Ratio of Expression of p16INK4a to p14ARF Correlates with the Progression of Non-small Cell Lung Cancer

The CDKN2 gene is located on the short arm of chromosome 9p and encodes two unrelated proteins, p16^INK4a and p14^ARF, through the use of independent first exons and shared exons 2 and 3. p16^INK4a is a cyclin‐dependent kinase inhibitor, whereas p14^ARF regulates the cell cycle through a p53 and MDM2–dependent pathway. We have examined the expression of p16^INK4a and p14^ARF using competitive RT‐PCR in 60 non‐small cell lung cancers (NSCLCs) and matching normal lung tissues. The intensities of bands for p16^INK4a and p14^ARF were nearly equal or the intensity of the p16^INK4a band slightly exceeded that of p14^ARF in the normal lung tissues (n=60). In 38 tumors the intensity of the p16^INK4a band was similar to or slightly weaker than that of p14^ARF. In 6 tumors the intensity of the p16^INK4a band was weaker than that of p14^ARF. In 15 tumors the intensity of the p14^ARF band was very strong and the p16^INK4a band was barely visible. In only one tumor was the intensity of the p16^INK4a band very strong, while the band of p14^ARF was barely visible. The ratio of the intensity of p16^INK4a to p14^ARF had an interesting correlation with the tumor's clinicopathological characteristics. The p stage II‐IV tumors had significantly lower p16^INK4a to p14^ARF ratios than the p stage I tumors (P=0.036). The T2–4 tumors had significantly lower p16^INK4a to p14^ARF ratios than the T1 tumors (P=0.005). The N1–3 tumors had significantly lower p16^INK4a to p14^ARF ratios than the NO tumors (P=0.014). Our results suggest that the ratio of expression of p16^INK4a to p14^ARFtends to decrease during the progression of NSCLC.     

p14ARF expression in invasive breast cancers and ductal carcinoma in situ – relationships to p53 and Hdm2

Introduction  

p14^ARF stabilises nuclear p53, with a variable expression of p14^ARF mRNA in breast cancers. In vitro, nuclear p14^ARF binds Hdm2 to block Hdm2-dependent nucleocytoplasmic shuttling of p53, which is required before cytoplasmic degradation of p53. p14^ARF is negatively regulated by p53 and through p53-independent pathways. No studies have yet examined levels of p14^ARF protein expression in breast cancer and their relationship to Hdm2/p53 immunoreactivity or subcellular localisation. Previously, immunohistochemical expression of cytoplasmic p14^ARF, p53 and Hdm2 has been described. HER-2 (c-erbB2/neu) predicts prognosis and interacts with the p14^ARF/Hdm2 pathway to inactivate p14^ARF and to influence Hdm2 activity and localisation. This study examined p14^ARF and p53/Hdm2 expression and subcellular localisation by using immunohistochemistry in a series of invasive ductal breast cancers (IDCs) with concomitant ductal carcinoma in situ (DCIS), to evaluate whether findings in vitro were related to clinicopathological parameters such as HER-2 and their effect on patient outcome.     

Deregulation of the p14ARF/Mdm2/p53 pathway and G1/S transition in two glioblastoma sets

Sixty-one glioblastomas have been studied, subdivided into the categories of classic glioblastomas (GBM) and glioblastomas with astrocytic (GBA) and oligodendroglial (GBO) differentiated areas. On surgical samples, TP53, Mdm2, CDKN2A/p16–p14 alterations were studied by molecular biology techniques and by immunohistochemistry. It has been found that Mdm2 amplification was more frequent in GBM than in GBA and GBO, that p14^ARF was inactivated in a high percentage of cases in the three tumor categories. Both these and other alterations did not reach a statistical significance, with the exception of CDKN2A/p16 homozygous deletion which showed the highest frequency in GBO. The latter finding could be in line with the observation that CDKN2A/p16 inactivation is a step in the molecular pathway to tumor progression in oligodendrogliomas. TP53 mutations and Mdm2 amplifications were mutually exclusive, whereas TP53 mutations and CDKN2A/p14 inactivation coexisted in 5 cases. The alterations of the p53/Mdm2/p14^ARF pathway occurred in 73% of cases and in 80% of cases if CDKN2A homozygous deletions were associated. All glioblastomas with gemistocytic areas showed p14^ARF inactivation. Immunohistochemistry showed higher percentages of positivity in comparison with molecular genetics, but with similar variations.     

