Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.     

Inactivation of the p14(ARF), p15(INK4B) and p16(INK4A) genes is a frequent event in human oral squamous cell carcinomas.

The p14(ARF), p15(INK4B) and p16(INK4A) genes were localized to 9p21, where genetic alterations have been reported frequently in various human tumors. We performed a molecular analysis of the mechanism of inactivation in cell lines and 32 oral squamous cell carcinoma (OSCC), using deletion screening, PCR-SSCP, methylation-specific-PCR and cycle sequencing. We detected homozygous deletion of p14(ARF)-1Ebeta in 9 (26.5%), of p15(INK4B) in one (3.1%), and of p16(INK4A) in 22 (56.3%) tumor samples. Three mutations were detected in the p16(INK4A) genes. We detected aberrant methylation of the p14(ARF) genes in 14 (43.8%), of the p15(INK4B) gene in 9 (28.1%), and of the p16(INK4A) gene in 16 (50.0%) tumor samples. Altogether, 87.5% of the samples harbored at least one of the alterations in the p14(ARF), p15(INK4B), and p16(INK4A) genes, indicating that the frequent inactivation of these genes may be an important mechanism during OSCC development.     

Alterations of p16(INK4a) and p14(ARF) in patients with severe oral epithelial dysplasia

A number of genetic aberrations have been reported in end-stage squamous cell carcinoma of the head and neck, including p16(INK4a) and p14(ARF) (INK4a/ARF) inactivation rates of 70-85%. Still, the cell cycle-regulatory genes p16(INK4a) and p14(ARF) remain poorly understood in oral cavity premalignant lesions. This study evaluated INK4a/ARF locus alterations in 26 patients (28 samples) deemed to be at increased risk for malignant transformation to squamous cell carcinoma due to the diagnosis of severe oral epithelial dysplasia. Microscopically confirmed dysplastic oral epithelium and matching normal tissue were laser capture-microdissected from paraffin sections, DNA was isolated, and molecular techniques were used to evaluate p16(INK4a) and p14(ARF) gene deletion, mutation, loss of heterozygosity (LOH), and hypermethylation events. Deletion of exon 1beta, 1alpha, or 2 was detected in 3.8%, 11.5%, and 7.7% of patients, respectively. INK4a and ARF mutations were detected in 15.4% and 11.5% of patients with severe dysplasia of the oral epithelium. All identified mutations occurred in the INK4a/ARF conserved exon 2. Allelic imbalance was assessed using three markers previously reported to show high LOH rates in head and neck tumors. LOH was found in 42.1%, 35.0%, and 82.4% of patients for the markers IFNalpha, D9S1748, and D9S171, respectively. Hypermethylation of p16(INK4a) and p14(ARF) was detected in 57.7% and 3.8% of patients, respectively, using nested, two-stage methylation-specific PCR. The highest rates of p16(INK4a) hypermethylation occurred in lesions of the tongue and floor of the mouth. In addition, p16(INK4a) hypermethylation was significantly linked to LOH in two or more markers. These data support that INK4a/ARF locus alterations are frequent events preceding the development of oral cancer and that p16(INK4a) inactivation occurs to a greater extent in oral dysplasia than does p14(ARF) inactivation.     

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in actinic keratosis

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14(ARF), p15(INK4b), p16(INK4a) and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16(INK4a) (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14(ARF) gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16(INK4a) and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15(INK4b), independently from the intracellular pathway mediated by p16(INK4a), and of p14(ARF) in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.     

Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.     

The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas

Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.     

Selective deletion of p14(ARF) exon 1beta of the INK4a locus in oral squamous cell carcinomas of Indians

The tumor suppressor gene - p16 INK4/CDKN2/MTS1 and its alternate splice product p14 (ARF), constitute the INK4a locus. We have examined the integrity of exon 1beta of p14(ARF) gene of oral squamous cell carcinomas (n=58) in untreated Indian patients. No mutations were detected in this region by PCR-SSCP analysis of the tumor DNA's. Further, PCR-based analysis revealed homozygous deletions of exon 1beta in 14 of the 58 tumors; these results were confirmed by hybridization of tumor DNAs with exon 1beta specific probe. The deletions were limited to the exon 1beta while the exons coding p16/INK4 were not affected. Except in two cases these deletions were mutually exclusive to the p53 inactivating mutations. These observations suggest an alternate mechanism of loss of p14(ARF) in the genesis of oral squamous cell carcinomas.     

