Increased expression of the bcl6 and CD10 proteins is associated with increased apoptosis and proliferation in diffuse large B-cell lymphomas.

There is increasing evidence that bcl6 and CD10 expression may be related to apoptosis and cell cycle progression. Therefore, 79 cases of de novo diffuse large B-cell lymphomas were studied for the expression of bcl6 and CD10 proteins in relation to 1) the apoptotic index; 2) the proliferation-associated proteins Ki67, cyclin A, and cyclin B1; and 3) the expression of the bcl2, p53, Rb, p16, and p27 proteins. Expression of bcl6, CD10, and bcl2 proteins was found in 54/79 (68%), 28/79 (35%), and 47/74 (63%) cases, respectively. The bcl6/CD10 patterns were as follows: bcl6+/CD10+ (26 cases, 32%), bcl6+/CD10- (28 cases, 33%), bcl6-/CD10- (23 cases, 31%), and bcl6-/CD10+ (2 cases, 4%). Significant positive correlations were found between bcl6/Ki67 (r =.328, P =.003), bcl6/cyclin A (r =.265, P =.018), bcl6/apoptotic index (r =.327, P =.010), CD10/Ki67 (r =.296, P =.008), and CD10/apoptotic index (r =.397, P =.001). In addition, high expression of bcl6 showed significant correlation with negative (null/low) bcl2 expression (chi(2) test, P =.002). The above findings indicate that increased expression of the bcl6 and CD10 proteins is associated with increased apoptosis and proliferation in diffuse large B-cell lymphomas. The association between increased bcl6 expression and enhanced apoptosis might be due, at least in part, to the null/low bcl2 expression because previous in vitro data showed that bcl6 overexpression induces apoptosis accompanied by bcl2 and bcl-xl downregulation. Moreover, significant correlation was found between increased apoptotic index and the bcl6+/CD10+ pattern (t test: P =.014, Mann-Whitney test: P =.046). This finding and the positive correlation of the apoptotic index with bcl6 and CD10 expression may be related to previous results showing that the expression of these proteins has favorable effects on the clinical outcome of diffuse large B-cell lymphomas.     

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.     

Expression of p21Waf1, p27Kip1 and cyclin D1 proteins in breast ductal carcinoma in situ: Relation with clinicopathologic characteristics and with p53 expression and estrogen receptor status

p21Waf1 (p21), p27Kip1 (p27) and cyclin D1 have recently been reported as useful prognostic markers for patients with breast carcinoma. However, studies on these cell cycle regulators in ductal carcinoma in situ (DCIS) have been extremely limited. Therefore, we studied the immunohistochemical expression of p21, p27 and cyclin D1 proteins in 49 DCIS cases and compared the findings with the clinicopathologic parameters (age, tumor size, gross type, histologic type, histologic grade, necrosis and mitotic index), p53 and estrogen receptor (ER) status. A significant correlation was found between positive p21 immunoreactivity (67.3% of the cases) and well-differentiated histologic grade, non-comedo type, ER-positive and p53-negative (p53-) status. DCIS with p21+/p53- is likely to be the non-comedo type. The overexpression of cyclin D1 (59.2% of the cases) correlated positively with the ER expression (P = 0.001). The p27 protein expression (46.9% of the cases) correlated with the cyclin D1 immunopositivity (P = 0.0003) and ER expression (P = 0.005). No significant associations were seen in the p27 or cyclin D1 expression and other clinicopathologic parameters. Our results suggest that p21 might be more related to the useful biologic markers in DCIS than p27 or cyclin D1. The significant positive association between p21, p27 or cyclin D1 and ER status, and close association of p27 and cyclin D1 expression might be implicated in the tumor biology of DCIS.     