Pathological classification and molecular genetics of meningiomas

Meningiomas are extremely common adult brain tumors originating from meningeal coverings of the brain and spinal cord. While most are slowly growing Word Health organization (WHO) grade I tumors, rare variants (clear cell, chordoid, papillary, and rhabdoid), as well as brain invasive (WHO grade II), atypical (WHO grade II), and anaplastic (WHO grade III) meningiomas are considerably more aggressive. This review summarizes the histopathological and genetic features of meningiomas, including differential diagnosis, pitfalls, and grading challenges. Early stages of meningioma tumorigenesis are closely linked to inactivation of one or more members of the 4.1 superfamily, including the neurofibromatosis type 2 (NF2) and 4.1B (DAL-1) genes, which interact with the 14-3-3 protein family. Other chromosome 22q genes implicated include BAM22, BCR (breakpoint cluster region), and TIMP-1, the last of which is implicated in higher-grade meningiomas. Atypical meningiomas also commonly show chromosomal losses of 1p, 6q, 10, 14q, and 18q, as well as multiple chromosomal gains. While most relevant genes remain unknown, two chromosome 14q candidates (MEG3 and NDRG2) have recently been identified. In addition to alterations of CDKN2A, p14 ^ARF , and CDKN2B tumor suppressor genes on 9p21, a contribution of the wingless (wnt) pathway with alterations of the E-cadherin and beta-catenin proteins, as well as alterations of the hedgehog signaling pathway have been implicated in anaplastic meningiomas. The integration of histopathological appearance, complex genetic/genomic data, and outcome will likely result in the identification of clinically distinct meningioma subgroups, which in turn can facilitate the development of targeted therapeutic strategies.     

Molecular pathogenesis of meningiomas

Meningiomas are common central nervous system tumors that originate from the meningeal coverings of the brain and the spinal cord. Most meningiomas are slowly growing benign tumors that histologically correspond to World Health Organization (WHO) grade I. However, certain rare histological variants (clear cell, chordoid, papillary, and rhabdoid), as well as atypical (WHO grade II) and anaplastic (WHO grade III) meningiomas show a more aggressive biological behavior and are clinically associated with a high risk of local recurrence and a less favorable prognosis. This review summarizes the most important features of meningioma pathology and provides an up-to-date overview about the molecular mechanisms involved in meningioma initiation and progression. Current data indicate that meningioma initiation is closely linked to the inactivation of one or more members of the highly conserved protein 4.1 superfamily, including the neurofibromatosis type 2 gene product merlin/schwannomin, protein 4.IB (DAL-1) and protein 4.1R. The genetic alterations in atypical meningiomas are complex and involve losses on 1p, 6q, 10, 14q and 18q, as well as gains on multiple chromosomes. The relevant genes are still unknown. Anaplastic meningiomas show even more complex genetic alterations, including frequent alteration of the CDKN2A, p14 ^ARF , and CDKN2Btumor suppressor genes at 9p21, as well as gene amplification on 17q23. A better understanding of the molecular mechanisms involved in meningioma pathogenesis may not only lead to the identification of novel diagnostic and prognostic marker but will also facilitate the development of new pathogenesis-based therapeutic strategies.     

Frequent but borderline methylation of<Emphasis Type="Italic"> p16</Emphasis><Superscript>INK4a</Superscript> and<Emphasis Type="Italic"> TIMP3</Emphasis> in medulloblastoma and sPNET revealed by quantitative analyses

Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 ^INK4A, p14 ^ARF, TIMP3, CDH1, p15 ^INK4B and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 ^INK4A (17/43), p14 ^ARF (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 ^INK4A and TIMP3 were methylated consistently in medulloblastomas (p16 ^INK4A 14%, TIMP3 11%) and p16 ^INK4A also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1–5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.     