Alterations of the p16INK4a/p14ARF pathway in clear cell sarcoma

Clear cell sarcoma (CCS) is a very rare soft tissue sarcoma with a poor prognosis. It has become apparent through immunohistochemical, ultrastructural, and microarray analyses that CCS is a soft tissue melanocytic neoplasm. Alterations in the p16INK4a/p14ARF gene are common in malignant melanoma, which is the prototypical melanocytic neoplasm. In the present study, we performed a clinicopathologic analysis and investigated p16 and cyclin D1 expression by immunohistochemistry in 14 cases. Furthermore, we investigated genetic changes of various tumor suppressor genes and an oncogene, including p16INK4a/p14ARF, p53, beta-catenin, and APC, in 11 cases. The 5-year overall survival rate in all the patients was 33.3%. A high mitotic rate was a significant adverse prognostic factor (P = 0.004). Decreased expression of p16 was observed in 4 (28.6%) of 14 cases. Overexpression of cyclin D1 was observed in 9 cases (64.3%). SSCP analysis followed by DNA direct sequencing revealed point mutations of the p16INK4a gene in 2 of 11 cases (18.2%). In addition, one case with the p14ARF mutation and 2 cases with the p53 mutation were observed. None of the cases harbored mutation of the beta-catenin or APC gene. Homozygous deletion of the p16INK4a/p14ARF gene was detected in one case. Methylation-specific PCR did not reveal hypermethylation of the p16INK4a/p14ARF promoter region in any of the cases. Three cases harbored genetic alterations of the p16INK4a/p14ARF gene (27.3%). All tumors with genetic alterations of the p16INK4a/p14ARF or p53 gene showed a high mitotic rate or tumor necrosis. These alterations were considered to be influential in the poor prognosis of CCS patients.     

Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.

The 9p21 gene cluster, harboring growth suppressive genes p14ARF, p15INK4b, and p16INK4a, is one of the major aberration hotspots in human cancers. It was shown that p14ARF and p16INK4a play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15INK4b is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14ARF, p15INK4b, and p16INK4a genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methylations were exclusive, whereas all but one of the p14ARF/p15INK4b methylations were accompanied by concomitant p16INK4a methylation. We detected homozygous deletion of p16INK4a in 7 (17.5%), of p14ARF-E1beta in 13 (33%), and of p15INK4b in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1beta-p15INK4b loci. Two samples contained p14ARF deletion but with p16INK4a and p15INK4b intact. No mutation was detected in the p14ARF and p16INK4a genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15INK4b and p16INK4a genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14ARF mRNA expression contained no detectable E1beta alteration but aberrations in the p16INK4a locus. Our results show that in human ESCCs, p14ARF is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.     

Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer

The CDKN2A locus on human chromosome 9p21 encodes two tumor suppressors, p14(ARF) and p16(INK4A), which enhance the growth-suppressive functions of the retinoblastoma (Rb) and the p53 proteins, respectively. Conversely, the E6 and E7 oncoproteins of the high-risk human papillomaviruses (HPVs) causally associated with carcinogenesis of the uterine cervix contributes to tumor development by inactivating p53 and Rb. Nevertheless, a correlation between expression of p14(ARF)/p16(INK4A) and HPV infection in uterine cervix is less clear. To clarify this, we examined 25 cervical cancers and 11 normal uterine cervixes. HPV was detected in 21 of 25 cervical cancers (84%) and their subtype was determined by PCR-RFLP. Quantitative real-time RT-PCR assays showed overexpression of p14(ARF) mRNA in all 21 HPV-positive cases (100%). p16(INK4A) mRNA was overexpressed in 17 cases of the HPV-positive cases (81%). In four HPV-negative cancers, reduced expression of p14(ARF) mRNA was detected in two cases (50%) and reduced p16(INK4A) mRNA in three cases (75%). Our data indicate that the overexpression of p14(ARF) and p16(INK4A) strongly associates with HPV-positive cervical cancers and that reduced expression of p14(ARF) and p16(INK4A) correlates with HPV-negative cervical cancers. These findings may indicate that impaired p14(ARF) and p16(INK4A) mRNA expression contribute to tumor development in HPV-negative cervical cancers by failure to support p53 and Rb instead of their inactivation by HPV E6 and E7.     

