Concomitant analysis of p16/INK4, cyclin D1, and retinoblastoma protein expression in esophageal squamous cell carcinoma

BACKGROUND/AIMS: Cyclin D1 expression is one of the important biologic factors and its close association with p16/INK4 and retinoblastoma protein expression has been proven in patients with esophageal squamous cell carcinoma, however, the clinical implication of these associations is still unknown. The objective of this study is to clarify its clinical significance. METHODOLOGY: We studied p16, cyclin D1, and pRB expression using immunohistochemistry in the resected specimens of 156 patients who underwent curative esophagectomy. RESULTS: Alteration of p16 expression, positive cyclin D1 expression and loss of pRB expression were demonstrated in 108 (69%) patients, 47 (30%) patients, and 48 (31%) patients, respectively. An inverse correlation was found between p16 expression and pRB expression (P < 0.0001). Of 47 cyclin D1-positive tumors, 39 (83%) tumor exhibited alteration of p16 and 43 (92%) tumors were positive for pRB. Based on p16, cyclin D1, and pRB expression, 138 (88%) patients were divided into the following three groups: p16-/cyclin D1+/pRB+ (n = 44), p16+/cyclin D1-/pRB- (n = 38), p16-/cyclin D1-/pRB+ (n = 56). The three groups were the most influential prognostic factors in a Cox's proportional regression model. The p16-/cyclin D1+/pRB+ group had frequent hematogenous recurrence, while the other groups had frequent lymph node recurrence. CONCLUSIONS: Assessment of p16, cyclin D1, and pRB expression may be helpful for determining postoperative therapeutic strategies in patients with esophageal squamous cell carcinoma.     

Alterations in cyclin D1 expression in esophageal squamous cell carcinoma in the Indian population

Purpose: The p16/cyclin D1/pRb pathway plays a critical role in tumourigenesis. We recently reported alterations in expression of tumour suppressor gene products, p16 and pRb in esophageal cancer. Knowledge of alterations in cyclin D1, a vital component of this pathway in esophageal carcinomas from the Indian subcontinent, where the etiology and pathogenesis may be confounded by various unique dietary and environmental factors, is presently scanty. In order to bridge the gap between the accentuating incidence of esophageal cancer and aberrations in the components of this vital pathway, we analysed cyclin D1 expression in esophageal squamous cell carcinoma in the Indian population. Method: Immunohistochemical analysis of cyclin D1 expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinomas (ESCC) (70 patients) and matched with histopathologically normal esophageal tissues from a distant site. The findings were correlated with clinicopathological parameters. Results: Overexpression of cyclin D1 was observed in the tumour nuclei in 41 out of 70 (59%) patients. We found concomitant alterations in 16 and cyclin D1 (p16⁻/CycD1⁺ phenotype) in 16 of the 70 patients (23%), while alterations of pRb and cyclin D1 (pRb⁻/CycD1⁺) were observed in 36 of the 70 (51%) patients of ESCCs. Cyclin D1 overexpression was significantly associated with the loss of p16 immunoreactivity (P=0.005). The pRb⁻ and p16⁻/pRb⁻/Cyc D⁺ phenotypes showed significant association with differentiation of the tumour (P=0.005, 0.05, respectively). Kaplan-Meier analysis for disease recurrence showed increased disease recurrence in cyclin D1 overexpressed patients. Median time to disease recurrence in the cyclin D1⁺ group was 15 months as against 18 months observed in the cyclin D1⁻ patients (P=0.067; log-rank test). Conclusion: Alterations in at least one of the components of the p16/cyclin D1/pRb pathway in majority of the 70 patients analysed herein, and concomitant alterations in all the three proteins in 19 patients (35%) underscore the critical role of this pathway in esophageal tumourigenesis. The results of the present study taken together with our previous findings on p16 and pRb alterations in ESCCs suggest that these alterations are not mutually exclusive and may cooperatively provide greater tumour growth advantage. The prognostic significance of alterations in the expression of these components cyclin D1, p16, and pRb remains to be established in a larger cohort. Received: 3 May 2000 / Accepted: 10 July 2000     