Cytogenetic analyses in 81 patients with brain gliomas: correlation with clinical outcome and morphological data

Specific gene mutations, loss of heterozygosity, deletions and/or amplifications of entire chromosomal regions and gene silencing have been described in gliomas. 82 samples from 81 patients were investigated to detect the deletion of TP53, RB1, CDKN2A genes, deletion of 1p36 and 19q13.3 region, amplification of EGFR gene, trisomy of chromosome 7 and monosomy of chromosome 10 in glial cells. Dual-colour interphase fluorescence in situ hybridization (I-FISH) with locus-specific and/or chromosome enumeration DNA probes were used for cytogenetic analyses. In the study, molecular cytogenetic analyses were successfully performed in 74 patients (91.3%) and were uninformative in 7 only (8.7%). The cytogenetic analyses were correlated with morphological data and clinical outcome. I-FISH was the essential part of diagnostics. In comparison with the clinical data, the patients’ age seems to be a factor more important for the overall survival, rather than cytogenetic findings in glial tumours. The combined deletion of 1p36 and 19q13.3 chromosomal regions predicts longer overall survival for patients with oligodendroglial tumours.     

Characterization of arsenic-induced cytogenetic alterations in acute promyelocytic leukemia cell line,NB4

Gain or loss of genes plays important roles in leukemogenesis of APL via cooperation with PML-RARA. Fluorescence in situ hybridization (FISH) was applied to investigate the DNA copy number changes of hTERT, ERG, CDKN1B (P27), CDKN2A (P16), and TP53 genes in an acute promyelocytic leukemia (APL) cell line (NB4). Five bacterial artificial chromosome probes (BAC) for 9p21.3, 17p13.1, 12p13.2, 5p15.33, 21q22.2 regions were prepared using sequence independent amplification (SIA) and were hybridized to NB4 cells treated with different doses of arsenic trioxide (As₂O₃; ATO) at various time intervals. NB4 cells were also karyotyped by G-banded chromosome analysis 24 h after culture initiation. FISH analysis prior to treatment showed CDKN1B, CDKN2A, and TP53 gene deletion but ERG and hTERT gene amplification. After treatment with ATO, the number of the NB4 cells with deleted CDKN1B and CDKN2A as well as the counts of the cells with hTERT amplification was significantly reduced in time- and does-dependent manners. In addition, we observed expressive increase in signal patterns of CDKN1B and CDKN2A along with significant decline in hTERT signal patterns in ATO-treated cells as compared with the control group (in time- and dose-dependent manners). On the other hand, no difference in signal patterns for Erg and p53 was observed in response to ATO exposure. The results of the present study show the cytogenetic alteration in hTERT, CDKN1B, and CDKN2A in NB4 cells after treatment with ATO might introduce a new mechanism of antitumor activities of ATO in APL cell line, NB4.     

Promoter hypermethylation of p14 ARF,RB, and INK4 gene family in hepatocellular carcinoma with hepatitis B virus infection

Both hepatitis B virus (HBV) and gene methylation play important roles in hepatocarcinogenesis. However, their association between HBV infection and gene methylation is not fully understood. Cell cycle control involving RB1 gene-related cell inhibitors is one of the main regulatory pathways were reported to be altered in hepatocellular carcinoma (HCC). The purpose of this research is to assess the methylation status of p14 ^ARF and INK4 gene family (p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and p18 ^INK4C ) in HCC with HBV infection and HCC without it, and discuss possible role of HBV-induced hypermethylation in the mechanism of hepatocarcinogenesis. Methylation status of RB, p14 ^ARF , and INK4 gene family in 64 case of HCC with HBV infection and 24 cases without it were detected by methylation-specific polymerase chain reaction, and HBV-DNA of the plasma were detected by quantitative real-time polymerase chain reaction. p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and RB hypermethylation were observed in 30 (34.1 %), 50 (56.8 %), 62 (70.5 %), and 24(27.3 %) of 88 hepatocellular carcinomas, respectively. Methylation frequencies of them between HCC with HBV infection and HCC without it were 43.8 % versus 8.3 % (p14 ^ARF ), 68.9 % versus 25 % (p15 ^INK4B ), 90.6 % versus 16.7 % ( p16 ^INK4A ), and 28.1 % versus 25 % (RB), respectively. In HBV-associated HCC, the numbers of methylated genes were also more than HCC without virus infection, more than two methylated genes were seen in 48 of 64 (75 %) cases; more than three methylated genes were found in 32 of 64 (50 %); correspondently, no one case has more than two genes methylated. p18 ^INK4C methylation product was not found in cancerous or non-cancerous tissues of 88 HCC. HBV infection is associated with p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and RB gene methylation (P?=?0.048, 0.035, 0.02); HBV-DNA replication is associated with p14 ^ARF , p15 ^INK4B , p16 ^INK4A , and RB gene methylation (P?=?0.048, 0.035, 0.02); high rate of p14 ^ARF , p15 ^INK4B , and p16 ^INK4A in HCC with HBV infection suggests that HBV-induced hypermethylation may be one of the mechanisms of HBV involved in hepatocellular carcinogenesis.     

Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line,FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization

Background

Pleomorphic malignant fibrous histiocytoma (MFH) is one of the most frequent malignant soft tissue tumors in adults. Despite the considerable amount of research on MFH cell lines, their characterization at a molecular cytogenetic level has not been extensively analyzed.

Methods and results

We established a new permanent human cell line, FU-MFH-2, from a metastatic pleomorphic MFH of a 72-year-old Japanese man, and applied multicolor fluorescence in situ hybridization (M-FISH), Urovysion™ FISH, and comparative genomic hybridization (CGH) for the characterization of chromosomal aberrations. FU-MFH-2 cells were spindle or polygonal in shape with oval nuclei, and were successfully maintained in vitro for over 80 passages. The histological features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor. Cytogenetic and M-FISH analyses displayed a hypotriploid karyotype with numerous structural aberrations. Urovysion™ FISH revealed a homozygous deletion of the p16^INK4A locus on chromosome band 9p21. CGH analysis showed a high-level amplification of 9q31-q34, gains of 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X, and losses of 1q43-qter, 4q32-qter, 5q14-q23, 7q32-qter, 8p21-pter, 8q23, 9p21-pter, 10p11.2-p13, and 10q11.2-q22.

Conclusion

The FU-MFH-2 cell line will be a particularly useful model for studying molecular pathogenesis of human pleomorphic MFH.     

p16INK4, pRB,p53 and cyclin D1 expression and hypermethylation of CDKN2 gene in thymoma and thymic carcinoma

There have been few reports on genetic alterations in thymomas. To investigate the expression of p16^INK4A, RB, p53 and cyclin D1 in thymomas, we first examined 36 thymomas (non-invasive type, 16 cases; invasive type, 20 cases) and 3 thymic carcinomas, using immunohistochemistry. Abnormal expression of p16^INK4A, RB, p53 and cyclin D1 was observed in 18, 8, 10 and 7 cases, respectively. Only a subgroup of invasive thymomas and thymic carcinomas showed an inverse correlation between p16^INK4A and RB expression. Subsequently, we examined the 36 thymomas and 4 thymic carcinomas for mutations in p53 and CDKN2 genes, using PCR-SSCP and direct-sequencing analyses. No mutation of these genes was detected in the thymomas and thymic carcinomas examined. A polymorphism in the 3′ untranslated region of exon 3 of CDKN2 was detected in 5 cases of thymoma. We searched for hypermethylation in the promoter region of CDKN2, observing it in 4 thymomas and 1 thymic carcinoma. Our data suggest that, unlike other more common cancers, alteration of the p53 gene may not play a significant role in the tumorigenesis of thymoma. However, inactivation of p16^INK4A and RB may play a role in the progression of thymoma and thymic carcinoma. Int. J. Cancer 73:639–644, 1997. © 1997 Wiley-Liss, Inc.     

p19INK4d mRNA and protein expression as new prognostic factors in ovarian cancer patients