Association between INK4a-ARF and p53 mutations in skin carcinomas of xeroderma pigmentosum patients

BACKGROUND: The INK4a-ARF locus encodes two tumor suppressor proteins, p16(INK4a) and p14(ARF), that act through the Rb-CDK4 and p53 pathways, respectively. Data from murine models and sporadic human skin carcinomas implicate p16(INK4a) and p14(ARF) in the development of skin carcinomas. We examined the frequency of INK4a-ARF, p53, and CDK4 mutations in skin carcinomas from patients with xeroderma pigmentosum (XP), a rare autosomal disease that is associated with a defect in DNA repair and that predisposes patients to skin cancer. METHODS: DNA from skin cancers of 28 unrelated XP patients was screened for mutations in p53, INK4a-ARF, and CDK4 coding exons by single-strand conformation polymorphism analysis and automated sequencing. Data were evaluated with the use of the exact unconditional test derived from Fisher's test. All statistical tests were two-sided. RESULTS: Eight of 28 XP-associated tumors had mutations in the INK4a-ARF locus. Three XP-associated tumors had multiple mutations at this locus. In all, 13 mutations in the INK4a-ARF locus were detected in XP-associated tumors, of which seven (54%) were signature UV radiation-induced mutations, i.e., tandem CC : GG-->TT : AA transitions. p53 mutations, mostly of the type induced by UV radiation, were present in 12 tumors (43%). Statistically significant positive associations were found between the frequency of mutations in p53 and in p16(INK4a) (P =.008) and between the frequency of mutations in p53 and in p14(ARF) (P<.001). No mutations were detected within the CDK4 gene. CONCLUSIONS: We have demonstrated for the first time the occurrence of UV radiation-induced mutations in INK4a-ARF in XP-associated skin carcinomas. The simultaneous inactivation of p53 and INK4a-ARF may be linked to the genetic instability caused by XP and could be advantageous for tumor progression.     

Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancer

Inactivation of the P16 (INK4A)/retinoblastoma (RB) or TP53 biochemical pathway is frequent event in most human cancers. Recent evidence has shown that P14ARF binds to MDM2 leading to an increased availability of wild type TP53 protein. Functional studies also support a putative tumor suppressor gene function for p14ARF suggesting that p14ARF or p53 inactivation may be functionally equivalent in tumorigenesis. To study the relative contribution of each pathway in tumorigenesis, we analysed and compared alterations of the p16, p14ARF and p53 genes in 38 primary non-small cell lung cancers (NSCLCs) (19 adenocarcinomas and 19 squamous carcinoma). The p16 tumor suppressor gene was inactivated in 22 of 38 (58%) tumors. Twelve of these samples (31%) had homozygous deletions by microsatellite analysis; eight of them (21%) had p16 promoter hypermethylation detected by Methylation Specific PCR (MSP) and the remaining two (5%) harbored a point mutation in exon 2 by sequence analysis. The absence of P16 protein in every case was confirmed by immunohistochemistry. Fourteen of the 22 tumors with p16 inactivation also inactivated the p14ARF gene (12 with homozygous deletions extending into INK4a/ARF and two with exon 2 mutations). Mutations of p53 were found in 18 (47%) of the tumors and nine of them (50%) harbored p14ARF inactivation. Thus, an inverse correlation was not found between p14ARF and p53 genetic alterations (P=0.18; Fisher Exact Test). Our data confirm that the p16 gene is frequently inactivated in NSCLC. Assuming that 9p deletion occurs first, the common occurrence of p53 and p14ARF alterations suggests that p14ARF inactivation is not functionally equivalent to abrogation of the TP53 pathway by p53 mutation.     

Aberrations of the p14(ARF) and p16(INK4a) genes in renal cell carcinomas.

The INK4a / ARF locus on chromosome 9p21, which encodes two distinct genes, p14(ARF) and p16(INK4a), is frequently altered in human neoplasms. To investigate the potential roles of p14(ARF) and p16(INK4a) genes in human renal cell carcinomas (RCCs), we analyzed 6 human RCC cell lines and 91 primary RCCs for homozygous deletion, promoter hypermethylation and expression of the p14(ARF) and p16(INK4a) gene products using differential PCR, methylation-specific PCR, and immunohistochemistry, respectively. Five cell lines showed homozygous co-deletion of both genes and one demonstrated promoter hypermethylation of the p16(INK4a) gene only. Eight of 91 RCCs showed aberrations of p14(ARF) or p16(INK4a) status and six of these featured gross extension into the renal vein. The results suggest that p14(ARF) and p16(INK4a) aberrations may play roles in the relatively late stage of renal tumorigenesis associated with tumor progression.     