采用组织芯片技术探讨p16和cyclin D1在胆囊癌中的表达及临床意义

目的探讨多肿瘤抑制蛋白（p16）和细胞周期蛋白（cyclin D1）的表达在胆囊癌发生发展中的作用及其对预后的影响,研究应用组织芯片在大规模高效检测临床组织样本的可行性。方法取40例胆囊癌、24例胆囊良性病变和16例癌旁黏膜标本,采用组织芯片技术制作成组织芯片,同时用S-P免疫组织化学方法检测组织芯片中p16和cyclinD1的表达。结果p16和cyclin D1在胆囊癌中的阳性表达率分别为40.0（和57.5（,与胆囊良性病变（75.0（,29.2（）和癌旁黏膜（93.8（,12.5（）相比有显著性差异（P〈0.005）。p16的低表达和cyclin D1的高表达与胆囊癌的分化程度（P（0.025,P〈0.01）、浸润深度（P〈0.01,P〈0.025）、淋巴结转移（P〈0.01,P〈0.01）以及患者的5年生存率（P〈0.025,P〈0.05）密切相关。p16和cyclinD1在胆囊癌中的表达呈负相关（r=-0.54）。结论p16和cyclin D1的异常表达在胆囊癌的发生、发展过程中起重要作用。p16和cyclin D1的表达水平可以作为评估胆囊癌预后的参考指标。应用组织芯片大规模高效检测临床组织样本是可行的,具有快速、方便、经济、准确的特点。     

肝癌细胞p53、p16及细胞周期蛋白D1表达的时间生物学对比

目的对比肝癌细胞中p53、p16和细胞周期蛋白D1(Cyclin D1)表达强度的时段特征.方法按年周期3个时段采集肝癌组织标本,每1个时段20例,均为肝细胞型肝癌及Edmondson-Steinet分级控制在Ⅱ～Ⅲ级内,用免疫组织化学S-P法进行p53、p16和Cyclin D1蛋白表达实验,表达强度按等级数据用秩和检验方法进行时段对比.结果p16表达出现时段差异有显著性(H=10.334,P＜0.05),即4～7月份为表达高峰期.结论肝细胞癌变及其生长的基因调控的时间生物学机制,p16可能起到重要作用.     

Prognostic and clinicopathological features of E-cadherin, α-catenin,β-catenin, γ-catenin and cyclin D1 expression in human esophageal squamous cell carcinoma

AIM: To investigate the expression of E-cadherin, α-catenin,β-catenin, γ-catenin and cyclin D1 in patients with esophageal squamous cell carcinoma (ESCC), and analyze their interrelationship with clinicopathological variables and their effects on prognosis. METHODS: Expression of E-cadherin, α-catenin, β-catenin, γ-catenin and cyclin D1 was determined by EnVision or SABC immunohistochemical technique in patients with ESCC consecutively, their correlation with clinical characteristics was evaluated and analyzed by univariate analysis. RESULTS：The reduced expression rate of E-cadherin, α-catenin, β-catenin and γ-catenin was 88.7%, 69.4%, 35.5% and 53.2%, respectively. Cyclin D1 positive expression ratewas 56.5%. Expression of γ-catenin was inversely correlated with the degree of tumor differentiation and lymph node metastasis (x^2=4.183 and x^2=5.035, respectively, P&lt;0.05), whereas the expression of E-cadherin was correlated only with the degree of differentiation (x^2=5.769, P&lt;0.05). Reduced expression of E-cadherin and γ-catenin was associated with poor differentiation of tumor, reduced expression of γ-catenin was also associated with lymph node metastasis. There obviously existed an inverse correlation between level of E-cadherin and γ-catenin protein and survival. The 3-year survival rates were 100% and 56% in E-cadherin preserved expression group and inreduced expression one and were 78% and 48% in γ-catenin preserved expression group and in reduced expression one, respectively. The differences were both statistically significant. Correlation analysis showed the expression level of α-catenin correlated with that of E-cadherin and β-catenin (P&lt;0.05). CONCLUSION: The reduced expression of E-cadherin and γ-catenin, but not α-catenin, β-catenin and cyclin D1, implies more aggressive malignant behaviors of esophageal carcinoma cells and predicts the poor prognosis of patients.     