p19^INK4d (CDKN2D) is a negative regulator of the cell cycle. Little is known of its role in cancer development and prognosis. We aimed to evaluate the clinical significance of p19^INK4d expression in ovarian carcinomas with respect to the TP53 accumulation status, as well as the frequency of CDKN2D mutations. p19^INK4d and TP53 expression was evaluated immunohistochemically in 445 ovarian carcinomas: 246 patients were treated with platinum–cyclophosphamide (PC/PAC), while 199 were treated with taxane–platinum agents (TP). CDKN2D gene expression (mRNA) was examined in 106 carcinomas, while CDKN2D mutations in 68 tumors. Uni- and multivariate statistical analyses (logistic regression and the Cox proportional hazards model) were performed for patient groups divided according to the chemotherapeutic regimen administered, and in subgroups with and without TP53 accumulation. High p19^INK4d expression increased the risk of death, but only in patients with the TP53-negative carcinomas (HR 1.61, P = 0.049 for PC/PAC-treated patients, HR 2.00, P = 0.015 for TP-treated patients). This result was confirmed by the mRNA analysis (HR 4.24, P = 0.001 for TP-treated group). High p19^INK4d protein expression associated with adverse clinicopathological factors. We found no alterations in the CDKN2D gene; the c.90C>G (p.R30R; rs1968445) polymorphism was detected in 10% of tumors. Our results suggest that p19^INK4d expression is a poor prognostic factor in ovarian cancer patients. Analyses of tumor groups according to the TP53 accumulation status facilitate the identification of cancer biomarkers.     

p14ARF genetic polymorphisms and susceptibility to second primary malignancy in patients with index squamous cell carcinoma of the head and neck

BACKGROUND:

p14^ARF, an alternate reading frame (ARF) product of the cyclin‐dependent kinase inhibitor 2A locus, plays a critical role in crosstalk between the tumor protein 53 (p53) and retinoblastoma (Rb) pathways and in cellular anticancer mechanisms. Therefore, the authors of this report investigated the association between single nucleotide polymorphisms (SNPs) of the p14^ARF gene and the risk of developing a second primary malignancy (SPM) after an index squamous cell carcinoma of the head and neck (SCCHN).

METHODS:

The log‐rank test and Cox proportional hazards models were used to assess the association of 2 p14^ARF SNPs (reference SNP [rs]3731217 and rs3088440) with SPM‐free survival and with the risk of developing an SPM among 1287 patients who had SCCHN.

RESULTS:

Patients with either p14^ARF variant genotypes of the 2 polymorphisms had a significantly reduced SPM‐free survival compared with patients with no variant genotypes (log‐rank test; P = .006). Compared with the p14^ARF thymine‐thymine (TT) and guanine‐guanine (GG) genotypes, the variant genotypes of p14^ARF TG/GG and guanine‐adenine (GA)/AA were associated with a significantly moderately increased risk of developing an SPM (p14^ARF rs3731217: adjusted hazard ratio [aHR], 1.48; 95% confidence interval [CI], 1.00‐2.19; p14^ARF rs3088440: aHR, 1.61; 95% CI, 1.07‐2.43). Moreover, after combining the variant genotypes of the 2 SNPs, patients who had variant genotypes were at significantly greater risk of developing an SPM compared with patients who had no variant genotypes (aHR, 3.07; 95% CI, 1.54‐6.12), and the risk was particularly pronounced in several subgroups.

CONCLUSIONS:

The current results suggested that there is a modestly increased risk of developing an SPM after an index SCCHN with each p14^ARF polymorphism, and there is an even greater risk of developing an SPM for patients with combined variant genotypes of the 2 SNPs. Therefore, p14^ARF polymorphisms may be susceptible markers of the risk of developing an SPM in patients with SCCHN. Cancer 2011. © 2010 American Cancer Society.     

CDKN2A but not TP53 mutations nor HPV presence predict poor outcome in metastatic squamous cell carcinoma of the skin

Genetic alterations in metastatic cutaneous squamous cell carcinoma (CSCC) which might serve as prognostic biomarkers are not well investigated. We investigated the mutation status and protein expression of the CDKN2A (INK4a‐ARF) and TP53 genes in metastatic CSCCs and correlated this with clinicopathological variables, HPV presence, and survival data. Sequence analysis was performed on formalin‐fixed and paraffin‐embedded tissue of 35 metastases and their primary tumors, and was correlated with immunohistochemical stainings for p53, p16 and p14. Beta‐PV and alpha‐PV DNA was detected using PCR‐based assays. Kaplan–Meier and Cox regression methods were used for survival assessment. CDKN2A was mutated in 31% of the metastases and their primary tumors, while the TP53 gene was mutated in 51% of the metastases. P53 protein expression was significantly associated with missense type of mutations (p = 0.002). No persistent HPV types were detected. CDKN2A mutations were significantly associated with disease‐specific death (p = 0.001). A significant difference was observed in disease‐specific survival between patients with or without a CDKN2A mutation (p = 0.010), while this was not the case for TP53. At univariate Cox's regression analysis tumor size (p = 0.010), invasion depth (p = 0.030) and CDKN2A mutations (p = 0.040) were significantly related to shorter disease‐specific survival. At multivariate Cox's regression only tumor size had an adverse effect on survival (p = 0.002). In conclusion, our study indicates that the CDKN2A mutation status might be of prognostic value in metastatic CSCCs. In most cases, CDKN2A and TP53 mutations are early genetic events in CSCC tumorigenesis. The possible role of HPV in metastatic CSCC needs further exploration.     

Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas

Aberrant hypermethylation of CpG islands in the promoter region plays a causal role in the inactivation of various key genes involved in the cell cycle regulatory cascade, which could result in a loss of cell cycle control. The aim of the present study was to examine in more detail the prevalence and role of the promoter methylation of genes with a proven involvement in the cell cycle regulation of pituitary adenomas, since their tumorigenesis has not yet been clearly defined. We profiled the CpG island methylation status of a series of well-characterized cell cycle regulation genes: the RB1, p14 ^ARF , p15 ^INK4b , p16 ^INK4a , p21 ^Waf1/Cip1 , p27 ^Kip1 , and p73 genes, in 34 pituitary adenomas as determined by a methylation-specific polymerase chain reaction assay. Promoter hypermethylation of the RB1, p14 ^ARF , p15 ^INK4b , p16 ^INK4a , p21 ^Waf1/Cip1 , p27 ^Kip1 , and p73 genes was detected in 12 (35%), 2 (6%), 11 (32%), 20 (59%), 1 (3%), 0 (0%), and 4 (12%) of the adenomas, respectively. In total, 88% (30 of 34) of the adenomas displayed methylation of at least one of such cell cycle regulatory genes, especially methylation of the member genes of the RB1 pathway (29 of 34; 85%). Promoter hypermethylation of p15 ^INK4b coincided with RB1 and/or p16 ^INK4a methylation, whereas RB1 and p16 ^INK4a methylations tended to be mutually exclusive (p = 0.0048). Furthermore, promoter hypermethylations of p14 ^ARF , p21 ^Waf1/Cip1 , and p73 (not belonging to the member genes of the RB1 pathway) were also coincident with RB1 and/or p16 ^INK4a methylation except in one p73 methylated case. In contrast, none of the clinicopathological features, including the cell proliferation index, was significantly correlated with any particular methylation status. Our results suggested that aberrant hypermethylation of the key cell cycle regulatory genes occurs at a relatively high frequency in pituitary adenomas, especially in RB1 pathway genes with promoter hypermethylation of the p16 ^INK4a gene being the most common deregulation. We further obtained evidence to indicate that RB1 and p16 ^INK4a methylations tended to be mutually exclusive, but did occasionally coincide with other cell cycle regulation gene methylations.     

Loss of heterozygosity at 1p-19q induces a global change in oligodendroglial tumor gene expression

Oligodendroglial tumors presenting loss of heterozygosity (LOH) at 1p and 19q have been shown to be sensitive to chemotherapy, thus making 1p-19q status testing a key aspect in oligodendroglioma diagnosis and prognosis. Twenty-nine tumor samples (19 oligodendrogliomas, 10 oligoastrocytomas) were analyzed in order to obtain a molecular profile identifying those bearing 1p-19q LOH. Other genomic anomalies usually present in gliomas, such as EGFR amplification, CDKN2A/ARF deletion, 10q LOH and TP53 mutation, were also studied. Tumors with 1p-19q LOH overexpressed genes related to neurogenesis. Genes linked to immune response, proliferation and inflammation were overexpressed in the group with intact 1p-19q; this group could in turn be further divided in two subgroups: one overexpressing genes involved in immune response and inflammation that did not show major genetic aberrations other than the TP53 mutation and EGFR trisomy in a few cases, and another overexpressing genes related to immune response and proliferation that had a predominance of samples carrying several anomalies and presenting worse outcomes. This molecular signature was validated by analyzing a set of ten tumor samples (three oligodendrogliomas, seven oligoastrocytomas); all ten samples were correctly assigned. LOH at 1p-19q results in haploinsufficiency and copy number reduction of several genes, including NOTCH 2; this phenomenon produces a global change in gene expression inducing a pro-neural status that results in restrictions to cell migration and proliferation. Tumors without LOH at 1p-19q exhibit the opposite characteristics, explaining their more aggressive behavior.     

