Abnormal expression of pRb, p16, and cyclin D1 in gastric adenocarcinoma and its lymph node metastases: relationship with pathological features and survival

The retinoblastoma (Rb) pathway controls the G1-S checkpoint of the cell cycle. Inactivating mutations and deletions of p16 and Rb and up-regulation of cyclin D1 disrupt this pathway and occur in many cancers. However, the concurrent expression of these genes in primary and metastatic gastric cancer is unknown, and the prognostic value of their expression is unclear. In this study, the expression of cyclin D1, retinoblastoma protein (pRb), and p16 in 67 resected gastric adenocarcinomas, and of pRb and p16 in 40 associated lymph node metastases, was determined using a streptavidin-biotin-peroxidase immunohistochemical method. Relationships with clinical and pathological features were analyzed. Cyclin D1 overexpression (>/=5% expression) was seen in 55% of cancers; pRb loss (<20% expression), in 33%; p16 loss (<10% expression), in 49%; and at least 1 of these abnormalities, in 92.5%. Cyclin D1 overexpression was associated with poor differentiation (P = 0.027) and signet ring cell type (P = 0.029). pRb expression was lower in lymph node metastases than in the corresponding primary tumors (P <0.001). Univariate and multivariate survival analysis (minimum follow-up 72 months or until death) revealed that <20% pRb expression, <30% pRb expression, and International Union Against Cancer stage >2 were associated with worse overall survival. The results suggest that Rb pathway disturbances play an important role in gastric carcinogenesis. The poor prognosis of cancers with low pRb expression and the reduced pRb expression in lymph node metastases raise the possibility that Rb and related genes also influence progression.     

软组织平滑肌肉瘤中p16,细胞周期蛋白D1和pRb蛋白的表达及

目的 探讨软组织平滑肌肉瘤中ｐ１６、细胞周期蛋白Ｄ１和ｐＲｂ的蛋白表达、相互关系及其意义。方法 应用免疫组化方法检测４２例ＬＭＳ患者组织中ｐ１６、ｃｙｃｌｉｎ Ｄ１和ｐＲｂ的蛋白表达情况。结果 ４２例中ｐ１６、ｃｙｃｌｉｎ Ｄ１、ｐＲｂ的总异常率为９７．６％,其中ｐ１６和ｐＲｂ蛋白表达缺失分别为３５．７％和４７．６％,ｃｙｃｌｉｎ Ｄ１过表达５７．１％。ｃｙｃｌｉｎ Ｄ１在平滑肌瘤中也有５５％呈过     

Analysis of the Cycilin D1/p16/pRb Pathway in Parathyroid Adenomas

Cyclin D1/p16/pRb pathway controlling G1→S cell cycle checkpoint is frequently altered in human tumors. Currently, scarce data are available for parathyroid tumors. We have studied 46 parathyroid adenomas (PTAs) and 12 normal parathyroid glands (PTGs) by immunohistochemistry with cyclin D1 (CD1), p16, pRb, and Ki-67 antibodies. We observed CD1 expression in 89%, p16 in 70%, and pRb in 100% of PTAs. Statistically significant differences with normal PTGs were found only concerning p16 expression (p<0.05). Proliferating rate (Ki-67) was always low, although significantly higher than in normal PTGs. Our findings demonstrate the presence of alterations in the CD1/p16/pRb pathway in PTAs, consisting in p16 overexpression apparently unrelated to pRb downregulation. On the other hand, we did not find significant differences in CD1 expression between PTAs and normal PTGs, suggesting CD1 overexpression could be a physiological event in parathyroid tissue.     

