and neck squamous cell carcinoma affects more than 500,000 people worldwide each year. Despite optimal treatment with surgery, irradiation, and chemotherapy, disease recurrence and progression remains a common and challenging oncological problem. Recently, interest has developed in identifying novel molecular markers that allow identification of those patients at increased risk for locoregional recurrence and death. This article reviews several such molecular markers studied in head and neck cancer, including p53, angiogenesis-related markers, cyclin D1, and epidermal growth factor receptor. The biological function of these markers and the potential clinical implications are discussed. The purpose of this review is to update the otolaryngologist on a rapidly emerging segment of applied translational research in our field.

Topics: Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Genes, p53; Genetic Markers; Head and Neck Neoplasms; Humans; Neovascularization, Pathologic; Prognosis

A central issue in the study of neoplastic transformation is to understand how proto-oncogene products deregulate normal processes of cell growth and differentiation: an intrinsic aspect of this is to probe the sequence of events leading to altered expression of proto-oncogenes. In the past few years, studies aimed at understanding the regulation and function of protein synthesis initiation factors, eIF4E initially, culminated in the unexpected finding that a moderate overexpression of this factor results in dramatic phenotypic changes, including rapid proliferation and malignant transformation. Conversely, the tumorigenic properties of cancer cells can be strongly inhibited by antisense-RNA against eIF4E, or overexpression of the inhibitory proteins: 4E-BPs. Furthermore, eIF4E is elevated in carcinomas of the breast, head and neck (HNSCC) and prostate, but not in typical benign lesions. This is a strong indication that elevated eIF4E expression may mark a critical transition in cancer progression. Establishing a greater protein synthesis output may be a necessary step for cancer cells in order to sustain their rapid proliferation. However, analysis of cells transformed by eIF4E revealed that the synthesis of only a few proteins was greatly enhanced, while synthesis of most was minimally increased. One possible explanation is that eIF4E causes these effects by specifically increasing the translational efficiency of several oncogene transcripts, leading to overexpression of their products. The feasibility of this hypothesis was confirmed experimentally with the identification of several important products that are specifically upregulated in eIF4E-overexpressing cells. These include: c-Myc, cyclin DI and ODC, which control cycle progression and tumorigenesis; basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF), which are powerful promoters of cell growth and angiogenesis. A deeper understanding of the mRNAs that are strongly dependent on excess eIF4E/F for efficient translation will eventually result in fuller understanding of the fundamental role of translational control in different pathophysiological conditions, including malignancy.

Topics: Animals; Apoptosis; Breast Neoplasms; Carcinoma; Cell Division; Cyclin D1; Disease Progression; Endothelial Growth Factors; Eukaryotic Initiation Factor-4E; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Genes, myc; Head and Neck Neoplasms; Humans; Lymphokines; Metalloendopeptidases; Neoplasm Proteins; Neoplasms; Peptide Initiation Factors; Proto-Oncogene Mas; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Squamous cell carcinoma of the head and neck affects more than 500,000 people worldwide each year. Local-regional recurrence of disease is a common and challenging oncological problem in patients affected by this disease. Identification of risk factors for local relapse after appropriate local therapy with surgery, radiation, or combination therapy remains an active area of clinical research. The recent development of novel molecular markers has resulted in numerous studies evaluating the prognostic significance and potential clinical utility of these markers in identifying patients at risk for local-regional relapse. This article reviews recent studies evaluating molecular markers, including p53, angiogenesis-related markers, cyclin D1, epidermal growth factor receptor, loss of heterozygosity, DNA ploidy, and cell kinetic markers. The potential clinical utility of these markers and future directions along this avenue of investigation are discussed.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; DNA, Neoplasm; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Male; Neoplasm Recurrence, Local; Prognosis; Radiation Tolerance; Sensitivity and Specificity; Tumor Suppressor Protein p53

As new knowledge of the molecular mechanisms of tumor development and progression is gained, studies are required to determine whether specific molecular genetic changes might be useful indicators in selecting patients for specific therapies. Alternatively, molecular or genetic changes may identify the patients at greatest risk for progression and relapse so that they can be treated with adjuvant therapy. p53 has an important function in the regulation of the cell cycle, DNA repair, and programmed cell death pathways. The Bcl-2 protein family has gained recent attention for its role in permitting or blocking apoptosis. Interaction between p53, Bcl-2, and other products of tumor suppressor genes or oncogenes are probably critical in tumor progression and response to treatment.

Topics: Apoptosis; Biomarkers, Tumor; Cell Cycle; Cyclin D1; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; Neoplasm Staging; Predictive Value of Tests; Prognosis; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

The significance of prognostic factors that may predict the clinical outcome of patients with head and neck cancer was discussed. Many indicators can be grouped into three categories, patient factors, tumor factors and treatment factors. The most significant indicator of prognosis seems to be pathological nodal stage. Factors such as clinical stage, resectability, and depth of invasion may also affect the patient outcome. Recent research development has revealed biological phenotypes of cancer cells to predict the effect of cancer treatment and the clinical course in head and neck cancer. Possible predictive indicators include DNA ploidy, Tpot, EGFR and cyclin D1. C erbB2 and p53 may not predict the survival of patients with head and neck cancer.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclins; DNA, Neoplasm; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Oncogene Proteins; Ploidies; Prognosis

Topics: Cadherins; Chromosome Aberrations; Cyclin D1; ErbB Receptors; Gene Expression; Genes, myc; Genes, p53; Genes, ras; Head and Neck Neoplasms; Humans; Mutation; Oncogenes; Papillomaviridae; Proto-Oncogene Proteins; Receptor, ErbB-2

To determine the prognostic and predictive values of molecular marker expression profiles based on data from a randomized clinical trial of postoperative conventional fractionation (p-CF) therapy versus 7-day-per-week postoperative continuous accelerated irradiation (p-CAIR) therapy for squamous cell cancer of the head and neck.. Tumor samples from 148 patients (72 p-CF and 76 p-CAIR patients) were available for molecular studies. Immunohistochemistry was used to assess levels of EGFR, nm23, Ki-67, p-53, and cyclin D1 expression. To evaluate the effect of fractionation relative to the expression profiles, data for locoregional tumor control (LRC) were analyzed using the Cox proportional hazard regression model. Survival curves were compared using the Cox f test.. Patients who had tumors with low Ki-67, low p-53, and high EGFR expression levels and oral cavity/oropharyngeal primary cancer sites tended to benefit from p-CAIR. A joint score for the gain in LRC from p-CAIR based of these features was used to separate the patients into two groups: those who benefited significantly from p-CAIR with respect to LRC (n = 49 patients; 5-year LRC of 28% vs. 68%; p = 0.01) and those who did not benefit from p-CAIR (n = 99 patients; 5-year LRC of 72% vs. 66%; p = 0.38). The nm23 expression level appeared useful as a prognostic factor but not as a predictor of fractionation effect.. These results support the studies that demonstrate the potential of molecular profiles to predict the benefit from accelerated radiotherapy. The molecular profile that favored accelerated treatment (low Ki-67, low p-53, and high EGFR expression) was in a good accordance with results provided by other investigators. Combining individual predictors in a joint score may improve their predictive potential.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Ki-67 Antigen; Laryngeal Neoplasms; Male; Middle Aged; Mouth Neoplasms; Neoplasm Recurrence, Local; NM23 Nucleoside Diphosphate Kinases; Oropharyngeal Neoplasms; Proportional Hazards Models; Radiotherapy; Radiotherapy Dosage; Risk Factors; Tumor Suppressor Protein p53

The current study aimed to evaluate the significance of the cell-cycle-control proteins cyclin D1 and p16 as prognostic markers in head and neck squamous cell carcinoma (HNSCC) patients treated with docetaxel and radiotherapy.. Cyclin D1 and/or p16 protein expression was retrospectively evaluated by immunohistochemistry in 53 patients with stage T1-3N0-2M0 (except T1N0 glottis) HNSCC who were treated with 10 mg/m(2)/week docetaxel 4 to 6 times and received concurrent chemoradiotherapy.. Kaplan-Meier univariate analysis revealed that patients with cyclin D1-positive tumors or p16-negative tumors had a worse prognosis compared with those with cyclin D1-negative tumors or p16-positive tumors (p = .0004 and p = .025, respectively). The prognostic significance of cyclin D1 expression, not p16 expression, was confirmed using a proportional hazard regression model.. An assessment of cyclin D1 and p16 levels might be of clinical use in defining subgroups of patients with poor prognosis.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Docetaxel; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Prognosis; Proportional Hazards Models; Radiotherapy, Adjuvant; Taxoids

The epidermal growth factor receptor (EGFR) and cyclin D1 are overexpressed in lung carcinogenesis. The rexinoid, bexarotene, represses cyclin D1 and EGFR expression in vitro. It was hypothesized that combining bexarotene with the EGFR inhibitor, erlotinib, would augment clinical activity.. In vitro studies and a phase I clinical trial were performed. Twenty-four patients with advanced aerodigestive tract cancers were enrolled; 79% had non-small-cell lung cancer (NSCLC). The primary objective was to determine the maximum-tolerated dose. Clinical activity was a secondary objective.. Combining erlotinib with bexarotene enhanced growth suppression in vitro compared with each single-agent treatment. This cooperatively repressed cyclin D1 expression. Clinically, the most frequent toxicities were mild hypertriglyceridemia and skin rash. Two serious treatment-related adverse events occurred (creatine phosphokinase elevation attributed to antilipid therapy and a case of generalized pain). Five objective responses (four partial and one minor) were observed in NSCLC patients. Responses were observed in males and smokers. EGFR sequence analyses did not reveal activating mutations in tumors from assessable responding patients. Median time to progression was 2.0 months; overall survival time was 14.1 months; and 1-year survival rate was 73.8%.. The recommended phase II doses are erlotinib 150 mg/d and bexarotene 400 mg/m2/d orally. These agents can be administered in combination at the recommended single-agent doses without added toxicity. Overall survival and clinical features of responding patients differ from prior reports of single-agent erlotinib treatment. These findings are encouraging and warrant further investigation of this regimen.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bexarotene; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cell Survival; Cyclin D1; Digestive System Neoplasms; ErbB Receptors; Erlotinib Hydrochloride; Exanthema; Female; Head and Neck Neoplasms; Humans; Hypertriglyceridemia; Lung Neoplasms; Male; Middle Aged; Mouth Mucosa; Protein Kinase Inhibitors; Quinazolines; Sequence Analysis, DNA; Tetrahydronaphthalenes; Treatment Outcome; Tumor Cells, Cultured

Identification of factors that assist prediction of tumor response to radiotherapy may aid in refining treatment strategies and improving outcome. Possible association of molecular marker expression profiles with locoregional control of head and neck squamous cell carcinoma was investigated in a randomized trial of conventional versus continuous hyperfractionated accelerated radiotherapy (CHART).. Tumor material was obtained from 402 patients. Immunohistochemistry was used to assess Ki-67, CD31, p53, Bcl-2, and cyclin D1 expression. A hierarchical clustering algorithm with a Bayesian information criterion was used to group tumors with similar marker expression; resulting expression profiles were then compared in terms of their difference in outcome after CHART and conventionally fractionated radiotherapy.. Molecular marker profile was an independent prognostic factor for locoregional control. This was confirmed in multivariate analysis, including clinical variables such as tumor and nodal status, primary site, histological grade, age, and gender (P < 0.001 and P = 0.006 for local and nodal relapse, respectively). In particular, Bcl-2-positive tumors responded significantly better than average in both arms of the trial. Tumors negative for p53- and Bcl-2, with high and randomly patterned Ki-67 expression, responded worse than average with no benefit from CHART. Tumors with similarly negative p53 and Bcl-2, but low Ki-67 staining, with an organized pattern, benefit significantly from CHART schedule.. This study demonstrates the potential of molecular profiles to predict radiotherapy response of head and neck squamous cell carcinoma and for treatment stratification. Distinct expression profiles correlate with three distinct clinical phenotypes, including good locoregional control, poor locoregional control, and an outcome strongly dependent upon fractionation schedule.

Topics: Algorithms; Bayes Theorem; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cluster Analysis; Cyclin D1; Dose Fractionation, Radiation; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Platelet Endothelial Cell Adhesion Molecule-1; Proportional Hazards Models; Proto-Oncogene Proteins c-bcl-2; Radiotherapy; Radiotherapy Dosage; Statistics as Topic; Time Factors; Tumor Suppressor Protein p53

The aim of this study was to investigate the value of p53 and cyclin D1 gene expression in predicting the risk of occult lymph node metastases in patients with head and neck squamous cell carcinoma (HNSCC).. The expression of cyclin D1 and p53 was evaluated by means of immunohistochemical analysis in 32 HNSCC patients with clinically and radiologically negative lymph nodes in whom metastatic involvement was subsequently demonstrated at histologic examination (pN+). A group of 64 head and neck cancer patients with histologically negative laterocervical lymph nodes (pN0) was used as a control.. Cyclin D1 and p53 expression were observed respectively in 42 (43.7%) and 48 cases (50%). Cyclin D1 expression significantly correlated with tumor extension and advanced clinical stage (p =.002 and p =.001, respectively). At univariate regression analysis, cyclin D1 expression significantly correlated with the presence of occult lymph node metastases (p =. 0007), and it remained an independent predictor at multivariate regression analysis (p =.0059).. Our study indicates that the expression of cyclin D1 correlates with the presence of occult cervical metastases in head and neck carcinoma patients, thus suggesting that its immunohistochemical evaluation in biopsy samples may be used as an additional tool for identifying patients to be treated with elective neck dissection.

Topics: Adult; Aged; Analysis of Variance; Biomarkers, Tumor; Biopsy, Needle; Carcinoma, Squamous Cell; Cyclin D1; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Logistic Models; Lymph Node Excision; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Predictive Value of Tests; Sensitivity and Specificity; Survival Rate; Tumor Suppressor Protein p53

While 15 to 20% of cancers are associated with microbial infection, the relationship between oral microorganisms and oral squamous cell carcinoma (OSCC) remains unclear. The location of bacteria in a tumor is closely related to its carcinogenic mechanism. The aim of this study was to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues, locate target bacteria, and search for proteins that may interact with target bacteria.. The 16S rDNA method was used to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues. Correlations between Fusobacterium abundance and clinicopathological characteristics were analysed using the χ2 test. The position of target bacteria was analysed by fluorescence in situ hybridization (FISH), and the expression of CK, CD31, CD45, CD68, cyclin D1, β-catenin, E-cadherin, NF-κB, and HIF-1α was analysed by immunohistochemistry (IHC) in OSCC tumor tissues and tumor distant normal tissues.. The 16S rDNA results showed that the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues. High expression of Fusobacterium was significantly correlated with the lifestyle-related oral risk habits, including smoking (p=0.036) and alcohol consumption (p=0.022), but did not correlate with patient sex, age, tumor laterality, tumor size, grade or TNM stage. Fusobacterium nucleatum was enriched in tumor stroma, where CD31+ blood vessels and inflammatory cells (including CD45+ leukocytes and CD68+ macrophages) were densely distributed. Cyclin D1 was mainly expressed in the nucleus of tumor cells. β-catenin was expressed in the tumor cell membrane and was positively expressed in tumor interstitial vascular endothelial cells. E-cadherin was mainly expressed in tumor cell membranes. NF-κB was positively expressed in the cytoplasm of tumor cells, tumor interstitial cells and myo-fibrocytes. HIF-1α was mainly expressed in the cytoplasm of tumor interstitial cells. HIF-1α was highly expressed where Fusobacterium nucleatum was densely distributed.. According to our study, the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues, and Fusobacterium nucleatum might aggravate inflammation and hypoxia by interacting with NF-κB and HIF-1α in OSCC.

Topics: beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cyclin D1; DNA, Ribosomal; Endothelial Cells; Fusobacterium nucleatum; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Mouth Neoplasms; NF-kappa B; Squamous Cell Carcinoma of Head and Neck

The aim of this study was to determine, by immunohistochemical methods, the expression of nEGFR and markers of cell proliferation (Ki-67), cell cycle (mEGFR, p53, cyclin D1), and tumor stem cells (ABCG2) in 59 pathohistological samples of healthy oral mucosa, 50 oral premalignant changes (leukoplakia and erythroplakia), and 52 oral squamous cell carcinomas (OSCC). An increase in the expression of mEGFR and nEGFR was found with the development of the disease (

Topics: Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Head and Neck Neoplasms; Humans; Ki-67 Antigen; Leukoplakia; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; Tumor Suppressor Protein p53

Head and neck squamous cell carcinoma (HNSC) is one of the leading causes of cancer death globally, yet there are few useful biomarkers for early identification and prognostic prediction. Previous studies have confirmed that CCND1 amplification is closely associated with head and neck oncogenesis, and the present study explored the ceRNA network associated with CCND1. Gene expression profiling of the Head and Neck Squamous Cell Carcinoma (HNSC) project of The Cancer Genome Atlas (TCGA) program identified the TPRG1-AS1-hsa-miR-363-3P-MYO1B gene regulatory axis associated with CCND1. Further analysis of the database showed that MYOB was regulated by methylation in head and neck tumors, and functional enrichment analysis showed that MYO1B was involved in "actin filament organization" and "cadherin binding ". Immune infiltration analysis suggested that MYO1B may influence tumorigenesis and prognosis by regulating the immune microenvironment of HNSC. MYO1B enhanced tumor spread through the EMT approach, according to epithelial mesenchymal transition (EMT) characterisation. We analyzed both herbal and GSCALite databases and found that CCND1 and MYO1B have the potential as predictive biomarkers for the treatment of HNSC patients. RT-qPCR validated bioinformatic predictions of gene expression in vitro cell lines. In conclusion, we found a CCND1-related ceRNA network and identified the novel TPRG1-AS1-hsa-miR-363-3p-MYO1B pathway as a possible HNSC diagnostic biomarker and therapeutic target.

Topics: Carcinogenesis; Cell Transformation, Neoplastic; Critical Pathways; Cyclin D1; Head and Neck Neoplasms; Humans; MicroRNAs; Myosin Type I; Prognosis; RNA, Long Noncoding; Squamous Cell Carcinoma of Head and Neck; Tumor Microenvironment

The aberrant upregulation of MCTS1 Re-Initiation and Release Factor (also known as Malignant T-cell-amplified sequence 1, MCTS1) can promote laryngeal squamous cell carcinoma (LSCC). It might act as a binding partner of multiple proteins. In this study, we further explored the expression of potential interaction between MCTS1 and OTU domain-containing protein 6B (OTUD6B) and its influence on the ubiquitination and degradation of OTUD6B's substrate in LSCC. LSCC cell lines AMC-HN-8 and TU177 were utilized for assessing protein-protein interaction, protein degradation and tumor growth in vitro and in vivo. The results showed that MCTS1 interacts with OUTD6B isoform 1 (OTUD6B-1) in the cell lines. Higher OTUD6B-1 expression is associated with significantly shorter progression-free interval in LSCC patients. OTUD6B positively modulated the expression of cyclin D1, cyclin E1 and c-Myc and LSCC cell proliferation in vitro and in vivo. MCTS1 negatively modulated the degradation of LIN28B in G1/S cells, via enhancing OTUD6B-mediated cleaving of K48-branched ubiquitin chains from LIN28B. OTUD6B or LIN28B shRNA weakened MCTS1 overexpression-induced cyclin D1 and c-Myc protein expression and LSCC cell proliferation. In summary, this study revealed that MCTS1 could enhance LSCC proliferation partially via the OTUD6B-LIN28B axis.

Topics: Cell Cycle Proteins; Cell Line; Cell Proliferation; Cyclin D1; Head and Neck Neoplasms; Humans; Oncogene Proteins; RNA-Binding Proteins; Squamous Cell Carcinoma of Head and Neck

We aimed to establish image recognition and survival prediction models using a novel scoring system of cyclin D1 expression pattern in patients with human papillomavirus-negative oral or oropharyngeal squamous cell carcinoma.. The clinicopathological data of 610 patients with human papillomavirus-negative oral/oropharyngeal squamous cell carcinoma were analyzed retrospectively. Cox univariate and multivariate risk regression analyses were performed to compare cyclin D1 expression pattern scoring with the traditional scoring method-cyclin D1 expression level scoring-in relation to patients' overall and progression-free survival. An image recognition model employing the cyclin D1 expression pattern scoring system was established by YOLOv5 algorithms. From this model, two independent survival prediction models were established using the DeepHit and DeepSurv algorithms.. Cyclin D1 had three expression patterns in oral and oropharyngeal squamous cell carcinoma cancer nests. Superior to cyclin D1 expression level scoring, cyclin D1 expression pattern scoring was significantly correlated with the prognosis of patients with oral squamous cell carcinoma (p < 0.0001) and oropharyngeal squamous cell carcinoma (p < 0.05). Moreover, it was an independent prognostic risk factor in both oral squamous cell carcinoma (p < 0.0001) and oropharyngeal squamous cell carcinoma (p < 0.05). The cyclin D1 expression pattern-derived image recognition model showed an average test set accuracy of 78.48% ± 4.31%. In the overall survival prediction models, the average concordance indices of the test sets established by DeepSurv and DeepHit were 0.71 ± 0.02 and 0.70 ± 0.01, respectively.. Combined with the image recognition model of the cyclin D1 expression pattern, the survival prediction model had a relatively good prediction effect on the overall survival prognosis of patients with human papillomavirus-negative oral or oropharyngeal squamous cell carcinoma.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Deep Learning; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Oropharyngeal Neoplasms; Papillomavirus Infections; Prognosis; Retrospective Studies; Squamous Cell Carcinoma of Head and Neck

As an important member of Wnt/β-catenin signaling pathway, the aberrant expression of β-catenin has been implicated in many cancers. Chibby, a β-catenin binding partner, is an antagonist involved in this pathway. In contrast, thyroid cancer 1 (TC1) as an activator of this pathway can relieve the antagonistic activity of Chibby on the β-catenin-mediated transcription and is high expressed in human tumors. The objectives of this study were to examine the expression of TC1, Chibby, and β-catenin and investigate the association among them in laryngeal squamous cell carcinoma (LSCC). The expression of TC1, Chibby, β-catenin, c-Myc, Cyclin D1, and matrix metalloproteinase-7 (MMP-7) were examined by immunohistochemistry in samples from 53 LSCC patients. Compared with normal laryngeal squamous epithelium (NLSE), there were upregulated expression of TC1, downregulated expression of Chibby, and aberrant cytoplasmic expression of β-catenin in the LSCC tissues (P < .001). The high expression of TC1 was correlated with the tumor site, advanced TNM and T stage, lymphovascular invasion, and poor differentiation in LSCC tissues (P < .050). There were correlations between the aberrant expression of β-catenin and the tumor site, advanced TNM and T stage, lymphovascular invasion, perineurial invasion, and poor differentiation in LSCC tissues (P < .050). Upregulated TC1 and downregulated Chibby were correlated with aberrant expression of β-catenin (P < .001), but no correlation between them (P = .076). The percent of abnormal expression of β-catenin in LSCC was 96.00% in TC1+/Chibby-, 73.68% in TC1+/Chibby+, 0.00% in TC1-/Chibby-, and 0.00% in TC1-/Chibby + group (P < .001). High expression of c-Myc, Cyclin D1, and MMP-7 was observed in LSCC tissues (P < .001). There was statistically significant about the expression of Cyclin D1 and MMP-7 among the groups of TC1+/Chibby-, TC1+/Chibby+, TC1-/Chibby-, and TC1-/Chibby + (P < .001), but was not significance about the expression of c-Myc among them (P = .339). No association was found between overall survival and the expression of TC1, Chibby, and β-catenin (P > .05). The upregulated expression of TC1 and downregulated expression of Chibby were correlated with the aberrant expression of β-catenin and the high expression of Cyclin D1 and MMP-7 in LSCC tissues.

Topics: beta Catenin; Carrier Proteins; Cyclin D1; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; Matrix Metalloproteinase 7; Nuclear Proteins; Squamous Cell Carcinoma of Head and Neck

This study investigated the role of lncRNA CASC15 in laryngeal squamous cell carcinoma (LSCC).. This study included 58 LSCC patients. Both tumor (LSCC) and adjacent (within 3 cm around tumors) non-tumor tissues from 3 different sites of each patient were collected. CCK-8 assay was used to determine cell proliferation. The expression levels of proteins and mRNAs were determined by Western blotting analysis and qRT-PCRs, respectively.. CASC15 was upregulated in LSCC and high expression levels of CASC15 predicted poor survival. In LSCC tissues, CASC15 was negatively correlated with miR-365 but positively correlated with cyclin D1. In LSCC cells, overexpression of CASC15 resulted in downregulation of miR-365 and upregulation of cyclin D1. Overexpression of miR-365 did not affect the expression of CASC15 but downregulated cyclin D1. Overexpression of Cyclin D1 did not affect the expression of miR-365 and CASC15. Overexpression of CASC15 and cyclin D1 led to promoted, while overexpression of miR-365 led to inhibited LSCC cell proliferation. In addition, overexpression of miR-365 reduced the effects of overexpression of CASC15.. Therefore, CASC15 upregulates cyclin D1 by downregulating miR-365 in LSCC to promote cell proliferation.

Topics: Carcinoma, Squamous Cell; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; MicroRNAs; RNA, Long Noncoding; Squamous Cell Carcinoma of Head and Neck; Up-Regulation

Emerging evidence has demonstrated that long noncoding RNA (lncRNAs) play a vital role in the development of head and neck squamous cell carcinoma (HNSCC); however, the biological effects and underlying mechanisms of human leukocyte antigen complex group-18 HCG18 (HCG18) have not yet been reported in HNSCC. In this study, we detected the expression of the HCG18 in HNSCC cell lines and patient tissues. We observed that HCG18 was upregulated in HNSCC patient tissues and cell lines. Furthermore, silencing of HCG18 significantly inhibited proliferation, migration, and invasion of HNSCC cells, whereas the opposite effects were detected in the HCG18-overexpressed group. We also found that HCG18 directly binds to the functional protein cyclin D1. Upregulated cyclin D1 reversed the inhibitory effects of HCG18 in HNSCC cell lines and activated the WNT pathway-related proteins (AXIN2, survivin, c-Myc, and β-catenin) simultaneously. Knockdown of cyclin D1 could accelerate the inhibitory effects of HCG18 and decrease the expression of AXIN2, survivin, c-Myc, and β-catenin. This indicated that lncRNA HCG18 might be involved in the tumorigenesis of HNSCC via the cyclin D1-WNT pathway. These results suggest that lncRNA HCG18 could act as a promising prognostic biomarker and potential therapeutic target in HNSCC patients.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; RNA, Long Noncoding; Squamous Cell Carcinoma of Head and Neck; Survivin; Wnt Signaling Pathway

Periodontitis is a risk factor for the development of oral squamous cell carcinomas (OSCC). Both DNA damage response (DDR) and activation of inflammasomes induced by the microbiome might play important roles in the development of tumors, in relation to genome stability of tumor cells. Herein, we explored whether periodontitis negative-associated bacteria (

Topics: Animals; Corynebacterium; Cyclin D1; Genomic Instability; Head and Neck Neoplasms; Inflammasomes; Mice; Mouth Neoplasms; Neisseria sicca; NLR Family, Pyrin Domain-Containing 3 Protein; RNA, Messenger; Squamous Cell Carcinoma of Head and Neck

Dysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies.. We performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(-) HNSCC cell lines.. We found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3-selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1.. Our results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials.