Expression of cell cycle regulators p57(KIP2), cyclin D1, and cyclin E in epithelial ovarian tumors and survival

The search for new prognostic indicators is especially important in the diagnosis and treatment of ovarian cancer because clinicopathologic criteria currently used to predict survival are largely inadequate. We examined 2 groups of patients with epithelial ovarian cancer, 1 group of long-term survivors (>5 years), and 1 group of short-term survivors (<2 years) for levels of expression of the cell cycle regulators p57(KIP2), cyclin D1, and cyclin E and their relationship with survival. Our findings show that p57(KIP2) is not associated with prognosis, in contrast to p27(KIP1) expression, which is previously shown to be positively associated with long-term survival in univariate analysis (P =.001). Cyclin E expression, in contrast to cyclin D1 expression, is marginally associated with short-term survival in univariate analysis for a group of 53 women. Among the short-term survivors, 15 (65%) of 23 were positive for cyclin E expression, compared with only 11 (37%) of 30 long-term survivors (P = 0.054). This association remained significant (P =.04) in a logistic regression analysis adjusted simultaneously for performance status and extent of residual disease, the 2 strongest predictors of survival in our study. We also found a significant difference in the frequency of the cyclin E staining pattern between nonserous and serous ovarian tumor subtypes (P =.0002). Immunostaining for levels of cyclin E and p27(KIP1) expression may have potential as prognostic markers in the management of ovarian cancer.     

Multiparameter immunohistochemical analysis of the cell cycle proteins cyclin D1, Ki-67, p21WAF1, p27KIP1, and p53 in mantle cell lymphoma

BACKGROUND: Mantle cell lymphoma (MCL) is characterized by overexpression of cyclin D1, a G1 cyclin that participates in the control of cell cycle progression at the G1 to S phase transition. In addition to cyclin D1, other cell cycle regulatory molecules may be involved in the proliferation and progression of MCL. Mutation of p53, deletion of p16(INK4a), and loss of p21(WAF1) expression have been reported in some cases of blastoid MCL. OBJECTIVE: We sought to examine levels of expression of these proteins in typical and blastoid MCL and to determine whether differences were present between these subtypes of lymphomas. DESIGN: A retrospective series of typical and blastoid MCLs was evaluated for expression of the cell cycle-related proteins cyclin D1, p21(WAF1), p27(KIP1), Ki-67, and p53, as well as mitotic index. Paraffin-embedded archival tissues from 24 MCL specimens (17 typical, 7 blastoid) were immunostained with antibodies to p21(WAF1), p27(KIP1), p53, Ki-67, and cyclin D1. The percentage of positive cells for each specimen was estimated by counting 1500 cells under oil immersion microscopy. Levels of antigen expression were compared for the typical and blastoid MCLs. The mitotic index was estimated using twenty 100x oil immersion fields (OIFs) for each specimen. RESULTS: Cyclin D1 expression was seen in 22/24 specimens (92%). Blastoid MCLs were characterized by a significantly higher mean mitotic index (>20 mitoses/20 OIFs) and Ki-67 index (>45%) when compared with typical MCLs (P <.001 and P <.008, respectively; Fisher's exact test). High expression of p27(KIP1) (>25% staining) was seen more frequently in typical MCLs than in the blastoid variants (P =.03; Fisher's exact test). No significant differences were found between typical and blastoid MCLs for the expression of p21(WAF1) or p53. CONCLUSIONS: A significantly higher mitotic index and Ki-67 index were found in blastoid MCLs as compared with typical MCLs. Low p27(KIP1) expression was associated with the blastoid MCL variant. These findings confirm the high proliferative nature of blastoid MCL and suggest a role for p27(KIP1) in the negative regulation of the cell cycle in MCL.     

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.     