乳腺癌cyclin D1、Rb蛋白表达的意义

目的：探讨cyclin D1蛋白和Rb蛋白的表达状况与乳腺癌发生发展的关系。方法：采用S-P免疫组化法检测52例乳腺癌和20例良性乳腺病变组织中cyclin D1蛋白和Rb蛋白的表达。结果：乳腺癌中cyclin D1过表达率为34．60％（18／52），显著高于良性乳腺病变组织的10．00％（2／20），P〈0．05，cyclin D1过表达出现于导管内癌中并持续于浸润、转移等进展过程中，与患者年龄、肿瘤大小、腋窝淋巴结转移及组织学分级均无相关性。乳腺癌中Rb蛋白阴性表达率为76．91％（40／52），显著高于良性乳腺病变组织的15．00％，P〈0．05；Rb蛋白阴性表达与乳腺癌临床病理特征间无相关性，Rb蛋白表达阴性者中，cyclin D1过表达率为20．00％，而Rb蛋白表达阳性者中，其cyclin D1过表达率为83．33％，两者比较有显著差异（P〈0．05）。结论：cyclin D1蛋白和Rb蛋白与乳腺癌发生、发 展密切相关；cyclin D1、P16及Rb通路失活可能与乳腺癌的发生密切相关。     

Correlation of cyclin D1 and Rb gene expression with apoptosis in invasive breast cancer.

BACKGROUND: In vitro studies have shown that amplification and overexpression of the cyclin D1 gene can accelerate the progress of cells through the G1 phase. Therefore, cyclin D1 may have an apoptosis inhibiting effect. The retinoblastoma (Rb) gene was shown recently to be an important regulator of apoptosis. AIMS: To evaluate whether expression of the cyclin D1 and Rb genes correlated with apoptotic counts in a group of 97 invasive breast cancers. METHODS: Expression of the cyclin D1 and Rb genes was detected by standard immunnohistochemistry using paraffin wax embedded sections. Apoptotic cells were counted according to a strict protocol, in 10 fields of vision systematically spread over the most poorly differentiated area of the tumour, at a magnification of x630. Apoptotic cells counts were expressed as apoptotic cells/mm2. RESULTS: Cyclin D1 overexpression was found in 49% of cases. Loss of Rb expression was found in 44% of cases, and occurred particularly in poorly differentiated tumours. Cyclin D1 and Rb expression showed a positive correlation (p = 0.003). Apoptotic counts varied from 1 to 62/mm2. There were no significant correlations between cyclin D1 overexpression and apoptotic counts in the total group or in the retinoblastoma protein (pRb) positive tumours. Loss of Rb expression also showed no correlation with apoptotic counts. CONCLUSIONS: Cyclin D1 is frequently overexpressed in pRb positive tumours, but no evidence has been found for an anti-apoptotic effect of cyclin D1 overexpression or Rb expression in invasive breast cancer.     

Expression of p16, Rb, and cyclin D1 gene products in oral and laryngeal squamous carcinoma: biological and clinical implications.

Cyclin D1, p16, and Rb genes play a critical role in the regulation of the G1-S transition of the cell cycle and are frequently altered in several neoplastic entities. Analysis of the protein products of these genes by molecular and immunohistochemical methods provides information on their functional status and allows for the phenotypic evaluation of tumor cells. We performed Western blotting and immunohistochemical analysis on tissues from 35 primary oral and laryngeal squamous carcinoma specimens with previous molecular analysis of the p16 gene and correlated the results with relevant clinicopathologic factors. Our study shows significant concordance between Western blotting and immunostaining results for cyclin D1 (P = .01), p16 proteins (P = .01), and Rb (P = .04). Heterogeneous staining of tumor cells and the positivity of non-neoplastic host elements for Rb by immunohistochemistry contributed to the discrepancy noted in some tumors by Western blotting. Significant reciprocal relationship between p16 and Rb proteins was observed (P < .001); in most tumors, absence of p16 (89%) and detectable Rb (94%) proteins were found. Two tumors had negative cyclin D1 expression, and one third overexpressed this protein. There was a lack of correlation between cyclin D1 overexpression and the clinicopathologic factors studied. Our results indicate that the absence of p16 in most of these tumors may constitute an early tumorigenic event and that the loss of the Rb function plays a minor role in HNSC.     