Topics: Carcinoma, Squamous Cell; Cohort Studies; Cyclin D1; DNA Methylation; Fibroblast Growth Factors; Head and Neck Neoplasms; Humans; Papillomavirus Infections; Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Statistics, Nonparametric

In situ mantle cell neoplasia (isMCN) and leukemic non-nodal mantle cell lymphoma (nnMCL) are classified as an indolent subtype of mantle cell lymphoma (MCL). The tumor cells of isMCN are restricted to the inner layer of the lymphoid tissue mantle zone, exhibiting an in situ pattern histologically. On the other hand, nnMCL is distributed in the peripheral blood, bone marrow and sometimes the spleen, but lymphadenopathy or systemic organ involvement is rare. We report a case of isMCN in a submandibular lymph node resected from a 65-year-old Japanese male. The tumor cells were positive for cyclin D1 (CCND1) and SOX11 expression, and were restricted to the mantle zone area of the lymph node. However, tumor cells were also detected in the stomach mucosa, bone marrow tissue and peripheral blood, suggesting nnMCL. isMCN and nnMCL may have a partly overlapping disease spectrum, although the correlation between these two subtypes has not been well described. This present case demonstrated characteristics overlapping between isMCN and nnMCL.

Topics: Aged; Cyclin D1; Head and Neck Neoplasms; Humans; Lymph Nodes; Lymphoma, Mantle-Cell; Male; SOXC Transcription Factors

Despite advances in treatment, head and neck squamous cell carcinoma (HNSCC) remains difficult to treat and the overall survival rate has only modestly improved over the past years. Therefore, there is a need to understand the molecular mechanism of HNSCC. Zinc finger protein 703 (ZNF703) is an oncogenic transcription factor, and ZNF703 gene expression is altered in many cancers as a result of chromosome 8p12 amplification. The purpose of this study was to investigate the expression pattern of ZNF703 in HNSCC in association with CCND1 expression and Akt phosphorylation.. Prospective study.. University hospital.. One hundred and five patients with HNSCC.. Fifty HNSCC tumour and non-cancerous tissue samples were investigated by qRT-PCR and Western blotting.. ZNF703 gene expression was increased in 22.9% of tumour tissues compared with its normal counterparts. The results were correlated with clinicopathological features, copy number variation and survival data.. ZNF703 over-expression is associated with copy number variation and this over-expression may activate PI3K/Akt signalling pathway in HNSCC.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Carrier Proteins; Chromosomes, Human, Pair 8; Cyclin D1; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Phosphorylation; Prospective Studies; Proto-Oncogene Proteins c-akt

Head and neck squamous cell carcinoma (HNSCC) is a deadly disease in which precision medicine needs to be incorporated. We aimed to implement next-generation sequencing (NGS) in determining actionable targets to guide appropriate molecular targeted therapy in HNSCC patients.. Ninety-three tumors and matched blood samples underwent targeted sequencing of 244 genes using the Illumina HiSeq 2500 platform with an average depth of coverage of greater than 1,000×. Clinicopathological data from patients were obtained from 17 centers in Korea, and were analyzed in correlation with NGS data.. Ninety-two of the 93 tumors were amenable to data analysis. TP53 was the most common mutation, occurring in 47 (51%) patients, followed by CDKN2A (n=23, 25%), CCND1 (n=22, 24%), and PIK3CA (n=19, 21%). The total mutational burden was similar between human papillomavirus (HPV)-negative vs. positive tumors, although TP53, CDKN2A and CCND1 gene alterations occurred more frequently in HPV-negative tumors. HPV-positive tumors were significantly associated with immune signature-related genes compared to HPV-negative tumors. Mutations of NOTCH1 (p=0.027), CDKN2A (p < 0.001), and TP53 (p=0.038) were significantly associated with poorer overall survival. FAT1 mutations were highly enriched in cisplatin responders, and potentially targetable alterations such as PIK3CA E545K and CDKN2A R58X were noted in 14 patients (15%).. We found several targetable genetic alterations, and our findings suggest that implementation of precision medicine in HNSCC is feasible. The predictive value of each targetable alteration should be assessed in a future umbrella trial using matched molecular targeted agents.

Topics: Adult; Aged; Aged, 80 and over; Cadherins; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Feasibility Studies; Female; Genetic Predisposition to Disease; Head and Neck Neoplasms; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Molecular Targeted Therapy; Mutation; Papillomaviridae; Papillomavirus Infections; Receptor, Notch1; Republic of Korea; Sequence Analysis, DNA; Squamous Cell Carcinoma of Head and Neck; Tumor Suppressor Protein p53

Human immunodeficiency virus-infected individuals (HIVIIs) have a higher incidence of head and neck squamous cell carcinoma (HNSCC), and clinical and histopathological differences have been observed in their tumors in comparison with those of HNSCC patients without a human immunodeficiency virus (HIV) infection. The reasons for these differences are not clear, and molecular differences between HIV-related HNSCC and non-HIV-related HNSCC may exist. This study compared the mutational patterns of HIV-related HNSCC and non-HIV-related HNSCC.. The DNA of 20 samples of HIV-related HNSCCs and 32 samples of non-HIV-related HNSCCs was sequenced. DNA libraries covering exons of 18 genes frequently mutated in HNSCC (AJUBA, CASP8, CCND1, CDKN2A, EGFR, FAT1, FBXW7, HLA-A, HRAS, KEAP1, NFE2L2, NOTCH1, NOTCH2, NSD1, PIK3CA, TGFBR2, TP53, and TP63) were prepared and sequenced on an Ion Personal Genome Machine sequencer. DNA sequencing data were analyzed with Ion Reporter software. The human papillomavirus (HPV) status of the tumor samples was assessed with in situ hybridization, the MassARRAY HPV multiplex polymerase chain reaction assay, and p16 immunostaining. Mutation calls were compared among the studied groups.. HIV-related HNSCC revealed a distinct pattern of mutations in comparison with non-HIV-related HNSCC. TP53 mutation frequencies were significantly lower in HIV-related HNSCC. Mutations in HIV+ patients tended to be TpC>T nucleotide changes for all mutated genes but especially for TP53.. HNSCC in HIVIIs presents a distinct pattern of genetic mutations, particularly in the TP53 gene. HIV-related HNSCC may have a distinct biology, and an effect of the HIV virus on the pathogenesis of these tumors should not be ruled out. Cancer 2018;124:84-94. © 2017 American Cancer Society.

Topics: Adult; Aged; Cadherins; Carcinoma, Squamous Cell; Case-Control Studies; Caspase 8; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; ErbB Receptors; F-Box-WD Repeat-Containing Protein 7; Female; Head and Neck Neoplasms; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; HIV Infections; HLA-A Antigens; Humans; In Situ Hybridization; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; LIM Domain Proteins; Male; Middle Aged; NF-E2-Related Factor 2; Nuclear Proteins; Papillomaviridae; Papillomavirus Infections; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Receptor, Notch1; Receptor, Notch2; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Squamous Cell Carcinoma of Head and Neck; Transcription Factors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

We aimed at identifying deleterious genomic alterations from untreated head and neck squamous cell carcinoma (HNSCC) patients, and assessing their prognostic value.. We retrieved 122 HNSCC patients who underwent primary surgery. Targeted NGS was used to analyse a panel of 100 genes selected among the most frequently altered genes in HNSCC and potential therapeutic targets. We selected only deleterious (activating or inactivating) single nucleotide variations, and copy number variations for analysis. Univariate and multivariate analyses were performed to assess the prognostic value of altered genes.. A median of 2 (range: 0-10) genomic alterations per sample was observed. Most frequently altered genes involved the cell cycle pathway (TP53 [60%], CCND1 [30%], CDKN2A [25%]), the PI3K/AKT/MTOR pathway (PIK3CA [12%]), tyrosine kinase receptors (EGFR [9%], FGFR1 [5%]) and cell differentiation (FAT1 [7%], NOTCH1 [4%]). TP53 mutations (p = 0.003), CCND1 amplifications (p = 0.04), CDKN2A alterations (p = 0.02) and FGFR1 amplifications (p = 0.003), correlated with shorter overall survival (OS). The number of genomic alterations was significantly higher in the HPV-negative population (p = 0.029) and correlated with a shorter OS (p < 0.0001). Only TP53 mutation and FGFR1 amplification status remained statistically significant in the multivariate analysis.. These results suggest that genomic alterations involving the cell cycle (TP53, CCND1, CDKN2A), as well as FGFR1 amplifications and tumour genomic alterations burden are prognostic biomarkers and might be therapeutic targets for patients with HNSCC.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chi-Square Distribution; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; DNA Copy Number Variations; Female; Gene Amplification; Gene Expression Profiling; Genetic Predisposition to Disease; Head and Neck Neoplasms; High-Throughput Nucleotide Sequencing; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Phenotype; Polymorphism, Single Nucleotide; Predictive Value of Tests; Proportional Hazards Models; Receptor, Fibroblast Growth Factor, Type 1; Retrospective Studies; Risk Factors; Squamous Cell Carcinoma of Head and Neck; Time Factors; Transcriptome; Treatment Outcome; Tumor Suppressor Protein p53; Young Adult

Head and neck cancers include carcinomas of the oral cavity, larynx, sinonasal tract and nasopharynx. Studies on molecular expression of prognostic tumour markers in Ghana are scarce. The purpose of this study was to determine the expression of p53, p16, EGFR, Cyclin-D1 and HER2 among patients with non-oropharyngeal head and neck squamous cell carcinoma (HNSCC).. Tissue microarrays from 154 histologically confirmed non-oropharyngeal HNSCC at the Komfo Anokye Teaching Hospital from 2006-2014 were constructed using duplicate cores of representative and viable areas from tumours. Expression of EGFR, p53, p16, Cyclin-D1 and HER2 was evaluated using immunohistochemistry.. For non-oropharyngeal HNSCC, majority of the cases (66.2%; 102/154) had stage IV disease. EGFR was the most expressed molecular marker (29.4%; 25/85) followed by p53 (24.0%; 29/121), p16 (18.3%; 23/126) and Cyclin-D1 (10.0%; 12/120). HER2 was not expressed in any of the cases. There was a significantly (p = 0.022) higher expression of Cyclin-D1 in tumours of the oral cavity (19.6%; 9/46) than in those of the larynx (4.7%; 2/43) and nose (3.2%; 1/31). Tumours in stages I-III were more frequently positive for p16 (28.6%; 12/42) than tumours in stage IV (13.1%; 11/84).. Expression of p53, EGFR, p16 and Cyclin-D1 in non-oropharyngeal HNSCC in Ghana is largely similar to what has been reported in published studies from other countries.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Ghana; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Staging; Receptor, ErbB-2; Retrospective Studies; Squamous Cell Carcinoma of Head and Neck; Tumor Suppressor Protein p53; Up-Regulation; Young Adult

The clinical impact of next-generation sequencing (NGS) in patients with head and neck squamous cell carcinoma (HNSCC) has not been described. We aimed to evaluate the clinical impact of NGS in the routine care of patients with HNSCC and to correlate genomic alterations with clinical outcomes.. Single-center study examining targeted NGS platform used to sequence tumor DNA obtained from 213 HNSCC patients evaluated in outpatient head and neck oncology clinic between August 2011 and December 2014. We correlated tumor genomic profiling results with clinical outcomes.. PI3K/RTK pathway activation occurred frequently [activating PIK3CA mutation or amplification (13%), PTEN inactivation (3%), RAS activation (6%), EGFR or ERBB2 activation (9%)]. Alterations in pathways affecting cell-cycle regulation [CCND1 amplification (9%), CDKN2A inactivation (17%), BRCA2 inactivation (2%)] and squamous differentiation [NOTCH1 inactivation (8%) andEP300 inactivation (6%)] were identified. PIK3CA amplification (n = 43), not PIK3CA mutation, was associated with significantly poorer progression-free survival (P = 0.0006). Oncogenic RAS mutations (n = 13) were associated with significantly poorer progression-free survival (P = 0.0001) and lower overall survival (P = 0.003). Eight patients with advanced, treatment-refractory HNSCC enrolled on clinical trials matched to tumor profiling results, and 50% achieved a partial response.. Incorporation of NGS clinical assays into the routine care of patients with HNSCC is feasible and may readily facilitate enrollment into clinical trials of targeted therapy with a higher likelihood of success. Data can be utilized for discovery of genomic biomarkers of outcome. PIK3CA amplification and RAS mutations were frequently identified and associated with poorer prognosis in this cohort. Clin Cancer Res; 22(12); 2939-49. ©2016 AACR.

Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Biomarkers, Tumor; Carcinoma, Squamous Cell; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; Disease-Free Survival; DNA-Binding Proteins; ErbB Receptors; Female; Gene Expression Profiling; Head and Neck Neoplasms; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Neoplasm Proteins; Proto-Oncogene Proteins p21(ras); Receptor, Notch1; Squamous Cell Carcinoma of Head and Neck; Treatment Outcome; Tumor Suppressor Protein p53; Young Adult

We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation.. Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results.. A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America.. HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cross-Sectional Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA, Viral; Female; Genotype; Head and Neck Neoplasms; Human papillomavirus 16; Humans; Immunohistochemistry; International Cooperation; Male; Middle Aged; Oropharyngeal Neoplasms; Papillomaviridae; Papillomavirus Infections; Predictive Value of Tests; Salivary Proline-Rich Proteins; Tumor Suppressor Protein p53

To investigate the effects of a dual phosphoinosmde-3-kinase (PI3K)/ mammalian target of rapamycin (mTOR) inhibitor, PI-103, cooperating with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the laryngeal squamous carcinoma Hep-2 cells.. Hep-2 cells were divided into 7 groups: LY294002 group, Rapamycin group, PI-103 group, LY294002+ TRAIL group, Rapamycin+ TRAIL group, PI-103+ TRAIL group and control group.The cell cycle and apoptosis of Hep-2 cells were assessed by flow cytometry.For PI-103 group, PI-103+ TRAIL group and control group, migration and invasion ability were measured by transwell migration and invasion assay respectively.The expression of relative proteins in apoptosis and PI3K/AKT/mTOR signal pathway was examined by Western blotting.. Combination of PI-103 and TRAIL could make cell cycle arrest at S phase (G1: 1.80%±0.30%; G2: 0.00), inhibit cell proliferation, and enhance apoptosis (66.78%±2.93%) (P<0.05). Combination of PI-103 and TRAIL could statistically decrease the migration and invasion number of Hep-2 cells (17.0±3.4, 18.4±5.4) than that of PI-103 group (41.2±3.8, 41.6±4.7). PI-103 could inhibit PI3K/AKT/mTOR signal pathway by decreasing the protein expression of p-AKT and p-4E-BP1.Comparing with the control group, the expression of cysteinyl aspartate specific proteinase (Caspase) 9, 8, 3 were increased while the expression of Cyclin D1, Cyclin E1, p-AKT, p-4E-BP1 were decreased in PI-103 and PI-103+ TRAIL group (P<0.05).. Enhanced anti-tumor effects was observed by combination of PI-103 and TRAIL on laryngeal cancer cells in vitro and this combined administration might be a promising strategy for clinical treatment of laryngeal cancer.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Cyclin D1; Cyclin E; Furans; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; Morpholines; Oncogene Proteins; Phosphatidylinositol 3-Kinases; Pyridines; Pyrimidines; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; TNF-Related Apoptosis-Inducing Ligand; TOR Serine-Threonine Kinases

Oral squamous cell carcinomas comprise a heterogeneous tumor cell population with varied molecular characteristics, which makes prognostication of these tumors a complex and challenging issue. Thus, molecular profiling of these tumors is advantageous for an accurate prognostication and treatment planning. This is a retrospective study on a cohort of primary locally advanced oral squamous cell carcinomas (n = 178) of an Indian rural population. The expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in a cohort of primary locally advanced oral squamous cell carcinomas was evaluated. A potential biomarker that can predict the tumor response to treatment was identified. Formalin-fixed paraffin-embedded tumor blocks of (n = 178) of histopathologically diagnosed cases of locally advanced oral squamous cell carcinomas were selected. Tissue microarray blocks were constructed with 2 cores of 2 mm diameter from each tumor block. Four-micron-thick sections were cut from these tissue microarray blocks. These tissue microarray sections were immunohistochemically stained for EGFR, p53, Bcl-2, cyclin D1 and p16. In this cohort, EGFR was the most frequently expressed 150/178 (84%) biomarker of the cases. Kaplan-Meier analysis showed a significant association (p = 0.038) between expression of p53 and a poor prognosis. A Poisson regression analysis showed that tumors that expressed p53 had a two times greater chance of recurrence (unadjusted IRR-95% CI 2.08 (1.03, 4.5), adjusted IRR-2.29 (1.08, 4.8) compared with the tumors that did not express this biomarker. Molecular profiling of oral squamous cell carcinomas will enable us to categorize our patients into more realistic risk groups. With biologically guided tumor characterization, personalized treatment protocols can be designed for individual patients, which will improve the quality of life of these patients.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cohort Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Mouth Neoplasms; Neoplasm Proteins; Prognosis; Proto-Oncogene Proteins c-bcl-2; Squamous Cell Carcinoma of Head and Neck; Tissue Array Analysis; Treatment Outcome; Tumor Suppressor Protein p53; Young Adult

Multiple primary tumors can occur in up to 35 % of the patients with head and neck cancer, however its clinicopathological features remain controversial. Deregulation of epithelial-mesenchymal transition (EMT) signaling has been associated with aggressive malignancies and tumor progression to metastasis in several cancer types. This study is the first to explore EMT process in multiple primary oral squamous cell carcinomas (OSCC). Immunohistochemical analysis of E-cadherin, catenin (α, β, and γ), APC, collagen IV, Ki-67, cyclin D1, and CD44 were performed in a tissue microarray containing multiple representative areas from 102 OSCC patients followed-up by at least 10 years. Results were analysed in relation to clinicopathological characteristics and survival rates in patients presenting multiple primary tumors versus patients without second primary tumors or metastatic disease. Significant association was observed among multiple OSCCs and protein expression of E-cadherin (P = 0.002), β-catenin (P = 0.047), APC (P = 0.017), and cyclin D1 (P = 0.001) as well as between lymph nodes metastasis and Ki-67 staining (P = 0.021). OSCCs presenting vascular embolization were associated with negative β-catenin membrane expression (P = 0.050). There was a significantly lower survival probability for patients with multiple OSCC (log-rank test, P < 0.0001), for tumors showing negative protein expression for E-cadherin (log-rank test, P = 0.003) and β-catenin (log-rank test, P = 0.031). Stratified multivariate survival analysis revealed a prognostic interdependence of E-cadherin and β-catenin co-downexpression in predicting the worst overall survival (log-rank test, P = 0.007). EMT markers have a predicted value for invasiveness related to multiple primary tumors in OSCC and co-downregulation of E-cadherin and β-catenin has a significant prognostic impact in these cases.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Catenins; Cyclin D1; Epithelial-Mesenchymal Transition; Female; Head and Neck Neoplasms; Humans; Hyaluronan Receptors; Ki-67 Antigen; Male; Middle Aged; Mouth Neoplasms; Neoplasms, Multiple Primary; Paraffin Embedding; Prognosis; Squamous Cell Carcinoma of Head and Neck; Tissue Preservation

Human head and neck squamous cell carcinoma (HNSCC) is the sixth most malignant cancer worldwide. Despite significant advances in the delivery of treatment and surgical reconstruction, there is no significant improvement of mortality rates for this disease in the past decades. Radiotherapy is the core component of the clinical combinational therapies for HNSCC. However, the tumor cells have a tendency to develop radiation resistance, which is a major barrier to effective treatment. HIV protease inhibitors (HIV PIs) have been reported with radiosensitizing activities in HNSCC cells, but the underlying cellular/molecular mechanisms remain unclear. Our previous study has shown that HIV PIs induce cell apoptosis via activation of endoplasmic reticulum (ER) stress. The aim of this study was to examine the role of ER stress in HIV PI-induced radiosensitivity in human HNSCC.. HNSCC cell lines, SQ20B and FaDu, and the most commonly used HIV PIs, lopinavir and ritonavir (L/R), were used in this study. Clonogenic assay was used to assess the radiosensitivity. Cell viability, apoptosis and cell cycle were analyzed using Cellometer Vision CBA. The mRNA and protein levels of ER stress-related genes (eIF2α, CHOP, ATF-4, and XBP-1), as well as cell cycle related protein, cyclin D1, were detected by real time RT-PCR and Western blot analysis, respectively. The results demonstrated that L/R dose-dependently sensitized HNSCC cells to irradiation and inhibited cell growth. L/R-induced activation of ER stress was correlated to down-regulation of cyclin D1 expression and cell cycle arrest under G0/G1 phase.. HIV PIs sensitize HNSCC cells to radiotherapy by activation of ER stress and induction of cell cycle arrest. Our results provided evidence that HIV PIs can be potentially used in combination with radiation in the treatment of HNSCC.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Head and Neck Neoplasms; HIV Protease Inhibitors; Humans; Models, Biological; Radiation Tolerance; Radiation-Sensitizing Agents; RNA, Messenger; Squamous Cell Carcinoma of Head and Neck; Unfolded Protein Response

To observe the effect of metastasis-associated gene 1 (MTA1) on the growth and metastasis of laryngeal squamous cell carcinoma in nude mice using RNA interference technology (RNAi).. Lentiviral vector of short hairpin RNA (shRNA) targeting MTA1 was constructed and packaged to transfect Hep-2 cells. Hep-2 cells transfected with scramble shRNA and MTA1 shRNA were injected into the paw pad of nude mice (n=5 per group). Nine weeks after modeling of the lymphatic metastasis of laryngeal carcinoma, the mice were sacrificed, and tumor tissues and inguinal lymph node were harvested to be subjected to HE staining, reverse transcription PCR and Western blotting.. The gene screening showed the lentiviral vector of MTA1 shRNA was constructed successfully, and those tumor cells were transplanted and grew well in all mice. The size of tumor in the mice of MTA1 shRNA tansfected group was obviously smaller compared to the scramble shRNA transfected group at the same time point. No inguinal lymph node metastasis was found in the mice of MTA1 shRNA group. In contrast, the tumor cells were seen in the inguinal lymph nodes of the scramble shRNA infected mice. Reverse transcription PCR and Western blotting showed that the mRNA and protein levels of MTA1, β-catenin, matrix metalloproteinase-9 (MMP-9), cyclin D1 were obviously reduced in MTA1 shRNA infected mice compared with the scramble shRNA infected mice.. The inhibition of MTA1 gene could depress the growth and metastasis of laryngeal squamous cell carcinoma in nude mice.

Topics: Animals; beta Catenin; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Head and Neck Neoplasms; Laryngeal Neoplasms; Lymphatic Metastasis; Matrix Metalloproteinase 9; Mice; Mice, Nude; Repressor Proteins; RNA, Small Interfering; Squamous Cell Carcinoma of Head and Neck; Trans-Activators; Transcription Factors

Oral squamous cell carcinoma (OSCC) ranks as the fifth most common cancer worldwide with poor prognosis. Recently, tumor necrosis factor receptor-associated factor 4 (TRAF4) has attracted increasing attenuation due to its overexpression in certain cancers. However, its function and underlying mechanism in OSCC remains elusive. In this study, the high expression of TRAF4 mRNA and protein levels was noted in OSCC cell lines. Its overexpression with pcDNA3.1-TRAF4 vector transfection dramatically promoted cell proliferation and inhibited cell apoptosis, indicating a pivotal role of TRAF4 in OSCC cell growth. Simultaneously, TRAF4 elevation also increased cell invasion and migration. Mechanism analysis confirmed that TRAF4 up-regulation induced the expression of β-catenin and the downstream target molecules of cyclinD1, c-myc, Bcl-2, MMP-9 and MMP-2, indicating that TRAF4 could induce the activation of Wnt/β-catenin pathway. After pretreatment with β-catenin siRNA, the pathway was remarkably silenced. Simultaneously, cell growth, invasion and migration induced by TRAF4 were strikingly abrogated, suggesting that TRAF4 may promote OSCC cell growth, invasion and migration by Wnt/β-catenin pathway. Together, this study confirmed that TRAF4 acts as an oncogene for the development and progression of OSCC. Therefore, our study may support a promising therapeutic target for the treatment of OSCC.

Topics: Apoptosis; beta Catenin; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Head and Neck Neoplasms; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA Interference; Squamous Cell Carcinoma of Head and Neck; TNF Receptor-Associated Factor 4; Tongue Neoplasms; Transfection; Wnt Signaling Pathway

Gene products, which show a significant association to cell proliferation and cell cycle control, are of high scientific interest, because genes as well as gene products could be possible targets for a specific therapeutic approach and eventually be prognostic markers.. Cyclin D1 expression and amplification as well as the Ki-67 expression status were examined in a two tissue microarray analysis for head and neck squamous cell carcinoma (HNSCC) including 546 patients. A tumour site-specific analysis and a survival analysis of 222 oral squamous cell carcinoma (OSCC) patients were performed. Cyclin D1 amplification status was examined with fluorescence in situ hybridisation analysis, while cyclin D1 expression and Ki-67 expression status were examined with IHC.. Amplification of the CCND1 gene and immunohistochemical expression of cyclin D1 and Ki-67 were examined in 546 tumours of the head and neck region in two tissue microarrays. CCND1 amplification was significantly more frequent in pharyngeal carcinomas (63%) than in laryngeal (37%) and oral (25%) carcinomas. Among the 222 cases of OSCCs, both CCND1 amplification and cyclin D1 expression were significantly associated with overall survival of the patients (p = 0.0127 and p = 0.0004, respectively). Ki-67 expression was significantly associated with cyclin D1 expression and with amplification of the CCND1 gene (p = 0.0002 and p = 0.0015, respectively) but not with patient overall survival.. Our results suggest the prognostic value of CCND1 amplification and cyclin D1 expression for patients with OSCC and highlight the genetic differences in HNSCC of different subanatomic localisation.. Cyclin D1 expression and CCND1 amplification seem to have a prognostic value for OSCC. Further studies of HNSCC should always consider subanatomic genetic differences.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Head and Neck Neoplasms; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Ki-67 Antigen; Lymphatic Metastasis; Survival Analysis

Head and neck squamous cell carcinoma (HNSCC) progression and metastasis have previously been associated with the activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) and Wnt signalling pathways, which lead to the activation of pro-proliferative genes, such as cyclin D1. The current study aims to investigate whether there is a crosstalk between these pathways in HNSCC and which pathway is more likely to regulate cyclin D1.. Two HNSCC and a control keratinocyte cell lines were treated with EGF and wortmannin to respectively activate and block the PI3K-Akt and Wnt pathways. Partial and total levels of cyclin D1, beta-catenin and Akt were evaluated by Western blotting and immunofluorescence. Twenty-four paraffin-embedded samples of human HNSCC, as well as normal oral mucosa biopsies, were also immunohistochemically evaluated for beta-catenin and cyclin D1 expression.. Following both treatments, change in cyclin D1 protein was correlated with Akt levels only. Cytoplasmic staining for beta-catenin and loss of its membranous expression in the HNSCC invasive areas were found in 92% of the HNSCC biopsies.. Taken together, we show that the change in cyclin D1 levels is more likely to be due to the EGFR-Akt pathway activation than due to beta-catenin nuclear translocation.

Topics: beta Catenin; Carcinoma, Squamous Cell; Cyclin D1; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Phosphatidylinositol 3-Kinases; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Tumor Cells, Cultured

The p16INK4a protein is a principal cyclin-dependent kinase inhibitor that decelerates the cell cycle. Abnormally high levels of p16INK4a are commonly observed in human papillomavirus (HPV)-positive head and neck squamous cell carcinomas (HNSCC). We and others found that p16INK4a overexpression is associated with improved therapy response and survival of patients with HNSCC treated with radiotherapy. However, the functional role of p16INK4a in HNSCC remains unexplored. Our results implicate p16INK4a in regulation of homologous recombination-mediated DNA damage response independently from its role in control of the cell cycle. We found that expression of p16INK4a dramatically affects radiation sensitivity of HNSCC cells. p16INK4a overexpression impairs the recruitment of RAD51 to the site of DNA damage in HPV-positive cells by downregulating of cyclin D1 protein expression. Consistent with the in vitro findings, immunostaining of HNSCC patient samples revealed that high levels p16INK4a expression significantly correlated with decreased cyclin D1 expression. In summary, these findings reveal an unexpected function of p16INK4a in homologous recombination-mediated DNA repair response and imply p16INK4a status as an independent marker to predict response of patients with HNSCC to radiotherapy.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Chemoradiotherapy; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; DNA Damage; Head and Neck Neoplasms; Humans; Kaplan-Meier Estimate; Papillomavirus Infections; Rad51 Recombinase; Radiation Tolerance; Recombinational DNA Repair; Treatment Outcome

While aberrant expression of cyclin-dependent kinase-4 (CDK4) has been found in squamous cell carcinoma of the head and neck (SCCHN), the associations between CDK4 and its regulators, namely, cyclin D1, cyclin E, gankyrin, SEI1, and BMI1 in gene expression remain to be explored. Herein we investigated the mRNA profiles of these oncogenes and their interrelations in different oral lesion tissues.. Thirty SCCHN specimens and patient-matched high at-risk mucosa (HARM) and 16 healthy control specimens were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses.. The mRNA levels of CDK4, cyclin D1, gankyrin, SEI1, BMI1 were significantly elevated in both HARM and SCCHN (in comparison with control specimens), and statistically significant correlations were found among these markers in gene expression.. Up-regulation of CDK4 and its regulators takes place in oral cancer progression in a coordinate manner, and HARM and SCCHN share a similar molecular signature within the CDK4-pRB pathway.