Human colorectal cancers with an intact p16/cyclin D1/pRb pathway have up-regulated p16 expression and decreased proliferation in small invasive tumor clusters

A systematic spatial heterogeneity with high proliferative activity at the luminal border and low activity at the invasive margin is an unexpected behavior that has been observed in colorectal cancer (CRC). To clarify this phenomenon and possible underlying regulatory mechanisms, we have by immunohistochemistry elucidated the proliferative activity and the expression of G1/S regulatory proteins in small and large tumor cell clusters at the invasive margin in 97 CRCs. By identifying small tumor clusters at the tumor front, actually invading cancer cells could be characterized and analyzed separately. These cells could then be compared with the main tumor mass represented by the larger tumor clusters. The proliferation was significantly lower in small tumor clusters compared with larger clusters (P < 0.001) and the decrease in proliferation was correlated with a p16 up-regulation (r(s) = -0.41, P < 0.001). Interestingly, CRCs lacking p16 expression (18%) or tumors with other aberrations in the p16/cyclin D1/pRb pathway had a less pronounced decrease in proliferation between large and small clusters (P < 0.001), further strengthening the association between p16 and ceased proliferation at the invasive margin. This contrasts to tumors with low p27 or abnormal p53 levels showing sustained proliferation in small tumor clusters. Our findings imply that invading CRC cells generally have low proliferative activity, and this phenomenon seems to be mediated through p16 and the p16/cyclin D1/pRb pathway.     

Expression of cell cycle regulatory proteins in the multistep process of oesophageal carcinogenesis: stepwise over-expression of cyclin E and p53, reduction of p21(WAF1/CIP1) and dysregulation of cyclin D1 and p27(KIP1)

AIMS: Cell cycle regulatory proteins were analysed by immunohistochemistry in order to clarify how their expression changes with the degree of atypia as oesophageal surface squamous epithelium progresses from normal mucosa, through reactive change, low-grade dysplasia, and high-grade dysplasia to mucosal invasive carcinoma. METHODS AND RESULTS: Immunostaining for cyclin D1, cyclin E, p21, p27, p53 and Ki67 proteins was performed using 22 normal mucosa, 17 reactive change, 22 low-grade dysplasia, 15 high-grade dysplasia and 22 mucosal invasive carcinoma specimens. Normal mucosa, low-grade dysplasia and high-grade dysplasia samples were taken from patients without any oesophageal invasive carcinoma by endoscopic biopsy or endoscopic mucosal resection, and reactive change and mucosal invasive carcinoma were obtained from oesophagectomy material. Stepwise over-expression of cyclin E (P < 0.0001) and p53 (P < 0.0001), reduction of p21 (P=0.0189) and dysregulation of cyclin D1 and p27 were observed in the multistep process of oesophageal carcinogenesis. Significant differences in expression of p27 (P < 0.0001), p53 (P=0.0299) and Ki67 (P=0.0101) were observed between reactive change and low-grade dysplasia. Furthermore, expression of cyclin D1, cyclin E, p27 and p53 in mucosal invasive carcinoma were significantly different from those in high-grade dysplasia (P=0.0079, P=0.0237, P=0.0042 and P= 0.0299, respectively). CONCLUSIONS: Cell cycle regulatory proteins, cyclin E, p53 and p21 show stepwise over-expression or reduction with progression of oesophageal carcinogenesis, correlating with the increased cell proliferation observed with Ki67 labelling. We conclude that immunohistochemical analysis for p27, p53 and Ki67 is practically useful for the discrimination between low-grade dysplasia and reactive change. Cyclin D1, cyclin E, p27 and p53 help to distinguish high-grade dysplasia from mucosal invasive carcinoma.     

Prognostic factors in survival of patients with stage Ta and T1 bladder urothelial tumors: the role of G1-S modulators (p53, p21Waf1, p27Kip1, cyclin D1, and cyclin D3), proliferation index, and clinicopathologic parameters

We studied 159 cases of superficial (stage Ta or T1) bladder tumors to determine the significance on survival of a subset of regulators of transition from G1 to S phase of the cell cycle (p53, p21Waf1, p27Kip1, cyclin D1, cyclin D3) and tumor proliferation (Ki-67 [MIB-1]). Clinical findings (patient age, sex, tumor size, grade, stage [Ta or T1]) were included in the analysis. Univariate analysis revealed association of tumor size (P = .0353), grade in stage Ta tumors (P = .0074), cyclin D1 expression (P = .0182), and Ki-67 index (P = .0033) with disease-free survival and of tumor size (P = .0005), stage (P = .0494), cyclin D3 expression (P = .0105), and Ki-67 index (P = .0272) with overall survival. Cox multivariate analysis revealed cyclin D1 expression and high proliferation index (disease-free) and tumor size, cyclin D3 expression, and high proliferation index (overall survival) as independent predictors. Results suggest that alterations of the progression from the G1 to S phase of the cell cycle are common in papillary urothelial bladder tumors. High tumor proliferation, expression of cyclins D1 and D3, and tumor size at diagnosis might be relevant predictors of survival in patients with stage Ta and T1 bladder urothelial tumors.     