Genetic status of cell cycle regulators in squamous cell carcinoma of the oesophagus: the CDKN2A (p16(INK4a) and p14(ARF) ) and p53 genes are major targets for inactivation

We determined inactivation of the CDKN2A (p16(INK4a) and p14(ARF)) gene in 21 cases of oesophageal squamous cell carcinoma (OSCC). The tumours were also analysed for mutations in exons 5-8 and allelic losses in the p53 gene. In addition, we screened the CDKN2B (p15 INK4b), CDKN2C (p18 INK4c), CDK4 and p53R2 genes for mutations in the tumour tissues. Besides concomitant alterations in the CDKN2A and p53 loci in more than half of the cases, our results showed that in 18 OSCC (86%) the CDKN2A (p16(INK4a) and p14(ARF) ) gene was affected through mutations, homozygous/hemizygous deletions and promoter hypermethylation. Eight out of 10 tumours with mutations or promoter hypermethylation specific to the CDKN2A/p16 INK4a gene showed loss of the wild-type allele. One tumour with a single base deletion in the N-terminus (codon 8) of the CDKN2A/p16(INK4a) gene carried a novel germ-line mutation or a rare polymorphism (Ile51Met) in exon 2 of the CDK4 gene. Promoter hypermethylation in the CDKN2A/p14 ARF gene was detected in 11 tumours. In the p53 gene 15 mutations were detected in 14 tumours. We detected an inverse relationship between CDKN2A/p16 INK4a inactivation and frequency of loss of heterozygosity at the p53 locus (OR 0.09, 95% CI 0.01-0.98; Fisher exact test, P-value approximately 0.03). Screening of nine exons of the p53R2 [Human Genome Organisation (HUGO) official name RRM2B] gene resulted in identification of a novel polymorphism in the 5' untranslated region, which was detected in four cases. Our results suggest that the CDKN2A (p16(INK4a) and p14(ARF) ) and p53 genes involved in the two cell cycle pathways are major and independent targets of inactivation in OSCC.     

Growth arrest and suppression of tumorigenicity of bladder carcinoma cell lines induced by the P16/CDKN2 (p16INK4A,MTS1) gene and other loci on human chromosome 9

Wild-type P16/CDKN2 (p16^INK4A, MTSI) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequences. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32–33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor-suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression. © 1996 Wiley-Liss, Inc.     

High incidence of unbalanced chromosomal changes in mantle cell lymphoma detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) was carried out in 30 mantle cell lymphoma (MCL) patients at the time of diagnosis. CGH results were supported by conventional cytogenetics (CC), FISH, molecular genetic PCR methods and 2 patients were examined by array CGH. Using all cytogenetic, molecular cytogenetic and PCR methods, chromosomal changes were detected in 28 (93%) patients. Using CGH, unbalanced chromosomal changes were detected in 24 (80%) cases. The most frequent aberrations were losses of 1p (8 cases), 8p (10 cases), 9q (6 cases), 11q (11 cases), 13q (10 cases) and 17p (9 cases), and gains of chromosome 3 and 3q (12 cases) and 8q (7 cases). Total number of 60 gains and 116 losses were detected. The primary chromosomal change t(11;14) was detected using FISH and/or PCR in 20 (66.6%) patients, and in 9 of them, the breakpoint was determined using PCR in the major translocation cluster (MTC). The evaluation of the frequencies of CGH changes in groups of patients with and without t(11;14) revealed the differences only in losses 6q and 9q, which were only found in patient with t(11;14). An important result was obtained using array CGH method. In a patient without the primary t(11;14), the gain of CCND1 gene was found. Our results show high heterogeneity of the additional chromosomal changes in MCL cases, which involved specific chromosomal subregions. We did not confirm the importance of subdividing of MCL cases with and without t(11;14). Also, statistical significance in survival rates between both subgroups was not confirmed.