Topics: Adult; Carcinoma, Squamous Cell; Case-Control Studies; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 7; Mouth Neoplasms; Nuclear Proteins; Principal Component Analysis; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; RNA, Messenger; Squamous Cell Carcinoma of Head and Neck; Trans-Activators; Transcription Factors; Up-Regulation

Galanin and its receptors, GALR1 and GALR2, are tumor suppressors and represent therapeutic targets in head and neck squamous cell carcinoma (HNSCC). In the present study, it was demonstrated that the re‑expression of GALR1 in GALR1 and GALR2‑negative HNSCC cells suppresses tumor cell proliferation. This is mediated via extracellular‑regulated protein kinase‑1/2 (ERK1/2)‑dependent effects on the cyclin‑dependent kinase inhibitors (CKI) and cyclin D1. In combination with galanin, GALR2 also suppressed proliferation by increasing CKI and decreasing cyclin D1 levels. In contrast to GALR1, overexpression of GALR2 also induced caspase‑3‑dependent apoptosis. It was identified that in GALR2‑transfected cells, galanin induced activation of ERK1/2 and suppressed cell proliferation. Galanin stimulation also decreased the expression of cyclin D1 and induced apoptotic DNA ladder formation in GALR2‑transfected cells. Pretreatment with the ERK1/2‑specific inhibitor U0126 and pertussis toxin prevented the suppression of cyclin D1 expression, however did not affect DNA ladder formation. In conclusion, GALR2 expression in the presence of galanin exerts antitumor effects via cell cycle arrest and apoptotic pathways, and reactivation of these pathways may have therapeutic benefits in HNSCC.

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Carcinoma, Squamous Cell; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Galanin; Gene Expression Regulation; Head and Neck Neoplasms; Humans; MAP Kinase Signaling System; Nitriles; Receptor, Galanin, Type 2; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

The molecular mechanism of p16-mediated senescence in cisplatin-treated cancer cells is not fully understood. Here we show that cisplatin treatment of head and neck cancer cells results in nuclear transport of p16 leading to a molecular modification of NFκB. Chromatin immunoprecipitation assays show that this modification is associated with the inhibition of NFκB interacting with its DNA binding sequences, leading to decreased expression of NFκB-transcribed proteins. LCMS proteomic analysis of LAP-TAP-purified proteins from HeLa cells containing a tetracycline-inducible GFP-S peptide-NFκB expression system identified gigaxonin, an ubiquitin E3 ligase adaptor, as an NFκB-interacting protein. Immunoblotting and siRNA studies confirmed the NFκB-gigaxonin interaction and the dependence of this binding on p16-NFκB binding. Using gel shift assays, we have confirmed p16-NFκB and gigaxonin-NFκB interactions. Furthermore, we have observed increased NFκB ubiquitination with cisplatin treatment that is abolished in the absence of p16 and gigaxonin expression. Analysis of 103 primary tumors has shown that increased nuclear p16 expression correlates with enhanced survival of head and neck cancer patients (p < 0.0000542), indicating the importance of nuclear p16 expression in prognosis. Finally, p16 expression is associated with reduced cytokine expression and the presence of human papilloma virus in chemoradiation-sensitive basaloid tumors. However, the absence of p16 expression is associated with enhanced cytokine expression and the absence of human papilloma virus in aggressive tumors. These results clearly demonstrate that nuclear p16 and gigaxonin play an important role in chemosensitivity of head and neck cancers through ubiquitination of NFκB.

Topics: Active Transport, Cell Nucleus; Antineoplastic Agents; Cell Line, Tumor; Cell Nucleus; Cellular Senescence; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Human papillomavirus 16; Humans; NF-kappa B; Prognosis; Ubiquitination

Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Silencing; Genetic Therapy; Head and Neck Neoplasms; Humans; RNA, Guide, Kinetoplastida; RNA, Messenger; Squamous Cell Carcinoma of Head and Neck

Galangin, an active flavonoid component extracted from the propolis and root of Alpinia officinarum Hance, has anti-tumor activity, but the mechanisms by which galangin affects various cancers, including human head and neck squamous cell carcinoma (HNSCC) remain unclear. In this study, we demonstrated for the first time that galangin suppressed the growth of HNSCC in vivo. With the cell culture system, galangin inhibited the proliferation and colony formation of HNSCC cells in a dose-dependent manner. Galangin induced significant cell cycle arrest of the tumor cells at the G0/G1 phase, which was accompanied by reduced AKT phosphorylation and mammalian target of rapamycin and S6 kinase activation. Decreased expression of cyclin D1, cyclin-dependent kinase (CDK)4, CDK6 and phosphorylation of retinoblastoma protein was observed in galangin-treated HNSCC cells. In addition, galangin induced apoptosis of HNSCC cells, downregulating antiapoptotic protein Bcl-2 and Bcl-xL and upregulating proapoptotic protein Bax and cleaved caspase 3. Immunohistochemical analysis showed a dose-dependent reduction in cyclin-D1-positive cancer cells and an increase in TUNEL-positive cancer cells in galangin-administrated mouse tumor sections. Therefore, galangin may be a novel therapeutic option in human HNSCC treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Flavonoids; G1 Phase Cell Cycle Checkpoints; Head and Neck Neoplasms; Humans; Male; Mice; Mice, Inbred BALB C; Protein Kinase Inhibitors; Retinoblastoma Protein; Xenograft Model Antitumor Assays

Tumors of the head and neck present aggressive pathological behavior in patients due to high expression of CDK/CCND1 proteins. P276-00, a novel CDK inhibitor currently being tested in clinic, inhibits growth of several cancers in vitro and in vivo. The pre clinical activity of P276-00 in head and neck cancer and its potential mechanisms of action at molecular level are the focus of the current studies.. We have investigated the anti-cancer activity of P276-00 in head and neck tumors in vitro and in vivo. Candidate gene expression profiling and cell based proteomic approaches were taken to understand the pathways affected by P276-00 treatment.. It was observed that P276-00 is cytotoxic across various HNSCC cell lines with an IC₅₀ ranging from 1.0-1.5 μmoles/L and culminated in significant cell-cycle arrest in G1/S phase followed by apoptosis. P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets. Further, we observed that apoptosis was mediated through P53 activation leading to higher BAX/BCL-2 ratio and cleaved caspase-3 levels. It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8. Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis.. In summary, we have observed that P276-00 inhibits cyclin-D/CDK4/P16/pRB/E2F axis and induces apoptosis by increased P53 phosphorylation in HNSCC cells. These results suggest a novel indication for P276-00 in head and neck cancer with a potential role for IL-6 and HSPA8 as candidate serum biomarkers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D; Cyclin D1; Cyclin-Dependent Kinases; E2F1 Transcription Factor; ErbB Receptors; Flavones; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; HSC70 Heat-Shock Proteins; Humans; Inhibitory Concentration 50; Interleukin-6; Mice; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Protein Kinase Inhibitors; Retinoblastoma Protein; Xenograft Model Antitumor Assays

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.

Topics: Aged; Carcinoma, Squamous Cell; Chromosome Aberrations; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; DNA Copy Number Variations; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; Male; Middle Aged; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinases; SOXB1 Transcription Factors; Squamous Cell Carcinoma of Head and Neck; Transcription Factors; Tumor Suppressor Proteins

To determine the biological characteristics of oropharyngeal squamous cell carcinoma (OpSCC) and related outcome.. Retrospective study.. Patients (N=60) with primary OpSCC from 2000 to 2005 were retrospectively identified from Pathology database and the outcome was confirmed through chart review. Among these, 41 biopsy samples with enough tissues were retrieved to construct a tissue microarray for detection of the presence of high-risk human papillomavirus (HPV) using Chromogenic in situ hybridization (CISH) as well as the expression of p16 and cyclin D1 using immunohistochemistry.. Disease-free survival.. Among 60 patients, 39 (65%) patients had no recurrence or died without disease at the last follow-up (disease-free survival or Group 1), and 21 (35%) patients had persistent disease or died of disease (progression-free survival or Group 2). Although follow-up time was twice as long in group 1 (4.7 ± 2.2 vs. 2.0 ± 1.6 years; P < 0.0001), there was no difference between the 2 groups in age, gender, smoking/alcohol habits, TNM staging and treatment modalities. Among those 41 cases with available tumour tissues, there was no difference in HPV status and p16 expression between the 2 groups but a significant difference in cyclin D1 expression (P = 0.05). Using Kaplan-Meir survival analysis and log-rank test, cyclin D1 overexpression was highly associated with a poor prognosis when comparing time to outcome (P < 0.0001).. Cyclin D1 overexpression is a potential prognostic marker of OpSCC.

Topics: Aged; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; In Situ Hybridization; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Proteins; Oropharyngeal Neoplasms; Prognosis; Retrospective Studies; Squamous Cell Carcinoma of Head and Neck; Tissue Array Analysis; Treatment Outcome

Celecoxib, a non-steroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2 (COX-2), has shown an important anticarcinogenic effect for the treatment of squamous cell carcinoma. The use of COX-2 inhibitors has effectively inhibited the growth of Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines, while a recent phase 1 trial demonstrated good response rate of cancer cells to this drug with minimal toxicity. Possible targets of celecoxib include proteins involved in cell proliferation and apoptosis control. Additionally, celecoxib antitumoral activity has been linked with a COX-2-independent event.. To better understand which cellular mechanisms are targeted by celecoxib, its effects upon the Akt signaling pathway using two different HNSCC cell lines were analyzed through cell viability assay, immunofluorescence, and Western blotting.. The results showed decreased levels of Cyclin D1 and pAkt protein expression in vitro. The number of viable cells was also diminished after celecoxib treatment.. As Akt pathway seems to be a valuable target for the HNSCC therapy, the results presented herein confirm that celecoxib can be considered as an alternative adjuvant drug for HNSCC treatment.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Celecoxib; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclooxygenase 2 Inhibitors; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Head and Neck Neoplasms; Humans; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; Sulfonamides

It was proved that cyclin D1-positive status in surgical margins was an independent prognostic indicator of local recurrence. The expression of cyclin D1 in tumor-free surgical margins may better predict local recurrence in patients with head and neck squamous cell carcinoma (HNSCC) after surgical treatment with curative intent.. This retrospective study aimed to determine the prognostic indicators for local recurrence in HNSCC.. A total of 116 HNSCC patients who underwent surgical treatment with curative intent and had histopathologically tumor-free margins were eligible for this study. The expression of p53 and cyclin D1 was assessed by immunohistochemical staining in surgical margins as well as in tumor specimens.. In all, 63 patients (54.3%) had p53-positive tumor specimens and 34 patients (29.3%) had p53-positive margins. Seventy-six patients (65.6%) had cyclin D1-positive tumor specimens and 54 patients (46.6%) had cyclin D1-positive margins. A significant difference in local control rates was observed between patients with cyclin D1-positive and -negative margins (77.2% vs 91.5%, log rank test, p = 0.0139). Multivariate Cox proportional hazards testing indicated that the hazard ratio of cyclin D1-positive margins for local recurrence was 4.58 (95% confidence interval 1.14-21.69, p = 0.0304).

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Female; Head and Neck Neoplasms; Humans; Japan; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Retrospective Studies; Tumor Suppressor Protein p53

Desmoglein 3 (DSG3) is a component of the desmosome, which confers strong cell-cell adhesion. Previously, an oncogenic function of DSG3 has been found in head neck cancer (HNC). Here, we investigated how this molecule contributes to the malignant phenotype. Because DSG3 is associated with plakoglobin, we examined whether these phenotypic alterations were mediated through the plakoglobin molecule. Immunoprecipitation and immunofluorescence staining revealed that DSG3 silencing disrupted its interaction with plakoglobin and induced plakoglobin translocation from the cytoplasm to the nucleus. Knockdown of DSG3 significantly increased the interaction of plakoglobin with the transcriptional factor TCF and suppressed the TCF/LEF transcriptional activity. These effects further conferred to reduced expression of the TCF/LEF downstream target genes, including c-myc, cyclin D1, and MMP-7. Functional analyses showed that DSG3 silencing reduced cell growth and arrested cells at G0/G1 phase. Besides, cell migration and invasion abilities were also decreased. These cellular results were confirmed using tumor xenografts in mice, as DSG3 silencing led to the suppressed tumor growth, plakoglobin translocation and reduced expression of TCF/LEF target genes in tumors. Therefore, our study shows that the desmosomal protein DSG3 additionally functions to regulate malignant phenotypes via nuclear signaling. In conclusion, we found that DSG3 functions as an oncogene and facilitates cancer growth and invasion in HNC cells through the DSG3-plakoglobin-TCF/LEF pathway.

Topics: Active Transport, Cell Nucleus; Animals; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Desmoglein 3; DNA-Binding Proteins; G1 Phase Cell Cycle Checkpoints; gamma Catenin; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Head and Neck Neoplasms; Humans; Male; Matrix Metalloproteinases; Mice; Neoplasm Invasiveness; Phenotype; Proto-Oncogene Proteins c-myc; Resting Phase, Cell Cycle; Signal Transduction; TCF Transcription Factors

Merkel cell carcinomas (MCC) are very aggressive tumors of the sun-exposed skin with a high potential to metastasize. Little is known about the genesis of MCC and very few prognostic markers have been detected so far. The Wnt pathway protein β-catenin and the cell cycle protein cyclin D1 are two promotors of tumor growth and are expressed in a variety of malignant neoplasms such as lymphomas, thyroid, breast cancer, and many others.. Tissue samples of 27 patients with MCC were immunohistochemically stained for β-catenin and cyclin D1 and correlated with overall survival of patients. In addition, western blot analysis was carried out in the two MCC cell lines MCC-13 and MCC-26.. β-catenin showed a cytoplasmatic expression of 10-30 % in 11 samples and an expression lower than 10 % in eight samples. Nuclear staining was visible in two samples. None of the 27 samples expressed cyclin D1.. Neither cyclin D1 nor β-catenin was expressed in a statistically significant manner, concluding that the development of MCCs is independent of β-catenin and cyclin D1 expression and these proteins are not suitable as prognostic markers. We could describe the expression pattern of cyclin D1 for the first time.

Topics: Aged; Aged, 80 and over; beta Catenin; Biomarkers, Tumor; Carcinoma, Merkel Cell; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Male; Middle Aged; Reproducibility of Results; Sensitivity and Specificity

Cyclin D1 plays a key role in cell cycle control, particularly in the transition from G1 to S phase, regulated by cyclin-dependent kinases. The objective of the present study was to screen the cyclin D1 gene (CCND1) for polymorphisms in patients with head and neck cancer (HNC). Genomic DNA was isolated from blood samples of 380 HNC patients and 350 controls. In a hospital-based case-control study using the PCR-SSCP technique we found 3 novel germline mutations: g3578C>A, g3475G>C and g3383delA. The commonly reported guanine to adenine polymorphisms in exon 4 g7656G>A (rs9344) and g10861C>A (rs7177) in 3'UTR of CCND1 were also observed. The calculated frequencies of the g7656G>A (rs9344) polymorphism in GG, GA and AA genotypes were 27.3%, 38.6%, and 33.9% in HNC cases, and 44.2%, 29.4%, and 26.2% in normal healthy controls, respectively. Adjusted by age (in years), sex and smoking status, multivariate logistic regression analysis showed that the AA and GA genotypes were associated with a significantly increased risk (OR 1.34, 95% CI 1.03-1.64, p=0.028) for HNC. The CCND1 AA genotype variant was associated with an increased risk in individuals who were <40 years old (OR 1.45, 95% CI 1.02-2.08, p=0.04). In conclusion, it is suggested that the CCND1 G/A polymorphism is associated with the early onset of HNC and may contribute to HNC susceptibility in a Pakistani population.

Topics: Adult; Base Sequence; Case-Control Studies; Cyclin D1; Female; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Germ-Line Mutation; Head and Neck Neoplasms; Humans; Male; Multivariate Analysis; Mutation, Missense; Polymorphism, Single Nucleotide; Risk Factors; Sequence Analysis, DNA

Head-Neck Squamous Cell Carcinoma (HN-SCC) is a clinically challenging disease associated with a high mortality rate. The chemo-radiotherapy treatments that aim to preserve the organ represent the current gold standard therapy for advanced laryngeal disease, reserving surgery only for non-responsive or relapsed cases. Despite these aggressive approaches, local persistent or recurrent disease remains the primary cause of treatment failure but we still do not have known factors and/or markers able to predict the outcome of the disease and in particular the risk of local relapse. Here we address this point on a series of 54 cases of HN-SCC for whom the presence of local relapse was known. Using immunohistochemistry (IHC) analysis to evaluate protein expression and localization in the recurrence free and recurrence positive samples, we studied the expression of key cell cycle regulators including p53, p16, p27, pRB, Cyclin D1, Cyclin D3, and Stathmin. Overall by analyzing seven different cell cycle regulators we can hypothesize that the alteration of G1/S regulation represents a fundamental event in the onset/progression of HN-SCC cancers and that the associate use of Cyclin D1/p16 expression should be considered as a possible biomarker toward the identification of those patients that will probably develop a recurrent disease and thus should benefit of a more aggressive treatment.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin D1; Cyclin D3; Female; G1 Phase; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; S Phase

The authors previously observed that enhancer of zeste homolog 2 (EZH2) overexpression was associated significantly with the development of oral cancer. In the current study, they investigated whether EZH2 can function as a prognostic predictor for patients with head and neck squamous cell carcinoma (HNSCC).. Expression levels of EZH2 in HNSCC cells were detected using reverse transcriptase-polymerase chain reaction (PCR) and Western blot analyses. In addition, the effects of EZH2 ablation on the proliferation and invasion of HNSCC cells were investigated through small interfering RNA (siRNA)-mediated knockdown. Real-time PCR and immunohistochemistry were used to evaluate EZH2 and cyclin D1 expression in 46 HNSCC samples, and the expression levels also were re-evaluated in 124 independent samples by immunohistochemistry.. EZH2 expression was elevated remarkably in HNSCC specimens and cell lines. Upon EZH2 silencing, the proliferation and invasion of HNSCC cells were remarkably suppressed. EZH2 expression frequently was correlated with cyclin D1 expression (P = .034) and tumor differentiation (P = .020). In addition, both EZH2 messenger RNA levels and EZH2 protein levels were strongly associated with signs of histologic severity (P = .012 and P = .032, respectively). Univariate analysis revealed that high EZH2 expression was associated with worse overall survival (P = .001) and disease-free survival (P = .002). The combined expression of EZH2 and cyclin D1 had superior prognostic ability for patients with HNSCC than the expression of either marker alone. In multivariate analysis, EZH2 expression was identified as an independent predictor of overall and disease-free survival.. The current results indicated that EZH2 is an independent prognostic indicator for patients with HNSCC. In addition, an analysis of the combined expression of EZH2 and cyclin D1 can serve as a more powerful prognostic predictor for patients with HNSCC.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclin D1; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Female; Gene Silencing; Head and Neck Neoplasms; Humans; Male; Middle Aged; Polycomb Repressive Complex 2; Prognosis; Squamous Cell Carcinoma of Head and Neck; Transcription Factors; Transfection; Up-Regulation

The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC.. The status of pAkt, NF-κB and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control.. The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1.. The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Head and Neck Neoplasms; Humans; NF-kappa B; Proto-Oncogene Proteins c-akt; Squamous Cell Carcinoma of Head and Neck; Tumor Cells, Cultured; Tumor Microenvironment

Overexpression of cyclin D1 is associated with resistance to chemotherapy and radiotherapy in several types of cancer including head and neck squamous cell carcinoma (HNSCC).. Cyclin D1 was silenced in an HNSCC cell line and its effect tested in sensitizing the cells to cisplatin, in vitro as well as in vivo. The HNSCC cell line NT8e, which is a chemoresistant, cyclin D1 over-expressing cell line, was used in the study. RNAi (shRNA) against cyclin D1 was designed and cloned in a vector.. Stable silencing of cyclin D1 resulted in delayed cell cycle progression and significantly sensitized the cells to cisplatin. Effective cell kill was achieved at a much lower therapeutic dose in vivo.. Suboptimal concentrations of cisplatin could be used in vivo to eradicate xenograft tumors indicating the promise of combining vector-based cyclin D1 silencing with chemotherapy to achieve maximum tumor regression.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Cisplatin; Colony-Forming Units Assay; Cyclin D1; Drug Resistance, Neoplasm; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; RNA, Small Interfering; Tumor Cells, Cultured

Cyclin D1 and FADD (Fas-associated protein with death domain) regulate the cell cycle and apoptosis, respectively, and are located on chromosome 11q13, which is frequently amplified in head and neck squamous cell carcinoma (HNSCC). This study evaluates these proteins as predictors of clinical outcomes for HNSCC.. Historical cohort study.. Academic tertiary care center.. Two hundred twenty-two patients with upper aerodigestive HNSCC.. Patients with tumors that were strongly positive for cyclin D1 and FADD had reduced overall (OS; P = .003 and P < .001), disease-specific (DSS; P = .039 and P < .001), and disease-free (DFS; P = .026 and P < .001) survival, respectively. Together, the 2 markers effectively stratified OS (P < .001), DSS (P < .001), and DFS (P = .002). Strong FADD staining correlated with greater alcohol consumption and varied significantly with primary tumor site: 56% of hypopharynx tumors expressed high levels of FADD but only 7% of glottis tumors. Using Cox regression analysis, FADD and N stage were significant independent predictors of DSS and DFS, whereas cyclin D1, FADD, and N stage were independently significant for OS.. Cyclin D1 and FADD may have utility as predictors of long-term outcomes for patients with HNSCC. Further study is needed to determine if these proteins predict response to different treatment approaches or assist in selecting patients for multimodality therapy.

Topics: Biomarkers; Carcinoma, Squamous Cell; Cohort Studies; Cyclin D1; Fas-Associated Death Domain Protein; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Survival Rate

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer in the world; the main risk factors are alcohol and tobacco use. Advancements in therapies have yet to improve the prognosis of HNSCC. The connection between diabetes and cancer is being recognized, and metformin has been shown to decrease cancer incidence in diabetic patients. Accordingly, here, for the first time, we investigated metformin's efficacy on the growth and viability of human HNSCC FaDU and Detroit cells. Our results show that metformin treatment (5-20 mM) dose-dependently inhibits the growth of both cell lines. In FaDU cells, metformin caused 18-57% and 35-81% growth inhibition after 48 and 72 h treatments, respectively. Similarly, in Detroit 562 cells, 48 and 72 h metformin treatment resulted in 20-57% and 33-82% inhibition, respectively. Mechanistically, metformin caused G 1 arrest, which coincided with a decrease in the protein levels of CDKs (2, 4 and 6), cyclins (D1 and E) and CDK inhibitors (p15, p16, p18 and p27), but no change in p19 and p21. Metformin also decreased the levels of oncogenic proteins Skp2 and β-Trcp. In other studies, metformin decreased the phosphorylation of 4E-BP1 at Ser65, Thr37/46 and Thr70 sites, but drastically increased the phosphorylation of EF2 at Thr56 and AMPK at Thr172, which results in global translational inhibition. In summary, the observed wide spectrum of mechanistic effects of metformin on HNSCC cells provides support for the anticancer capability of the drug and its potential use in future therapies.

Topics: beta-Transducin Repeat-Containing Proteins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase Cell Cycle Checkpoints; Head and Neck Neoplasms; Humans; Metformin; Neoplasm Proteins; Protein Biosynthesis; rho GTP-Binding Proteins; S-Phase Kinase-Associated Proteins; Squamous Cell Carcinoma of Head and Neck

Squamous cell carcinoma(SCC) is a significant cause of cancer morbidity and mortality worldwide, with an incidence of up to 166 cases per 100 000 population. It arises in the skin, upper aerodigestive tract, lung, and cervix and affects more than 200 000 Americans each year. We report here that a microarray experiment comparing 41 SCC and 13 normal tissue specimens showed that Id2, a gene that controls the cell cycle, was significantly up-regulated in SCC. Enforced expression of Id2 in vitro stimulated the proliferation of SCC cells and up-regulated the transcription of nuclear factor kappa B (NF-κB) and cyclin D1. Enhancement of the NF-κB activity with p65 significantly increased the cell proliferation and the transcription of cyclin D1, whereas inhibition of the NF-κB activity with I kappa B alpha mutant (IκBαM) and pyrroline dithiocarbamate (PDTC) abrogated cell proliferation and transcription of cyclin D1. Furthermore, a mutated NF-κB binding site in the cyclin D1 promoter fully abrogated the Id2-induced transcription of cyclin D1. Taken together, these data indicate that Id2 induces SCC tumor growth and proliferation through the NF-κB/cyclin D1 pathway.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Head and Neck Neoplasms; Humans; I-kappa B Proteins; Inhibitor of Differentiation Protein 2; NF-kappa B; NF-KappaB Inhibitor alpha; RNA, Messenger; Signal Transduction; Transcription Factor RelA; Transcription, Genetic; Up-Regulation

Malignant melanoma is a relatively rare but potentially aggressive tumor in children and adolescents. We report the case of a metastatic malignant melanoma in a 17-year-old girl, first diagnosed on cytological features of a fine-needle lymph node aspiration and then histologically confirmed by both examination of the metastatic adenopathy and a clinically harmless skin lesion of the scalp, which harbored focal microscopic pattern of melanoma. A fluorescent in situ hybridization study revealed that both metastatic and primary cutaneous tumours contained the same and pejorative chromosomal aberration consisting in CCND1 amplification (11q13). This observation raises actual limits and challenges in the fields of diagnosis and treatment of fast-killing melanomas.