p53, p21(Waf1/Cip1) and cyclin D1 protein expression and prognosis in esophageal cancer

Recently, various cell cycle regulators have been investigated as biological markers of malignant potential. These regulators might influence the survival rate and the effect of adjuvant therapies. In this study, we analyzed p53, p21(Waf1/Cip1) and cyclin D1 expression in 64 esophageal cancer patients and the relationship between clinicopathologic parameters and patient survival. The positive expression rate was 48.4%, 42.2% and 43.8% in the p53, p21 and cyclin D1 groups respectively. Multivariant analysis revealed that tumor depth, chemotherapy, p53, p21 and cyclin D1 expression showed significant values. p53- and cyclin D1-negative patients had a worse prognosis. p21-positive patients had a better prognosis. In stage 0, I and II patients, there was a significant difference between p53-positive and -negative, p21-positive and -negative, and cyclin D1-positive and -negative groups. In stage III and IV patients, there was no significant difference between any two groups. However, a significant difference was seen in the p21 group: among patients who received adjuvant chemotherapy, the p21-positive group had a 5-year survival rate of 50% compared with 13.4% in the p21-negative group (not significant).     

Clinicopathological and prognostic role of cyclin D1 in esophageal squamous cell carcinoma: a meta-analysis

Cyclin D1 is one of the most commonly over-expressed oncogenes; however, its role in esophageal squamous cell carcinoma (ESCC) remains controversial. We conducted a meta-analysis of 20 studies, comprising 2,041 patients to clarify this issue. In all studies, paraffin-embedded surgical specimens were collected and the status of cyclin D1 was determined by immunohistochemistry (IHC). The combined odds ratios (Ors) for cyclin D1 expression were 0.74 (95% confidence interval [CI]: 0.58-0.93) for well and moderately differentiated versus poorly differentiated tumors, 0.65 (95% CI: 0.45-0.94) for T1/T2 versus T3/ T4 tumors, 0.59 (95% CI: 0.39-0.90) for N0 versus N1 tumors, and 0.48 (95% CI: 0.33-0.71) for stage I/II versus stage III/IV diseases, respectively. The association between cyclin D1 expression and prognosis was examined in 10 studies, and the combined hazard ratio was 1.78 (95% CI: 1.49-2.12). Cyclin D1 expression level detected by IHC is associated with worst clinicopathological features and prognosis for ESCC.     

食管内反流物成分与p53、细胞周期素D1、p21、p16表达的相关性研究

目的 研究胃及十二指肠液食管反流对食管内抑癌基因、癌基因表达的影响及与黏膜损伤的关系。方法 制作单纯胃食管反流（G组）、单纯十二指肠食管反流（D组）、十二指肠胃混合食管反流（DG组）及无反流对照组（C组）动物模型,用免疫组化法（SABC）检测各组不同时期食管上皮P53、细胞周期素D1（Cyclin D1）、p21、p16等基因的表达。结果 反流组食管组织中p53、CyclinD1基因表达均显著高于C组,并随病程延长而明显增强,D组表达又强于G组;各组不同时期p16表达无明显差异;p21表达在D组、DG组较C组为低,且与p53蛋白表达呈负相关。结论 食管内胃及十二指肠反流物均可改变食管上皮p53、Cyclin、p21基因的表达,十二指肠内容物的作用更明显。但反流对p16基因的表达影响不大。p53、CyclinD1、p21等癌基因或抑癌基因的表达改变可能参与反流性食管炎及其并发症的发生。     