Altered expression of G1 regulatory proteins in human soft tissue sarcomas

CONTEXT: Soft tissue sarcomas constitute a heterogeneous group of tumors for which tumorigenesis is not fully understood. Altered cell-cycle regulation may underlie the development and/or progression of human malignancies. However, data concerning the occurrence of cell-cycle aberrations in soft tissue sarcomas are very limited. OBJECTIVES: To detect the abnormal features of cell-cycle regulatory proteins in soft tissue sarcomas and to determine the potential role of these proteins in clinical behavior. DESIGN: The p53 and Rb-cyclin D pathways were investigated by immunohistochemical studies of p53, mdm2, pRb, p16, cyclin D1, and cdk4 proteins, respectively. RESULTS: Of the 67 sarcomas analyzed, nuclear accumulation of p53 was detected in 25 samples (37%), and overexpression of mdm2 was found in 16 samples (24%). Both p53 and mdm2 expression correlated with tumor grade. Abnormalities involving the Rb-cyclin D pathway were identified in all of the tumors by the altered expression of either pRb (72%) or p16 (94%). Fourteen (21%) and 64 (96%) cases demonstrated cyclin D1 or cdk4 expression, respectively. Overexpression of cyclin D1 showed an association with pRb and p53. There was no correlation between pRb, p16, cyclin D1, or cdk4 and tumor grade or relapse. CONCLUSION: Disturbance in the cell-cycle regulatory system involving the p53 pathway and the Rb-cyclin D pathway is relatively frequent in soft tissue sarcomas and may be a contributing factor in the tumorigenesis of these tumors. The alterations in the Rb-cyclin D pathway probably constitute an early event, whereas the abnormalities in the p53 pathway seem to be involved in tumor progression. It is noteworthy that cyclin D1 may play a key role in linking both pathways.     

Alterations of expression of Rb,p16INK4A and cyclin D1 in non-small cell lung carcinoma and their clinical significance

Inactivation of the Rb pathway in non-small cell lung carcinoma (NSCLC) occurs mostly through inactivation of the cyclin-dependent kinase inhibitor p16^INK4A and/or up-regulation of cyclin D1. In order to assess the frequency and the prognostic value of these abnormalities in NSCLC, immunohistochemical analysis of Rb, p16^INK4, and cyclin D1 has been performed on 168 cases of NSCLC including 77 squamous cell carcinomas, 43 adenocarcinomas, and 48 basaloid carcinomas. The reduced survival rate of basaloid carcinoma (stage I–II) compared with other histological types of NSCLC was confirmed (p = 0·008). Loss of protein expression of Rb and p16^INK4A was observed in 12 per cent and 58 per cent of NSCLC cases respectively and cyclin D1 overexpression in 43 per cent. There was an inverse correlation between Rb and p16 expression ( p < 0·0001) and a direct correlation between Rb and cyclin D1 expression ( p = 0·0007). In univariate analysis, Rb-negative adenocarcinomas at stages I–II had a significantly shorter survival than Rb-positive cases ( p = 0·04) and stages I–II p16-positive cases had a shorter survival than p16-negative cases ( p = 0·02), which was more significant in basaloid carcinoma ( p = 0·003). p16 status retained its influence on survival in multivariate analysis at stage I–II for all cases ( p = 0·01) and for basaloid carcinoma ( p = 0·005). Cyclin D1 overexpression did not influence survival. Combined Rb/p16/cyclin D1 phenotypes in univariate analysis showed a shorter survival for Rb-negative/p16-positive/cyclin D1-negative tumours ( p = 0·002). These results, linked to previous data, indicate that the Rb pathway of G1 arrest is initially disrupted in the vast majority of NSCLCs (83 per cent), but could not confirm an unfavourable role for each individual event (p16^INK4A loss or cyclin D1 up-regulation) in prognosis. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.

Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb.     

Solid pseudopapillary neoplasms of the pancreas show an interruption of the Wnt-signaling pathway and express gene products of 11q.