Malignant transformation of mature cystic teratoma to squamous cell carcinoma involves altered expression of p53‐ and p16/Rb‐dependent cell cycle regulator proteins

Ovarian mature cystic teratomas (MCT) uncommonly undergo malignant transformation to squamous cell carcinoma (SCC). While alterations in the p53 tumor suppressor gene and protein have been shown, few studies have analyzed other molecular changes leading to this malignant conversion. The purpose of the present study was to investigate 21 samples of SCC arising in MCT for altered expression in known p53‐ and p16/Rb‐dependent cell cycle regulatory proteins, and the association between their expression and cellular proliferation and histological features. Overexpression of the p53 protein was observed in 14 SCC (67%), while four (19%) had point mutations in the p53 gene. Reduced expression of the p16 protein was observed in 18 SCC (86%), while p16 gene alterations (hypermethylation (29%) and point mutation (33%)) were found in 11 (52%). Furthermore, a statistically significant correlation was observed between p53 and Rb overexpression (P = 0.0010), and the overexpression of both p53 and Rb was respectively significantly correlated with increased cellular proliferation. The results indicate that alterations in both the p53 and p16‐Rb pathways are associated with SCC arising in MCT.     

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.     

Overall Survival in Aggressive B-Cell Lymphomas Is Dependent on the Accumulation of Alterations in p53, p16, and p27

Different studies have already shown that the isolated inactivation of p21, p16, or p27 cyclin-dependent kinase inhibitors (CKIs) is associated with increased growth fraction, tumor progression, or decreased overall survival in cases of non-Hodgkin's lymphoma. In this study we linked molecular study of the p53 and p16 genes with immunohistochemical analysis of p27 expression in a group of aggressive B-cell lymphomas [large B-cell lymphomas (LBCLs) and Burkitt's lymphomas]. This was done to analyze the relationship between p53 and p16 silencing, p27 anomalous overexpression, and clinical follow-up, testing the hypothesis that the accumulation of CKI alterations could confer to the tumors a higher aggressivity. In a group of 62 patients, p53 inactivation as a result of mutation was observed in 11 cases (18%) and p16 silencing was seen in 27 cases (43.5%) as a result of methylation (20 of 62), 9p21 deletion (7 of 44), or p16 mutation (2 of 62). The simultaneous inactivation of p53 and p16 was detected exclusively in five LBCL cases. Anomalous expression of p27, which has been proven to be associated with the absence of p27/CDK2 complexes and the formation of p27/cyclin D3 complexes where p27 is inactivated, was detected in 19 of 61 cases (31%). Cases characterized by p27 anomalous expression display concurrent inactivation of p21 (provided by p53 mutations) and/or p16 CKIs in 11 of 14 LBCL cases (P = 0.040). When the relationship between the association of inactivated CKIs and overall survival was considered, a significant relationship was found between a lower overall survival probability and an increased number of inactivated CKIs in LBCL cases, with the worst prognosis for the cases displaying concurrent p53, p16, and p27 alterations. This proves that simultaneous inactivation of different tumor suppressor pathways does indeed take place, and that tumor aggressiveness takes advantage of this CKI-concerted silencing. In this same series of data, Burkitt's lymphoma patients seem to behave in a different way than LBCLs, with p53 and p16 alteration being mutually exclusive and the association with p27 anomalous expression not being clinically significant. These facts seem to support that the additive effect of the inactivation of different CKIs could be dependent of the histological type.     