Cyclin B1、CDK1在食管鳞癌组织中的表达及临床意义

目的: 探讨食管鳞癌组织中细胞周期蛋白Cyclin B1和细胞周期蛋白依赖性激酶CDK1表达及其临床病理学意义.方法: 应用免疫组织化学SP法对52例食管鳞癌(组织学Ⅰ级8例, Ⅱ级20例, Ⅲ级24例;有淋巴结转移20例, 无淋巴结转移32例;原位癌16例, 侵袭性癌36例包括浸润至黏膜下层、肌层、全层)及其配对的癌旁正常组织进行Cyclin B1、CDK1的检测, 分析其阳性表达与食管鳞癌患者临床病理因素的关系.结果: 食管鳞癌组织中Cyclin B1、CDK1的阳性表达高于癌旁正常食管黏膜组织, 2组差异都有统计学意义(71.2% vs 2.0%, 65.4%vs 3.9%, 均P <0.05). 食管鳞癌组织中CyclinB1、CDK1的表达都与性别、年龄无关;与组织学分级、浸润深度及淋巴结转移有关(均P <0.05). Cyclin B1阳性表达强度与CDK1的阳性表达强度之间呈正相关(r = 0.697, P <0.05) .结论: Cyclin B1、CDK1的高表达会促进食管鳞癌的发生与发展. 而且食管鳞癌中CyclinB1与CDK1的表达密切相关, 可作为食管鳞癌生物学行为预测的参考指标.     

Over-expression of p53 protein in esophageal squamous cell carcinoma of women

BACKGROUND/AIMS: Most physicians naturally accept the etiological aspect that the incidence of esophageal squamous cell carcinoma (ESCC) is excessively more frequent in men than that in women. However, a definitely scientific confirmation to explain it has not been found. In the current study, we investigated the relationship between gender and p53 over-expression, which might resolve the difference between the genders in the mechanism for carcinogenesis in ESCC. METHODOLOGY: Immunohistochemical expression of p53 was examined for 134 ESCCs, and the correlation of the gender with the clinicopathologic features and over-expression of p53 was compared. RESULTS: The proportion of p53 over-expression in women was 23.8% (5 out of 21) and this incidence proportion was significantly lower than that in men (48.7%, 55 out of 113; p=0.031). CONCLUSIONS: This biological modulation might be correlated with the lower incidence of ESCC in women.     

The clinicopathological significance of p21 and p53 expression in esophageal squamous cell carcinoma: an analysis of 153 patients

OBJECTIVE: The p21 gene is thought to play a central role in tumor suppression. The aim of this study was to examine the clinicopathological role of p21 and p53 in esophageal squamous cell carcinomas. METHODS: The expression of p21 and p53 proteins in 153 Chinese patients (131 men, 22 women) with resected esophageal squamous cell carcinomas was investigated by the immunohistochemical method. Correlation between p21 and p53 expression and clinicopathological features was examined. RESULTS: The expression of p21 and p53 was detected in 70% and 64% of the tumors, respectively. The staining of p21 and p53 was also found in squamous carcinoma in situ, dysplasia, and nontumor epithelium. p21 expression was often weak in the suprabasal cells and found in better differentiated tumors. There was no significant correlation between the expression of p21 and the abnormal accumulation of p53. The prognosis of the patients depended on the size, stage, and p21 expression of the lesion. In stage III lesions with tumor diameter < or = 7.5 cm (n = 93), patients with loss of p21 expression had better survival. The survival rates of patients were worse if they had expression of both p21 and p53. CONCLUSIONS: Thus, p21 and p53 had prognostic value for esophageal squamous cell carcinomas. Loss of p21 expression was shown without p53 alternations, indicating that other mechanisms are also involved in turning off the gene. The pattern of p21 and p53 expression predicts an aggressive clinical course of esophageal squamous cell carcinomas.     

Correlation of p53, MDM2 and p14ARF protein expression in human esophageal squamous cell carcinoma

Purpose  

To determine the interrelationships of p53, MDM2, and p14^ARF protein expression in primary esophageal squamous cell carcinoma (ESCC) and their prognostic value in ESCC.     