Solid pseudopapillary neoplasms of the pancreas almost consistently show a beta-catenin mutation activating the Wnt-signaling pathway, resulting in overexpression of cyclin D1, but not in overt malignancy of this tumor. Besides cyclin D1, a set of markers (ie FLI-1, CD56 and progesterone receptor), whose genes map to chromosome 11q, are frequently expressed in solid pseudopapillary neoplasms. Chromosome 11q is a region that is also often affected in pancreatic neuroendocrine tumors. This immunohistochemical study was undertaken to gain insights into the downstream regulation of the Wnt-signaling pathway and the significance of overexpressed gene products belonging to chromosome 11q for the tumorigenesis in solid pseudopapillary neoplasms. Fourteen solid pseudopapillary neoplasms were analyzed for the expression of cyclin-dependent kinase inhibitors p21, p27, p16 and hyperphosphorylated retinoblastoma (pRb) proteins. In an extended series of 93 solid pseudopapillary neoplasms, beta-catenin, cyclin D1, FLI-1 and CD56 expression was examined and compared with that in 22 pancreatic neuroendocrine tumors. Solid pseudopapillary neoplasms (98%) showed aberrant expression of beta-catenin with a concomitant cyclin D1 expression in 69% of the cases, but no expression of pRb (0%) was found. p27 and p21 were expressed in 100% (14/14) and 86% (12/14) of the cases, but only 2/14 (14%) were positive for p16. FLI-1 was expressed in 63% of solid pseudopapillary neoplasms, but only in 1/22 pancreatic neuroendocrine tumors (5%), cyclin D1 expression was present in 14% of the latter. We conclude that in solid pseudopapillary neoplasms the activated Wnt-signaling pathway is disrupted, and that p21 and p27 are contributing to this fact by blocking of the hyperphosphorylation of the Rb protein, thus causing the very low proliferation rate characterizing the solid pseudopapillary neoplasms. The accumulation of high expression of proteins whose genes are located on chromosome 11q is characteristic of solid pseudopapillary neoplasms, but not of pancreatic neuroendocrine tumors.     

Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.

A total of 111 pulmonary neuroendocrine tumors comprising 13 typical carcinoids, five atypical carcinoids, 44 large-cell neuroendocrine carcinomas and 49 small-cell carcinomas were immunohistochemically studied for dysregulated cyclin B1 expression and disruption of the Rb/p16/cyclin D1 pathway (Rb pathway), and the results were correlated with tumor proliferation activity and clinical outcome. Overexpression of cyclins B1 and D1, respectively, was detected in no and 15% typical carcinoids, 20 and 20% atypical carcinoids, 84 and 32% large-cell neuroendocrine carcinomas, 84 and 10% small-cell carcinomas. Loss of Rb and p16 expression, respectively, was observed in no and 14% typical carcinoids, no and 40% atypical carcinoids, 49 and 18% large-cell neuroendocrine carcinomas, 84 and 8% small-cell carcinomas. In summary, 29% typical carcinoids, 20% atypical carcinoids, 78% large-cell neuroendocrine carcinomas and 93% small-cell carcinomas had Rb pathway aberrations. Rb pathway aberration was mostly attributed to Rb loss in small-cell carcinomas, while p16 loss and/or cyclin D1 overexpression besides Rb loss also played an important role in large-cell neuroendocrine carcinomas, while cyclin D1 overexpression was the only cause of Rb pathway aberration in carcinoid tumors. Thus, both cyclin B1-associated G2/M arrest and Rb-mediated G1 arrest are consistently compromised in high-grade large-cell neuroendocrine carcinoma and small-cell carcinoma, but are generally intact or occasionally altered in carcinoid tumor; the mechanisms involved in Rb pathway aberration among the tumor categories are different, reflecting a genetic divergence among the individual tumor categories. Cyclin B1 expression closely correlated with the Ki-67 labeling index either in the individual tumor categories or overall tumors (P < 0.0001, r = 0.742), suggesting that cyclin B1 is one of the key factors regulating cell proliferation in pulmonary neuroendocrine tumors. Neither cyclins B1 and D1, Rb, p16, nor Ki-67 correlated with patient survival in individual tumor categories, suggesting that the prognostic significance of these factors is tumor-type specific.     