Topics: Adolescent; Antibodies, Monoclonal; Antineoplastic Agents, Alkylating; Back Pain; Combined Modality Therapy; Cyclin D1; Dacarbazine; Drug Resistance, Neoplasm; Fatal Outcome; Female; Gene Amplification; Head and Neck Neoplasms; Humans; Immunotherapy; In Situ Hybridization, Fluorescence; Ipilimumab; Lymphatic Metastasis; Melanoma; Nausea; Neoplasm Proteins; Neoplasms, Second Primary; Nevus; Osteolysis; Scalp; Skin Neoplasms; Weight Loss

In this study, we analyzed Ki67 and cyclin D1 immunoexpression in 44 oral squamous cell carcinomas from various anatomical sites. Ki67 immunoreaction was identified in all analyzed cases and presented an index of proliferation of 22% for well-differentiated carcinomas, 32% for moderately differentiated and 53% for the poorly differentiated ones. In case of cyclin D1, the mean positivity index was 8% for well-differentiated carcinomas, 18% for moderately differentiated and 34% for the poorly differentiated carcinomas. The analyzed biomarkers prove useful to identify lesions with poor differentiation and invasive behavior.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Female; Head and Neck Neoplasms; Humans; Ki-67 Antigen; Male; Middle Aged; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

A subgroup of head and neck squamous cell carcinomas (HNSCCs) contains high-risk human papillomavirus-type 16 (HPV16). The viral E6 and E7 oncoproteins inactivate the p53 and pRb proteins, respectively. We examined the causative effect of HPV16 E6 and E7 expression on the immortalization of normal oral keratinocytes (OKCs) and compared the resulting phenotype with alternative ways of p53- and pRb-pathway abrogation frequently found in HNSCCs without HPV. Primary OKCs were conditionally immortalized with temperature-sensitive SV40 large T-antigen and human telomerase, allowing these cells to return to their senescent primary state after temperature shift. HPV16 E6 and E7 were introduced to overcome senescence, determined with population doubling (PD) as read-out. For comparison, we downregulated p53 and p16 by short hairpin RNA genes and expressed mutant p53R(175)H and cyclinD1. Expression of HPV16 E6 caused an extended life span similar to expression of mutant p53R(175)H or p53 knockdown. Expression of mutant p53R(175)H seemed to cause additional activation of the hypoxia and WNT signaling pathways. HPV16 E7 expression had no direct effect on lifespan, similar to p16 knockdown or cyclinD1 expression. In combination with HPV16 E6 or other functional inactivations of p53, abrogation of the pRb-pathway by either HPV16 E7 or other manipulations caused an immortal phenotype. Our data show the causative role of HPV16 E6/E7 in early squamous carcinogenesis. Activity of each gene could be mimicked by other genetic events frequently found in HNSCC without HPV. This data provides the experimental proof of causal association of HPV in HNSCC carcinogenesis and further support the crucial role of the p53- and pRb-pathways.

Topics: Calcium; Cell Line, Tumor; Cyclin D1; Gene Expression Profiling; Gene Expression Regulation; Genetic Vectors; Head and Neck Neoplasms; Humans; Keratinocytes; Oncogene Proteins, Viral; Papillomaviridae; Phenotype; Repressor Proteins; Retinoblastoma Protein; Signal Transduction; Tumor Suppressor Protein p53

Germline variation in DNA damage response may explain variable treatment outcomes in squamous cell carcinoma of the head and neck (SCCHN). By grouping patients according to stage and radiation treatment, we compared SCCHN survival with regard to ERCC2 A35931C (Lys751Gln, rs13181) and CCND1 G870A (Pro241Pro, rs9344) genotypes.. In a hospital-based SCCHN case series (all white, 24.7% female, mean age 58.4 years), this treatment-outcome cohort study genotyped 275 stage III-IV cases that were initially treated with radiation (with or without chemotherapy) and 80 stage III-IV and 130 stage I-II cases that were initially treated without radiation or chemotherapy and used Kaplan-Meier and Cox regression analyses to compare genotype groups on the basis of overall, disease-specific, progression-free, and recurrence-free survival rates.. ERCC2 35931 AA predicted worse survival in stage III-IV cases treated with radiation [multiply-adjusted HR = 1.66, 95% confidence interval (CI), 1.15-2.40; HR over the first 3 follow-up years = 1.92; 95% CI, 1.28-2.88] and better survival in stage III-IV cases not treated with radiation (HR = 0.26; 95% CI, 0.11-0.62). Although not associated with survival in stage III-IV cancers treated with radiation (HR = 1.00; 95% CI, 0.67-1.51), CCND1-870 GG predicted better survival in stage III-IV cancers not treated with radiation (HR = 0.14; 95% CI, 0.04-0.50). Survival in stage I-II did not depend on ERCC2 A35931C or CCND1 G870A genotype.. Although promoting tumor progression in untreated patients, germline differences in DNA-repair or cell-cycle control may improve treatment outcome in patients treated with DNA-damaging agents.. ERCC2 A35931C may help distinguish advanced stage SCCHN with better outcomes from radiation treatment.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cohort Studies; Combined Modality Therapy; Cyclin D1; DNA Damage; DNA Repair; Female; Genetic Predisposition to Disease; Genotype; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Staging; Polymorphism, Single Nucleotide; Squamous Cell Carcinoma of Head and Neck; Survival Analysis; Treatment Outcome; Xeroderma Pigmentosum Group D Protein

Cyclin D1 (CCND1) has been associated with chemotherapy resistance and poor prognosis. In this study, we tested the hypothesis that CCND1 expression determines response and clinical outcomes in locally advanced head and neck squamous cell carcinoma (HNSCC) patients treated with neoadjuvant chemotherapy followed by surgery and radiotherapy.. 224 patients with HNSCC were treated with either cisplatin-based chemotherapy followed by surgery and radiotherapy (neoadjuvant group, n = 100) or surgery and radiotherapy (non-neoadjuvant group, n = 124). CCND1 expression was assessed by immunohistochemistry. CCND1 levels were analyzed with chemotherapy response, disease-free survival (DFS) and overall survival (OS). There was no significant difference between the neoadjuvant group and non-neoadjuvant group in DFS and OS (p = 0.929 and p = 0.760) when patients treated with the indiscriminate administration of cisplatin-based chemotherapy. However, in the neoadjuvant group, patients whose tumors showed a low CCND1 expression more likely respond to chemotherapy (p<0.001) and had a significantly better OS and DFS than those whose tumors showed a high CCND1 expression (73% vs 8%, p<0.001; 63% vs 6%, p<0.001). Importantly, patients with a low CCND1 expression in neoadjuvant group received more survival benefits than those in non-neoadjuvant group (p = 0.016), however patients with a high CCND1 expression and treated with neoadjuvant chemotherapy had a significantly poor OS compared to those treated with surgery and radiotherapy (p = 0.032). A multivariate survival analysis also showed CCND1 expression was an independent predictive factor (p<0.001).. This study suggests that some but not all patients with HNSCC may benefit from neoadjuvant chemotherapy with cisplatin-based regimen and CCND1 expression may serve as a predictive biomarker in selecting patients undergo less than two cycles of neoadjuvant chemotherapy.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cisplatin; Cyclin D1; Demography; Female; Head and Neck Neoplasms; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Neoadjuvant Therapy; Neoplasm Staging; Prognosis; Proportional Hazards Models; Risk Factors; Treatment Outcome

Head and neck squamous cell carcinoma (HNSCC) is a solid malignant neoplasm exhibiting aggressive phenotypes and high recurrence rates. To improve its clinical management, understanding the molecular basis of HNSCC development is of critical importance. For the investigation of tumor-associated genes, functional analyses in well-characterized tumor cell systems are required. To establish an experimental platform, a set of 20 HNSCC cell lines was screened for genetic imbalances by chromosomal comparative genomic hybridization (cCGH). Frequent DNA copy number gains were detected on 3q26.3-qter, 5p, 7p11-p13, 8q23-qter, 9p11-p13, 9q31-qter, 11q13 and 20q13.1, whereas copy number losses were found on 3p, 4p, 4q32.1-qter, 8p11-p12 and 18q22 in agreement with previous observations on genetic aberrations detected in primary HNSCC specimens. Subsequent mRNA expression analysis of 11q13 candidate genes CCND1 and CTTN revealed that HNSCC cell lines exhibiting a DNA copy number gain on 11q13 had a higher transcript level of CCND1 and CTTN compared with HNSCC cell lines without 11q13 copy number gain (P = 0.014 and P = 0.009, respectively). Furthermore, CCND1 and CTTN amplification as detected by fluorescence in situ hybridization correlated with protein expression as assessed by immunocytochemistry. In summary, the cytogenetic characterization illustrates that this set of HNSCC cell lines is representative for the HNSCC genome and provides tumor model systems for detailed analysis of genes with a possible role in the pathomechanism of head and neck tumors.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Chromosomes, Human, Pair 11; Comparative Genomic Hybridization; Cortactin; Cyclin D1; Gene Dosage; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Models, Genetic; Neoplasm Proteins; Polymerase Chain Reaction; RNA, Messenger

The number of targeted small molecules being developed in oncology is increasing rapidly. Many of these are designed to inhibit multiple kinases, and thus the mechanisms of responsiveness and predictive biomarkers can be difficult to discern. In fact, with few exceptions, multi-kinase inhibitors are developed with limited mechanism-based patient selection. Enzastaurin is a multi-kinase inhibitor being studied in several malignancies that we hypothesized would be active in squamous cell carcinoma of the head and neck, because it inhibits classic and novel protein kinase C isoforms. Indeed, enzastaurin reduced the growth of SQ-20B and CAL27 tumor xenografts, decreased proliferation in these cell lines, inhibited putative target phosphorylation, and induced cell cycle arrest. Gene expression arrays confirmed that expression of cell cycle genes, including cyclins D and E, were significantly altered by exposure to enzastaurin. However, testing a panel of squamous cell carcinoma of the head and neck cell lines revealed variable sensitivity to enzastaurin, which correlated significantly with baseline cyclin D1 protein expression. Moreover, sensitivity and resistance could be reversed, respectively, by expression or depletion of cyclin D1. Furthermore, analysis of sensitive and resistant cell lines revealed distinct differences in cyclin D1 regulation. Enzastaurin modulated cyclin D1 synthesis through an Akt-regulated pathway in the former, whereas high-level CCND1 gene amplification was present in the latter. These results underscore the critical relevance of cellular signaling context in developing cancer therapies in general and suggest that enzastaurin in particular would be most effective in tumors where baseline cyclin D1 expression is low to moderate and physiologically regulated.

Topics: Animals; Base Sequence; Carcinoma, Squamous Cell; Cell Cycle; Cell Line; Cell Proliferation; Cyclin D1; DNA Primers; Female; Head and Neck Neoplasms; Humans; Indoles; Mice; Mice, Nude; Oligonucleotide Array Sequence Analysis; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

The aim of this study was to investigate the effect of epidermal growth factor receptor (EGFR) blockade on cell survival and on downstream signalling pathways using the monoclonal antibody cetuximab.. We used three colon cancer cell lines, of which one was EGFR-negative, and two head and neck squamous cell carcinoma (HNSCC) lines. EGFR expression and gene copy number were measured by immunohistochemistry and FISH analysis, respectively. The effect of cetuximab, irradiation or the combination of both on cell growth was estimated by SRB assay. Western blotting was used to determine the phosphorylation of intracellular signalling proteins and cell cycle phase distribution was measured by flow cytometry.. The addition of cetuximab had only limited effects on cell growth, with a maximum inhibition of approximately 30%, but was correlated with the amount of protein expression and gene copy number of EGFR. When combined with irradiation, the effect of cetuximab was only additive and not dependent on the inherent radio-sensitivity of the cell lines. Persistent phosphorylation of Akt and/or p44/42 MAPK was detected by western blot in all of the cell lines, whereas there was no phosphorylation of Jak2 or STAT3.. None of these factors alone could predict the sensitivity to cetuximab. Rather, the results suggest that it might be necessary to determine the activation status of several intracellular signalling proteins to better predict the sensitivity to cetuximab treatment.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line, Tumor; Cetuximab; Colonic Neoplasms; Cyclin D1; ErbB Receptors; Fluorescent Dyes; Gene Amplification; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Mutation; Rhodamines; Signal Transduction

Amplification of the 11q13 region is a prevalent genetic alteration in head and neck squamous cell carcinoma (HNSCC). We investigated the clinical significance of cortactin (CTTN) and cyclin D1 (CCND1) amplification in both malignant transformation and tumour progression. CTTN and CCND1 amplification was analysed by differential and real-time PCR in a prospective series of laryngeal/pharyngeal carcinomas and archival premalignant tissues. CTTN mRNA and protein expression were respectively determined by real-time RT-PCR and immunohistochemistry, and correlated with gene status. Molecular alterations were associated with clinicopathological parameters and disease outcome. CTTN and CCND1 amplifications were respectively found in 75 (37%) and 90 (45%) tumours. Both correlated with advanced disease; however, only CTTN amplification was associated with recurrence and reduced disease-specific survival (p = 0.0022). Strikingly, CTTN amplification differentially influenced survival depending on tumour site (p = 0.0001 larynx versus p = 0.68 pharynx) and was an independent predictor of reduced survival in the larynx (p = 0.04). CCND1 amplification was detected in early tumourigenesis and increased with the severity of dysplasia. Importantly, CTTN amplification was only found in high-grade dysplasias that progressed to invasive carcinoma. CTTN gene status strongly correlated with mRNA and protein expression. Furthermore, CTTN overexpression correlated significantly with reduced disease-specific survival (p = 0.018). Taken together, these data indicate that CTTN may serve as a valuable biomarker to identify patients with laryngeal tumours at high risk of recurrence and poor outcome.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 11; Cortactin; Cyclin D1; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate

Galanin and its three receptors (GALR1-3) are expressed in many normal tissues, but silenced in some tumors. Contradictory roles for galanin and its receptors in various tumors have been reported. To understand their function, investigations of individual galanin receptors are necessary. In head and neck squamous carcinoma cells (HNSCC) with silenced GALR1 and GALR2, we showed that reexpressed GALR1 suppresses tumor cell proliferation via Erk1/2-mediated effects on cdk inhibitors and cyclin D1. Others showed that GALR2 could induce apoptosis in neuroblastoma cells with wild-type p53, whereas GALR2 stimulated proliferation in small cell lung cancer. In this study, we investigated the role of GALR2 in HNSCC cells that have mutant p53 and do not express GALR1.. UM-SCC-1, a human oral carcinoma cell line with a splice site mutation causing a 46-bp p53 off-frame deletion, was stably transfected to express GALR2 (UM-SCC-1-GALR2).. Galanin treatment of UM-SCC-1-GALR2 caused morphologic changes and a marked decrease in cell number that were not observed in UM-SCC-1-mock cells. Galanin and GALR2 resulted in decreased bromodeoxyuridine incorporation, p27(Kip1) and p57(Kip2) up-regulation, and decreased cyclin D1 expression. These effects were similar to GALR1 signaling in HNSCC, but GALR2 also induced caspase-3-dependent apoptosis, which was confirmed by Annexin-V staining and DNA fragmentation analysis. These were not observed with GALR1.. This study shows that GALR2 reexpression can inhibit cell proliferation and induce apoptosis in HNSCC cells with mutant p53. GALR2 may be a feasible target for HNSCC therapy.

Topics: Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinase Inhibitor p57; Genes, p53; Head and Neck Neoplasms; Humans; Mutation; Receptor, Galanin, Type 2

MicroRNAs are the endogenous small non-coding RNA molecules capable of silencing protein coding genes at the posttranscriptional level. Based on computer-aided predictions, a single microRNA could have over a hundred of targets. On the other hand, a single protein-coding gene could be targeted by many potential microRNAs. However, only a relatively small number of these predicted microRNA/mRNA interactions are experimentally validated, and no systematic validation has been carried out using a reporter system.. In this study, we used luciferease reporter assays to validate microRNAs that can silence cyclin D1 (CCND1) because CCND1 is a well known proto-oncogene implicated in a variety of types of cancers. We chose miRanda http://www.microRNA.org as a primary prediction method. We then cloned 51 of 58 predicted microRNA precursors into pCDH-CMV-MCS-EF1-copGFP and tested for their effect on the luciferase reporter carrying the 3'-untranslated region (UTR) of CCND1 gene.. Real-time PCR revealed the 45 of 51 cloned microRNA precursors expressed a relatively high level of the exogenous microRNAs which were used in our validation experiments. By an arbitrary cutoff of 35% reduction, we identified 7 microRNAs that were able to suppress Luc-CCND1-UTR activity. Among them, 4 of them were previously validated targets and the rest 3 microRNAs were validated to be positive in this study. Of interest, we found that miR-503 not only suppressed the luciferase activity, but also suppressed the endogenous CCND1 both at protein and mRNA levels. Furthermore, we showed that miR-503 was able to reduce S phase cell populations and caused cell growth inhibition, suggesting that miR-503 may be a putative tumor suppressor.. This study provides a more comprehensive picture of microRNA/CCND1 interactions and it further demonstrates the importance of experimental target validation.

Topics: 3' Untranslated Regions; Carcinoma; Cell Line, Tumor; Cyclin D1; Gene Silencing; Genes, Reporter; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; MicroRNAs; Proto-Oncogene Mas; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S Phase

Genetic alteration in oral premalignant lesions (OPLs), the precursors of oral squamous cell carcinomas (OSCCs), may represent key changes in disease initiation and development. We ask if DNA amplification occurs at this early stage of cancer development and which oncogenic pathways are disrupted in OPLs. Here, we evaluated 50 high-grade dysplasias and low-grade dysplasias that later progressed to cancer for gene dosage aberrations using tiling-path DNA microarrays. Early occurrences of DNA amplification and homozygous deletion were frequently detected, with 40% (20/50) of these early lesions exhibiting such features. Expression for 88 genes in 7 recurrent amplicons were evaluated in 5 independent head and neck cancer datasets, with 40 candidates found to be overexpressed relative to normal tissues. These genes were significantly enriched in the canonical ERK/MAPK, FGF, p53, PTEN and PI3K/AKT signaling pathways (p = 8.95 x 10(-3) to 3.18 x 10(-2)). These identified pathways share interactions in one signaling network, and amplification-mediated deregulation of this network was found in 30.0% of these preinvasive lesions. No such alterations were found in 14 low-grade dysplasias that did not progress, whereas 43.5% (10/23) of OSCCs were found to have altered genes within the pathways with DNA amplification. Multitarget FISH showed that amplification of EGFR and CCND1 can coexist in single cells of an oral dysplasia, suggesting the dependence on multiple oncogenes for OPL progression. Taken together, these findings identify a critical biological network that is frequently disrupted in high-risk OPLs, with different specific genes disrupted in different individuals.

Topics: Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Fibroblast Growth Factors; Gene Amplification; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Oligonucleotide Array Sequence Analysis; Precancerous Conditions; RNA, Messenger; Signal Transduction

To assess radiosensitivity of neck metastases of squamous cell carcinoma of the head and neck (SCCHN) by immunocytochemical profiling of fine-needle aspiration biopsy (FNAB) cell specimens.. Immunocytochemical reactions to p53, cyclin D1, stefin A and Ki-67 were determined in FNAB cell samples of neck metastases from 21 patients treated with concomitant chemoradiotherapy and correlated to clinical characteristics and response to therapy.. Six (28.6%), eight (38.1%), 15 (71.4%) and nine (42.9%) FNAB cell samples were classified as p53, cyclin D1, stefin A and Ki-67 positive, respectively. Statistically significant predictors of favorable nodal response to chemoradiation were p53 (P=0.025) and cyclin D1 (cytoplasmic fraction, P=0.048) negativity and Ki-67 positivity (P=0.045). Regional recurrence correlated with low Ki-67 immunoreactivity. A favorable profile of cyclin D1 and Ki-67 (one or both of the two) further improved the predictive strength of these markers.. FNAB is a non-invasive, simple and cheap procedure, which could serve simultaneously for diagnostic purposes and for radiosensitivity testing. Immunocytochemical determination of cyclin D1 and Ki-67 in FNAB cell samples from neck metastases of SCCHN seems to be a valuable marker for predicting regional response to radiotherapy and might assist when deciding on appropriate primary therapy.

Topics: Adult; Aged; Biopsy, Fine-Needle; Carcinoma, Squamous Cell; Cyclin D1; Cystatin A; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Ki-67 Antigen; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neck; Radiation Tolerance; Tumor Suppressor Protein p53

The variant allele of CCND1 G870A encodes a splice variant of the cyclin D1 protein, which possesses an increased half-life. To confirm the phenotypic effect of the variant allele, we examined the immunohistochemical staining pattern of the protein in tumors from a case population of head and neck squamous cell carcinoma (HNSCC) and compared it with the genotype of these individuals. We also examined how this genotype was associated with the risk of HNSCC and if this genotype-phenotype association was related to patient outcome.. In a population-based case-control study of 698 cases and 777 controls, we both genotyped all participants for the CCND1 gene and did immunohistochemical staining of the cyclin D1 protein in the HNSCC tumors.. The variant AA genotype was significantly associated with positive immunohistochemical staining (P < 0.02), and this variant genotype was associated with a significantly elevated odds ratio of 1.5 (95% confidence interval, 1.1-2.0) for HNSCC overall, with risk greatest in oral and laryngeal sites. Positive immunohistochemical staining was inversely related to human papillomavirus 16 DNA present in the tumor (P < 0.03). The AA genotype and superpositive immunohistochemical staining for cyclin D1 also had independent and significant effects on patient survival.. These results strongly suggest that this splice variant, when present in two copies, is a significant predictor of both the occurrence of HNSCC as well as patient survival after treatment. These data further indicate that this variant protein is an important determinant of individual response to therapy for this disease.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; DNA, Viral; Female; Genotype; Head and Neck Neoplasms; Human papillomavirus 16; Humans; Immunohistochemistry; Male; Middle Aged; Phenotype; Risk Factors

Protein microarrays are of increasing importance for high-throughput screening of fresh tissues. In our study, protein microarrays were generated by printing antibodies onto membranes to characterize protein profiles expressed by head and neck squamous cell carcinomas (HNSCCs). Cellular proteomes of 30 matched normal squamous epithelial cells and carcinoma specimens were analyzed after tissue microdissection using microarrays composed of 83 different antibodies. As controls, Western blot analysis and tissue microarrays (TMAs) containing 98 HNSCC specimens were used. Of the 83 proteins examined, 14 showed differential expression between HNSCCs and normal epithelium. The protein microarray approach revealed an upregulation of 8 proteins and a downregulation of 6 proteins. Bag-1, Cox-2, Hsp-70, Stat3, pescadillo, MMP-7 (matrilysin), IGF-2, and cyclin D1 were identified to be significantly upregulated, whereas suppressor of cytokine signaling 1, thrombospondin, TGF-beta1, Jun, Fos, and Fra-2 were downregulated. The differential expression of these proteins was confirmed using Western blot and TMA. Upon correlation of differentially regulated proteins with the clinicopathologic data of our patients, MMP-7 (matrilysin) was found to be associated with survival in univariate, but not multivariate, analysis. These data indicate that our protein arrays provide protein information in a systematic, reproducible, and also high-throughput fashion.

Topics: Biomarkers, Tumor; Blotting, Western; Carcinoma, Squamous Cell; Cyclin D1; Head and Neck Neoplasms; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Matrix Metalloproteinase 7; Multivariate Analysis; Protein Array Analysis; Proteome; Reproducibility of Results; STAT3 Transcription Factor; Tissue Array Analysis

DNA amplification of the 11q13 region is observed frequently in many carcinomas. Within the amplified region several candidate oncogenes have been mapped, including cyclin D1, TAOS1 and cortactin. Yet, it is unknown which gene(s) is/are responsible for the selective pressure enabling amplicon formation. This is probably due to the use of low-resolution detection methods. Furthermore, the size and structure of the amplified 11q13 region is complex and consists of multiple amplicon cores that differ between different tumor types. We set out to test whether the borders of the 11q13 amplicon are restricted to regions that enable DNA breakage and subsequent amplification. A high-resolution array of the 11q13 region was generated to study the structure of the 11q13 amplicon and analyzed 29 laryngeal and pharyngeal carcinomas and nine cell lines with 11q13 amplification. We found that boundaries of the commonly amplified region were restricted to four segments. Three boundaries coincided with a syntenic breakpoint. Such regions have been suggested to be putatively fragile. Sequence comparisons revealed that the amplicon was flanked by two large low copy repeats known as segmental duplications. These segmental duplications might be responsible for the typical structure and size of the 11q13 amplicon. We hypothesize that the selection for genes through amplification of the 11q13.3 region is determined by the ability to form DNA breaks within specific regions and, consequently, results in large amplicons containing multiple genes.

Topics: Animals; Carcinoma; Cell Line, Tumor; Chromosome Mapping; Chromosomes, Human, Pair 11; Cortactin; Cyclin D1; DNA Damage; Gene Duplication; Head and Neck Neoplasms; Humans; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Neoplasm Proteins; Nucleic Acid Hybridization

Histone deacetylase inhibitors (HDI) can inhibit proliferation and enhance apoptosis in a wide range of malignancies. However, HDIs show relatively modest activity in head and neck squamous cell carcinomas (HNSCC), in which we have shown the activation of nuclear factor-kappaB (NF-kappaB; NF-kappaB1/RelA or p50/p65), a transcription factor that promotes expression of proliferative and antiapoptotic genes. In this study, we examined if HDIs enhance activation of NF-kappaB and target genes and if genetic or pharmacologic inhibition of NF-kappaB can sensitize HNSCC to HDIs. Limited activity of classic HDIs trichostatin A and sodium butyrate was associated with enhanced activation of NF-kappaB reporter activity in a panel of six HNSCC cell lines. HDIs enhanced NF-kappaB p50/p65 DNA binding and acetylation of the RelA p65 subunit. Transfection of small interfering RNAs targeting p65 strongly inhibited NF-kappaB expression and activation, induced cell cycle arrest and cell death, and further sensitized HNSCC cells when combined with HDIs. The p65 small interfering RNA inhibited HDI-enhanced expression of several NF-kappaB-inducible genes implicated in oncogenesis of HNSCC, such as p21, cyclin D1, and BCL-XL. Bortezomib, an inhibitor of proteasome-dependent NF-kappaB activation, also increased sensitization to trichostatin A, sodium butyrate, and a novel HDI, PXD101, in vitro, and to the antitumor effects of PXD101 in bortezomib-resistant UMSCC-11A xenografts. However, gastrointestinal toxicity, weight loss, and mortality of the combination were dose limiting and required parenteral fluid administration. We conclude that HDI-enhanced NF-kappaB activation is one of the major mechanisms of resistance of HNSCC to HDIs. The combination of HDI and proteasome inhibitor produced increased antitumor activity. Low starting dosages for clinical studies combining HDIs with proteasome inhibitors and IV fluid support may be warranted.