Prognostic implications of cyclin B1, p34cdc2, p27(Kip1) and p53 expression in gastric cancer

PURPOSE: Cell cycle progression is regulated by interactions of specific cyclins and cyclin dependent kinases (CDKs) at the G1-S and G2-M checkpoints and cell cycle deregulation plays a major role in carcinogenesis of human cancers. PATIENTS AND METHODS: To investigate the role of cell cycle regulators in the pathogenesis and progression of human gastric cancers, 23 cases of gastric carcinomas were examined for the expression of cyclin B1, p34cdc2, p27(Kip1) and p53 by immunohistochemical methods, and gene expression was correlated with various clinicopathologic findings. RESULTS: Out of 23 cases studied, cyclin B1 was diffusely expressed in 20 cases (87.0%), p34cdc2 in 14 cases (60.9%) and p53 in 12 cases (52.2%), whereas in normal gastric tissues, cyclin B1 and p34cdc2 were weakly expressed and p53 was not expressed. In contrast, p27(Kip1) was expressed in only 8.7% of gastric carcinomas compared with 78.3% of normal gastric tissues. There was correlation between the expression of cyclin B1 and expression of p34cdc2 (p=0.002), between the expression of cyclin B1 and loss of p27(Kip1) (p=0.025), and between the expression of p34cdc2 and loss of p27(Kip1) (p=0.065). In addition, expression of cyclin B1 was correlated with regional lymph node metastasis (p=0.032). CONCLUSION: Our results indicate that cyclin B1 and p34cdc2 are involved in the genesis or progression of gastric cancers. Furthermore, overexpression of cyclin B1 may play an important role in lymph node metastatic potential of gastric cancer. Thus, abnormal expression of cyclin B1 and CDKs, overexpression of p53 and loss of p27(Kip1) expression may play important roles in human gastric carcinogenesis.     

Expression of the G1-S modulators in hepatitis B virus-related hepatocellular carcinoma and dysplastic nodule: association of cyclin D1 and p53 proteins with the progression of hepatocellular carcinoma

Deranged expression of cell cycle modulators has been reported to contribute to the development and progression of hepatocellular carcinoma (HCC). However, their expression patterns remain poorly understood in hepatitis B virus (HBV)-related HCC, which constitutes about 65-70% of HCC in Korea. The aims of this study were to evaluate the expressions of G1-S modulators in HBV-related HCCs and dysplastic nodules (DNs), and to correlate with the histopathologic features of HCCs. Immunohistochemical expressions of cyclin D1, cyclin E, p53, p27, p21, p16, Rb, and PCNA proteins were investigated in 80 HCCs and 22 DNs. Cyclin D1 overexpression showed positive relationships with advanced tumor stage, poor differentiation, larger tumor size, microvascular invasion, intrahepatic meta-stasis, no tumor capsule formation, infiltrative growth, aberrant p53 expression, and high PCNA labeling index (LI) of HCC (p<0.05). Aberrant p53 expression showed positive relationship with poor differentiation of HCC (p<0.01). Expression of cyclin D1 or p53 was not observed in DNs. The p27 LI and p16 LI were lower in HCCs with intrahepatic metastasis (p<0.05). Cyclin D1 overexpression and aberrant p53 expression could be associated with the progression of HBV-related HCC, and might have a less crucial role in the DN-HCC sequence. In addition, elevated expression of p27 and p16 proteins might have inhibitory action to the intrahepatic metastasis of HBV-related HCC.     

Cell cycle regulators in bladder cancer: a multivariate survival study with emphasis on p27Kip1

