Alterations of Rb, p16(INK4A) and cyclin D1 in the tumorigenesis of oral squamous cell carcinomas

Immunohistochemical analysis of Rb, p16(INK4A) and cyclin D1 expression was performed on 78 oral squamous cell carcinoma (SCC), 46 leukoplakia, and 20 normal mucosa. Rb and p16(INK4A) expression were observed in all normal mucosa and most of leukoplakia. Lack of Rb and p16(INK4A) was observed in 56.4 and 67.9% of SCC, respectively. The overexpression of cyclin D1 was not observed in normal mucosa and was observed in 35.9% of SCC. A strong reciprocal relationship between Rb and p16(INK4A) expression was observed in oral SCC, and all these SCC cases have at least one of the alterations in the Rb pathway.     

Cyclin D1在口腔鳞癌中的表达及临床意义

目的 探讨口腔鳞状细胞组织中Cyclin D1的表达与临床和病理学特征之间的关系。方法 应用免疫组化Envision二步法检测43例口腔鳞癌组织中Cyclin D1的表达情况，并将其阳性表达率与口腔鳞癌的患者性别、临床分期、病理分化程度、淋巴结转移情况以及预后做统计学分析。结果 Cyclin D1在口腔鳞癌中的阳性表达率增加，其阳性表达率与颈淋巴结转移和预后相关，但与患者性别、病理分化程度以及临床分期之间无相关性。结论 Cyclin D1异常表达在口腔鳞癌的发生、发展中发挥了重要作用，可作为判断口腔鳞癌颈淋巴结转移和预后的一个重要指标。     

Alterations of p53, cyclin D1 and pRB expression in the carcinogenesis of esophageal squamous cell carcinoma

Many molecular alterations occur in esophageal carcinogenesis; however, little is known about the molecular genetic events responsible for the development of carcinoma. We investigated the expression of ki67, p53, cyclin D1 and pRB in 105 biopsy specimens using immunohistochemistry from iodine unstained lesions as indicators of carcinogenesis of the esophagus. Also, the genetic alternation of esophageal dysplasia from patients with accompanying esophageal squamous cell carcinoma (ESCC) was examined to study the evidence for field carcinogenesis in the esophagus. The expression of p53, cyclin D1 and pRB was detected in 31, 0 and 51.7% respectively of mild dysplasia; 40, 0 and 70% of moderate dysplasia; 40, 20 and 70% of severe dysplasia; and 48, 32 and 80% of carcinoma specimens. p53 expression was significantly increased in mild dysplasia, whereas cyclin D1 and pRB expression were significantly increased in carcinoma as compared to both normal epithelium and esophagitis. The ki67 LI and the rate of p53 expression were significantly higher in dysplasia with ESCC than in dysplasia without ESCC. Ki67, p53, cyclin D1 and pRB expression may be useful biomarkers for assessing the risk of developing esophageal cancer. Dysplasia observed at screening for secondary lesions has a highly malignant potential and careful follow-up studies are required.     

Prognostic significance of cyclin D1 amplification and overexpression in oral squamous cell carcinomas

Amplification and overexpression of cyclin D1 (CCND1) have been reported as independent prognostic indicators of several tumors. To investigate the association between CCND1 amplification and overexpression in oral squamous cell carcinomas (OSCCs), and to determine which is more reliable as a prognostic indicator, fluorescence in situ hybridization (FISH) on fine-needle aspiration (FNA) biopsies and immunohistochemistry were performed on 41 primary OSCCs (26 males, 15 females; mean age; 58.4 years, range 21-89 years). Thirteen patients were stage I, 13 were stage II, nine were stage III, and six were stage IV. CCND1 amplification and overexpression was detected in 13 (31.7%) and 27 (65.9%) of 41 cases. CCND1 was overexpressed in all cases showing CCND1 amplification. On the other hand, CCND1 overexpression was also detected in 14 of 28 cases (50.0%) lacked such amplification. Statistical analysis showed that the correlation between CCND1 overexpression and decreased survival just failed to reach statistical significance, and CCND1 amplification and nodal status were independent prognostic indicators. In conclusion, it will be necessary to investigate the other pathways that regulate CCND1 expression besides CCND1 amplification. From the present study, CCND1 amplification is a more reliable prognostic indicator than CCND1 overexpression in OSCCs.     