Expression of pRb, p53, p16 and cyclin D1 and their clinical implications in urothelial carcinoma

The aim of this study was to assess immunohistochemical expression of p53, pRb, p16, and cyclin D1, alone or in combination, as prognostic indicators and to investigate their correlation with clinocopathologic features of urothelial carcinoma. Immunohistochemical staining for p53, pRb, p16, and cyclin D1 was performed on a tissue microarray from 103 patients with urothelial carcinoma who underwent radical cystectomy. Of the patient samples analyzed, 36 (35%), 61 (59%), 47 (46%) and 30 (29%) had altered expression of p53, pRb, p16, and cyclin D1, respectively. Abnormal expression of p53 and pRb correlated with depth of invasion (P=0.040 and P=0.044, respectively). Cyclin D1 expression was associated with tumor stage and recurrence (P=0.017 and P=0.036, respectively). Altered pRb was significantly correlated with overall survival (P=0.040). According to the expression pattern of pRb and p53, p53/pRb (altered/normal) had worse survival than p53/pRb (normal/altered) (P=0.022). Alteration of all markers had worse survival than all normal (P=0.029). As determined by multivariate analysis, tumor stage, lymph node metastasis and the combined expression of p53 and pRb are independent prognostic factors. In conclusion, immunohistochemical evaluation of cell cycle regulators, especially the p53/pRb combination, might be useful in planning appropriate treatment strategies.     

Hyperphosphorylation of pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder cancer

Loss of heterozygosity, mutations or deletions of the RB1 gene usually result in loss of pRb expression, which has been regarded as an indicator of loss of pRb function in human tumours. It has previously been shown that in addition to loss of pRb expression, aberrantly high (pRb2+) pRb expression also indicates loss of pRb function in bladder tumours compared with moderate (normal, pRb1+) pRb expression. The aim of this study was to elucidate the mechanism by which pRb is functionally inactivated in bladder tumours expressing aberrantly high levels of pRb. Constitutive phosphorylation was therefore investigated as a mechanism of pRb inactivation in bladder tumours. Of 28 bladder tumours examined, western blotting demonstrated pRb hyperphosphorylation in 5/7 (71%) pRb2+ bladder tumours compared with only 4/11 (36%) pRb1+ tumours (p = 0.002). All cases with undetectable pRb showed moderate to high p16 expression and none showed cyclin D1 expression by immunohistochemistry. All pRb1+ tumours with underphosphorylated pRb showed p16 but not cyclin D1 expression. All pRb2+ tumours with hyperphosphorylated pRb showed loss of p16 expression and/or cyclin D1 overexpression. Thus, elevated pRb expression was associated with pRb hyperphosphorylation, which, in turn, was associated with loss of p16 expression and/or increased cyclin D1 expression. In order to analyse this association in vitro, T24 cells, which express high levels of pRb, were transfected with p16 cDNA. Transfection with p16 cDNA resulted in a marked decrease in pRb phosphorylation, decreased cell proliferation, and a change in expression of pRb from high to moderate phenotype as assessed by immunohistochemistry. This paper gives the biological basis for constitutive alteration of pRb function in human tumours in the presence of an intact, expressed pRb protein; the mechanism of pRb inactivation is through hyperphosphorylation, which results from loss of p16 expression and/or cyclin D1 overexpression. Immunohistochemical expression of pRb appears to be a reliable indicator of pRb function.     

The role of the cyclin D1-dependent kinases in ErbB2-mediated breast cancer

Intact cyclin D1 functions are essential for transformation by erbB2 in tissue culture and murine models. Because cyclin D1 may alter cell proliferation through a variety of mechanisms, we used transgenic models and human tumor samples to particularly address the role of cyclin D1-cyclin-dependent kinases in transformation by erbB2. The p16 tumor suppressor specifically blocks cyclin-dependent kinase 4 and 6 activity. Here we show that an MMTV-p16 transgene blocked tumorigenesis by erbB2, demonstrating that deregulation of the cyclin-dependent kinase partner of cyclin D1 is an essential target of erbB2. ErbB2 overexpression was a determining factor in deregulation of cyclin D1-cdk4/6 interactions because neither transgenic cyclin D1 nor loss of p16 accelerated tumorigenesis in MMTV-erbB2-transgenic mice. ErbB2 was also a deciding factor in deregulation of cyclin D1-cdk4/6 in human tumors because no loss of pRb or p16 was found in tumors overexpressing erbB2, although erbB2-negative invasive breast adenocarcinomas frequently lacked expression of p16 or pRb. We conclude that deregulation of cyclin D1-Cdk4/6 interactions is a critical target of erbB2 function in human and mouse breast tumors, and erbB2's overexpression may be sufficient to deregulate cyclin D1-cdk4/6 activity in breast cancer.     