Topics: Acetylation; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; bcl-X Protein; Boronic Acids; Bortezomib; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA, Neoplasm; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitor of Apoptosis Proteins; Mice; Mice, SCID; NF-kappa B p50 Subunit; Proteasome Inhibitors; Protein Binding; Pyrazines; RNA, Small Interfering; Sulfonamides; Transcription Factor RelA; Xenograft Model Antitumor Assays

Nuclear/cytoplasmic accumulation of beta-catenin is mainly regulated by its degradation, which is initiated by interaction with adenomatous polyposis coli (APC) protein. Accumulation of beta-catenin activates the transcription of 1 of the target oncogenic genes, cyclin D1, in the Wnt/Wingless pathway. The role of beta-catenin and cyclin D1 in this pathway has not been previously studied in head and neck mucoepidermoid carcinoma (MEC). This study investigates abnormalities of beta-catenin and the APC gene in MEC and correlates the patterns of cyclin D1 overexpression and nuclear/cytoplasmic accumulation of beta-catenin with the clinical outcome.. Mutations of the beta-catenin and APC genes, as well as overexpression of cyclin D1, were investigated by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in tissue samples from 44 cases of MEC. In addition, we employed differential PCR method to detect amplification of the cyclin D1 gene. Furthermore, the overexpression of cyclin D1 and nuclear/cytoplasmic accumulation of beta-catenin was examined by immunohistochemistry, and any correlation with clinicopathologic parameters was evaluated.. Nuclear/cytoplasmic accumulation of beta-catenin was observed in 6 of 44 MEC cases (13.6%), 5 of which were high-grade MEC, while the other 1 case was intermediate-grade tumor. Mutational analysis of exon 3 of the beta-catenin gene revealed that 4 of 26 cases (15.4%) contained point mutations (3 in codon 32, GAC [Asp] to GGC [Gly]; 1 in codon 42, ACA [Thr] to ATA [Ile]), and all these 4 cases showed beta-catenin accumulation immunohistochemically. The nuclear/cytoplasmic accumulation of beta-catenin was significantly correlated with the adverse outcome of patients (p = .011). Two APC gene alterations were detected in 2 cases of low-grade MEC, where there was no beta-catenin nuclear accumulation. Amplification of the cyclin D1 gene was observed in 10 of 26 cases (38.5%). Cyclin D1 overexpression was recognized in 19 of 44 cases (43.2%) and was significantly correlated with beta-catenin accumulation (p = .003).. These findings suggest that beta-catenin, which, in cooperation with cyclin D1, plays crucial role in the Wnt-signaling pathway, may also contribute to the adverse outcome and high-grade tumor staging of MEC.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Carcinoma, Mucoepidermoid; Codon; Cyclin D1; Disease Progression; Exons; Female; Genes, APC; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Survival Analysis

Human lactoferrin is a naturally occurring glycoprotein that inhibits cancer growth. Our purpose was to evaluate recombinant human lactoferrin as a chemotherapeutic agent against head and neck squamous cell carcinoma.. Controlled experiments both in vitro and in the murine model evaluating both the effect and mechanism of lactoferrin on cancer growth.. In both human and murine cell lines, lactoferrin induced dose-dependent growth inhibition. Using flow cytometric analysis, lactoferrin was shown to induce G(1)-G(0) growth arrest. This arrest seemed to be modulated by down-regulation of cyclin D1. In the in vitro model, luminex data revealed that lactoferrin inhibited cellular release of proinflammatory and prometastatic cytokines, including interleukin-8, interleukin-6, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-alpha. Lactoferrin up-regulated the cellular activation of nuclear factor-kappaB within 4 h of cellular exposure. In C3h/HeJ mice implanted with SCCVII tumors, orally delivered lactoferrin inhibited tumor growth by 75% compared with control mice. Immunohistochemical analysis of harvested tumors revealed up to 20-fold increases of lymphocytes within treated animals. When mice were depleted of CD3(+) cells, all lactoferrin-induced tumor inhibition was abrogated.. We conclude that human recombinant lactoferrin can inhibit the growth of head and neck squamous cell carcinoma via direct cellular inhibition as well as systemically via immunomodulation. Our data support the study of human lactoferrin as an immunomodulatory compound with therapeutic potential.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytokines; Flow Cytometry; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lactoferrin; Mice; Recombinant Proteins; T-Lymphocytes

Head and neck squamous cell carcinoma (HNSCC) represents 5.5% of malignancies worldwide, with approximately 30,000 new cases and approximately 11,000 deaths reported in the United States annually. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth regulating system; the OGF-OGFr axis influences the G(0)-G(1) phase of the cell cycle in HNSCC. Cells treated with small interfering RNA (siRNA) for OGFr no longer responded to the growth inhibitory effects of OGF or the growth stimulatory effects of naltrexone, indicating that these activities are entirely mediated by OGFr. In this investigation, we examined the precise target of OGF in the cell cycle. Using SCC-1 cells, OGF decreased the phosphorylation of retinoblastoma protein. This change was correlated with reduced Cdk4, but not Cdk2, kinase activity. OGF treatment increased cyclin-dependent kinase inhibitor p16 protein expression. Importantly, p16 complexed with Cdk4 was increased by OGF treatment at all time points, consistent with the hypothesis that OGF mediated growth inhibition through p16. Blockade of OGF-OGFr interactions with naloxone abolished the increased expression of p16 protein by OGF. Inhibition of p16 (INK4a) activation by p16-specific siRNA blocked OGF's repressive action on proliferation of SCC-1, CAL-27, and SCC-4 HNSCC cells. These data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human HNSCC is a cyclin-dependent kinase inhibitory pathway, and this may be useful in the diagnosis and treatment of HNSCC.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Enkephalin, Methionine; Head and Neck Neoplasms; Humans; Phosphorylation; Receptors, Opioid; Retinoblastoma Protein

Alcohol consumption and cigarette smoking play a key role in the development and progression of head and neck cancer. Additionally, epidemiologic studies have given evidence that other environmental and genetic factors are relevant. We present a prospective study including 465 head and neck cancer patients. All patients were recruited between 1994 and 1998 during the initial tumor diagnosis. Three hundred twelve patients could be followed over 5 years after histologically proven curative surgical treatment. All clinical data were recorded (i.e., age, gender, TNM stage, histological grading, smoking and drinking habits) and genetic variations at loci encoding detoxifying enzymes (glutathione S-transferase and cytochrome P450); immune modulating cytokines (tumor necrosis factor) and cell cycle regulating proteins (cyclin D1) were determined. Parameters with an impact on recurrence-free survival were analyzed. A strong influence could be attributed to the tumor size at the time of presentation. Additionally, the grading of the tumor showed a strong influence (5 years recurrence free: G1: 87% and G1: 61%). Furthermore, it could be shown that the recurrence-free survival was significantly influenced by cyclin D1 genotypes (CCND1GG: P =0.01; HR=3.72) and TNF microsatellite haplotypes (TNFB1D5: P =0.043; HR=2.05). These findings are compatible with the view that genetic predisposition is important in determining recurrence-free survival after surgical treatment of head and neck cancer.

Topics: Alcohol Drinking; Carcinoma, Squamous Cell; Cyclin D1; Cytochrome P-450 Enzyme System; Disease-Free Survival; Glutathione Transferase; Head and Neck Neoplasms; Humans; Microsatellite Instability; Neoplasm Recurrence, Local; Polymorphism, Genetic; Risk Factors; Smoking; Survival Rate; Tumor Necrosis Factor-alpha

Recent studies provide evidence that the constitutive activation of nuclear factor-kappa B, NF-kappaB plays a critical role in enhancing the growth of several types of malignancies, including head and neck squamous cell carcinoma (HNSCC).. In this study, we examined the effects of a newly synthesized NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on growth, induction of apoptosis, gene expression, and chemosensitivity in two HNSCC cell lines (YCU-H891 and KB), which expressed high levels of nuclear NF-kappaB protein.. DHMEQ showed strong growth inhibitory effects on these two cell lines, with a 50% cell growth inhibition (IC50) concentration of approximately 20 microg/mL. These growth inhibitory effects were associated with inhibition of the NF-kappaB activity. Treatment with DHMEQ induced apoptosis in a dose-dependent manner accounting, at least in part, for the growth inhibition by DHMEQ. DHMEQ strongly inhibited cyclin D1 and vascular endothelial growth factor (VEGF) promoter activity and decreased the levels of cyclin D1 protein and VEGF mRNA in KB cells. In addition, low concentrations of DHMEQ (1.0 or 5.0 microg/mL) synergistically enhanced the cellular sensitivity of YCU-H and KB cells to cisplatin, which is a key chemotherapeutic agent in the treatment of HNSCC.. These results suggest that DHMEQ may be effective when used alone or in combination with other agents in the treatment of HNSCC.

Topics: Apoptosis; Benzamides; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclohexanones; Gene Expression; Head and Neck Neoplasms; Humans; NF-kappa B; Vascular Endothelial Growth Factor A

To investigate the response of tumour growth to cisplatin treatment, in relation to p53 mutation and cyclin D1 dysregulation on DNA and protein level, biopsies from seven xenografted human squamous cell carcinomas from the head and neck were analysed with immunohistochemistry for p53 expression and cyclin D1 expression. Polymerase chain reaction-singlestranded conformation polymorphism was used to determine p53 mutations. Fluorescence in situ hybridization was performed to analyse cyclin D1 amplification. The mice were injected i.p. with NaCl (controls) or cisplatin. After injection the tumour volume were measured. The inhibition of tumour growth by cisplatin was defined as the area under the growth curves, and compared with the growth curves of the tumours in the control group. Xenografts with p53 mutation showed significantly higher resistance to cisplatin (p < 0.001) and also tumours with cyclin D1 amplification showed significantly higher resistance (p < 0.001).

Topics: Animals; Carcinoma, Squamous Cell; Cheek; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Flow Cytometry; Gene Amplification; Gingival Neoplasms; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; Mice; Mouth Neoplasms; Mutation; Neoplasms, Unknown Primary; Statistics as Topic; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Adenoid cystic carcinoma (ACC) is an uncommon salivary gland malignancy characterized by indolent yet relentless growth that exhibits inherent resistance to systemic chemotherapy, surgical salvage and conventional radiotherapy. We used microarray analysis to characterize gene expression changes associated with ACC. Eight ACC patient specimens were compared with normal parotid gland tissue and the ACC3 cell line. Differentially expressed genes were identified (512 total) using supervised analysis methods and functional categories assigned using OntoExpress. Genes associated with morphogenesis, neurogenesis, proliferation and apoptosis characterized ACC tumors. Genes associated with saliva production and immune response characterized normal parotid tissues while the ACC3 cell line expressed genes primarily associated with proliferation, chromosome maintenance and the cell cycle. These results demonstrate that ACC tumors express genes associated with early developmental processes including morphogenesis and neurogenesis implicating oncogenic events that result in dedifferentiation of normal salivary glands.

Topics: Adult; Apoptosis; Carcinoma, Adenoid Cystic; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, cdc; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Parotid Gland; Polymerase Chain Reaction; Salivary Gland Neoplasms; Salivation

In recent studies, green tea components have been shown to induce cell growth arrest and apoptosis in head and neck squamous cell carcinoma (HNSCC) cells. In this report, we have investigated the effects of epicatechin gallate (ECG), one of the catechins in green tea, on anti-cancer activity in vitro. We found that cyclin D1 was highly expressed in HNSCC cells, and ECG suppressed 90% of cyclin D1 expression in SCC7 cells. We have also evaluated the effect of ECG on cell growth and apoptosis, showing that ECG (50 microM) exhibited a significant inhibition (50%) on the growth of SCC7 cells via G1 cell cycle arrest. ECG suppressed cyclin D1 in SCC7 cells in a dose- and time-dependent manner, and the suppression of the beta-catenin pathway by ECG is one of the mechanism to facilitate ECG-induced cell growth arrest. These results suggest that ECG has a potential usage as a chemopreventive agent in HNSCC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Catechin; Cell Line, Tumor; Cyclin D1; Cyclooxygenase 1; Cyclooxygenase 2; Cytokines; Flavonoids; Flow Cytometry; Growth Differentiation Factor 15; Growth Inhibitors; Head and Neck Neoplasms; Humans; Neoplasm Proteins; Phenols; Polyphenols

Overexpression of cyclin D1 in head and neck cancer has been suggested to be a poor prognostic factor. To understand the role of cyclin D1 expression in head and neck cancer, we overexpressed cyclin D1 in TU182 (a cell line derived from pharyngeal cancer) using a retroviral vector. Stable transfectants were isolated by neomycin (G418) selection. Compared to the parental and control-vector transfected cells, the cyclin D1 transfected cells revealed a decrease of the G1/G0 population and resulted in continuous proliferation under low serum conditions. Proliferation assays revealed an increase in resistance to cisplatin in cyclin D1 overexpressing cells. These observation suggest that deregulation of cyclin D1 may reduce growth factor requirements and contribute to the resistance to some chemotherapeutic agents among head and neck cancer patients.

Topics: Antineoplastic Agents; Blotting, Northern; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Line; Cell Survival; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; In Vitro Techniques; Prognosis; Transfection; Tumor Cells, Cultured

The development of more effective prevention and treatment strategies for solid tumors is limited by an incomplete understanding of the critical growth pathways that are activated in carcinogenesis. Signal transducers and activators of transcription (STAT) proteins have been linked to transformation and tumor progression. Several approaches have been used to block STAT3 in cancer cells resulting in reduced proliferation and apoptosis. We tested the hypothesis that blocking STAT3 activation using a transcription factor decoy approach would decrease tumor growth and STAT3 target gene expression in vivo. In a xenograft model of squamous cell carcinoma of the head and neck (SCCHN), daily administration of the STAT3 decoy (25 microg) resulted in decreased tumor volumes, abrogation of STAT3 activation, and decreased expression of STAT3 target genes (VEGF, Bcl-xL, and cyclin D1) compared to treatment with a mutant control decoy. Blockade of STAT3 with the STAT3 decoy also induced apoptosis and decreased proliferation, an effect that was augmented when the STAT3 decoy was combined with cisplatin, both in vitro and in vivo. These results suggest that a transcription factor decoy approach may be used to target STAT3 in cancers that demonstrate increased STAT3 activation including SCCHN.

Topics: Acute-Phase Proteins; Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Cisplatin; Cyclin D1; DNA-Binding Proteins; Down-Regulation; Genetic Therapy; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transcription Factors; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

DNA repair enzyme genetic polymorphisms have been postulated to increase the risk of certain cancers in the presence of tobacco carcinogen exposures. The XPD protein is an important component of the TFIIH transcription factor complex. XPD genetic polymorphisms resulting in amino acids substitutions may lead to alterations in TFIIH helicase activity, resulting in repair and transcription defects. Cyclin D1 is a key regulatory protein for the transition of cells from the G(1)-S cell cycle phase. The CCND1 G870A polymorphism has been reported to enhance alternate splicing of a stable mRNA variant, which may result in the bypass of the G(1)/S cell cycle checkpoint. In this study, XPD G23591A (Asp312Asn) and A35931C (Lys751Gln) polymorphisms and the CCND1 G870A splice variant frequencies were determined in 273 upper aero-digestive tract cancer cases and 269 controls. The XPD Asp312Asn variant frequency was significantly different among cases and controls and conferred an odds ratio (OR) of 1.3 (95% CI 1.0-1.8). However, individuals with the CCND1 G870A and XPD Lys751Gln variants had higher age adjusted ORs of 3.2 (95% CI 2.2-4.6) and 2.2 (95% CI 1.5-3.2), respectively. Furthermore, a significant gene-gene interaction was observed among cases with at least two variant alleles for both CCND1 and XPD genes [OR 7.09 (95% CI 4.03-12.5)]. Smokers with a combination of at least one variant allele of both CCND1 and XPD genes also had an elevated risk as compared to nonsmokers. This is the first study to suggest an associative interaction between XPD and CCND1 genetic polymorphisms, tobacco exposure, and cancer risk.

Topics: Alternative Splicing; Carcinoma, Squamous Cell; Cyclin D1; DNA Helicases; DNA Primers; DNA-Binding Proteins; Exons; Female; Genetic Variation; Head and Neck Neoplasms; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Reference Values; Smoking; Transcription Factors; Xeroderma Pigmentosum Group D Protein

beta-catenin, depending on subcellular localization, plays a dual role in carcinogenesis: as a signaling factor (in the nucleus) and as an adhesion molecule (in cell membrane). In this study, we sought to determine the role of beta-catenin in head and neck carcinogenesis.. First, we studied the incidence of mutations of beta-catenin in a cohort of 60 head and neck squamous cell cancers (HNSCC). We subsequently evaluated the protein expression levels of beta-catenin in a cohort of oropharyngeal squamous cell cancer tissue microarray using a novel in situ method of quantitative protein analysis and correlated those with cyclin D1 levels and clinical and pathologic data.. The mean follow-up time for survivors was 45 months and for all patients was 35 months. We found no mutations in the cohort of 60 HNSCC. beta-catenin displayed primarily membranous expression pattern. Patients with high tumor-node-metastasis stage were more likely to have high expression of beta-catenin (P = 0.040). Patients with low beta-catenin expression had a local recurrence rate of 79% compared with 29% for patients with high beta-catenin tumors (P = 0.0021). Univariate Cox regression revealed a hazard ratio for low beta-catenin tumors of 3.6 (P = 0.004). Kaplan-Meier analysis showed that patients with low beta-catenin expressing tumors trended toward worse 5-year disease-free survival (P = 0.06). In multivariate analysis, only beta-catenin expression status was an independent prognostic factor (P = 0.044) for local recurrence. Tumors with high beta-catenin had low cyclin D1 and vice versa (P = 0.007).. The absence of activating beta-catenin mutations combined with the inverse correlation between beta-catenin levels with cyclin D1 levels and outcome suggest that beta-catenin mainly functions as an adhesion and not signaling molecule in HNSCC.

Topics: Adult; Aged; beta Catenin; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cyclin D1; Cytoskeletal Proteins; DNA Mutational Analysis; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Multivariate Analysis; Mutation; Neoplasm Staging; Survival Analysis; Trans-Activators

Head and neck squamous cell carcinomas (HNSCCs) exhibit an increased expression of the translation initiation factor eIF4E and the cell-cycle regulator cyclin D1 (CCND1). Both stimulate cell cycle progression and transform squamous epithelial cells. We used small interfering RNAs (siRNAs) to silence the expression of eIF4E and CCND1 in HNSCC UMSCC22B cells and analyzed the effects of reduced levels of these proteins on colony formation. Transfection of either eIF4E or CCND1 siRNAs decreased the levels of their targeted proteins and inhibited cell growth. siRNA-mediated decrease of eIF4E has led to decreases in the expression of CCND1, basic fibroblast growth factor and vascular endothelial growth factor. Combination of these siRNAs and cisplatin showed more than additive inhibition of colony formation. These findings demonstrate that siRNA silencing of either eIF4E or CCND1 leads to inhibition of the growth of HNSCC cells and suggest that these siRNAs alone or combined with conventional cytotoxic agents may be useful for therapy of HNSCCs.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cisplatin; Combined Modality Therapy; Cyclin D1; Down-Regulation; Eukaryotic Initiation Factor-4E; Gene Expression; Gene Targeting; Genes, Neoplasm; Head and Neck Neoplasms; Humans; RNA, Small Interfering; Transfection

The purpose of this study was to determine whether curcumin would trigger cell death in the head and neck squamous cell carcinoma (HNSCC) cell lines CCL 23, CAL 27, and UM-SCC1 in a dose-dependent fashion.. HNSCC cells were treated with curcumin and assayed for in vitro growth suppression using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide and fluorescence-activated cell sorting analyses. Expression of p16, cyclin D1, phospho-Ikappabeta, and nuclear factor-kappabeta (NF-kappabeta) were measured by Western blotting, gel shift, and immunofluorescence.. Addition of curcumin resulted in a dose-dependent growth inhibition of all three cell lines. Curcumin treatment resulted in reduced nuclear expression of NF-kappabeta. This effect on NF-kappabeta was further reflected in the decreased expression of phospho-Ikappabeta-alpha. Whereas the expression of cyclin D1, an NF-kappabeta-activated protein, was also reduced, there was no difference in the expression of p16 at the initial times after curcumin treatment. In vivo growth studies were done using nude mice xenograft tumors. Curcumin was applied as a noninvasive topical paste to the tumors and inhibition of tumor growth was observed in xenografts from the CAL27 cell line.. Curcumin treatment resulted in suppression of HNSCC growth both in vitro and in vivo. Our data support further investigation into the potential use for curcumin as an adjuvant or chemopreventive agent in head and neck cancer.

Topics: Animals; Annexin A5; Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Nucleus; Cell Separation; Cell Survival; Curcumin; Cyclin D1; Dose-Response Relationship, Drug; Female; Flow Cytometry; Head and Neck Neoplasms; Humans; I-kappa B Proteins; In Vitro Techniques; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasm Transplantation; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Tetrazolium Salts; Thiazoles

infection by oncogenic subtypes of human papillomavirus (HPV) and cyclin D1 gene (CCND1) amplification are frequent events in head and neck squamous cell carcinomas. The objective of this paper is to establish the relationship between the presence of human papillomavirus (HPV) gene sequences and the development of CCND1 gene amplification in these tumours.. 59 squamous cell carcinomas of the head and neck were studied for HPV types 6b and 16 and CCND1 gene amplification by polymerase chain reaction.. HPV DNA was detected in 14 tumors (24%). Ten of them were positive for the HPV type 6b and 4 for the HPV type 16. CCND1 gene amplification was found in 15 cases (25%). Although we have found a higher frequency of CCND1 amplification in the HPV-positive cases (36%, versus 22% in the HPV-negative cases), these differences were not statistically significant (P= 0,32).. The presence of HPV gene sequences does not seem to be related to a significative higher incidence of CCND1 gene amplification in the squamous cell carcinomas of the head and neck.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Gene Amplification; Head and Neck Neoplasms; Human papillomavirus 16; Human papillomavirus 6; Humans; Male; Middle Aged; Papillomavirus Infections

Squamous cell carcinoma of the upper aerodigestive tract (UADT) is associated with environmental factors, especially tobacco and alcohol consumption. Genetic factors, including cyclin D1 (CCND1) polymorphism have been suggested to play an important role in tumorigenesis and progression of UADT cancer. To investigate the relationship between CCND1 polymorphism on susceptibility for UADT cancers, 147 cancer and 135 non-cancer subjects were included in this study. CCND1 genotype at codon 242(G870A) in exon 4 was undertaken using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. Significant odds ratio (OR) of the AA+GA genotypes [OR=7.5 (95% CI: 1.4-39.7)] was observed in non-drinkers but for non-smokers a non-significant [OR=5.4 (95% CI: 0.9-31.4)] was found in the adjusted model. These results suggest that allele A may be a risk factor for UADT cancer, especially in non-alcoholics. However, further epidemiological studies are needed to establish the exact role of CCND1 polymorphism and the development of UADT cancers.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Alleles; Carcinoma, Squamous Cell; Cyclin D1; Female; Genotype; Head and Neck Neoplasms; Humans; Hypopharyngeal Neoplasms; Laryngeal Neoplasms; Male; Middle Aged; Mouth Neoplasms; Oropharyngeal Neoplasms; Polymorphism, Genetic; Risk Factors

Esophageal cancer has been reported to be frequently associated with cancer of the head and neck. The iodine dye method is reportedly useful to detect early esophageal cancer. The aim of this study was to clarify clinicopathological and biological characteristics of esophageal squamous cell carcinoma associated with head and neck cancer (HN group).. Thirty-seven patients of the HN group who underwent esophagectomy were examined clinicopathologically compared to 42 patients with esophageal cancer alone (SE group). All resected specimens were histologically studied after iodine dye staining, and p53 and cyclin D1 (CD1) expression were immunohistochemically examined in esophageal cancer.. The HN group had more multiple iodine-unstained lesions and multiple primary cancers within the esophagus compared with the SE group (p = 0.0027, p = 0.067, respectively). There was no significant difference in smoking, drinking, family history and the other clinicopathological factors between the HN and SE groups. Coexpression of p53 and CD1 was found to be significant in the HN group (p = 0.03).. The coexistence of multiple iodine-unstained lesions, multiple cancers in the esophagus and overexpression of p53 and CD1 is suggested as a risk factor for the HN group.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Esophagectomy; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasms, Multiple Primary; Predictive Value of Tests; Risk Factors; Tumor Suppressor Protein p53; Up-Regulation

Despite promising initial results, recent Phase III trials of the selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib ("Iressa"; AstraZeneca, Wilmington, Delaware) in advanced head and neck squamous cell carcinoma (HNSCC) have been equivocal. Cyclin D1, an EGFR target gene, is frequently overexpressed in HNSCC, has been implicated in its pathogenesis, and is strongly associated with poor prognosis in this disease. Therefore, we examined the relationship between deregulated cyclin D1 expression and sensitivity to gefitinib to determine whether this frequently occurring oncogenic change affected the cellular response to gefitinib.. A panel of six EGFR-overexpressing HNSCC cell lines was used to correlate CCND1 gene copy number, cyclin D1 expression, and response to gefitinib. The effect of constitutive overexpression of cyclin D1 was assessed by establishing stably transfected clonal SCC-9 cell lines.. Three of six cell lines displayed cyclin D1 amplification and/or overexpression, and these cell lines were resistant to gefitinib. SCC 9 clones overexpressing cyclin D1 continued to proliferate and maintained their S-phase fraction when treated with gefitinib, whereas empty vector control clones and the parental SCC 9 cells were profoundly inhibited and displayed marked reductions in S-phase. The resistance of cyclin D1-overexpressing clones and cyclin D1-amplified cell lines was associated with maintenance of cyclin D1 expression after gefitinib treatment.. These data suggest that deregulated cyclin D1 overexpression may be associated with resistance of HNSCC to EGFR inhibitors. Therefore, the role of cyclin D1 as a marker of therapeutic response and its utility as a prognostic marker in HNSCC warrant additional analysis.

Topics: Antineoplastic Agents; Blotting, Southern; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA; Dose-Response Relationship, Drug; ErbB Receptors; Flow Cytometry; G1 Phase; Gefitinib; Gene Expression Regulation, Neoplastic; Genetic Vectors; Head and Neck Neoplasms; Humans; Inhibitory Concentration 50; Prognosis; Protein Kinase Inhibitors; Quinazolines; S Phase; Time Factors

The difficult cancer development and the insufficient knowledge of the biology of the existing cancers lead for the constant search for the new techniques of diagnosis. The aim of this study was to analyze the selected prognostic factors such as p53, Ki67 and cyclin D1 in patients with head and neck cancer in connection with histological differentiation (G), TNM classification and the clinical advancement of the disease (S). The material comprised of the tissue specimens from 32 patients with squamous cell carcinomas of head and neck, who underwent the primary surgery. The immunohistochemical study was performed with avidin-biothin method. A 5-point grading system was adopted to assess the level of the immunohistochemical staining of the carcinoma cells. Through our study we found a statistically significant correlation between the staining intensity of carcinoma cells for p53, Ki67, cyclin D1 and the TNM classification and the clinical advancement of the disease (S). Such correlation for the histological differentiation (G) has not been observed. We think that the immunohistochemical techniques may constitute an important supplement of evaluation of the malignancy in the squamous cell carcinomas of head and neck.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Staging; Tumor Suppressor Protein p53

Fluorescence in situ hybridization was applied on a collection of 609 squamous cell carcinomas of the head and neck (HNSCCs),including 511 primary carcinomas of different clinical stage and anatomical localization and 98 recurrent carcinomas, second primary carcinomas, and regional metastases on a tissue microarray. The overall prevalence of amplifications of five oncogenes analyzed was 34.5% for CCND1, 12.7% for EGFR, 8.8% for MYC, 6.2% for ZNF217, and 3.6% for ERBB2. CCND1 amplifications were associated with the pharyngeal site in primary carcinomas (P < 0.001), whereas amplifications of ZNF217 were less frequent in pharyngeal carcinomas as compared with primary oral and laryngeal carcinomas (P = 0.02). The amplification pattern of these oncogenes suggests that different molecular pathways are involved in HNSCCs of different localizations.