Alterations of the cyclin D1/pRb/p16(INK4A) pathway in multiple myeloma.

The retinoblastoma protein (pRb), p16(INK4A), D-type cyclins, and their partners cyclin-dependent kinase (CDK) 4 and 6 constitute a G(1) regulatory pathway commonly targeted in tumorigenesis. Several malignancies show a reciprocal correlation between genetic alterations of single members of the pRb pathway. Therefore, we determined the frequency of Rb deletions and cyclin D1 alterations by fluorescence in situ hybridization as well as 5' CpG island hypermethylation of the p16(INK4A)gene using methylation-specific polymerase chain reaction in bone marrow mononuclear cells from 82 individuals with plasma cell disorders. Alterations in at least one of the components of the pathway were found in 75%. Cyclin D1 translocations or amplifications were detected in 14/82 (17.1%), Rb deletions at 13q14 in 23/82 (28%) of the cases, including three (3.6%) homozygous deletions. p16(INK4A) was hypermethylated in 33/57 (57.9%) of the samples. Further analysis revealed a highly significant correlation between cyclin D1 alterations and extramedullar or leukemic myeloma manifestations (P = 0.014; Fisher's test). Whereas Rb deletions seemed to occur alternatively to cyclin D1 alterations, no reciprocal correlation was found between p16(INK4A) hypermethylations and cyclin D1 or Rb locus aberrations. Cyclin D1 locus alterations and Rb deletions were associated with a significantly worse prognosis whereas p16(INK4A) hypermethylation had no impact on survival. We conclude that cyclin D1 and Rb aberrations seem to occur as alternative events in plasma cell malignancies and contribute to clinical course and prognosis. In contrast, although p16(INK4A) hypermethylation is frequent, inactivation of p16(INK4A) seems not to be involved in the pathogenesis of plasma cell disorders.     

p53 mutation and cyclin D1 amplification correlate with cisplatin sensitivity in xenografted human squamous cell carcinomas from head and neck

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).     

Fluorescence in situ hybridization for detecting genomic alterations of cyclin D1 and p16 in oral squamous cell carcinomas

BACKGROUND: Cyclin D1 (CCND1) and p16 alterations have been detected in oral squamous cell carcinomas (SCCs), suggesting that abnormalities of these genes may play an important role in the genesis or progression of oral SCCs and serve as independent prognostic indicators. The detection of CCND1 and p16 aberrations using a simple and sensitive method would be valuable for the development of effective treatment modalities for oral cancer. The objective of the current study was to determine whether CCND1 numerical aberrations and p16 deletions in oral SCCs detected by fluorescence in situ hybridization (FISH) have any impact on clinical outcome. METHODS: Using genomic DNA probes for CCND1 and p16, FISH was performed on specimens that were obtained by fine-needle aspiration (FNA) from 57 primary oral SCCs. RESULTS: The CCND1 numerical aberration was observed in 28 of 57 patients (49%) with oral SCCs and was associated significantly with reduced disease-free survival (P = .0004) and overall survival (P = .0179). Conversely, p16 deletion was detected in 22 of 57 patients (39%). The disease-free and overall survival rates for patients with p16 deletion were lower than those among patients without the p16 deletion, although the difference just failed to reach statistical significance (P = .0516 and P = .1878, respectively). The p16 deletion in the presence of the CCND1 numerical aberration conferred significantly worse disease-free survival (P = .0002) and overall survival (P = .0153). CONCLUSIONS: Although the CCND1 numerical aberration was a good predictor of aggressive tumors, recurrence, and poor prognosis in patients with oral SCCs, the authors were able to identify subgroups of patients that had early disease recurrence and a poor prognosis more efficiently by assessment of p16 deletion in addition to CCND1 genetic status using FISH on FNA biopsy samples compared with the analysis of either alteration alone.     

p53 and cyclin D1 protein expression in glottic laryngeal squamous cell carcinomas involving the anterior commissure (pT1bN0M0)