Progressive deregulation of the cell cycle with higher tumor grade in the stroma of breast phyllodes tumors

We studied cell cycle-regulating proteins in phyllodes tumor pathogenesis by immunohistochemical analysis for Ki-67, cyclin A, cyclin D1, retinoblastoma protein (pRb), p53, p16INK4A, bcl-2, and p21waf1 in the epithelium and stroma of 40 primary (benign, 21; borderline, 8; malignant, 11) and 7 recurrent tumors of different grades. In most cases, the epithelium showed no altered expression of cell cycle regulators. Stromal overexpression of p16INK4A, p53, cyclin A, pRb, and p21waf1 correlated significantly with tumor grade. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation. Stromal cyclin A expression was the best separating marker between tumor grades. Correlations existed between stromal overexpression of p16NK4A and p21waf1, p16INK4A and p53, and p53 and pRb. No immunostaining differences were detected between primary tumors and recurrences. Four or more altered proteins and p53 expression in the stromal component were independent negative prognosticators for disease-free survival. The stromal component of mammary phyllodes tumors displays an increasing level of cell cycle deregulation with higher tumor grade; the epithelial compartment mostly remains inconspicuous. Several combinations of aberrantly expressed cell cycle proteins seem important in the stromal progression of phyllodes tumors. The number of stromal cell cycle aberrations and stromal p53 expression might predict clinical behavior.     

The P16/cyclin D1/Rb pathway in neuroendocrine tumors of the lung

Rb protein in its hypophosphorylated form acts as a cell cycle regulator for G1 arrest. Both cyclin D1 overexpression and P16(INK4) loss of protein produce persistent hyperphosphorylation of Rb with resultant evasion of cell cycle arrest. To better establish the mechanisms of loss of Rb function in neuroendocrine lung tumors, we performed an immunohistochemical analysis of the P16(INK4)/cyclin D1/Rb pathway in the spectrum of neuroendocrine tumors, including 34 typical carcinoids (TCs), 25 atypical carcinoids (ACs), 42 large cell neuroendocrine carcinomas (LCNECs), and 79 small cell lung carcinomas (SCLCs). Absence of Rb expression was not observed in TCs but was seen in 21% of ACs, 68% of LCNECs, and 87% of SCLCs. P16 was expressed in 91% of TCs, 77% of ACs, 78% of LCNECs, and 93% of SCLCs. Cyclin D1 was overexpressed in 6% of TCs, 20% of ACs, 9.5% of LCNECs, and 1.3% of SCLCs. There was an inverse relationship between Rb and P16 in high-grade tumors (P < 0.001) and a direct relationship between cyclin D1 and Rb (P < 0.001) in all tumors, demonstrating that P16 and cyclin D1 act exclusively on the Rb pathway for cell cycle regulation. Overall, the Rb pathway (Rb/P16(INK4)/cyclin D1) was altered more frequently in ACs than in TCs (P = 0.001) and more frequently in LCNECs than in ACs (P = 0.001). Although Rb-negative tumors had shorter survival in the overall group (P < 0.001) as a result of lack of Rb in most SCLCs, cyclin D1 overexpression and P16 loss did not influence survival in any individual category. We conclude that Rb pathway of G1 arrest is consistently compromised in high-grade neuroendocrine lung tumors (92%), primarily through loss of Rb protein, and is intact in low-grade TCs. In ACs an intermediate level of alterations (59%) is seen, consistent with their less-aggressive behavior compared with high-grade tumors. The specific profile of the Rb pathway parameters might provide specific therapeutic targets in neuroendocrine lung tumors.     