Topics: Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Gene Amplification; Genes, erbB-2; Genes, myc; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Neoplasm Staging; Oncogenes; Trans-Activators

Tumor-Node-Metastasis classification does not fully predict outcome of treatment and prognosis in patients with squamous cell carcinoma of the head and neck. Different biomarkers have been suggested to yield additional prognostic information, but no single marker has thus far been introduced in the clinic. The objective of the present study was to analyze the copy number of the frequently amplified oncogenes CCND1 and c-MYC in relation to the commonly deleted tumor suppressor gene cyclin-dependent kinase (CDK)N2A (p16) to enhance the clinical significance.. Extracted DNA from diagnostic biopsies of 78 untreated patients were analyzed by real-time PCR with specific primers for c-MYC, CCND1, and CDKN2A. Gene copy number ratios were calculated by dividing the copy number of c-MYC or CCND1 with CDKN2A. Ratios > 2 were defined as enhanced. These data were related to disease-free interval and disease-specific survival.. Enhanced gene ratio of c-MYC:CDKN2A was detected in 35 of 78 (45%) and enhanced ratio of CCND1:CDKN2A in 36 of 78 (46%) of the cases. The c-MYC:CDKN2A and CCND1:CDKN2A ratios correlated with disease-specific survival with respect to death (P = 0.042 and 0.049, respectively; Log-rank test). Furthermore, enhancement of c-MYC:CDKN2A was associated with a shorter disease-free interval as marked by the development of recurrences or metastases (P = 0.014; Log-rank test).. We conclude that CCND1 and/or c-MYC amplification, when combined with CDKN2A deletion, yield additional prognostic information as compared with analysis of single genetic aberrations. These gene ratios, as analyzed by a sensitive method like real-time PCR on diagnostic biopsies, might help clinicians to individualize the treatment of squamous cell carcinoma of the head and neck as they reflect the biological properties of the tumors. This could be used as an adjunct to the Tumor-Node-Metastasis classification system.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Female; Gene Amplification; Gene Deletion; Gene Dosage; Genes, myc; Genes, p16; Head and Neck Neoplasms; Humans; Male; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Survival Rate

A microbial secondary metabolite, arisostatins A (As-A), was originally discovered as a substance carrying the antibiotic activity against Gram-positive bacteria and shown to possess potent anti-tumor properties. The mechanism by which arisostatins A initiates apoptosis remains poorly understood. In the present report we investigated the effect of arisostatins A on activation of the apoptotic pathway in HN-4 cells. Arisostatins A was shown to be responsible for the inhibition of HN-4 cell growth by inducing apoptosis. Treatment with 4 microM arisostatins A for 24h produced morphological features of apoptosis and DNA fragmentation in HN-4 cells. Arisostatins A caused dose-dependent apoptosis and DNA fragmentation of HN-4 cells used as a model. Treatment with caspase inhibitor significantly reduced the arisostatins A-induced caspase 3 activation. In addition, arisostatins A-induced apoptosis was associated with the generation of reactive oxygen species (ROS), which was prevented by an antioxidant NAC (N-acetyl-cysteine). These data indicate that cytotoxic effect of arisostatins A on HN-4 cells is attributable to the induced apoptosis and that arisostatins A-induced apoptosis is mediated by caspase-3 activation pathway, loss of mitochondrial transmembrane potential (DeltaPsi(m)), and release of cytochrome c into cytosol.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Division; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Activation; Head and Neck Neoplasms; Macrolides; Membrane Potentials; Mitochondria; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

Overexpression of the HER-2/neu receptor (HER-2) is associated with a poor prognosis in patients with breast carcinoma and also in patients with head and neck squamous cell carcinoma (HNSCC). In a previous study on HNSCC cell lines, we found that epigalocathechin-3-gallate (EGCG), a major biologically active component of green tea, inhibited activation of the epidermal growth factor receptor (EGFR) and thereby inhibited EGFR-related downstream signaling pathways in HNSCC cells. In the present study, we examined the effects of EGCG on activation of the HER-2 receptor in human HNSCC and breast carcinoma cell lines that display constitutive activation of HER-2. Treatment of these cells with 10 or 30 microg of EGCG, respectively, doses that cause 50% inhibition of growth, markedly inhibited the phosphorylation of HER-2 in both cell lines. This was associated with inhibition of Stat3 activation, inhibition of c-fos and cyclin D1 promoter activity, and decreased cellular levels of the cyclin D1 and Bcl-XL proteins. Although these concentrations of EGCG are quite high, we found that concentrations of 0.1-1.0 microg/ml, which are in the range of plasma concentrations after administering a single oral dose of EGCG or a green tea extract, markedly enhanced the sensitivity of both types of cell lines to growth inhibition by Taxol, a drug frequently used in the treatment of breast carcinoma and HNSCC. These results, taken together with previous evidence that EGCG also inhibits activation of the EGFR in carcinoma cells, suggest that EGCG may be useful in treating cases of breast carcinoma and HNSCC in which activation of the EGFR and/or HER-2 plays important roles in tumor survival and growth.

Topics: Antineoplastic Agents, Phytogenic; Blotting, Western; Breast Neoplasms; Carcinoma; Catechin; Cell Division; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Genes, Reporter; Head and Neck Neoplasms; Humans; Immunoblotting; Luciferases; Paclitaxel; Phosphorylation; Prognosis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Receptor, ErbB-2; Signal Transduction; Time Factors

Overexpression of the epidermal growth factor receptor (EGFR) in the cancers of the head and neck is well demonstrated. In addition, copy numbers of the EGFR mRNA were significantly higher in poorly differentiated tumors than in tumors that had a differentiated phenotype. Studies by others also showed that the constitutively activated signal transducer and activator of transcription-3 (STAT3), but not STAT1, is required for EGFR-mediated cell growth. Our aim was to reveal if STAT expression is differentiation-dependent and thus, might respond to exogenous stimuli in a differentiation-dependent manner. Both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry revealed that expression of STAT1 was high in well/moderately differentiated tumors in vivo. In contrast, STAT3 was expressed in poorly differentiated tumors. In vitro experiments showed that differentiated primary oral keratinocytes expressed higher levels of STAT1, but lower levels of STAT3 than did their undifferentiated counterparts. Epidermal growth factor treatment of oral keratinocytes with various degrees of differentiation showed the maximal induction of cyclin D1 in undifferentiated cells. Our findings suggest that the level of differentiation might modulate the outcome of EGFR signaling (i.e. cyclin D1 transcription), due to the differentiation-associated intracellular balance of transcriptional regulators (STAT1 versus STAT3).

Topics: Biopsy; Cell Differentiation; Cyclin D1; DNA-Binding Proteins; Growth Substances; Head and Neck Neoplasms; Humans; Immunohistochemistry; Keratinocytes; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators

The aim of this study was to analyse the alterations of the genes in the CDKN2A/CCND1/CDK4/RB1 pathway in the G1-S phase of the cell cycle during development of head and neck squamous cell carcinoma (HNSCC).. The alterations of these genes were analysed in 22 dysplastic lesions, 26 stage-I/II and 33 stage-III/IV HNSCC tumours of Indian patients.. The alterations [mutation, hypermethylation, homozygous deletion and loss of heterozygosity/microsatellite size alteration (LOH/MA)] in the CDKN2A were found to be highest in 57% of the samples, followed by CCND1 amplification and LOH/MA at the RB1 locus in 14% and 8.5% of the samples, respectively. No dominant CDK4 Arg24Cys mutation was seen in our samples. Comparatively high frequency of CDKN2A alterations (except homozygous deletion) was found in dysplastic head and neck lesions and remained almost constant or increased during progression of the tumour, whereas the homozygous deletion of CDKN2A and the alterations in CCND1 and RB1 genes were seen mainly in the later stages of the tumour.. Our study suggested that mutation/hypermethylation/allelic alterations (LOH/MA) of CDKN2A were associated with the development of dysplastic head and neck lesions. All the other alterations might provide some cumulative effect during progression of later stages of the tumour to have selective growth advantages.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Female; Genes, p16; Head and Neck Neoplasms; Humans; India; Loss of Heterozygosity; Male; Methylation; Mutation; Polymorphism, Genetic; Promoter Regions, Genetic; Proto-Oncogene Proteins; Retinoblastoma Protein

At present, the differences between head and neck basaloid squamous cell carcinoma (BSCC) and nonbasaloid squamous cell carcinoma (SCC) are mostly on the basis of histologic and immunohistologic findings.. In this study, we investigated 8 BSCCs and 59 SCCs for loss of heterozygosity (LOH) at chromosomes 5q, 9p, 9q, 10q, 11q, 13p, 17p, and 18q. In addition, we analyzed p16, PTEN, and CCND1 (cyclin D1) and investigated the HPV status. Immunohistochemically, the expression of MIB-1, p16, p53, and cyclin D1 was determined.. Aberrations in the BSCCs were especially frequent at 9p and in the CCND1 gene. In contrast, alterations at 10q occurred almost exclusively in conventional SCCs. Obvious differences could be determined concerning the HPV status: HPV-DNA was detected in all BSCCs but only in 17% of conventional SCCs.. Although the number of investigated BSCCs is rather low and did not allow statistical conclusions, our results focus on certain differences between the molecular pathogenesis of BSCCs and SCCs.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA, Viral; Genes, cdc; Head and Neck Neoplasms; Humans; Immunohistochemistry; Laryngeal Neoplasms; Loss of Heterozygosity; Microsatellite Repeats; Papillomaviridae; Phosphoric Monoester Hydrolases; PTEN Phosphohydrolase; Tumor Suppressor Proteins

The recent development of tissue array technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. This study evaluates the potential for using archival head and neck cancer tissue in such arrays.. Tissue array blocks were made from 184 head and neck cancer specimens. Three core tissue biopsies (0.6 mm x 3-4 mm) were taken from individual "donor" paraffin-embedded tumor blocks and arrayed into a new "recipient" paraffin block. Immunohistochemical (IHC) analyses were performed using antibodies recognizing cyclin-D1, Rb, and EGFR. IHC was scored on a 6-point scale for extent and a 3-point scale for intensity. We compared the staining of tissue array disks with staining of full tissue sections.. Seventy-four percent (475 of 640) of samples placed into tissue arrays were confirmed to represent tumor tissue. The remaining samples were lost during processing or contained too few tumor cells. Only 6% of cases were completely lost, whereas 55%, 28%, and 11% of cases were judged on 3, 2, or 1 disk, respectively. Cohen's kappa coefficient was 0.66 for cyclin-D1, 0.40 for EGFR, and 0.41 for Rb.. Tissue array technology is a rapid and efficient method for retrospective analysis of protein expression and is a promising tool for validation of prognostic markers in large series of head and neck squamous cell carcinomas. The agreement in scoring of the full section and the tissue arrays is reasonable. Discordance is probably due to intraobserver variation and lack of robustness of the scoring inherent of the proteins studied.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Middle Aged; Oropharyngeal Neoplasms; Prognosis; Protein Array Analysis; Retinoblastoma Protein; Statistics, Nonparametric

Cyclin D1, a cell cycle regulator localized to chromosome 11q13, is amplified in several human tumors including head and neck squamous cell carcinoma (HNSCC). Amplification and/or overexpression of cyclin D1 have been correlated to a poor prognosis. Deletion of the p16 gene, localized to 9p21, has also been observed in a significant proportion of HNSCC. The p16 gene regulates cyclin D1-CDK4 activity and prevents retinoblastoma tumor suppressor gene phosphorylation, thereby downregulating cellular proliferation. Detection of cyclin D1 amplification and p16 deletion using a simple and sensitive method will be valuable for the development of effective treatment modalities for head and neck cancer.. We have used fluorescence in situ hybridization (FISH) to study cyclin D1 amplification and p16 gene deletion in head and neck tumors. Both single- and dual-color FISH were performed.. Paraffin-embedded tissues from 103 patients with HNSCC were analyzed using genomic DNA probes for cyclin D1 and p16. Dual-color FISH was performed with chromosome 11 or 9 centromeric probes as a control. Twenty-eight of these samples were analyzed for p16 expression by immunohistochemistry.. Cyclin D1 amplification was observed in 30% (31/103) of patients, and p16 deletion in 52% (54/103). Lack of p16 expression was observed in 64% (18/28) of patients. There was a good correlation between the deletion of p16 sequences and the loss of p16 expression (P = .008). Amplification of cyclin D1 had a statistically significant association with recurrence, distant metastasis, and survival at 36 months. There was a significant association between p16 deletion and the development of distant metastases. Cyclin D1 amplification and p16 deletion together correlated with recurrence, distant metastasis, and survival.. We demonstrate that FISH is a simple and sensitive method for detecting cyclin D1 amplification and p16 deletion in head and neck cancer. Our results suggest that these two genetic aberrations together portend a poorer outcome than either of the abnormalities alone in head and neck cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chi-Square Distribution; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Amplification; Gene Deletion; Head and Neck Neoplasms; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Middle Aged; Prognosis; Survival Analysis

Head and neck squamous cell carcinomas of non-smoking patients may result from specific defects in cell cycle control. Expression of p53, pRb, p16(INK4a) and Cyclin D1 was examined by immunohistochemistry of biopsies obtained from 24 non-smoking and 25 smoking patients, both groups representing similar clinical features (tumor site, stage of disease, gender). Expression of p16(INK4a) was restricted to carcinomas of the tonsils (8/24), P=0.0069. In 6/8 p16(INK4a)-positive cases, expression of pRb was absent or reduced. p16 was the only marker showing a significant correlation with a negative smoking history (P=0.0208). Overexpression of Cyclin D1 was frequent in carcinomas of the tongue (6/14) but rare in tonsillar carcinomas (2/24), P=0.0303. Expression of p53 was independent of the smoking history and the tumor site. Our results implicate that there are factors other than tobacco consumption which may influence the development of head and neck cancers at distinct tumor sites.

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Head and Neck Neoplasms; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Retinoblastoma Protein; Smoking; Tumor Suppressor Protein p53

P53 and CCND1 (cyclin D1) genes play a critical role in the cell cycle regulation. Abnormalities of these genes are frequent in different types of cancers, including those of the head and neck. The aim of this work is to investigate whether P53 inactivation (determined by loss of heterozygosity analysis) is related to CCND1 gene amplification (determined by differential PCR analysis), and if these alterations are correlated with clinical outcome in a series of 56 patients with squamous cell carcinoma of the head and neck. Loss of heterozygosity of the P53 gene was found in 39 cases (70%) and CCND1 amplification in 17 cases (30%). Both abnormalities together were found in 11 cases (20%), without a significant association between them (P = 0.83). No relationship was found between P53 inactivation, the clinico-pathological parameters analyzed and the clinical outcome. CCND1 amplification was associated with advanced T-stages (P = 0.02), nodal metastases (P = 0.01) and a decreased survival (P = 0.002). The combination of both abnormalities shows a pattern that seems to be additive, since it was associated with an increase in tumor recurrences and a decrease in survival that was higher than for either of them individually. In conclusion, P53 and CCND1 abnormalities are frequent in squamous cell carcinomas of the head and neck. The combined analysis of these abnormalities seems to be more informative than either of them individually and may have a prognostic value in these carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Female; Gene Amplification; Genes, p53; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Staging

Epidermal growth factor receptor (EGFR) is up-regulated and contributes to the loss of growth control in squamous cell carcinoma of the head and neck (SCCHN). Previously, we reported an association between autocrine stimulation of EGFR and constitutive signal transducers and activators of transcription (STAT) 3 activation in SCCHN cells in vitro and in vivo. Here, we evaluated the role of activated STAT3 in tumor progression and EGFR-independent mitogenic signaling. We found that SCCHN cells stably transfected with a dominant active STAT3 construct expressed elevated levels of STAT3 target genes, including Bcl-X(L) and cyclin D1, and demonstrated increased proliferation in vitro and more rapid tumor growth rates in vivo. Cell cycle analysis demonstrated an increased proportion of STAT3 construct transfectants in G(2)-M. These findings provide evidence that constitutive STAT3 activation contributes to tumor growth in SCCHN, independent of the EGFR autocrine axis.

Topics: Autocrine Communication; bcl-X Protein; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cyclin D1; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

Verrucous carcinoma (VC) is a locally invasive, nonmetastasizing variant of squamous cell carcinoma (SCC) with distinct clinical and histologic features. Molecular alterations detectable by immunohistochemical analyses in VC have not been extensively studied. This study investigates the expression of p53, epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-alpha), and cyclin D1 in VC, verrucous hyperplasia (VH), and classic SCC of the head and neck. Twenty-six cases of VC, 12 cases of SCC of various differentiations, and 4 cases of VH were studied. Formalin-fixed, paraffin-embedded archival material was used for immunohistochemistry (avidin-biotin immunoperoxidase technique) to study the expression of oncogenes and their tumor markers. Identification of p53 protein was found in 100% of VH, 88% of VC, and 100% of SCC. EGFR expression was noted in 25% of VH, 54% of VC, 40% of well-differentiated SCC (WDSCC), and 100% of moderately and poorly differentiated SCC (MDSCC/PDSCC). TGF-alpha was detected in 25% of VH, 88% of VC, 80% WDSCC, and 100% of MDSCC/PDSCC. Cyclin-D1 expression was seen in 75% of VH, 35% of VC, 100% of WDSCC, 67% of MDSCC, and 50% of PDSCC. Correlation between the level of expression of all markers and the grade of this group of squamous lesions revealed statistically significant correlation coefficients for p53 and EGFR but not for TGF-alpha and cyclin D1.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Carcinoma, Verrucous; Cell Differentiation; Cyclin D1; Epithelium; ErbB Receptors; Head and Neck Neoplasms; Humans; Hyperplasia; Immunohistochemistry; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

CCND1 amplification results in cyclin D1 overexpression. However, other unidentified genetic mechanisms contribute to enhanced gene expression. In the present study, 32 squamous cell carcinoma of the head and neck (SCCHN) were investigated regarding chromosomal abnormalities involving 11q13 by cytogenetic analysis, genomic CCND1 amplification by differential PCR and FISH, and cyclin D1 expression on the mRNA and protein level by differential RT-PCR and immunohistochemistry, respectively. CCND1 amplification, observed in 11 of 32 (34%) tumours, resulted in overexpression of cyclin D1 on the mRNA and/or protein level, in 3 cases in association with chromosomal translocations. In cytogenetic analysis, 4 tumours had hsr(11)(q13), all of which showed CCND1 amplification and cyclin D1 overexpression. Overexpression of cyclin D1 was detected at the mRNA level in 23 tumours (72%) and on the protein level in 25 tumours (78%). In one case a translocation was seen together with cyclin D1 overexpression on the mRNA level, without any cytogenetic or molecular signs of amplification. Furthermore, cases with cyclin D1 overexpression were frequently observed in the absence of any genomic rearrangement. We conclude that, besides amplifications, chromosomal translocations and other transcriptional or translational regulatory mechanisms are involved in CCND1 deregulation.

Topics: Biopsy; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 11; Cyclin D1; DNA Primers; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Neoplasm Proteins; Paraffin Embedding; Polymerase Chain Reaction; RNA, Messenger; Translocation, Genetic; Tumor Cells, Cultured

Eight cytogenetically characterized head and neck squamous cell carcinomas (HNSCCs) with CCND1 amplification in the form of a homogeneously staining region (hsr) in 11q13 were studied by COBRA FISH and FISH with specific probes to identify and characterize chromosomal segments added to the derivative chromosomes 11. In 4 of the tumors, it could be recognized that the material added was derived from the long arm of chromosome 3. The rearrangements were interpreted as der(11)hsr(11)(q13)t(3;11)(q21;q13) in 3 cases and as der(11)hsr(11)(q13)t(3;11)(q14;q13) in 1 case. In the other 4 cases, material from chromosomes 1, 16, or 19 was added to the derivative chromosomes 11. By further FISH analysis with 14 YAC clones spanning 3q13-q21 in the 4 tumors with der(11)hsr(11)t(3;11), it could be shown that they had different breakpoints at the molecular level, excluding the possibility that a particular gene was rearranged by the translocations. More surprisingly, gain of the 3q21-q29 segment was found in all 8 tumors with hsr in 11q13 and loss of 3p was seen in 7 of the tumors. These findings strongly indicate a synergistic effect of CCND1 amplification, loss of distal 11q, 3q gain and 3p deletion in HNSCC development and also suggests a mechanistic link between intrachromosomal amplification at 11q13 and recombination with distal 3q.

Topics: Aged; Biopsy; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 3; Cyclin D1; DNA, Neoplasm; Female; Gene Amplification; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Translocation, Genetic

We have used a combination of vitamin A (all-trans-retinyl palmitate), 5-fluorouracil (5-FU) and radiation to treat human head and neck squamous cell carcinoma (HNSCC). This chemoradiotherapy is called "FAR therapy." In this study we examined the effects of all-trans-retinoic acid (ATRA), the active metabolite of vitamin A, and ATRA plus 5-FU on two HNSCC cell lines (YCU-N861 and YCU-H891) to gain insight into the molecular mechanisms of FAR therapy. ATRA at 1 mM (the order of concentration found in HNSCC tumors treated with FAR therapy) inhibited cell proliferation and caused G1 cell cycle arrest in both cell lines. This was associated with a decrease in cyclin D1, an increase in p27(Kip1) and a reduction in the hyperphosphorylated form of retinoblastoma protein (pRB). With YCU-N861 cells, ATRA also caused a decrease in Bcl-2 and Bcl-X(L) and an increase in Bax. Both ATRA and 5-FU activated c-Jun N-terminal kinase (JNK) 1 and the combination of both agents resulted in additive or synergistic activation of JNK1, and also enhanced the induction of apoptosis. The YCU-H891 cells, in which the epidermal growth factor receptor (EGFR)-signal transducer and activator of transcription 3 (Stat3) pathway is constitutively activated, were more resistant to treatments with ATRA, 5-FU and the combination of both agents than YCU-N861 cells. A dominant negative Stat3 construct strongly enhanced the cellular sensitivity of this cell line to 5-FU but not to ATRA. In addition there is evidence that activation of Stat3 is associated with cellular resistance to radiation in HNSCC. Therefore, the addition to FAR therapy of agents that inhibit activation of the Stat3 pathway may enhance the clinical response of patients with HNSCC to FAR therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Division; Cell Survival; Cyclin D1; DNA-Binding Proteins; Drug Synergism; ErbB Receptors; Flow Cytometry; Fluorouracil; Head and Neck Neoplasms; Humans; Immunoblotting; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transfection; Tretinoin; Tumor Cells, Cultured

To identify markers that are relevant as predictors of lymph node metastasis in head and neck squamous cell cancer.. Expression of p53, Rb, cyclin D1, E-cadherin, and epithelial cell adhesion molecule was examined using immunohistochemical analysis and traditional histological parameters, and the correlation of these markers with the histologically verified presence of regional metastases was determined.. The study sample comprised 121 patients with head and neck squamous cell cancer from whom paraffin-embedded material of primary tumors was used.. Lymph node metastasis was correlated with the loss of expression of Rb (P =.04) and marginally correlated with the loss of expression of E-cadherin (P =.06). If the results are broken down to subsites, loss of E-cadherin expression in oral cancer (P =.04) and absence of eosinophilic infiltration in laryngeal cancer (P =.003) correlated with nodal metastasis. None of the other markers correlated. A combination of relevant parameters did not result in a much stronger correlation.. The expression of the investigated genetic markers and histopathological features of primary tumors can supply limited information on the metastatic behavior of tumors. Although the use of markers for regional metastasis would be a welcome additional tool, these results do not warrant the use of these parameters for clinical decision making concerning the treatment of the neck in patients with head and neck squamous cell cancer.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cyclin D1; Epithelial Cell Adhesion Molecule; Female; Head and Neck Neoplasms; Humans; Lymphatic Metastasis; Male; Middle Aged; Retinoblastoma Protein; Sensitivity and Specificity; Tumor Suppressor Protein p53

Epidemiologic studies have provided evidence of an alcohol-associated increased risk of upper aerodigestive tract cancers. Recently we reported ethanol-induced proliferation in a squamous cell carcinoma of the head and neck (SCCHN) cell line, but the underlying mechanisms are unknown. In order to further clarify these findings, major G0/G1-regulating proteins were investigated in the present study. Synchronized cells of a SCCHN line (JP-PA) and a human immortalized keratinocyte line (HaCaT)-used as a control-were cultured with or without 10(-3) M ethanol for up to 96 h. At distinct time intervals the expression of cyclin D1 and the inhibitors p16, p18, p19 and p21 were determined by Western blot analyses. In both lines ethanol had no influence on the protein expression of cyclin D1. In contrast, distinct downregulations of p21, p18 and p19 were detectable at the protein level. The p16 protein was not expressed in the SCCHN line and was unchanged in the control line after the addition of ethanol. In these in vitro experiments the marked downregulation of important cell-cycle inhibitors may accelerate progression from the G1 to the S phase of the cell cycle. The relevance of our findings to in vivo conditions remains speculative, but the observed mechanisms of significantly reduced expression of cell-cycle inhibitor proteins may be involved in the carcinogenesis of head and neck malignancies.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Line; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; Enzyme Inhibitors; Ethanol; Flow Cytometry; Head and Neck Neoplasms; Humans; In Vitro Techniques; Tumor Cells, Cultured

The precise mechanism responsible for the frequent overexpression of cyclinD1 in human head and neck squamous cell carcinoma (HNSCC) is not known. In view of the fact that signal transducers and activators of transcription 3 (Stat3) is often activated in HNSCC cells, we examined the effects of Stat3 on cyclin D1 expression and cell proliferation in the YCU-H891 HNSCC cell line that displays constitutive activation of Stat3. Expression of a dominant negative Stat3 construct in YCU-H891 cells inhibited proliferation, cyclin D1 promoter activity, and cellular levels of cyclin D1 mRNA and protein. The levels of the antiapoptotic Bcl-2 and Bcl-X(L) proteins were also inhibited. In 51 primary tumor samples from patients with squamous cell carcinoma of the p.o. tongue, there was a significant correlation between increased levels of the activated form of Stat3, phosphorylated-Stat3, and increased levels of cyclin D1 (P < 0.0001). Increased tumor levels of phosphorylated-Stat3 were also associated with lower survival rates (P < 0.01). This study provides the first evidence that in HNSCC, constitutive activation of Stat3 plays a causative role in overexpression of cyclin D1, and in clinical studies, Stat3 activation may provide a novel prognostic factor. Furthermore, agents that target Stat3 may be useful in the treatment of HNSCC.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Division; Cyclin D1; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Neoplasm Metastasis; Neoplasm Staging; Prognosis; RNA, Messenger; STAT3 Transcription Factor; Tongue Neoplasms; Trans-Activators; Tumor Cells, Cultured

CCND1 gene amplification and cyclin D1 protein overexpression are indicators for poor prognosis in invasive head and neck carcinomas. Increased CCND1 gene dosage is a more sensitive prognostic factor than protein overexpression as evaluated by conventional immunohistochemical techniques. Qualitative immunohistochemistry cannot distinguish cyclin D1 overexpression accompanied by amplification of the CCND1 gene from overexpression associated with normal CCND1 gene copy number. To improve the sensitivity of cyclin D1 protein determination, we applied quantitative techniques of image analysis to evaluate cyclin D1 in 54 head and neck carcinomas. There was a significantly higher rate of occurrence of adverse events (P = 0.043) among patients with CCND1 gene amplification than among those without gene amplification. There was a strong association between CCND1 gene amplification (as detected by Southern blot analysis) and the highest nuclear score (by image cytometry of the immunostained tumor sections). The predominance of cells in the lowest nuclear score category was significantly associated with normal copy number (P = 0.005). Conversely, the highest nuclear score was a significant predictor of gene dosage (P = 0.02). Similarly, high nuclear score was a good predictor of death as the final outcome of the disease (P = 0.01). Although somewhat less accurate than Southern blotting, image cytometry of immunohistochemical cyclin D1 stain appears to be a promising tool that could be useful for other tumor marker expression studies.

Topics: Adult; Aged; Biomarkers, Tumor; Blotting, Southern; Carcinoma, Squamous Cell; Culture Techniques; Cyclin D1; Female; Gene Amplification; Head and Neck Neoplasms; Humans; Image Cytometry; Immunohistochemistry; Male; Middle Aged; Prognosis; Sensitivity and Specificity

Basal cell carcinomas (BCCs) may be subdivided into primary with a favorable biologic course (BCC1) and recurrent and/or metastatic (BCC2). No clear association between primary tumor location, histologic subtype, or other clinicopathologic variables and predisposition for BCC2 has been found. Histopathologic criteria are limited for prognostication. To identify prognostic factors useful for planning therapy, we studied cyclin D1 immunohistochemical expression, DNA ploidy, and epiluminescence light microscopic (ELM) patterns in 60 cases of BCC (30 BCC1 and 30 BCC2) in the head and neck region, half of which were hyperpigmented. Cyclin D1 was absent in 27 cases, expressed at low level in 4 cases, and overexpressed in 30 cases. Seven BCCs were euploid, 28 exhibited a mixed cellular population, and 25 were aneuploid. Among aneuploid tumors, hypodiploidy was found in 12. Among the 30 pigmented carcinomas, only 15 showed a typical ELM pattern. No association between pigmentation and more aggressive biologic behavior of BCC was found. These results and follow-up data seem to indicate that an unfavorable outcome can be predicted by hyperexpression of cyclin D1, aneuploidy, and an atypical ELM pattern for pigmented cases. A definite hypodiploid peak was associated with worse prognosis. The analysis of cyclin D1 expression and DNA ploidy may help identify BCC with an aggressive phenotype and a poor clinical outcome.