The prognosis of patients carrying glottic squamous cell carcinomas (GSCCs) involving the anterior commissure is often unpredictable. In order to assess the possible prognostic role of new and reliable parameters, p53 and cyclin D1 protein expression was immunohistochemically analysed in pathological samples from 27 patients with GSCG (pTlbNOMO) and a median follow-up of 90 months. p53 protein expression was observed in the majority of patients (15/27), but it did not correlate with their clinical outcome; p53 protein immunoreactivity was frequently observed in normal (9/14), mildy dysplastic (10/14) and highly dysplastic (3/7) mucosa samples, suggesting that its overexpression may be involved in the earliest phases of the multistep tumourigenesis of laryngeal squamous cell carcinomas (LSCCs); neither the non-neoplastic nor the neoplastic samples expressed any cyclin D1. As cyclin D1 protein expression has been associated with a high frequency of nodal metastases, its absence in our series could ba related to the rarity of nodal involvement in early glottic LSCCs.     

Coexpression of cyclin D1 and retinoblastoma gene product (pRb) in human squamous cell lung carcinomas is associated with increased tumor take rate in nude mice

Samples from 72 squamous cell lung carcinomas were analyzed immunohistochemically for the expression of cyclin D1 and pRb. Expression of cyclin D1 was found in 61% and expression of pRb in 39% of the cases. The take rate of human squamous cell lung carcinomas in nude mice was significantly different according to the expression of cyclin D1 and expression of pRb in primary human tumors [cyclin D1-negative (32%) vs. cyclin D1-positive (59%); P=0.026; pRb-negative (34%) vs. pRb-positive (71%); P=0.002]. The take rate was improved by coexpression of both proteins [both proteins negative/both proteins positive: 12% vs. 80% (P=0.00005)]. An association of the take rate and clinical parameters (age, extent of tumors, lymph node involvement, stage) could not be observed. Coexpression of cyclin D1 and pRb is also a prognostic factor for the patients' survival.     

p21~(WAF1)、cyclinD1和pRb在膀胱移行细胞癌中的表达及其意义

 目的　探讨 p2 1WAF1、细胞周期蛋白D1(cyclinD1)和 pRb在膀胱移行细胞癌 (BTCC)中的表达及相互关系和其意义。方法　应用免疫组织化学SP法检测 5 7例BTCC患者癌组织中 p2 1WAF1、CyclinD1和 pRb的蛋白表达。 结果　p2 1WAF1、cyclinD1和 pRb的阳性表达率分别为 36 .8%、4 9.1%和 4 5 .6 % ,p2 1WAF1随病理分级升高阳性率显著下降 ,cyclinD1和 pRb的表达与BTCC的病理分级、临床分期和有无转移均相关 ,p2 1WAF1与 pRb的表达呈负相关 ,cyclinD1和 pRb的表达呈正相关 ,而 p2 1WAF1与cyclinD1的表达无关。结论　p2 1WAF1/cyclinD1/ pRb通路异常与BTCC的发生发展密切相关 ,p2 1WAF1的改变可能为癌变的早期事件 ,联合检测 p2 1WAF1、cyclinD1和 pRb可较准确地评价BTCC的生物学特性 ,估计预后 ,指导治疗     

Aberrant expression of pRb, p16, p14ARF, MDM2, p21 and p53 in squamous cell carcinomas of lung.

Expression of cell cycle regulatory proteins in both the RB and p53 pathways was investigated in 50 cases of squamous cell carcinoma (SCC) of the lung using immunohistochemical techniques. Abnormality of pRb and p16 expression was seen at the frequencies of 16% and 78%, respectively, and appeared to be in a reciprocal relationship. On the other hand, strong and diffuse p53 immunoreactivity was seen in 60% of SCCs. MDM2 and p14ARF expressions were each observed in about half of the cases of SCC and were not significantly associated with strong p53 immunoreactivity. Statistical analysis revealed that p14ARF expression was significantly correlated with both p16 and MDM2 expression. Moreover, strong p53 expression was not correlated with the expression of p21. In comparing clinicopathological status with the immunohistochemical results, lack of p16 immunoreactivity was observed in the elderly group (over 65 years) as compared with the younger group (less than 65 years). Strong p53 expression was frequently observed in higher stages of SCC, with the developing tumor located in the central field of the lung. Similarly, the frequency of p14ARF expression was high in centrally developed SCC, but low in SCC developed in the periphery. These results suggest that disruption of the RB and p53 pathways is a frequent event in SCC, and that abnormal expression of p16 and p53 plays a more critical role than that of pRB, p14ARF and MDM2 in the development of SCC of the lung.     