Individuals with presumably hereditary uveal melanoma do not harbour germline mutations in the coding regions of either the P16INK4A, P14ARF or cdk4 genes

In familial cutaneous malignant melanoma (CMM), disruption of the retinoblastoma (pRB) pathway frequently occurs through inactivating mutations in the p16 (p16INK4A/CDKN2A/MTS1) gene or activating mutations in the G1-specific cyclin dependent kinase 4 gene (CDK4). Uveal malignant melanoma (UMM) also occurs in a familial setting, or sometimes in association with familial or sporadic CMM. Molecular studies of sporadic UMM have revealed somatic deletions covering the INK4A-ARF locus (encoding P16INK4A and P14ARF) in a large proportion of tumours. We hypothesized that germline mutations in the p16INK4A, p14ARF or CDK4 genes might contribute to some cases of familial UMM, or to some cases of UMM associated with another melanoma. Out of 155 patients treated at the Institut Curie for UMM between 1994 and 1997, and interviewed about their personal and familial history of melanoma, we identified seven patients with a relative affected with UMM (n = 6) or CMM (n = 1), and two patients who have had, in addition to UMM, a personal history of second melanoma, UMM (n = 1), or CMM (n = 1). We screened by polymerase chain reaction single-strand conformation polymorphism the entire coding sequence of the INK4A-ARF locus (exon 1alpha from p16INK4A, exon 1beta from p14ARF, and exons 2 and 3, common to both genes), as well as the exons 2, 5 and 8 of the CDK4 gene, coding for the functional domains involved in p16 and/or cyclin D1 binding. A previously reported polymorphism in exon 3 of the INK4A-ARF locus was found in one patient affected with bilateral UMM, but no germline mutations were detected, either in the p16INK4A, p14ARF or CDK4 genes. Our data support the involvement of other genes in predisposition to uveal melanoma.     

Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines.

The tumor-suppressor genes p14(ARF), p16(INK4a) and Tp53 are commonly inactivated in many tumors. We investigated their role in the pathogenesis of 9 bile tract cancer cell lines and 21 primary sporadic extrahepatic bile duct carcinomas. p53 and p16 protein expression was examined by Western blot analysis and immunohistochemistry. Mutation screening of p53 was done by SSCP and direct sequencing. Inactivating mechanisms of p14 and p16 were addressed by screening for mutations, homozygous deletions, chromosomal loss of 9p21 (loss of heterozygosity [LOH] analysis) and promoter hypermethylation of the p14/p16 genes. p53 overexpression could be detected in 7 of 9 cell lines and 7 of 21 primary tumors, but mutations were found in 3 cell lines only. p16 expression was absent in all cell lines, due to homozygous deletion of the gene in 8 of 9 cell lines and hypermethylation of the p16 promoter in one cell line (CC-LP-1). p14 exon 1beta was homozygously deleted in 6 of 9 cell lines, while retained in CC-LP-1 and 2 additional lines. No p14 promoter hypermethylation could be detected. p16 expression was lost in 11 of 21 primary tumors. p16 promoter hypermethylation was present in 9 of 21 primary tumors, all with lost p16 expression. Allelic loss at 9p21 was detected in 13 of 21 primary tumors, 10 of 11 with lost p16 expression and 8 of 9 with methylated p16 promoter. No p14 promoter hypermethylation or p14/p16 mutations could be detected. Neither Tp53 nor p16 alterations showed obvious association with histopathologic or clinical characteristics. In conclusion, inactivation of the p16 gene is a frequent event in primary sporadic extrahepatic bile duct cancers, 9p21 LOH and promoter hypermethylation being the principal inactivating mechanisms. Therefore, p16, but not p14, seems to be the primary target of inactivation at the INK4a locus in bile duct cancers. Other mechanisms than Tp53 mutations seems to be predominantly responsible for stabilization of nuclear p53 protein in bile duct cancers.     