Cyclin-dependent kinase inhibitors (CKIs) prevent cyclin-dependent kinases from phosphorylating critical substrates such as retinoblastoma gene protein (pRb), hence blocking the cascade of events leading to cell proliferation. Currently, the list of CKIs includes p21WAF1/Cip1, p27Kip1, p57Kip2 (the Cip/Kip family), p15/ INK4b, p16/INK4a, p18/INK4c, and p19/INK4d (the INK4 family). Among them, p27 plays a crucial role linking extracellular growth-regulatory signals to progression to or exit from the cell cycle. Unlike p53, p16, and Rb, mutations in Kip1 and WAF1 genes are distinctly rare in bladder cancer. We analyzed immunohistochemically the expression of p27 and other interacting G1 proteins (ie, p21, p16, pRb, p53) in 120 consecutive cases of transitional cell carcinomas (TCCs) and related it to proliferation rate, clinicopathologic parameters, and survival. p27 levels were significantly higher in low-grade (P = .001), superficial (Ta-T1) (P = .001), papillary (P < .001), and slowly proliferating TCCs (rs = -0.235, P = .05). p27 also positively correlated with p16 expression (rs = 0.212, P = .05). In univariate analysis, decreased p27 expression was associated with poor overall (P = .0109) and postrelapse (P = .0344) survival, especially if combined to increased Ki-67 expression (P = .0004 and P = .036, respectively). Furthermore, in multivariate analysis, Ki-67/p27 status had the strongest bearing on the overall survival of muscle-invasive TCCs (P = .0019). Our results indicate that low p27 expression is more common in poorly differentiated muscle-invasive TCCs and is a major player in cell cycle control in these neoplasms. More importantly, the combined Ki-67/p27 expression provides prognostic information beyond that provided by conventional parameters or other cell cycle-related proteins, concerning overall survival in muscle-invasive TCCs.     

p27KIP-1, cyclin A and cyclin D1 protein expression in ductal carcinoma in situ of the breast: p27KIP-1 correlates with hormone receptor status but not with local recurrence

Using whole sections of formalin-fixed paraffin-embedded material the expression of p27(KIP-1), cyclin A and cyclin D1 was examined in 60 cases of ductal carcinoma in situ (DCIS) using routine immunohistochemistry with a median follow up of 95 months (range 10-139 months) to identify any association with disease recurrence. Fifty-six patients were treated by local excision and radiotherapy and four by mastectomy without radiotherapy. There was a highly significant positive association between p27(KIP-1) and estrogen receptor/progesterone receptor (ER/PR) status (P = 0.002, P = 0.02) and with p27(KIP-1) and cyclin D1 expression (P = 0.002). A trend between cyclin A and PR status (P = 0.08) was also identified. These findings mirror those described in invasive ductal carcinoma, but there were no associations of any biomarker with histological parameters such as nuclear grade or with local recurrence on univariate analysis, which was present in four of the 56 locally excised group (7.1%). Further examination of a larger cohort may be worthwhile to explore the possible role as adjunctive predictive markers to aid clinical decision making.     

Cyclin D1, retinoblastoma, p53, and Her2/neu protein expression in preinvasive breast pathologies: correlation with vascularity.

OBJECTIVES: Preinvasive breast pathologies show a degree of vascularization that correlates with risk of invasion. Recently, numerous oncogenes and tumor suppressor genes have been shown to regulate neovascularization. Therefore, we examined archival tissues of preinvasive breast pathologies by immunohistochemistry for alterations in the expression of four proteins, cyclin D1, retinoblastoma (Rb), p53, and Her2/neu, known to be important in breast tumorgenesis, and correlated these data with tissue vascularity. METHODS: Vascularity was determined by immunologic detection of von Willebrand factor. For carcinoma in situ (CIS) both stromal vascularity (MVD) and vascular cuffing (MCD) were determined. RESULTS: We found that cyclin D1 expression was increased in usual hyperplasia (11% of cases). Atypical hyperplasia, noncomedo CIS and comedo CIS were positive in 43, 49, and 57% of cases, respectively. Changes in Rb and p53 were rare in hyperplasia but occurred in 8 and 10% of CIS, respectively. Her2/neu protein was identified rarely in atypical hyperplasia and in both noncomedo and comedo ductal CIS. Neither Rb nor Her2/neu expression correlated with vascularity. p53 immunoreactivity correlated positively with both MCD and MVD. Cyclin D1 was negatively associated with MVD. CONCLUSION: These data suggest that p53 and cyclin D1 proteins may regulate the microvessel density of preinvasive breast pathologies.     