Association of p53/p21 expression with cigarette smoking and prognosis in esophageal squamous cell carcinoma patients

factors,such as cigarette smoking,in esophageal squamous cell carcinoma(ESCC)in northeastern Iran,a region with a high incidence of ESCC.METHODS:The expression of p53 and p21 proteins was investigated immunohistochemically in tumor tissue from 80 ESCC patients and in 60 available paraffinembedded blocks of adjacent normal specimens from the cases,along with normal esophageal tissue from 80 healthy subjects.RESULTS:Positive expression of p53 protein was detected in 56.2%(45/80)of ESCC cases,and in none of the normal esophageal tissue of the control group(P<0.001).Furthermore,73.8%(59/80)of ESCC cases and 43.8%(35/80)of controls had positive expression of p21 protein(P<0.001).Cigarette smoking was significantly associated with p53 over-expression in ESCC cases(P=0.010,OR=3.64;95%CI:1.32-10.02).p21 over-expression was associated with poorer clinical outcome among the ESCC patients(P=0.009).CONCLUSION:Over-expression of p53 in association with cigarette smoking may play a critical role in ESCC carcinogenesis among this high-risk population of northeastern Iran.Furthermore,p21 over-expression was found to be associated with poor prognosis,specifically in the operable ESCC patients.     

食管鳞癌及不典型增生组织Fas/FasL的表达

目的研究Fas/FasL在食管正常粘膜、不典型增生组织、及鳞癌组织中的表达,并探讨其生物学意义.方法利用免疫组化方法对28例食管鳞癌组织、癌旁组织和正常组织的Fas/FasL表达进行检测,检验上述组织中Fas/FasL表达的差异.结果肿瘤组织与正常黏膜比较,Fas表达的阳性率(89%vs 100%)和平均积分(2.9 vs 5.0)均有显著性差异(P＜0.05).与不典型增生组织比较,肿瘤组织中Fas表达的阳性率(89%vs96%)无显著差异(P＞0.05),但其平均积分(2.9 vs 4.0)有显著差异(P＜0.05).肿瘤组织与正常黏膜比较,FasL表达的阳性率(100%vs86%)和平均积分(5.0vs2.7)均有显著差异(P＜0.05),3种组织中FasL表达平均积分(5.0 vs 3.6vs 2.7)相互比较均有显著差异(P＜0.05).而不同分化的肿瘤组织中Fas/FasL表达的平均积分无显著差异(P＞0.05).结论食管鳞癌中存在Fas表达的下调和FasL表达的上调,Fas/FasL系统可能参与了食管癌细胞的免疫逃避机制.     

Prognostic and clinicopathological features of E-cadherin, α-catenin, β-catenin, γ-catenin and D1 cyclin expression in human esophageal squamous cell carcinoma

AIM: To investigate the expression of E-cadherin, α-catenin,β-catenin, γ-catenin and cyclin D1 in patients with esophageal squamous cell carcinoma (ESCC), and analyze their interrelationship with clinicopathological variables and their effects on prognosis.METHODS: Expression of E-cadherin, α-catenin, β-catenin,γ-catenin and cyclin D1 was determined by EnVision or SABC immunohistochemical technique in patients with ESCC consecutively, their correlation with clinical characteristics was evaluated and analyzed by univariate analysis.RESULTS: The reduced expression rate of E-cadherin, α-catenin, β-catenin and γ-catenin was 88.7%, 69.4%, 35.5%and 53.2%, respectively. Cyclin D1 positive expression rate was 56.5%. Expression of γ-catenin was inversely correlated with the degree of tumor differentiation and lymph node metastasis (x2 = 4.183 and x2 = 5.035, respectively, P＜0.05),whereas the expression of E-cadherin was correlated only with the degree of differentiation (x2 = 5.769, P＜0.05).Reduced expression of E-cadherin and γ-catenin was associated with poor differentiation of tumor, reduced expression of γ-catenin was also associated with lymph node metastasis. There obviously existed an inverse correlation between level of E-cadherin and γ-catenin protein and survival. The 3-year survival rates were 100% and56% in E-cadherin preserved expression group and in reduced expression one and were 78% and 48% in γ-catenin preserved expression group and in reduced expression one,respectively. The differences were both statistically significant. Correlation analysis showed the expression level of α-catenin correlated with that of E-cadherin and β-catenin(P＜0.05).CONCLUSION: The reduced expression of E-cadherin and γ-catenin, but not α-catenin, β-catenin and cydin D1, implies more aggressive malignant behaviors of esophageal carcinoma cells and predicts the poor prognosis of patients.     