喉鳞癌中p53和cyclin D1基因表达的预后意义

目的探讨抑癌基因p53和细胞周期蛋白cyclin D1在喉鳞癌中表达的预后意义.方法应用免疫组化方法SP法对56例喉鳞癌cyclin D1和p53表达进行检测.结果56例喉鳞癌中p53和cyclin D1表达阳性率分别为51.8%、42.9%.Kaplan-Meier生存分析显示p53表达和较差的预后相关(P＜0.05),cyclin D1表达阳性患者有预后较差的趋势,但差异无显著性意义(P＞0.05).结论 p53阳性表达可作为判断喉鳞癌预后的一个独立的预后指标.     

A study on p16, pRb, cdk4 and cyclinD1 expression in non-small cell lung cancers

To observe the expression of p16, pRb, cdk4 and cyclinD1 in non-small cell lung cancers, 104 cases of resected lung cancers were collected, which included squamous cell carcinomas, adenocarcinomas and large cell carcinomas. Immunohistochemistry assay was carried out. The results showed that 67% of squamous cell carcinomas and 46% of adenocarcinomas expressed p16, 64% of squamous cell carcinomas and 85% of adenocarcinomas expressed pRb and 66% of cancers expressed p16 or pRb. About 70% of the tumors expressed cyclinD1. More than 90% of the tumors expressed cdk4 and there was an increased trend with decreasing differentiation of both squamous cell carcinomas and adenocarcinomas. Sixty-seven percent of the highly differentiated and 100% of the poorly differentiated squamous cell carcinomas expressed cdk4. The aberrant p16 and pRb gene product expression played a significant role in the development and histological subtype of lung cancers by conditioning the biological behavior of NSCLC. cdk4 was an important factor in histological differentiation.     

Expression of p53 and p21CIP1/WAF1 proteins in oral epithelial dysplasias and squamous cell carcinomas

The expression of tumor suppressor gene, p53 and cyclin-dependent kinase inhibitor, p21 in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC) was examined immunohistochemically and its relationship with clinicopathological findings was analyzed. Among 24 epithelial dysplasias, 4 cases (17%) expressed p53 protein and 23 cases (96%) expressed p21 protein. On the other hand, expression of p53 was observed in 64% of OSCCs, and expression of p21 was observed in 77% of OSCCs. In the analyses of the correlation between the expression of p53 and p21 in epithelial dysplasia and OSCC, 79% of epithelial dysplasias were p53-negative and p21-positive, compared to 25% of the OSCCs. p21 expression did not correlate with p53 expression. These results were also demonstrated in OSCC cell lines by western blot analysis. Cumulative survival rate of the patients p53-negative and p21-positive was higher than those p53-positive and p21-negative, those p53-negative and p21-negative and those p53-positive and p21-positive. These findings suggest that p53 expression and p21 negative expression may involve in neoplastic transformation of oral epithelium. In the present study, we did not observe correlation between the expression of p53 and p21 proteins in OSCC. p21 expression may be regulated by p53-independent pathways as well as p53-dependent ones. However, combination of the p21 and p53 expression may be useful as a prognostic marker.     