CDK4 and MDM2 gene alterations mainly occur in highly proliferative and aggressive mantle cell lymphomas with wild-type INK4a/ARF locus

Amplification of 12q13 locus occurs in some mantle cell lymphomas (MCL), potentially involving CDK4 and MDM2 genes. To determine the role of these genes in MCL, we have examined their gene status and expression and their relationship to INK4a/ARF and p53 gene aberrations in 69 tumors. Increased CDK4 gene copy number was detected in 4 of 19 (21%) highly proliferative blastoid variants and was associated with mRNA and protein overexpression. Three additional cases showed mRNA overexpression with no structural alterations of the gene. MDM2 gene overexpression was detected in three blastoid tumors (16%) with no relationship to gene copy gains. INK4a/ARF and p53 aberrations were observed in 13 and 12 tumors, respectively. Four of the seven lymphomas with CDK4 aberrations had concurrent inactivation of p53 gene, whereas only one case had a concomitant homozygous deletion of INK4a/ARF. No other gene alterations were found in the three cases with MDM2 overexpression. Patients with INK4a/ARF deletions or simultaneous aberrations of p53 and CDK4 had a significantly shorter median survival (17 months) than patients with isolated alterations of p53, MDM2, or CDK4 (32 months) and patients with no alterations in any of these genes (77 months). The prognostic impact of the concomitant oncogenic alterations of the p14ARF/p53 and p16INK4a/CDK4 pathways was independent of the proliferation of the tumors. These findings indicate that CDK4 and MDM2 gene alterations mainly occur in MCL with a wild-type INK4a/ARF locus and may contribute to the higher proliferation and more aggressive behavior of the tumors.     

Frequent alterations of the p14(ARF) and p16(INK4a) genes in primary central nervous system lymphomas

To elucidate the role of p53/p16(INK4a)/RB1 pathways in the tumorigenesis of primary central nervous system lymphomas (PCNSLs), we have analyzed p14(ARF), p16(INK4a), RB1, p21(Waf1), and p27(Kip1) status in a series of their 18 sporadic cases of diffuse large B-cell lymphoma, using methylation-specific PCR, differential PCR, and immunohistochemistry. Homozygous deletion or methylation of p14(ARF) was detected in 10 (56%) PCNSLs, and they were almost entirely deletions (except 1 case). A total of 11 (61%) PCNSLs demonstrated homozygous deletion (6 cases) or methylation (5 cases) of p16(INK4a). Six tumors showed both p14(ARF) and p16(INK4a) homozygous deletions. Hypermethylation of the RB1 and the p27(Kip1) promoter region was detected in 2 (11%) cases, whereas p21(Waf1) methylation was not detected in any. Immunohistochemistry revealed loss of p14(ARF) and p16(INK4a) expression in 10 (56%) samples, correlating with the gene status. Four cases showed independent negative immunoreactivity for pRB and p27(Kip1), and nearly one-half of cases (8 of 18; 44%) were characterized by lack of p21(Waf1) expression. These results indicate that inactivation of p14(ARF) and p16(INK4a) by either homozygous deletion or promoter hypermethylation represents an important molecular pathogenesis in PCNSLs. Hypermethylation of RB1, p21(Waf1), and p27(Kip1) appears to be of minor significance, these genes being independently methylated in PCNSLs.     

Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma.

The two gene products of the CDKN2A gene, p16 and p19ARF, have recently been linked to each of two major tumour suppressor pathways in human carcinogenesis, the RB1 pathway and the p53 pathway. p16 inhibits the phosphorylation of the retinoblastoma gene product by cyclin D-dependent kinases, whereas p19ARF targets MDM2, a p53 inhibitory protein, for degradation. A deletion of CDKN2A would therefore disturb both pathways. To explore the p53 pathway genes as a functional unit in diffuse large B cell non-Hodgkin's lymphomas (DLCL), we wanted to see whether there exists mutually exclusiveness of aberrations of CDKN2A, MDM2 and p53, since this has not been analysed previously. We investigated 37 DLCL for aberrations of p15, p16, p19ARF, MDM2, and p53 at the epigenetic, genetic and/or protein levels. Homozygous deletion of CDKN2A was detected in seven (19%) of 37 tumours, and another three cases were hypermethylated at the 5' CpG island of p16. No point mutations were found in CDKN2B or CDKN2A. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue for p16 confirmed these results, as all tumours with alterations of CDKN2A were p16 immunonegative. We found p53 mutations in eight (22%) cases and MDM2 overexpression in 16 (43%) tumours. Twenty-three (62%) tumours had alterations of one or more p53 pathway components (p53, p19ARF and MDM2). Furthermore, 7/9 (78%) p16-immunonegative tumours showed co-aberration of p53 and/or MDM2. The lack of correlation between these aberrations suggests that DLCL acquire additional growth advantage by inactivating both of these critical regulatory pathways.