Topics: Carcinoma, Basal Cell; Cyclin D1; DNA, Neoplasm; Follow-Up Studies; Head and Neck Neoplasms; Humans; Immunohistochemistry; Luminescent Measurements; Microscopy; Ploidies; Prognosis; Skin Neoplasms; Skin Pigmentation

A G-->A polymorphism (G870A) in exon 4 of the cyclin D1 (CCND1) gene creates an alternative splice site in its mRNA, encoding a protein with an altered C-terminal domain. It has been suggested that DNA damage in cells with the A allele bypasses the G(1)/S checkpoint of the cell cycle more easily than damage in cells without the A allele. Because CCND1 plays a critical role in cell cycle control and reduced DNA repair capacity is associated with an increased risk for squamous cell carcinoma of the head and neck (SCCHN), we hypothesize that this CCND1 polymorphism modulates individual susceptibility to SCCHN. To test this hypothesis we evaluated the frequency of the polymorphism in a hospital-based case-control study of 233 newly diagnosed SCCHN patients and 248 non-cancer controls. The cases and controls were frequency matched by age (+/-5 years), sex and tobacco use. All subjects were non-Hispanic whites. We found that the A allele frequency was slightly higher in the cases (0.485) than in the controls (0.425), but the difference was borderline statistically significant (P = 0.064). The frequencies of the CCND1 AA, GA and GG genotypes were 23.6, 49.8 and 26.6%, respectively, in cases and 16.5, 52.5 and 31.5%, respectively, in controls. Multivariate logistic regression analysis adjusting for age (in years), sex, smoking and alcohol use was performed to calculate odds ratios (OR) and 95% confidence intervals (CI). Compared with the wild-type CCND1 GG, the CCND1 A G genotype was associated with a non-significantly increased risk (adjusted OR 1.15, 95% CI 0.75-1.76), but the CCND1 AA genotype was associated with a significantly increased risk (adjusted OR 1.77, 95% CI 1.04-3.02) for SCCHN. Results from a trend test using a logistic regression model were statistically significant (P = 0.044). Among the cases the mean age of onset was 59.0, 56.8 and 55.5 years for the GG, GA and AA genotypes, respectively. In the stratification analysis the CCND1 AA variant genotype was associated with a >3-fold increased risk in individuals who were

Topics: Carcinoma, Squamous Cell; Case-Control Studies; Cyclin D1; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Polymorphism, Genetic; Risk Factors

The aim of this study was to investigate whether there is an association between overexpression of cyclin D1 and response to therapy. Immunohistochemical overexpression of cyclin D1 was determined in paraffin-embedded specimens from diagnostic biopsies of 89 primary cases of squamous cell carcinoma of the head and neck (SCCHN), using a polyclonal antiserum. The tumor response rates were estimated after curative treatment (i.e. surgery and/or radiotherapy and/or chemotherapy). Patients whose tumors were overexpressing cyclin D1 showed complete or partial response to neoadjuvant chemotherapy with cisplatin/5-FU. In addition, a majority of cyclin D1 negative tumors did not respond at all to this treatment (p = 0.02, Fisher's exact test). This study indicates that immunohistochemical assessment of cyclin D1 expression in SCCHN could be a new predictive marker to select a subgroup of patients that will benefit from neoadjuvant chemotherapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chemotherapy, Adjuvant; Cisplatin; Cobalt Radioisotopes; Combined Modality Therapy; Cyclin D1; Fluorouracil; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Life Tables; Neoadjuvant Therapy; Neoplasm Proteins; Particle Accelerators; Radioisotope Teletherapy; Radiotherapy, Adjuvant; Radiotherapy, High-Energy; Remission Induction; Survival Analysis

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cyclin D1; Genes, p53; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; Keratinocytes; Mutation; Telomerase

Cyclin D1 protein (encoded by the CCND1 gene) contributes to the progression of the cell cycle in the G1/S checkpoint. Cyclin D1 overexpression (for instance as a consequence of CCND1 amplification) might result in loss of control over genetic damage at this point and in an accumulation of chromosomal aberrations. In this work we analyze whether CCND1 amplification is associated with a higher incidence of alterations in cellular DNA content. 31 squamous cell carcinomas of the head and neck were studied. CCND1 amplification was determined by polymerase chain reaction. Cellular DNA content was determined by flow cytometry. CCND1 amplification was found in 6 (19%) cases. Thirteen (42%) cases were diploid and 18 (58%) were aneuploid. Two (33%) of the 6 cases with CCND1 amplification were aneuploid compared with 16 (64%) of the cases without CCND1 amplification (P = 0.36). We conclude that CCND1 amplification is not associated to a higher incidence of chromosomal aberrations in squamous cell carcinomas of the head and neck.

Topics: Adult; Aged; Aneuploidy; Carcinoma, Squamous Cell; Cyclin D1; DNA, Neoplasm; Gene Amplification; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Proteins; Oncogenes

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 second CCND1 polymorphism with gene expression and outcome in SCCHN patients. Analysis of a CCND1 G/C1722 polymorphism revealed that CCND1 CC1722 genotype 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 of CCND1 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 the CCND1 GG870 genotype (P = 0.04). Splicing of CCND1 mRNA in head and neck tissues was modulated by CCND1 A/G870 alleles, thus CCND1 transcript a was spliced equally from CCND1 A870 and G870 alleles, 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 two CCND1 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.

Topics: Alleles; Carcinoma, Squamous Cell; Cyclin D1; Female; Genotype; Head and Neck Neoplasms; Humans; Male; Polymorphism, Genetic; Sex Characteristics

Non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit tumorigenesis in colorectal cancer due to the induction of apoptosis. Disturbances of cellular pathways ultimately leading to apoptosis may contribute to the process of neoplastic transformation and immortalization. In this study we wanted to determine the influence of different NSAIDs (indomethacin, ibuprofen and sodium salicylate) and hydrocortisone on Bcl-2 expression and the apoptotic behavior of head and neck tumor cell lines and normal oral keratinocytes. Bcl-2 expression was determined by monoclonal antibody staining and fluorescence-activated cell-sorting measurement. Apoptotic cells were visualized with a epifluorescence microscope after staining with CytoDeath M30 antibody. Indomethacin (1 mM) and ibuprofen (1 mM) significantly reduced Bcl-2 expression in the cancer cell lines tested and might be thought responsible for the observed increase in apoptosis. At all concentrations tested the influence of sodium salicylate and hydrocortisone on Bcl-2 expression was not significant. In contrast, the NSAIDs tested had only a minor influence on normal oral keratinocytes. Our results demonstrate a significant reduction in growth and an increase in apoptosis, possibly due to a reduction in Bcl-2 expression. after exposure to indomethacin and ibuprofen in the head and neck cancer cell lines tested.

Topics: Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal; Apoptosis; Carcinoma, Squamous Cell; Cyclin D1; Head and Neck Neoplasms; Humans; Keratinocytes; Microscopy, Fluorescence; Mouth Mucosa; Steroids; Tumor Cells, Cultured

Local recurrence of squamous cell cancer (SCC) causes high morbidity and is often readily accessible, making such patients potential candidates for gene therapy. Cyclin D1 (CD1), critical in the G1-S transition in the cell cycle, is amplified in 20-50% and overexpressed in up to 80% of head and neck SCC. Our earlier studies indicated that CD1 expression increased with progression from low grade to high grade dysplasia, and that treatment of established tumors with antisense cyclin D1 (AS-cyclin D1) led to tumor regression during a one week evaluation period. We hypothesized that: 1) CD1 expression increases with disease progression to advanced SCC, and 2) AS-cyclin D1 therapy would lead to prolonged tumor regression in a xenograft model of human SCC. CD1 expression, evaluated by immunostain in 30 stage III/IV head and neck SCC, increased in the basal layer from normal-dysplasia (P = 0.06) and from dysplasia-carcinoma (P = 0.004). In the germinative layer CD1 expression increased from dysplasia-carcinoma (P = 0.002) but not from normal-dysplasia. Western blotting of eight SCC and two transformed keratinocyte cell lines demonstrated CD1 overexpression in 8/10 (80%) lines. An 11th cell line (A431) had previously been shown to overexpress cyclin D1. 8/9 (89%) cell lines overexpressing CD1 formed tumors in immunodeficient mice, whereas 0/2 cell lines without CD1 overexpression formed a tumor. Three established SCCs, one fast growing, one with moderate growth rate (with CD1 overexpression) and one slow growing (without increased CD1), shrank significantly for 2-4 weeks after AS-cyclin D1 treatment, while tumors transduced with control vector grew. Cyclin D1 expression increases in frequency with disease progression, and antisense cyclin D1 was effective in a xenograft model of human cancer, independent of tumor growth rate.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Line, Transformed; Cyclin D1; DNA, Antisense; Genetic Therapy; Head and Neck Neoplasms; Humans; Keratinocytes; Mice; Mice, SCID; Skin Neoplasms; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

To study the role of cyclin D1 in regulating the biological behavior of head and neck cancer.. Squamous cell carcinoma of the head and neck (SCCHN) cells were stably transfected with an antisense cyclin D1 using lipofectin-mediated transfection. In vitro growth assays, cell cycle analyses, cytotoxicity assays, and in vivo tumorigenicity assays were performed.. Human SCCHN cell lines TU138, TU167, TU177, TU182, MDA183, and MDA1386 and athymic nude mice were used for this study.. The antisense cyclin D1 transfected cells revealed decreased growth rates in vitro and decreased tumorigenicity in athymic nude mice. Furthermore, antisense cyclin D1 transfection enhanced the chemosensitivity against cisplatin.. These studies provided evidence that overexpression of cyclin D1 may play an important role in growth rates and biological behavior of human head and neck cancer. Additionally, expression of cyclin D1 may make human head and neck cancer cells resistant to platinum-based chemotherapeutic approaches. The ability to suppress the malignant phenotype by down-regulating cyclin D1 expression may provide a new gene therapy approach for patients with head and neck cancer.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; Antisense Elements (Genetics); Blotting, Western; Carcinoma, Squamous Cell; Cell Movement; Cell Transformation, Neoplastic; Cisplatin; Cyclin D1; Down-Regulation; Electrophoresis, Agar Gel; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, cdc; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; In Vitro Techniques; Mice; Transfection

The relationship between CCND1 and/or EMS1 amplification and disease outcome was studied in a prospective series of 104 head and neck squamous cell carcinomas treated by surgical resection. The CCND1 and EMS1 copy number in tumor samples was estimated by differential PCR. The presence or absence of amplification was analyzed in relation to clinicopathological variables, tumor recurrence, and patient survival. CCND1 amplification occurred in 32 cases (31%) and was associated with increased lymph node stage (P = 0.005) and advanced disease stage (P = 0.003). EMS1 amplification was identified in 21 cases (20%) and was related with advanced T stages (P = 0.001), increased lymph node stage (P = 0.02), advanced disease stage (P = 0.041), poor histological differentiation (P = 0.018), recurrent disease (P = 0.0004), and reduced disease-specific survival (P < 0.0001). Coamplification of both genes occurred in 11 cases (11.5%). Multivariate analysis confirmed that in addition to regional lymph node status, EMS1 amplification is an independent predictor of death from the tumor (P = 0.0027). CCND1 amplification was not prognostic. These data indicate that EMS1 amplification, but not CCND1 amplification, predicts early recurrence and reduced survival in squamous cell carcinoma of the head and neck. The prognostic significance previously attributed to CCND1 amplification may be attributable to its frequent coamplification with EMS1.

Topics: Carcinoma, Squamous Cell; Cortactin; Cyclin D1; DNA, Neoplasm; Female; Gene Amplification; Head and Neck Neoplasms; Humans; Male; Microfilament Proteins; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Polymerase Chain Reaction; Prognosis; Prospective Studies

Ex-chromate workers are frequently afflicted with lung cancers, especially central-type squamous cell carcinomas (SCCs) of the lung. However, little is known about the molecular and cellular biologic characteristics of chromate-induced lung cancers. We investigated expression of cyclin D1, bcl-2, and p53 proteins in chromate-induced lung cancers by immunohistochemistry, compared with those in lung cancers from nonexposed individuals and those in individuals with pneumoconiosis. Of 19 chromate-induced lung cancers, 16 tumors were SCCs, including 11 central and 5 peripheral types. Eleven (69%) of 16 chromate SCCs showed cyclin D1 expression. In contrast, cyclin D1 expression was observed in only 3 (12%) of 26 SCCs from nonexposed individuals and 6 (16%) of 37 SCCs that developed in patients with pneumoconiosis, respectively. The frequency of cyclin D1 expression proved to be significantly higher in chromate-induced SCCs than in SCCs from nonexposed individuals and from those with pneumoconiosis (P < .001). When comparisons were extended to all histologic types of lung cancer, cyclin D1 expression was observed significantly more often in chromate-induced lung cancers than in lung cancers from nonexposed subjects and those from patients with pneumoconiosis (11 [58%] of 19 v 5 [10%] of 52, P < .001, and 7 [11%] of 63, P < .001, respectively). Frequencies of bcl-2 and p53 expression were not significantly different among lung cancers from ex-chromate workers, nonexposed individuals and those with pneumoconiosis. The current study suggests that cyclin D1 expression may be involved in the development of chromate-induced lung cancers, although its underlying mechanism remains to be determined.

Topics: Adenocarcinoma; Carcinoma, Large Cell; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Chromates; Cyclin D1; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lung Neoplasms; Occupational Exposure; Proto-Oncogene Proteins c-bcl-2; Smoking; Tumor Suppressor Protein p53

Cyclin D1, p16, and Rb are key genes that play critical roles in the control of G1/S cell cycle progression and cellular homeostasis. To assess the differential expression of these genes in the biologic evaluation of head and neck squamous carcinoma (HNSC), we analyzed their protein expression in 11 cell lines and 46 primary tumors by Western blotting and correlated the results with clinicopathologic factors. In all cell lines, reciprocal expression between p16 and cyclin-D1 and Rb proteins was noted; p16 protein was detected in one (9%) cell line that lacked Rb and cyclin D1 and was absent in 10 of the cell lines (91%) that expressed both cyclin D1 and Rb proteins. Similar, albeit less striking, results were obtained in primary tumors: 30 tumors (65%) lacked p16 expression, and 33 tumors (72%) and 38 tumors (83%) expressed Rb and cyclin D1 proteins, respectively. p16 and Rb proteins were inversely expressed in 72% of tumors and in all cell lines. Except for gender and age, no significant correlation between protein expression and the clinicopathologic factors was found. Our results indicate that (1) loss of the p16 protein may constitute an early event in the development of these HNSC, (2) the reciprocal expression of p16 and Rb suggests a tight regulatory interaction between these genes in HNSC tumorigenesis, and (3) alteration in at least one of these genes might be required for HNSC development and progression.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Expression; Head and Neck Neoplasms; Humans; Male; Middle Aged; Retinoblastoma Protein; Tumor Cells, Cultured

Progression through the G1 phase of the cell cycle is mediated by phosphorylation of the retinoblastoma protein (pRb) resulting in the release of essential transcription factors such as E2F-1. The phosphorylation of pRb is regulated positively by cyclin D1/CDK4 and negatively by CDK inhibitors, such as p16 (CDKN2/MTS-1/INK4A). The p16/cyclin D1/Rb pathway plays a critical role in tumorigenesis and many tumor types display a high frequency of inactivation of at least one component of this pathway. In order to determine the overall contribution of these three components to progression of head and neck squamous cell carcinoma (HNSCC), we examined p16 inactivation, cyclin D1 amplification, and pRb expression in 23 primary HNSCC tumors and five cell lines. p16 inactivation was detected in 19/23 (83%) primary tumors by detailed genetic analysis and was confirmed by immunohistochemistry (IHC). Absence of Rb protein expression indicative of pRb inactivation was identified in 2/23 (9%) tumors. In this set of tumors, there was a perfect inverse correlation between p16 and pRb inactivation. Using fluorescence in situ hybridization (FISH) cyclin D1 amplification was identified in 4/5 (80%) cell lines and 4/11 (36%) primary tumors. However, 2/4 cell lines and all four primary tumors with cyclin D1 amplification contained a concomitant alteration of p16. Therefore 21/ 23 (91%) of primary HNSCC contained at least one alteration in the p16/cyclin D1/Rb pathway. Although p16 and Rb alteration are apparently exclusive, cyclin D1 amplification occurs concomitantly with the loss of p16 suggesting an additional role for this amplification in HNSCC.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Genes, Retinoblastoma; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Retinoblastoma Protein; Tumor Cells, Cultured

Time-dependent ladder-type DNA fragmentation and morphological alterations consistent with apoptosis were observed among A253 human head and neck squamous cell carcinoma (HNSCC) cells in nude mice from 15 to 18 days after transplantation, without any drug treatment. No evidence of ladder-type DNA fragmentation was detected in A253 cells in vitro or in normal nude mouse tissues (skin and muscle). Our aim was to explore molecular factors associated with such spontaneous apoptosis. Bcl-2 protein expression decreased, while bax protein expression increased from day 9 after transplantation. Moreover, altered expression of bcl-2 and bax was accompanied by the increased proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Time-dependent dephosphorylation of Rb, followed by proteolytic cleavage, was also observed from day 9 after transplantation. The data indicate that the caspase-3 activation and cleavage of Rb protein may represent important steps in the regulation pathway of bax-mediated spontaneous apoptosis. Interestingly, the time-dependent activation of spontaneous apoptosis was almost simultaneous with the induction of differentiation and increased expression of several differentiation-associated regulatory proteins. An increased expression of cyclin D1 and cyclin-dependent kinase-5 (cdk5) was observed from day 9 after transplantation, whereas only slight alteration of cdk4 expression was found. The time-dependent activation of cyclin D1 and cdk5 preceded both the induction of ladder-type DNA fragmentation and increased keratin pearl formation. Furthermore, MCM3 was cleaved early in spontaneous apoptosis and differentiation. Our observations suggest the involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-mediated spontaneous apoptosis and differentiation in A253 xenografts. P53 and WAF1 proteins were not expressed in the xenografts, indicating that the changes in the regulatory proteins during apoptosis and differentiation were not p53 or WAF1 dependent.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 1; Cell Cycle Proteins; Cell Differentiation; Cyclin D1; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; DNA Fragmentation; DNA-Binding Proteins; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Mice; Mice, Nude; Minichromosome Maintenance Complex Component 3; Neoplasm Transplantation; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transplantation, Heterologous; Tumor Cells, Cultured

Cyclin D1 plays an essential regulatory role in the G1 phase of the cell cycle. The cyclin D1 gene is amplified in 20-50% of squamous cell carcinomas (SCCs), and the protein is overexpressed in up to 80% of SCCs. Our hypothesis was that gene transduction of antisense (AS) cyclin D1 in human SCCs in vivo would result in tumor reduction. A cyclin D1 cDNA was inserted into an E1/E3-deficient serotype 5 adenovirus (AS cyclin D1) in an AS orientation using homologous recombination. AS cyclin D1 transduction suppressed cyclin D1 protein expression in both cultured cells and tumors. AS cyclin D1 significantly inhibited cell proliferation by both [3H]thymidine incorporation in six SCC cell lines (P = 0.01-0.001) and the conversion of tetrazolium salt to formazan in four SCC cell lines (P = 0.01-0.004). Apoptosis detected in >25% of cells in each cell line 48 h after AS cyclin D1 transduction paralleled the reduction in cyclin D1 protein. Preformed SCCs transduced with AS cyclin D1 were significantly inhibited (P = 0.002-0.005), and apoptosis was prominent in the AS cyclin D1-treated tumors, but not in tumors treated with the control vector. These data extend prior in vitro and ex vivo results and indicate that AS cyclin D1 suppresses SCC growth both in vitro and in vivo through suppression of cyclin D1 protein expression, leading to cellular apoptosis. Our findings suggest that cyclin D1 may have a role in cell survival and that cyclin D1 AS therapy may be useful as an adjunct to standard treatment for SCC.

Topics: Adenoviruses, Human; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cyclin D1; DNA, Antisense; Female; Gene Amplification; Genetic Vectors; Head and Neck Neoplasms; Humans; Open Reading Frames; Skin Neoplasms; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vulvar Neoplasms

Two hundred eight primary squamous cell carcinomas of the head and neck have been analyzed with respect to the presence of the retinoblastoma tumor suppressor protein, pRb. Of these, 23 tumors (11%) that preferentially localized to the tonsils revealed complete absence or dramatic reduction in the amount of pRb. Other cell cycle components, cyclin D1 and p16INK4A, which are intimately related to pRb through an autoregulatory loop, were also dramatically decreased or overexpressed, respectively, in these pRb-defective tumors. On the other hand, the majority of the pRb-defective tumors contained the wild-type p53 gene. No evidence was found for genetic defects at the Rb locus in these tumors. Very significantly, in 11 of 12 pRb-defective tonsillar tumors, but in none of 9 pRb-positive tonsillar tumors (P < 10[-7]), DNA of oncogenic human papillomavirus types was identified, providing a strong indication for a human papillomavirus-associated etiology of these tumors and suggesting the functional inactivation of the pRb protein by the viral E7 gene product. In comparison to all head and neck squamous cell carcinomas studied, the pRb-defective tonsillar tumors were in general more poorly differentiated (P = 0.0059), and they were all metastatic at the time of resection. Of particular clinical interest, despite these adverse histopathological factors, the clinical outcome for these patients was relatively favorable, strongly implying that the pRb-defective tumors responded uniformly well toward postoperative radiation therapy.

Topics: Carcinoma, Squamous Cell; Chromosome Aberrations; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease-Free Survival; DNA, Viral; Follow-Up Studies; Gene Deletion; Gene Expression; Genes, Retinoblastoma; Head and Neck Neoplasms; Humans; Neoplasm Proteins; Papillomaviridae; Retinoblastoma Protein; RNA, Messenger; Tonsillar Neoplasms; Tumor Suppressor Protein p53

p21(Waf1/Cipl) is a critical downstream effector in the p53-dependent pathway of growth control and causes growth arrest through inhibition of cyclin-dependent kinases. In this study 67% of 43 head and neck squamous cell carcinoma (HNSCC) and 60% of 15 tumour-adjacent oral dysplasias overexpressed p21 by immunohistochemical staining. Overexpression of p21 in HNSCC was independent of the presence of functional p53, as assessed by analysis of mutations and loss of heterozygosity and by immunohistochemisty. Rather, the expression pattern of p21 was associated with differentiation. Furthermore, in most tumours, the p21 positive cells did not incorporate bromodeoxyuridine (BrdU), which indicates inhibition of proliferation by p21 in these cells. In some tumours, p21 was also expressed in proliferating cells. In these latter tumour cells, cyclin D1 was frequently expressed as well. Therefore, we suggest that expression of cyclin D1 might overcome the inhibitory effect of p21 in these cells.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Mouth Diseases; Mouth Mucosa; Tumor Suppressor Protein p53

We have examined the correlation of a frequent A/G polymorphism within exon 4 of the cyclin D1 gene (CCND1) with genetic susceptibility and clinical outcome in 384 patients with squamous cell carcinoma (SCC) of the head and neck. CCND1 genotype frequencies were similar in the cases and 191 controls. Furthermore, the CCND1 genotype was not associated with susceptibility to SCC of the larynx, pharynx, or oral cavity. The influence of the CCND1 genotype on clinical outcome was also assessed. We found no correlation between genotype and tumor size (T1-T4), the involvement of nodes at presentation, or patient age and gender. However, the distribution of CCND1 genotypes in cases with poorly differentiated tumors was significantly different to that in patients with well-/moderately differentiated tumors (P = 0.016; chi2(2) = 8.71). Homozygosity for CCND1*G (GG genotype) was associated with poorly differentiated tumors (G3). We used Cox's proportional hazards model to investigate the influence of the CCND1 genotype on disease-free interval. CCND1 GG was associated with reduced disease-free interval [P = 0.001; hazard ratio (HR) = 2.95; 95% confidence interval (CI) = 1.54-5.63]. This remained significant after correction for tumor differentiation (P = 0.013; HR = 2.34; 95% CI = 1.2-4.6) and tumor stage (P = 0.005; HR = 2.64; 95% CI = 1.34-5.19). Analysis of the data from patients with tumors at different sites showed that the CCND1 GG genotype was associated with reduced disease-free interval in laryngeal (P = 0.004; HR = 3.63; 95% CI = 1.44-8.83) and pharyngeal (P = 0.006; HR = 3.48; 95% CI = 1.43-8.46) tumors. This is the first report of an association between CCND1 polymorphism and prognosis in SCC of the head and neck. These data show that the CCND1 GG genotype is an independent prognostic indicator of disease-free interval and supports initial observations in non-small cell lung cancer, that polymorphism within CCND1 influences tumor behavior.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Female; Genotype; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Polymorphism, Genetic; Prognosis

Flavopiridol (HMR 1275) has been identified recently as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Flavopiridol inhibits most cyclin-dependent kinases (cdks) and displays unique anticancer properties. Here, we investigated whether this compound was effective against head and neck squamous cell carcinomas (HNSCC). Exposure of HNSCC cells to flavopiridol diminished cdc2 and cdk2 activity and potently inhibited cell proliferation (IC50 43-83 nM), which was concomitant with the appearance of cells with a sub-G1 DNA content. Moreover, DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) reaction confirmed that flavopiridol induces apoptosis in all cell lines, even on certain HNSCC cells that are insensitive to apoptosis to DNA-damaging agents (gamma-irradiation and bleomycin). A tumorigenic HNSCC cell line was used to assess the effect of flavopiridol in vivo. Treatment (5 mg/kg per day, intraperitoneally) for 5 d led to the appearance of apoptotic cells in the tumor xenografts and caused a 60-70% reduction in tumor size, which was sustained over a period of 10 wk. Flavopiridol treatment also resulted in a remarkable reduction of cyclin D1 expression in HNSCC cells and tumor xenografts. Our data indicate that flavopiridol exerts antitumor activity in HNSCC, and thus it can be considered a suitable candidate drug for testing in the treatment of refractory carcinomas of the head and neck.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cyclin D1; Cyclin D3; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Female; Flavonoids; Gene Expression; Growth Inhibitors; Head and Neck Neoplasms; Mice; Mice, Nude; Piperidines; Protein Serine-Threonine Kinases; Transplantation, Heterologous; Tumor Cells, Cultured

Cyclin D1 proto-oncogene is a key regulator of the mammalian cell-cycle acting at the restriction point in late G1. Amplification of the cyclin D1 locus, located on chromosome 11q13, as well as cyclin D1 protein overexpression have been reported in several human malignancies. The purpose of this study was to evaluate cyclin D1 gene copy status and protein expression during the multistep process of head and neck tumorigenesis, using a combination of fluorescence in situ hybridization and immunohistochemistry techniques. From 29 selected patients presenting with head and neck squamous carcinoma and whose tumor cytospins had been previously screened for presence (16 cases) or absence (13 cases) of amplification at the 11q13 band, we analysed 46 paraffin-embedded tissue specimens that demonstrated, besides the primary tumor, the presence of contiguous adjacent normal tissue and/or premalignant lesions. Of the 16 amplified cases, nine demonstrated a continuous progression from premalignant to invasive carcinoma and seven (77.7%) of these cases showed cyclin D1 gene amplification in premalignant lesions prior to development of invasive carcinoma. Increased cyclin D1 protein expression was observed in all 16 amplified tumors and five of the 13 (38.4%) non-amplified tumors. Interestingly, dysregulated cyclin D1 expression was also found in the premalignant lesions adjacent to all 16 amplified tumors, and it appeared to precede cyclin D1 gene amplification. In contrast no dysregulated expression was detected in the premalignant lesions of the non-amplified tumors. In conclusion, these findings provide strong evidence for early dysregulation of cyclin D1 expression during the tumorigenesis process and suggest that dysregulated increased expression precedes and possibly enables gene amplification.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Gene Amplification; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Neoplasm Proteins; Precancerous Conditions; Proto-Oncogene Mas; Proto-Oncogene Proteins

The gene for Cyclin D1 (CCND1) lies within chromosomal region 11q13 and codes for a cell cycle regulator essential for G1 phase progression. This G1-cyclin is a putative protooncogene whose clonal rearrangement and/or amplification and mRNA overexpression occurs in several types of human neoplasias, including head and neck squamous cell carcinomas. Data from the literature suggest that amplification and overexpression of the CCND1 gene could lead to destabilisation of cell cycle control mechanisms and uncontrolled cell proliferation. We developed a differential PCR system for the determination of CCND1 gene amplification in head and neck squamous cell carcinomas. A 115 bp CCND1 fragment and a 150 bp gamma-interferon fragment are amplified simultaneously in the same reaction tube under optimized conditions. Statistical analysis of amplification data obtained by differential PCR revealed excellent correlation with amplification data obtained by conventional Southern hybridization.