P16 and p53 expression in (pre)malignant epidermal tumors of renal transplant recipients and immunocompetent individuals.

Ultraviolet (UV) radiation is a prevailing factor implicated in the etiology of keratinocytic intraepidermal neoplasia (KIN) and squamous cell carcinomas (SCCs), as evidenced by the high frequency of UV-related mutations in the p53 and p16 tumor suppressor genes. In renal transplant recipients (RTRs), immunosuppression is considered another important risk factor in the enhanced carcinogenesis in these patients. So far, effects of UV and immune status on p53 and p16 immunoexpression in SCCs and precursors have not been studied. The aims of this study were to assess (1) the relation between risk factors for carcinogenesis, sun exposure and immune status, and p16 or p53 expression, and (2) to assess differences in p16 and p53 expression between KINs and SCCs. Immunostaining for p16 and p53 was performed on paraffin-embedded sections of 23 low-grade KIN (LKIN) lesions, 28 high-grade KINs (HKINs), and 35 SCCs from 44 RTRs and 42 immunocompetent controls (ICIs). In 74/86 lesions (86%), p53 was expressed, and in 63/86 (76%) lesions, p16 expression was present. Negativity for both p16 and p53 was found in 4/86 (5%) cases, whereas combined p53/p16 staining was most prevalent (55/86 lesions, 64%). P16 staining proved independent of p53 expression (P =.8), and immune status, sun exposure, and histological diagnosis (LKIN-HKIN-SCC) had no influence on this independence. Transplantation was associated with p53 expression in SCCs (P =.02; power = 34%) caused by higher prevalence of p53-negative SCCs in RTRs than in ICIs (30% versus 0). In HKINs, p16 was more frequently positive than in LKINs (P =.003; power = 49%) and SCCs (P =.03; power = 53%). HKINs showed more frequent transepidermal p16 and p53 staining than LKIN lesions (P <.001; power >/= 99%). This study demonstrates that in KIN lesions and cutaneous SCCs, p16 expression is independent of p53 expression, and immune status, sun exposure, and histological diagnosis have no influence on this independence. Furthermore, HKIN lesions express significantly more p16 than LKINs and SCCs.     

Prognostic significance of expression patterns of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor in oesophageal adenocarcinoma: a tissue microarray study

AIMS: To correlate immunohistochemical expression patterns and prognosis in oesophageal adenocarcinoma. METHODS: The expression of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor (EGFR) was studied in a series of 137 primarily resected oesophageal adenocarcinoma samples. The expression analysis on protein level was performed on routine paraffin wax-embedded material, with immunohistochemical staining of the samples, assembled on a tissue microarray. The results were correlated with clinicopathological features (pT, pN and G) and survival. RESULTS: 22 (16%) tumours showed an overexpression of the c-erbB-2 oncoprotein. Expression of EGFR was observed in 72 (55%) cases, accumulation of p53 in 68 (52%) cases and of cyclin D1 in 102 (77%) cases. Loss of p16INK4A expression was observed in 101 (76%) cases and low expression of p27KIP1 in 91 (71%) cases. Expression of these proteins did not correlate with tumour stage, grade, Lauren's or World Health Organization classification or lymph node status. On univariate survival analysis, more advanced tumour stage (p = 0.002), lymph node involvement (p = 0.003), high tumour grade (p = 0.017) and lack of EGFR expression (p = 0.034) were found to be associated with poorer survival. On multiple regression analysis, only tumour stage (p = 0.03) and lymph node involvement (p = 0.004) were shown to have an association with the survival of the patient. CONCLUSION: The immunohistochemical expression of c-erbB-2 oncoprotein, cylin D1, p16INK4A, p27KIP1, p53 and EGFR in most oesophageal adenocarcinomas suggests their implication in the pathogenesis of this entity. None of the molecular markers assessed, however, was of prognostic value. Identification of any marker superior to or even approaching the prognostic value of conventional histopathological markers (pT and pN) was therefore not possible.