Cyclin D1 and associated proteins in mammary ductal carcinoma in situ and atypical ductal hyperplasia

Experimental studies suggest that cyclin D1 is a potential oncogene but in clinical studies of invasive breast cancer, overexpression of cyclin D1 is found to be associated with oestrogen receptor (ER) expression and low histological grade, both markers of good prognosis. Immunohistochemistry has been used to examine the relationship between cyclin D1 expression and differentiation in 36 cases of ductal carcinoma in situ (DCIS) and the interrelationship between expression of cyclin D1, its associated protein product of the retinoblastoma gene (pRb), and ER, in this group of cases. The expression of these markers has also been examined in nine cases of atypical ductal hyperplasia (ADH) and these results have been compared with the levels of expression seen in DCIS. Cyclin D1 overexpression was found in 23/36 (64 per cent) cases of DCIS and, in contrast to invasive carcinoma, there was no relationship with either differentiation or ER expression. The level of pRb expression was significantly associated with cyclin D1 expression (r_S=0·49, P=0·001) and only two cases (6 per cent) were pRb-negative. There was no association between pRb and differentiation of DCIS or ER status. In contrast to DCIS, only one case of ADH showed overexpression of cyclin D1 (Mann–Whitney U-test, P=0·02). All cases of ADH were ER-positive and showed moderate pRb staining, similar to that seen in well-differentiated DCIS. These results provide further evidence that overexpression of cyclin D1 plays a role early in carcinogenesis. © 1998 John Wiley & Sons, Ltd.     

Expression of G1-S modulators (p53, p16, p27, cyclin D1, Rb) and Smad4/Dpc4 in intrahepatic cholangiocarcinoma

Aberrations of G1-S cell cycle arrest and TGF-beta/Smad pathway are critical events in human carcinogenesis. We studied alterations of both pathways by immunohistochemical staining for p53, p16, p27, cyclin D1, Rb and Smad4/Dpc4 in 42 intrahepatic cholangiocarcinomas (ICCs). Abnormal nuclear overexpression of p53 and cyclin D1 was noted in 15 (35.7%) and 26 (61.9%) cases, respectively. Total loss of p16, p27, Rb and Smad4 was detected in 15 (35.7%), 13 (31.0%), 5 (11.9%) and 19 (45.2%) cases, respectively. Forty cases (95.2%) showed aberrations of at least one of the pathways, of which 21 (50%) revealed abnormality in G1-S pathway only, 17 (40.5%) had abnormalities in both pathways and 2 (4.8%) had an abnormality in TGF-beta/Smad pathway only. Among the examined genes, loss of Smad4 was found to have a positive relationship with the pTNM stage (P < 0.05). The overall stage of the high-altered group (alterations in 2 to 5 of the genes, n = 29) was significantly higher than that of the low-altered group (alteration of one or no gene, n = 13) (P < 0.01). We also examined the expression of above genes in the accompanying biliary dysplasia and found out abnormal expression of p53, cyclin D1 or p16 in 7 out of 13 dysplastic lesions. Our data suggest that abnormal G1-S cell cycle and altered TGF-beta/Smad pathway are major events in cholangiocarcinogenesis. Moreover, there might be a possible cumulative effect of the alterations in the examined genes upon the clinical outcome of patients with resectable ICCs.     

Differential expression of key cell cycle genes (p16/cyclin D1/pRb) in head and neck squamous carcinomas

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.     

Cell cycle alterations and their relationship to proliferation in apocrine adenosis of the breast

Aims:  Apocrine adenosis (AA) is generally considered a benign disease of the breast. However, recent studies have suggested a precancerous potential for some of these lesions. The aim was to investigate the status of cell cycle proteins previously reported to be deregulated in breast cancer to identify their possible role in AA.
Methods and results:  The cases were categorized into AA without atypia (NAA) and atypical AA (AAA). Using immunohistochemistry, the expression of cyclin D1, cyclin A, p27, p21, p16, pRb and Ki67 was determined in 29 NAA and 16 AAA cases. Cyclin D1, p21 and cyclin A were overexpressed in 58.6%, 51.7% and 31.8% of the NAA cases, respectively, whereas 81.3%, 62.5% and 41.7% of the AAA cases showed overexpression of cyclin D1, p21 and cyclin A, respectively. All cases were negative for p16, whereas pRb was expressed in all cases. Furthermore, proliferation in AA (4.5%) was significantly higher than that of normal breast epithelium (1%). There was no statistical significance in the degree of proliferation between the NAA (3.7%) and AAA (4.8%) groups.
Conclusions:  The study indicates that NAA and AAA are biologically similar. A subset of AA defined by increased proliferation and significant cell cycle alterations may be susceptible to oncogenesis.