p21 is associated with cyclin D1, p16INK4a and pRb expression in resectable non-small cell lung cancer

p21 (p21WAF1/CIP1) is involved in cell cycle regulation, as an inhibitor of cyclin dependent kinases (CDK2, CDK4 and CDK6). However, subsequent in vitro studies have suggested that p21 may influence this process by an additional mechanism, in particular through the regulation of cyclin D1 subcellular localisation. This study of primary resectable non-small cell lung cancer (NSCLC) was designed to examine p21 functions in association with the expression of cyclin D1 (including its subcellular localisation), p16INK4a and pRb. p21 expression was examined in 50 NSCLC (stage I-IIIA) and in several normal lung samples all of which had previously been studied for cyclin D1 (DNA, RT-PCR, immunostaining), p16INK4a (DNA, RT-PCR, immunostaining), and pRb (immunostaining). As assessed by immunoblotting and immunostaining, p21 was expressed at low levels in normal lung tissue with immunoreactivity seen in a small number of bronchial epithelial cells only. In NSCLC, p21 expression (> or =10% of positive cells) was observed in 42% (21/50) of cases. High p21 expression was associated with well differentiated tumours (p = 0.01) and cyclin D1 nuclear staining (p = 0.02). Furthermore, we found an inverse correlation with p16INK4a (p = 0.004) and a direct correlation with pRb expression (p = 0.02). Risk of relapse was associated with p16INK4a and p21 status with no relapse in patients with normal p16INK4a and p21. Our results confirm that a large number of NSCLC have a low level of p21 expression. The associations of p21 and nuclear cyclin D1, pRb, p16INK4a support the relevance of pathways linked to lung carcinogenesis that involve p21 but may act in addition to direct CDK inhibition.     

Cell cycle progression of chronic lymphocytic leukemia cells is controlled by cyclin D2, cyclin D3, cyclin-dependent kinase (cdk) 4 and the cdk inhibitor p27.

B-CLL cells are arrested in G0/early G1 phase of the cell cycle and are characterized by a marked hyporesponsiveness towards a variety of polyclonal B cell activators. We have previously demonstrated that costimulation with CpG-ODN and IL-2 can overcome this proliferative defect. Cyclin D3 is the principal D-type cyclin which mediates G1 progression in normal B cells, but in B-CLL cells both cyclin D2 and cyclin D3, were strongly upregulated upon stimulation. Both cyclins were associated with cdk4 but not with cdk6, which is the catalytic partner of D-type cyclins in normal B cells. Moreover, immune complexes consisting of cyclin D2 and cdk4 or cyclin D3 and cdk4 were both functional and phosphorylated the RB protein in vitro. The cell cycle inhibitor p27 plays a pivotal role in cell cycle progression of B lymphocytes and has been shown to be overexpressed in B-CLL cells. P27 was rapidly downregulated in B-CLL cells even when stimulated with a non-CpG-ODN or IL-2 alone, while only moderate regulation could be observed in normal B cells. Taken together, our findings demonstrate that regulation of early cell cycle progression differs between B-CLL cells and normal B cells. These findings do not only contribute to the understanding of B-CLL pathophysiology, but might ultimately lead to the identification of new therapeutic targets.     

Alteration of pRb/p16/cdk4 regulation in human osteosarcoma.

Cell-cycle regulation depends on a fine balance between cyclin-cyclin-dependent kinase complexes and a family of kinase inhibitors that bind cyclin-cdk complexes and block their activity. To investigate the role of mechanisms regulating cell-cycle progression in human osteosarcomas (OS), pRb/p16/cdk4 expression was analyzed in 39 high-grade OS; 19 of these developed metastasis during follow-up. Positive reaction for functional pRB was shown by 18/39 (46%) OS, while 21/39 (54%) were negative. A higher probability of metastasis was seen in patients with negative pRb expression (p < 0.05). Furthermore, while functional pRb and D1 expression are inversely associated to metastasis occurrence, the presence of D1/cdk4 complex in our study was related to poor prognosis. We found that 10/18 pRb-positive and 14/21 pRb-negative tumors were p16-positive. No significant correlation was found between pRb and p16 expression. On the other hand, high cdk4 levels in p16-positive tumors as compared with p16-negative tumors resulted in a positive association between p16 and cdk4 expression (Chi squared = 5.98; p = 0.01). No extensive p16INK4A genomic alterations were found in tumors lacking p16-protein expression. To determine which mechanisms are involved in the down-regulation of p16 protein, the methylation status of the p16INK4 gene was evaluated on the 15 p16-negative tumors: 8 samples showed 5' CpG-island methylation; 4/8 had a complete methylation status, while in the remaining 4 the gene was only partially methylated. These data confirm the role of the pRb/p16/cdk4 pathway in OS development.