Topics: Blotting, Southern; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 11; Cyclin D1; DNA, Neoplasm; Gene Amplification; Genes, bcl-1; Head and Neck Neoplasms; Humans; Polymerase Chain Reaction

Cyclin D1 and cyclin G are essential regulatory factors in the progression of the cell cycle from G0 through G1 and S phase. Aberrations in expression of these cyclins may lead to dysregulated cellular proliferation that could result in neoplasia. Amplification and overexpression of cyclin D1 have been observed in many human cancers, whereas cyclin G is a new cyclin recently described in osteosarcoma cells. This study was performed to determine whether these cyclins were amplified in head and neck squamous cell carcinoma (HNSCC) tumors. Polymerase chain reaction of DNA extracted from 22 HNSCC primary tumors and three HNSCC cell lines did not reveal amplification of cyclin D1 in any of the tumor samples. Southern blot analysis identified amplification of cyclin D1 in a single tumor. Amplification of cyclin G was not observed in any of the tumors by Southern blot hybridization with a cyclin G probe. HNSCC cell lines transfected with antisense cyclin D1 were tested for cell proliferation by the incorporation of 3H-thymidine into cells grown in serum-free media. By 72 hours of incubation, there was a greater than 30% reduction in proliferation of cells transfected with antisense cyclin D1 as compared with non-transfected control cells. The results indicate that cyclin D1 may play an important role in the growth and proliferation of HNSCC cells.

Topics: Adult; Aged; Blotting, Southern; Blotting, Western; Carcinoma, Squamous Cell; Cell Division; Cyclin D1; DNA, Neoplasm; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Staging; Polymerase Chain Reaction; Tumor Cells, Cultured

Abnormalities of chromosome band 11q13 are frequent in squamous cell carcinoma of the head and neck (SCCHN). The oncogene CCND1 is located at 11q13 and encodes cyclin D1, a cell cycle-regulating protein. The authors investigated the clinical relevance and associations between amplification and overexpression of cyclin D1 and 11q13 rearrangements.. The study involved two series of patients. In Series 1, overexpression of cyclin D1 and 11q13 rearrangements, assessed by immunohistochemistry and cytogenetics, respectively, were compared with clinical data in 75 patients with SCCHN. Patients were monitored for at least 18 months or until death. In another 23 patients (Series 2), the authors investigated the association between DNA amplification (by slot blot hybridization), overexpression of cyclin D1, and cytogenetics.. In Series 1, 9 of 75 tumors (12%) had 11q13 aberrations, 6 of which manifested elevated expression of cyclin D1. Patients with tumors strongly positive for cyclin D1 (n = 9) and those with tumors showing 11q13 rearrangements had poorer survival (P = 0.047 and 0.005, respectively). However, the correlation between these two variables was weak (P = 0.12). In Series 2, 17 of 23 tumors (74%) showed elevated cyclin D1 protein expression, and 6 of these showed gene amplification as well. Of these six, only one revealed 11q13 rearrangements.. Overexpression of cyclin D1 and 11q13 rearrangements are independent prognostic factors for SCCHN. In general, DNA amplification results in overexpression of cyclin D1, but additional genetic mechanisms are involved in the deregulation. Furthermore, oncogenes at 11q13 besides CCND1 may be involved in the tumorigenesis.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Chromosome Aberrations; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; Female; Gene Amplification; Gene Rearrangement; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins; Phenotype; Prognosis

Hypopharyngeal squamous cell carcinomas (HPC) has an extremely poor prognosis. Characteristics of cell lines of head and neck squamous cell carcinomas including HPC were studied by various methods, e.g., chemosensitivity test and the immunohistochemistry staining method, to determine whether this poor prognosis is due to the biological behavior of this cancer. An HPC cell line was found to be resistant to anti tumor drugs, i.e., PEP, MTX and CPM and moderately sensitive to CDDP, 5-FU and ADM. Thermoresistance to hyperthermatic treatment and weak expression of ICAM-1 on the HPC cell line were observed. DNA synthesis by the HPC cell line was induced by stimulation with a low concentration of EGF and the amount of EGFR on these HPC cells was very high. In addition, cyclinD1 overexpression was found in the HPC cell line. Based on the above findings, further analysis of hypopharyngeal carcinoma cells and the development of a new treatment modality to control tumor growth and metastatic factors influencing the poor outcome are necessary to improve the prognosis of this cancer.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; DNA, Neoplasm; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; ErbB Receptors; Head and Neck Neoplasms; Humans; Hypopharynx; Intercellular Adhesion Molecule-1; Oncogene Proteins; Pharyngeal Neoplasms; Tumor Cells, Cultured

Alterations, especially homozygous deletions, of the putative tumor suppressor gene, p16 (p16INK4A, MTS1, CDKN2) have been found in tumor cell lines from a variety of neoplasms. Recent studies have reported frequent p16 gene deletions in cell lines from squamous cell carcinomas of the head and neck (SCCHN), although the prevalence of alterations was variable in primary tumors. This study determined the prevalence of point mutations and deletions of the p16 gene in 33 SCCHN. In addition, the association of p16 gene alterations and abnormalities of p53, PRAD-1 (cyclin D1), and the presence of human papillomavirus (HPV) was examined. We found an overall prevalence of p16 alterations of 36% (nine deletions, three single base substitutions, including one polymorphism). Seven tumors (of 29, 24%) had an alteration of p16 and p53; five (of 33, 15%) had alterations of p16 and PRAD-1; three (of 29, 10%) had alterations of all three genes. In addition, of the five tumors with human papillomavirus detected, only one also had a p16 gene alteration. The results indicate a potentially important role for the p16 gene in head and neck tumorigenesis. In addition, the presence of tumors with multiple somatic gene alterations suggest a possible interaction in the dysregulation of the cell cycle.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclins; Female; Genes, p53; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; Male; Middle Aged; Mutation; Oncogene Proteins; Papillomaviridae

To evaluate the overexpression of cyclin D1 and p53 as a prognostic marker of squamous cell carcinoma of the head and neck and to investigate whether deregulation of these genes is associated with an unfavorable course of disease.. Retrospective study.. Formalin-fixed, paraffin-embedded tumor materials that were obtained from a well-characterized series of 115 patients with resectable head and neck cancer at The Netherlands Cancer Institute, Amsterdam, were analyzed by immunohistochemical methods using antiserum samples that were directed against 2 proteins (ie, cyclin D1 and p53), which are crucial in the regulation of the G1 phase of the cell cycle.. Overexpression of cyclin D1 protein was found in 49% of the patients with squamous cell carcinoma of the head and neck. This overexpression was not associated with known prognostic factors (eg, the T and N stages). Tumors recurred more frequently and in a shorter period in patients whose primary tumors showed an overexpression of cyclin D1 protein. This difference (P = .05) was statistically significant in a stepwise proportional hazard regression analysis. However, since a discrepancy in staining results was observed between the biopsy and resection materials that were taken from the same patient, this result may not have been applicable in the evaluation of biopsy specimens only. This discrepancy is most likely owing to tissue heterogeneity. The overexpression of p53 that was found in 42% of the patients was of no prognostic significance.. These data provide evidence that overexpression of cyclin D1 protein in resection material of squamous cell carcinomas of the head and neck is indicative of a poor prognosis, independently of other known prognostic factors. Whether overexpression of cyclin D1 may therefore be used to select patients for more intensive treatment should be examined in the context of a clinical trial.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Oncogene Proteins; Prognosis; Retrospective Studies

Mutations in the p53 tumour suppressor gene and amplification of the cyclin D1 (CCND1) oncogene have been commonly reported in various malignancies. In the present study of 39 squamous cell carcinomas of the head and neck, p53 mutations were manifest in 11 (28%) of the cases, whereas CCND1 amplification was seen in 6 (16%) of 37 analysed tumours. The 10 mutations occurring in coding sequences of p53 were found in exon 5 (4 cases), exon 6 (3 cases),f and exon 8 (3 cases). No mutation was found in exon 7. Eight of the 10 exon nucleotide substitutions were missense mutations and two were nonsense mutations. All six tumours with CCND1 amplification also had p53 mutations, while an additional five tumours manifested p53 mutations in the absence of CCND1 amplification. There was a statistically significant positive correlation between these two gene alterations. This raises the possibility that mutation of p53 precedes CCND1 amplification in carcinogenesis.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Exons; Female; Gene Amplification; Genes, p53; Head and Neck Neoplasms; Humans; Male; Middle Aged; Nucleic Acid Hybridization; Oncogene Proteins; Oncogenes; Point Mutation; Polymerase Chain Reaction

Cyclin D1 is thought to play a critical role in the G1/S phase transition of the cell cycle. Amplification of this gene has been reported in several types of human neoplasms including breast, lung, esophageal, and head and neck tumors. In this study, we have analyzed the relative level of expression of cyclin D1 messenger RNA (mRNA) in fresh specimens of head and neck squamous cell carcinoma (HNSCC), and investigated the concordance of the overexpression of cyclin D1 mRNA with gene amplification. Levels of cyclin D1 mRNA were analyzed by a modified method of competitive reverse transcription-polymerase chain reaction and levels of cyclin D1 gene amplification were evaluated by Southern blot hybridization in a series of 23 matched normal mucosas and HNSCC. Overexpression of cyclin D1 mRNA was observed in 10 of 23 cases (43.5%) of HNSCC, ranging from 2 to 50-fold higher than the normal control. Twelve of 23 cases could be evaluated by Southern blot hybridization, and gene amplification was found in only 2 of 12 cases (16.7%). These findings suggest that cyclin D1 plays an important role in tumorigenesis of HNSCC, and gene amplification is not one of the major mechanisms for overexpression of cyclin D1.

Topics: Aged; Aged, 80 and over; Blotting, Southern; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Female; Gene Amplification; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Head and Neck Neoplasms; Humans; Male; Middle Aged; Oncogene Proteins; Polymerase Chain Reaction; Reference Standards; RNA, Messenger; Transcription, Genetic

Recently, attention has focused on the potential of oncogenes, tumour suppressor genes, and assessment of cell proliferation as biological response indicators in human cancer. In this study, immunocytochemical analysis was used to evaluate the usefulness of Ki67, epidermal growth factor receptor (EGFr), and the protein products of c-Myc and Bcl-2 as indicators of radiosensitivity in primary cultures of head and neck tumours. Primary cultures established from tumours taken at surgery were divided into two groups; the control group remained untreated, and the treatment group received a single dose of 2 Gy. The cultures were incubated for 14 days, after which time they were fixed and examined immunocytochemically. The response to treatment of the cultures was measured as the percentage of growth inhibition (%GI) in the treated cultures relative to the untreated controls. Expression of Ki67 measured after a single dose of 2 Gy significantly differentiated the radioresistant and radiosensitive groups (P = 0.045); high percentages of Ki67+ cells correlated with radioresistance. A significant difference was found between the expression of EGFr in the resistant and sensitive groups, as measured in control cultures and after a dose of 2 Gy (P = 0.002 and P = 0.03, respectively); high levels of expression of EGFr correlated with radioresistance. The level of expression c-Myc+ cells, as measured in control cultures, significantly distinguished the radiosensitive group from the radioresistant group (P = 0.05). These results indicate a potential role for these proteins as indicators of radioresistance.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Ki-67 Antigen; Proto-Oncogene Proteins c-myc; Radiation Tolerance; Radiotherapy Dosage; Tumor Cells, Cultured

To evaluate the prognostic significance of cyclin D1 protein/gene expressions in human head-and-neck squamous-cell carcinoma (HNSCC), we examined amplification of the cyclin-D1 gene (CCND1) by the differential PCR method and over-expression of cyclin-D1 protein by immunohistochemistry in 45 paraffin-embedded sections from HNSCC. Amplification of CCND1 was found in 10 (22%) cases and over-expression of cyclin D1 was found in 24 (53%) cases. CCND1 amplification was also found in 3 (25%) of 12 cases of dysplastic lesions adjacent to HNSCC. The overall 5-year survival of patients with CCND1 amplification or with protein over-production was significantly lower than that of patients without (p < 0.0001 and p < 0.05, respectively). However, with multivariate analysis, only amplification of CCND1 retained an independent prognostic value (p = 0.0018). These suggest that CCND1 amplification occurs at early stages of HNSCC tumorigenesis and is a more useful prognostic factor than over-expression of cyclin D1 in HNSCC.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Female; Gene Amplification; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Paraffin Embedding; Polymerase Chain Reaction; Prognosis; Retrospective Studies

Deregulation of expression of the cell cycle regulator cyclin D1 (cD1) may be responsible for rapid proliferation of squamous cell carcinoma of the head and neck (SCCHN). We have studied the expression of cD1 in 46 SCCHNs using immunohistochemistry. Before biopsy, the patients received an in vivo infusion of iododeoxyuridine (IdUrd) for cell proliferation assessment. Additionally, the level of apoptosis was estimated using in situ end labeling (ISEL). Among 33 tumors, the proportion of cD1(+) cells varied from 0.5 to 51.3% (19.9 +/- 2.2%). Thirteen tumors did not express cD1. The fraction of S-phase (IdUrd-positive) cells was 26.3 +/- 1.8% in cD1(+) versus 20.0 +/- 2.4% in cD1(-) tumors (P = 0.06). The percentages of cD1(+) cells and of S-phase cells were not correlated (P = 0.37). Apoptosis was detected by ISEL in 15 of 33 tumors studied. ISEL-positive tumors contained a significantly higher proportion of cD1(+) cells (14.9 +/- 2.6%) than cD1(-) ones (7.9 +/- 2.8%; P = 0.03). There was a positive correlation between the percentage of cD1(+) cells and the degree of ISEL (r = 0.54; P < 0.001). In noninvolved oral mucosa, cD1(+) cells were located primarily in the suprabasal layers (29.3 +/- 3.8% versus 1.2 +/- 0. 2% in the basal layer). Only 23 of 44 mucosal specimens contained cD1(+) cells. All cD1(-) samples were proliferatively active and contained IdUrd-labeled cells. The percentage of cD1(+) cells in the oral epithelium from nontumor controls (uvula samples) was significantly higher than in the SCCHN group in both basal (2.4 +/- 0.4%; P = 0.008) and suprabasal (42.7 +/- 3.3%; P = 0.005) layers. Additionally, whereas in uvuli, cD1(+) cells were distributed evenly along the epithelial lining, in SCCHN samples the regions showing cD1 expression alternated with areas in which cD1 expression was undetectable. These data indicate that cD1 expression in SCCHN varies among tumors and is not correlated with cell proliferation. In noninvolved oral mucosa, cD1 expression differs from that in truly normal epithelium obtained from nontumor patients. A correlation between cD1 expression and the extent of ISEL positivity suggests a possible involvement of cD1 expression in the apoptotic pathways.

Topics: Adult; Aged; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Cyclin D1; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Tissue Distribution

Chromosome 11q13 amplification has been identified in a subset of head and neck squamous cell carcinomas (H&N SCCs). This region contains several putative oncogenes, including cyclin D1 (PRAD1, CCND1), which encodes for an important cell cycle regulatory protein, and the locus encoding for the drug-detoxifying enzyme glutathione-S-transferase-pi (GST-pi). To determine the relationship of cyclin D1 and GST-pi gene amplification to expression of the encoded proteins, the authors examined 64 H&N SCCs by both Southern blot hybridization and immunohistochemistry, using a recently described, affinity-purified, anticyclin D1 polyclonal antibody no. 19 as well as a polyclonal antibody against GST-pi. Anticyclin D1 antibody no. 19 labeled the tumor cell nuclei in 28 (44%) of the H&N SCCs, whereas cytoplasmic immunoreactivity for GST-pi was noted in 55 (86%) neoplasms. By Southern blot 24 tumors (37.5%) showed twofold to tenfold amplification of 11q13 loci; only two of these were coamplified for GST-pi. Immunopositivity with anticyclin D1 antibody no. 19 but not anti-GST-pi significantly correlated with 11q13 amplification (P < .0001). Of the 28 tumors positive with anticyclin D1 antibody no. 19, however, only 18 (64%) were amplified for 11q13, and six amplified tumors did not react with the no. 19 antibody. A strong trend was noted between anticyclin D1 antibody no. 19 reactivity and a hypopharyngeal primary site (P = .053), but no correlations were observed between immunoreactivity and cytological grade, architectural pattern, pathological stage, and disease-free or overall survival. The inconsistent association of cyclin D1 immunoreactivity with 11q13 amplification indicates that other mechanisms may exist for protein overexpression. Immunoreactivity for the GST-pi protein is prevalent in H&N SCC but is clearly unassociated with amplification. In this series, the presence or extent of cyclin D1 and GST-pi immunoreactivity was of no proven prognostic benefit in H&N SCC.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Southern; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Gene Amplification; Gene Expression Regulation, Neoplastic; Glutathione Transferase; Head and Neck Neoplasms; Humans; Immunohistochemistry; Middle Aged; Oncogene Proteins; RNA, Messenger

D-type cyclins are proto-oncogenic cell cycle regulators implicated in the pathogenesis of several types of cancer. Amplification of the cyclin D1 gene has been described in 30-50% of human head and neck squamous cell carcinoma (HNSCC). Using immunohistochemistry on archival specimens of human HNSCC and a mAb DCS-6, which is specific for cyclin D1, strong positivity was found in nuclei of 9 (17%) of 52, a moderately elevated signal in 16 (31%) of 52, and weak staining comparable with normal tissues in 27 (52%) of 52 patients. Immunoblotting analysis of five HNSCC-derived cell lines showed three distinct spectra of D-type cyclin proteins: cyclin D1 only (in UMSCC-2 and UMSCC-22b cell lines with 11q13 amplification), cyclins D1 and D3 (in HN5 and HN6), or cyclins D1, D2, and D3 (in UMSCC-1). Electroporation of neutralizing antibodies demonstrated requirement for cyclin D1 in cell cycle progression of all five HNSCC cell lines. Cyclin D2 was essential and showed a cooperative effect with cyclin D1 in positive regulation of G1 in UMSCC-1 cells. These data are consistent with the proposed oncogenic role of cyclin D1 in HNSCC and open up the way for immunohistochemical assessment of cyclin D1 aberrations in archival clinical specimens. It is also suggested that excessive levels of cyclin D1 alone or cooperative effects of several D-type cyclin proteins may lead to deregulation of G1 control in distinct subsets of human HNSCC. These results are discussed in the context of possible functional redundancy of D-type cyclins and the role of the D-type cyclin/p16-CDKN2/pRB pathway in tumorigenesis.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclin D2; Cyclins; G1 Phase; Head and Neck Neoplasms; Humans; Immunohistochemistry; Oncogene Proteins; Paraffin Embedding

To gain a better understanding of genetic changes in squamous cell carcinomas of the head and neck we used comparative genomic hybridization for the analysis of 13 primary tumors. Copy number increases were most frequently observed on chromosomes 3q (10 cases) and 5p (8 cases) and less frequently on 1q (4 cases), 2 (1 case), 7 (2 cases), 8q (2 cases), 9 (1 case), 10p (2 cases), 13q (2 cases), 14q (1 case), 16 (1 case), 17 (2 cases), 20p (2 cases), 21q (1 case) and 22q (1 case). Copy number decreases occurred most frequently at 3p (5 cases), 5q (4 cases), 19p (6 cases), and 19q (5 cases). Copy number decreases also were observed on 1p (2 cases), 2q (2 cases), 4p (2 cases), 4q (2 cases), 7q (2 cases), 8p (1 case), 10q (1 case), 11p (2 cases), 11q (3 cases), 13q (3 cases), 14q (1 case), 16p (1 case), 17p (3 cases), 17q (1 case), 18q (1 case), and 22 (2 cases). Eight sites exhibiting significant sequence amplification were mapped to 3q26-->qter (3 cases), 11q13 (2 cases), 12p (2 cases), 2q33-36 (1 case), 7q21-22 (1 case), 7q33-->qter (1 case), 9p (1 case), and 13q32-->qter (1 case). Our data suggest that the regions 3q26-->qter and 5p may harbor oncogenes important for initiation or progression of squamous cell carcinomas of the head and neck. In addition, comparative genomic hybridization defines a subgroup of tumors with 11q13 involvement, the location of the PRAD1/(CCND1)/cyclin D1 gene.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Chromosome Aberrations; Chromosome Mapping; Chromosomes, Human; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 3; Cyclin D1; Cyclins; DNA, Neoplasm; Female; Gene Amplification; Gene Deletion; Head and Neck Neoplasms; Humans; Male; Middle Aged; Nucleic Acid Hybridization; Oncogene Proteins

We evaluated the prognostic significance of overexpression of cyclin D1 in 47 patients with surgically resected squamous cell carcinomas of the head and neck. Overexpression of cyclin D1 was detected immunohistochemically using an affinity-purified polyclonal antibody directed against the carboxyl-terminal part of the cyclin D1 protein, applied to formalin-fixed, paraffin-embedded tissue sections. Overexpression of cyclin D1 was found in 30 of 47 head and neck squamous cell carcinoma (HNSCC) cases and was associated with a more rapid and frequent recurrence of disease (P = 0.027). There was a 5-year disease-free interval of 47% for HNSCC patients with a strong overexpression of cyclin D1 and of 80% for cyclin D1-negative HNSCC patients. Overexpression of cyclin D1 was also associated with a shortened overall survival of these patients (P = 0.0095), with a 5-year survival of 60% for the cyclin D1 strongly positive cases and of 83% for cyclin D1-negative cases. Overexpression of cyclin D1 appears to indicate poor prognosis in operable HNSCC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Cyclins; Female; Gene Amplification; Gene Expression; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Recurrence, Local; Oncogene Proteins; Paraffin Embedding; Prognosis; Retrospective Studies; Tumor Suppressor Protein p53

High proliferative fraction and cyclin D1/CCND1 gene amplification have been associated with certain aggressive features of head and neck squamous carcinoma in some studies, but not in others. The differences may be related to the intratumoral heterogeneity of these factors. Moreover, the interrelationship between these seemingly related factors has not been determined. In order to determine the correlation between tumor proliferative fractions and CCND1 gene amplification, 3 spatially different samples from each of 32 primary head and neck squamous cell carcinomas (HNSCC) were separately analyzed by flow cytometry. A mixture of these specimens was also minced and snap frozen for molecular studies. DNA was extracted from patient lymphocytes, normal appearing squamous epithelium, and tumors. A genomic DNA probe containing the first exon of CCND1 was used for hybridization by Southern technique. A 5.6 kb genomic DNA probe of immunoglobulin heavy chain was used as an internal standard for quantification of CCND1 gene amplification. Our results showed that 30% of the tumor cases manifested intratumoral heterogeneity (> 50% differences) of their proliferative activity. The highest value was used for correlation with gene amplification. Eleven (34.4%) of the 32 tumors showed CCND1 amplification (2-10-fold). When the proliferative fraction was dichotomized into high and low groups based on the mean value (> or = 13%), a highly statistical correlation between CCND1 amplification and tumor proliferation was obtained (P < 0.001). No significant correlation between gene amplification and other clinicopathologic parameters was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blotting, Southern; Carcinoma, Squamous Cell; Cell Division; Cyclin D1; Cyclins; Flow Cytometry; Gene Amplification; Gene Dosage; Genetic Heterogeneity; Head and Neck Neoplasms; Humans; Oncogene Proteins

Mutations of the tumor-suppressor gene TP53 and amplification of CCND1 gene have been reported to occur frequently in head and neck squamous cell carcinomas (HNSQCC). In experimental systems, TP53 mutations have been shown to lead to genomic instability, including an increased propensity for gene amplification. We have examined 16 HNSQCC cell lines for the association between TP53 over-expression/mutation and CCND1 amplification. p53 over-expression was detected in 50% of the cell lines by immunohistochemistry using the monoclonal antibody (MAb) PAb1801. TP53 mutations were also detected in 50% of the cell lines by analysis of single-strand conformation polymorphism (SSCP) and DNA sequencing of exons 4 through 9. Six cell lines showed TP53 mutations and over-expression of the protein, 2 cell lines showed TP53 mutations but no p53 expression, and 2 cell lines showed over-expression of p53 protein but no TP53 gene mutations. CCND1 amplification was found in 38% of the cell lines by Southern blot analysis. Only 1 cell line showed both TP53 mutation and CCND1 amplification, whereas 7 of 8 cell lines with TP53 mutations had no CCND1 amplification. pRb expression was detected by Western blot analysis, and the level of pRb did not correlate with either CCND1 amplification or TP53 mutation. Our findings suggest that TP53 mutation and CCND1 amplification are common genetic alterations in HNSQCC and that the occurrence of either genetic event may be sufficient to abrogate normal cell cycle control.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Cyclins; DNA, Neoplasm; Gene Amplification; Gene Expression; Genes, p53; Head and Neck Neoplasms; Humans; Immunoblotting; Immunoenzyme Techniques; Molecular Probe Techniques; Mutation; Oncogene Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Abnormalities in chromosome 11q13 regions have been frequently found in head and neck squamous carcinoma. Recent studies indicate that the PRAD-1 (also CCND1), which encodes cyclin D1, is a putative oncogene that is an important component of this region.. DNA was extracted from 32 snap-frozen specimens from primary head and neck squamous carcinomas. DNA from peripheral blood lymphocytes, normal mucosa, and salivary gland tissue were used as controls. A genomic DNA probe containing the first exon of PRAD-1 was used for hybridization with specimen DNAs by the Southern technique. A 5.6-kb genomic DNA probe of immunoglobulin heavy chain was used as an internal standard for assessing PRAD-1 amplification.. Eleven (34.4%) squamous carcinoma specimens showed PRAD-1 amplification (2- to 10-fold). Although no significant statistical correlation among amplification status, grade stage, and DNA ploidy was observed in this small cohort, amplification was more noted in high grade, high stage, and aneuploid tumors. A highly statistical correlation between PRAD-1 amplification and proliferative activity was noted (P > 0.001).. The results of this study indicate that PRAD-1 amplification appears to be a late event in the tumorigenesis of head and neck carcinoma and is associated often with a subset of aggressive tumors and high proliferation neoplasms.

Topics: Aneuploidy; Carcinoma, Squamous Cell; Chromosome Mapping; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; DNA, Neoplasm; Flow Cytometry; Gene Amplification; Head and Neck Neoplasms; Humans; Oncogene Proteins; Oncogenes

Amplification of 11q13 DNA markers, particularly hst-1/FGF4 oncogene and the bcl-1 locus, was evaluated in 178 head and neck squamous cell carcinomas (SCCs) by Southern blot and slot blot hybridisation. Coamplification of hst-1/FGF4 and bcl-1 genes was found in 57% of primary tumours and in 60% of the 89 metastatic lymph nodes tested. The pattern of amplification was significantly similar in matched sets of primary SCCs and metastatic lymph nodes. Levels of amplification, quantified by densitometric analysis of slot blots, ranged from 2 to 18-fold normal gene dosage. Also, c-myc oncogene (8q24) was found amplified less frequently, since 7% of 169 SCCs tested contained amplification of this gene, the level of which ranged from 2 to 8-fold. Hst-1/bcl-1 gene amplification was observed more frequently in the tumours arising from the hypopharynx. Coamplification of hst-1 and bcl-1 genes was significantly positively associated with tumours with nodal involvement (P = 0.001). Incidence of hst-1/bcl-1 gene amplification is higher in the tumours with a clinical stage III or IV. Hst-1/bcl-1 gene amplification was not related to tumour differentiation or local invasiveness. This prospective study shows that amplification of 11q13 DNA markers is a prominent event occurring in head and neck SCC and may contribute to the pathogenesis and evolution of a subset of patients bearing this type of cancer.

Topics: Blotting, Southern; Carcinoma, Squamous Cell; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; Female; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Amplification; Head and Neck Neoplasms; Humans; Male; Oncogene Proteins; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc