Altered expression of cell cycle regulatory proteins in gastrointestinal stromal tumors: markers with potential prognostic implications

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the digestive tract. The prediction of the malignant potential of GISTs is still difficult. Altered cell cycle regulation may underlie the tumorigenesis and/or the progression of human malignancies. Although p53 and Bcl-2 have been extensively investigated in GISTs, little is known about the frequency of expression and possible clinical implications of alterations of other cell cycle regulatory proteins in these neoplasms. We have previously investigated the role of loss of p16(INK4A) by loss of heterozygosity and immunohistochemistry in the progression of GISTs and found that loss of heterozygosity of 9p and loss of p16 expression are confined to malignant GISTs. This has led us to investigate the role of other cell cycle regulatory proteins in these tumors. Twenty-three cases of GIST (9 low malignant potential [LMP], 10 primary malignant, and 4 intra-abdominal recurrences) were examined. All cases were strongly positive for KIT (CD117). Immunohistochemical stains were carried out on tissue microarrays to evaluate the expression of proteins involved in the G(1)-S transition and proteins that regulate apoptosis including Rb, E2F1, cyclin D1, CDK4, CDK6, p27(KIP1), p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax. The positive phenotypes identified were as follows: Rb, 39.1%; E2F1, 69.6%; cyclin D1, 30.4%; CDK4, 100%; CDK6, 30.4%; 39.1%; p27(KIP1), 47.8%; p21(WAF1/CIP1), 39.1%; p53, 43.5%; Mdm2, 17.4%; Bcl-2, 91.3%; and Bax, 100%. Malignant GISTs are more likely to be associated with a positive E2F1 and p53 phenotype and a negative p16 and p27(KIP1) phenotype. It was concluded that aberration of the cell cycle regulators is a frequent finding and may be a contributing factor to the pathogenesis of GISTs. While some alterations are seen in LMP and malignant GISTs and therefore may represent an early event in molecular tumorigenesis of GISTs, other alterations are more common in malignant GISTs than LMP and therefore have potential utility as complementary tools for the prognostication of GISTs.     

脑膜瘤P16^INK48、RB基因的甲基化分析及对基因表达的影响

目的 检测脑膜瘤中发生P16^INK48和RB基因甲基化的情况及对蛋白表达的影响。方法 用甲基化特异性聚合酶链反应对50例脑膜瘤进行了P16^INK48和RB的甲基化分析；并对其中的25例检测了P16^INK48蛋白的表达。结果 良性脑膜瘤中没有检测到甲基化，分别有6例ⅡⅢ级(37．5％)和4例Ⅲ级(28．6％)肿瘤发生至少一种基因的甲基化，其中有1例不典型脑膜瘤同时发生了两种基因的甲基化。全部13例P16^INK48阳性表达的肿瘤都是没有检测到甲基化者。结论 P16^INK48或RB的甲基化与不典型和间变性脑膜瘤的发生发展有关，其机制可能是甲基化使蛋白表达丢失并导致P16^INK48细胞周期蛋白D1／CDK4／RB途径功能障碍。     

Incidence of p14ARF gene deletion in high-grade adult and pediatric astrocytomas

The INK4a-ARF locus encodes 2 separate proteins through differential splicing of alternative first exons to produce p16INK4a (exon 1alpha) and p14ARF (exon 1beta) products in human cells. The p16INK4a protein inhibits the cyclin D-dependent kinases (CDK) that control the phosphorylation of the Rb protein and cell proliferation. The p14ARF gene product can complex with and sequester the MDM2 protein within the nucleus, thus modulating the activity of the p53 protein. Loss of p16INK4a expression would disrupt the retinoblastoma (Rb)/p16INK4a/cyclin D-dependent kinase (CDK4) pathway, whereas loss of p14ARF expression would inactivate both the Rb and p53/ MDM2/p14ARF pathways through MDM2, which can complex with either Rb or p53. Loss of the p16INK4a gene on 9p21 has been documented in a wide range of human tumors, including one third of glioblastomas. However, in tumors showing homozygous loss of exon 2 of the p16INK4a gene, loss of exon 1beta of the p14ARF gene has not been established. In this study, we have assessed deletion of the p14ARF gene in 29 pediatric and 107 adult high-grade astrocytomas and 9 glioma cell lines, using multiplex PCR analysis for exon 1beta. We found homozygous deletions for exon 1alpha and exon 1beta in 3 of 29 (10%) of the pediatric cases (2 grade III, 1 grade IV), 25 of 107 (23%) of the adult cases (6 grade III and 19 grade IV), and 8 of 9 (89%) of the glioma cell lines. Therefore, loss of the INK4a-ARF locus in high-grade astrocytomas may contribute to the highly malignant behavior and treatment resistance of these tumors through elimination of multiple checkpoint cell cycle control proteins.     

Growth inhibitory effect on glioma cells of adenovirus-mediated p16/INK4a gene transfer in vitro and in vivo

The tumour suppressor gene p16/INK4a encodes a specific inhibitor of the cyclin D-dependent kinases CDK4 and CDK6. p16/INK4a prevents the association of CDK4 with cyclin D1, and subsequently inhibits phosphorylation of retinoblastoma tumour suppressor protein (pRb), thus preventing exit from the G1 phase. In human cancers, the estimated frequency of genetic alteration involving the p16/INK4a locus is believed to be second only to alteration of p53. A high frequency (greater than 50%) of homozygous p16/INK4a gene deletion has been demonstrated in glioblastoma tissues and p16/INK4a is altered in 80% of glioma cell lines. Therefore, restoration of p16/INK4a would suppress cell proliferation and induce cell growth arrest. We showed here that restoration of p16/INK4a expression in p16 negative U87MG, U251MG and partially deleted U373MG by Ad-CMV-p16/INK4a induced growth suppression in vitro and in vivo. Expression of p16 transferred by Ad-CMV-p16/INK4a in glioma cells was highly efficient and maintained for more than seven days. In addition, we found that the endogenous status of p16 and Rb might affect the expression of exogenous p16/INK4a gene and inhibitory effect of cell proliferation. Even though, there were several factors affecting the efficiency of Ad-CMV-p16/INK4 gene transfer, our results suggest that Ad-CMV-p16 gene therapy strategy is potentially useful and warrants further clinical investigation for the treatment of gliomas.     

Expression of p16INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis

Haller F, Agaimy A, Cameron S, Beyer M, Gunawan B, Happel N, Langer C, Ramadori G, von Heydebreck A & Füzesi L
(2010) Histopathology 56, 305–318 Expression of p16 ^INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis Aims: To determine the prognostic impact of p16^INK4A expression in gastrointestinal stromal tumours (GISTs), which is currently being questioned, with both loss and overexpression said to be correlated with poor prognosis. Methods and results: Two different forms of p16^INK4A were identified, presenting with predominantly nuclear and cytoplasmic expression pattern, respectively. The immunohistochemical expression of the two forms and their correlation with E2F1 and prognosis were analysed in a series of 120 GISTs with clinical follow‐up. Low nuclear p16^INK4A expression correlated with E2F1 up‐regulation, higher mitotic counts, and tumour progression. The prognostic value of nuclear p16^INK4A expression was only marginally significant (P = 0.05). Strong expression of the cytoplasmic p16^INK4A form was significantly associated with shorter disease‐free survival (P = 2 × 10^?5). The prognostic impact of strong expression of the cytoplasmic p16^INK4A form was independent of anatomical localization, tumour size and mitotic counts, and significant even among the cohort of tumours with high malignant potential. Conclusions: Low expression of the nuclear p16^INK4A form and strong expression of the cytoplasmic p16^INK4A form both represent two independent parameters each associated with tumour progression in GISTs. Low nuclear p16^INK4A expression enables E2F1 up‐regulation and consecutive accelerated cell proliferation. In contrast, strong cytoplasmic p16^INK4A expression probably reflects a negative feedback loop as a result of (as yet unknown) oncogenic events.     

No p16INK4A/CDKN2/MTS1 mutations independent of p53 status in soft tissue sarcomas

The p16^INK4A/CDKN2/MTS1 gene encodes a specific inhibitor of cyclin-dependent kinases (CDKs) 4 and 6. This study investigates p16^INK4A gene status and expression in mesenchymal tumours, in particular soft tissue sarcomas (STSs). Employing non-radioactive polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) sequencing, no p16^INK4A mutation was found in 86 samples taken from 74 mesodermal tumours with known p53 gene status. This suggests that p16^INK4A gene alterations, in contrast to p53, are not involved in the progression of STS. This finding is supported by the reports of a low frequency of deletions and intragenic mutations in STS. Furthermore, by immunohistochemistry (IHC), an inverse correlation was established between p16^INK4A and RB positivity for 62 per cent of the frozen tumour samples investigated. However, alterations in other components of the pRb/p16^INK4A/CDK4/cyclin D1/E2F pathway have been proven crucial for tumourigenesis in human sarcomas. © 1998 John Wiley & Sons, Ltd.     

Cyclin dependent kinase inhibitors and replicative senescence]

Replicative senescence is defined for human diploid fibroblasts in culture as a cell growth arrest appearing beyond 50 +/- 10 population doublings and associated with telomeres' shortening. This phenomenon shows an increased expression of growth cell inhibitors: p21Waf1 described as an universal CDK inhibitor and p16INK4a as a specific inhibitor for both G1 phase kinases CDK4 and CDK6. The cell proliferation inhibitor p14ARF, product of INK4a/ARF locus is involved in replicative senescence too. Overexpression or homozygotic deletion of these inhibitors demonstrated their role in senescence induction. These proteins are involved in two different metabolic pathways, the first including p53, represented by E2F, ARF, MDM2, p53, p21Waf1, and the second concerning pRb and p16INK4a. These two pathways present numerous interactions and the polymerase (PARP) in relation with p53 and activated by telomere shortening might represent via p21Waf1 a link between this shortening and cell cycle control. An another metabolic pathway involving PTEN and p27KIP1 is discussed in senescent-like phenotype induction, but its activity in replicative senescent is uncertain.     

Loss of p16 protein expression associated with methylation of the p16INK4A gene is a frequent finding in Hodgkin's disease

p16 protein binds and inactivates cyclin D-CDK4/6 complexes, stopping the cell cycle at the G1/S boundary. Loss of p16 expression is found frequently in human cancer tissues, often resulting from allelic loss or promoter region hypermethylation in non-Hodgkin's lymphomas. Hodgkin's disease has been shown to be a monoclonal neoplasm of B-cells in which a majority of cells are cycling. In the attempt to identify hypothetical CDK inhibitor inactivation that could explain the accumulation of proliferating cells, we decided to focus on the p16INK4A gene. To determine whether inactivation of this gene is implicated in the development of Hodgkin's disease, we immunostained 40 cases with a monoclonal antibody for the p16 protein. At the same time, we used a methylation-specific PCR technique to determine the methylation status of exon 1 of the p16INK4A gene in 23 cases in this series. Loss of p16 expression was found in 30 of 37 cases (absence of expression in most Hodgkin's/Reed-Sternberg cells, with a normal scattered pattern of p16 expression in the reactive background). Only seven samples showed nuclear p16 expression in a significant proportion of large tumoral cells. In agreement with this finding, hypermethylation of p16INK4A gene was found in 14 of 23 cases by PCR. All the p16 cases found positive by immunohistochemistry also showed unmethylated DNA. These results show that loss of p16 protein expression is usually observed in Hodgkin's/Reed-Sternberg cells in Hodgkin's disease, frequently associated with p16INK4A gene hypermethylation. The high frequency of abnormal methylation found in this study suggests that this genetic event may play an important role in the pathogenesis of the disease.     

Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors.

SUMMARY: The EWS-ETS rearrangements, and their respective fusion gene products, are specifically associated with histopathologically Ewing family tumors (EFT). These translocations are implicated in generating malignant transformation of EFT, but the presence of additional genetic alterations must be considered in the pathogenesis of such tumors. We analyzed 26 samples (biopsies and/or nude mice xenotransplants) collected from 19 patients with an EFT to determine whether molecular and cytogenetic alterations of the G(1)/S checkpoint genes are implicated in the pathogenesis of EFT. We found inactivating p53 mutations in three (16%) cases, which correlated with a loss of p21(WAF1/Cip1) expression and with a monosomy of chromosome 17 in two cases. Homozygous deletion of the p16(INK4A)/p14(ARF) gene was detected in four (21%) cases, three with codeletion of the p15(INK4B) gene and with chromosome 9 abnormalities. In all of these cases, expression of the implicated genes was absent. Hypermethylation of the p16(INK4A) and p15(INK4B) genes was detected in two (10%) and three (16%) cases, respectively, and was correlated with a low level of gene expression. Neither cyclin D1, nor MDM2 and CDK4 amplification was observed. Kaplan-Meier analysis showed that patients with tumors carrying homozygous deletion of the 9p21 locus, or point mutations of the p53 gene, had poorer outcomes than those without these molecular alterations (p = 0.005). In conclusion, 58% (11 of 19) of the analyzed patients showed genetic or epigenetic alterations in either the 9p21 locus or p53 tumor suppressor genes, defining a subgroup of patients with poor clinical outcome. This fact points to an important role of the G(1)/S cell cycle checkpoint dysregulation in the pathogenesis of EFT.     

Alterations of gene expression of RB pathway in Opisthorchis viverrini infection-induced cholangiocarcinoma

Opisthorchiasis has the significant relationship with the high prevalence of cholangiocarcinoma (CCA; a bile duct cancer) in the endemic areas in Southeast Asia. To reveal the molecular mechanism of the tumorigenesis induced by Opisthorchis viverrini infection, the present study investigated the kinetic expression of RB pathway genes, including RB1, p16^INK4, cyclin D1, and CDK4, during the development of opisthorchiasis-associated CCA in hamster model. The results of quantitative real-time polymerase chain reaction indicated that the expressions of RB1 and p16^INK4 were down-regulated during the development of CCA induced by infection plus N-nitrosodimethylamine treatment in a time-dependent manner. On the other hand, the expressions of cyclin D1 and CDK4 were up-regulated. The expression kinetics was corresponding to the pathological progression of the opisthorchiasis-associated CCA, revealed by histopathological observation. Moreover, the analysis of the expression of these genes in human opisthorchiasis-associated CCA cases showed the decreased expression of RB1 and p16^INK4 in 50% and 82.7% cases and overexpression of cyclin D1 and CDK4 in half cases, respectively. The results suggested that RB pathway is likely involved in the tumorigenesis of opisthorchiasis-induced CCA and proposed the potential application of some of these genes as biomarkers in predispose and molecular therapy of the parasite-associated cancer.     

Herpes simplex virus type 1 infection imposes a G(1)/S block in asynchronously growing cells and prevents G(1) entry in quiescent cells

Herpes simplex virus type 1 (HSV-1) infection disrupted cell cycle regulation in at least two ways. First, infection of quiescent human embryonic lung cells simultaneously with readdition of serum caused inhibition of cyclin D/cyclin-dependent kinase (CDK) 4,6-specific and cyclin E/CDK2-specific phosphorylation of the retinoblastoma protein pRb. The inhibition of cyclin D/CDK4,6 kinase activity corresponded to a loss of cyclin D1 protein and a failure of CDK4 and CDK6 to translocate to the nucleus. Failure to detect cyclin E/CDK2 kinase activity was accompanied by a loss of cyclin E protein and a failure of CDK2 to translocate to the nucleus. Levels of pocket protein p130 persisted, whereas p107 did not accumulate. As a result of these effects on cyclin kinase, G(0)-infected cells failed to reenter the cell cycle. The second type of HSV-induced cell cycle dysregulation was observed in asynchronously dividing cell cultures. A rapid inhibition of preexisting cyclin E/CDK2 and cyclin A/CDK2 activities was observed in human embryonic lung cells, as well as two other human cell lines: C33 and U2OS. HSV-1 immediate-early gene expression was necessary for the inhibition of CDK2 kinase activity. Cyclin and CDK subunit protein levels, intracellular localization, and complex stability were unaffected by infection. In addition, levels of cyclin-dependent kinase inhibitors, p27 and p21, were not affected by HSV-1. Previous experiments demonstrated that in asynchronous infected cells, hypophosphorylated pRb and pocket protein-E2F complexes accumulated, and cellular DNA synthesis was rapidly inhibited. Coupled with the present results, this indicates that HSV-1 has evolved mechanisms for preventing cells in G(1) from proceeding through the restriction point and for cells in S from completing a round of DNA replication.     

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.     

乳腺癌中p16INK4a和视网膜母细胞瘤基因甲基化状况及其表达

目的 研究乳腺癌及癌旁增生组织中p16INK4a和视网膜母细胞瘤(RB)基因启动子区域的甲基化状况,并探讨基因异常甲基化与蛋白表达及其临床意义.方法 采用甲基化特异性PCR方法 对46例乳腺癌、22例癌旁增生组织及7例正常乳腺组织中p16INK4a和RB基因启动子区域甲基化状况进行检测,并采用免疫组织化学SP法对p16INK4a蛋白表达情况进行相应检测.结果 乳腺癌、癌旁增生组织和正常乳腺组织中p16INK4a基因的甲基化率分别为23.9%(11/46)、18.2%(4/22)、1/7;RB基因的甲基化率分别为10.8%(5/46)、9.1%(2/22)、0(0/7);肿瘤组织、癌旁增生组织和正常乳腺组织中p16INK4a基因、RB基因甲基化率差异均无统计学意义(P>0.05).正常乳腺组织、癌旁增生组织、乳腺癌中p16INK4a蛋白表达阳性率分别为7/7、60.8%(28/46)和81.8%(18/22),三者之间差异无统计学意义(P>0.05);肿瘤组织中p16INK4a蛋白表达与肿瘤分级相关(P<0.05);肿瘤组织中p16INK4a甲基化状况与其蛋白表达、肿瘤分级、ER表达阴性具有相关性(P<0.05),与肿瘤大小、淋巴结转移、年龄均不相关;RB基因甲基化状态与肿瘤分级、肿瘤大小、ER表达及年龄均无相关性,但与淋巴结转移相关(P<0.05).结论 p16INK4a基因异常甲基化可能在乳腺癌发生过程中作用有限,但在肿瘤的演进中发挥作用;RB基因甲基化检测对于分析乳腺癌进展及预后情况可能有一定参考价值;p16INK4a基因甲基化是p16INK4a蛋白失表达的机制之一.     

Aberrant expression of the Rb pathway proteins in soft tissue sarcomas.

Cell cycle regulation depends on a fine balance between cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) that block the cycle progression. Alterations of the cell cycle regulators are a common feature of many malignant tumors, and some have been shown to have prognostic significance. In this study, 152 cases of different types of soft tissue sarcomas were evaluated for alterations of cell cycle regulator proteins that control the cell cycle progression from G1 to S phase and govern the Rb pathway. Immunohistochemical stains for proteins Rb, E2F1, cyclin D1, CDK4, CDK6, p16, and p27 were carried out on tissue microarrays. The relationship between the expression of these proteins and the histologic grade of the sarcomas was assessed. Altered expression for Rb and p16 proteins was identified in 67.8% and 65.1% of the cases, respectively. Overexpression of E2F1, cyclin D1, CDK4, and CDK6 was detected in 50.7%, 24.3%, 92.1%, and 10.5%, respectively. Overexpression of E2F1 was associated with altered expression of Rb protein. Overexpression of cyclin D1, CDK4, and CDK6 showed an association with normal Rb expression. CDK6 expression revealed a positive correlation with the histologic grade of the sarcoma, and p27 expression was inversely correlated with sarcoma grade. These results suggest that alterations of the Rb pathway proteins are common in soft tissue sarcomas and may participate in their tumorigenesis. CDK6 and p27 showed correlation with the histologic grade of the sarcomas, suggesting that these proteins could be used as prognostic markers.