Specific intronic p53 mutation in esophageal squamous cell carcinoma in Southern Thailand

AIM:To investigate p53 mutations in esophageal cancer in a high-risk population,and correlate them with smoking,alcohol consumption and betel chewing.METHODS:One hundred and sixty-five tumor samples of esophageal squamous cell carcinoma(ESCC) obtained from a university hospital in Songkhla province,Southern Thailand were investigated for p53 mutations in exons 5-8,using polymerase chain reaction-single strand conformation polymorphism analysis,followed by direct sequencing.A polymerase chain reactionrestric...     

食管黏膜凋亡增殖和p53表达的增龄变化与食管鳞癌的关系

目的探讨食管黏膜增殖凋亡和p53表达的增龄变化以及这些变化与食管鳞癌的关系。方法按年龄分层选择连续胃镜检查对象818例,男414例,女404例。分为青中年组（258例）、老年前期组（296例）和老年组（264例）,进行食管活组织检查,应用TUNEL法检测凋亡指数（AI）,免疫组化法检测增殖指数（PI）和p53表达。结果食管正常上皮→单纯增生→轻中重度异型增生→鳞癌的发展过程中,AI等级逐渐下降,PI等级和p53表达逐渐增高。在食管正常上皮中,AI等级青中年组〉老年前期组〉老年组;PI等级比较,老年前期组分别高于青中年组和老年组（P〈0.01）;p53表达在老年前期组和老年组均高于青中年组（P〈0.01）。非条件logistic回归分析,食管鳞癌独立危险因素为增龄、PI等级和p53表达。当食管黏膜PI等级明显增高伴p53阳性时,鳞癌检出率明显增高,轻中重度异型增生有增高趋势,老年人尤为突出。结论食管黏膜正常上皮随增龄出现增殖凋亡和p53表达变化,与食管黏膜癌变过程变化相似,老年人食管黏膜PI明显增高伴p53阳性可作为食管鳞癌和癌前病变进一步随访和筛查的线索。     

Epidermal growth factor receptor and cyclin D1 are independently amplified and overexpressed in esophageal squamous cell carcinoma

Purpose To assess the status of EGFR, HER-2, and CCND1 at the gene and protein levels in esophageal squamous cell carcinoma.Methods Dual-color FISH assays were performed using DNA probes for EGFR/CEP 7, HER-2/CEP 17, and CCND1/CEP 11. The respective proteins, furthermore, was assessed in IHC assays and correlated with patient and tumor characteristics.Results From 55 ESCCs, 8 (15%) tumors showed gene amplification and 20 (36%) had gene overrepresentation (balanced gene and chromosome 7 polysomy) for EGFR. High-level protein expression was frequent (49%), positively correlated with gene copy numbers (kappa=0.4), and associated with well-differentiated histology (p=0.02). For HER-2, gene amplification was detected in a single tumor (2%) and protein overexpression was rare (9%). CCND1 gene was amplified in 23 (42%) tumors; likewise, CCND1 protein overexpression was common (58%) and prevailed in gene overrepresentation or amplification. Only 1 patient showed gene amplification for both EGFR and CCND1. Survival was not associated with EGFR or CCND1 gene/protein status, whereas negative patients for HER-2 protein had a better survival than positive patients (p=0.04).Conclusions Frequent overexpression and gene amplification of EGFR and CCND1 make these molecules and their pathways potential therapeutic targets for ESCC. In addition, EGFR and CCND1 appeared to be independently altered suggesting alternative mechanisms for pathway activation. Therapeutic agents targeting these molecules are urged to be tested in clinical trials and comprehensive biological analyses should be included to properly interpret the outcome.