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.     

P16INK4Gene Mutations Are Relatively Frequent in Ampullary Carcinomas

A high incidence of gene mutations or deletions of P16INK4, a cell cycle regulator which inhibits the activity of cyclin-dependent kinase 4/cyclin D complex and blocks the Gl-to-S transition, has been reported in pancreato-biliary tract cancers. In order to investigate P16INK4 gene alterations in sporadic ampullary carcinomas, 17 sporadic ampullary carcinomas were examined. After histological diagnosis, DNA samples extracted separately from both cancerous and normal paraffin-embedded tissues were investigated. Loss of heterozygosity (LOH) was investigated utilizing 3 microsatellite markers on 9p21-22, and a mutationsl analysis was performed by cloning and sequencing. LOH was observed in 3 cases (17.6%) and somatic mutations with retention of heterozygosity were found in 7 cases (41.2%). Of note was that two mutations resulted in truncated incomplete proteins and one was a point mutation at the consensus site in the conserved ankyrin repeats, which would be crucial for function. Although two-hit inactivation was not evident in any of the mutation cases and further investigation would be needed to elucidate the role of altered p16INK4 , these results suggest that the pl6INK4 gene mutations are relatively frequent and its inactivation might be important in ampnllary carcinogenesis.     

High prevalence of p16 genetic alterations in head and neck tumours

Inactivation of the p16 gene is believed to contribute to the tumorigenic process of several neoplasms, including head and neck tumours. In the present study, DNA samples from paired tumour and adjacent normal tissue from 47 patients with squamous cell carcinoma of the head and neck were investigated for the occurrence of p16 genetic alterations. Single-strand conformation polymorphism and direct DNA sequence analysis led to the identification of p16 mutations in six cases (13%). Southern blot analysis showed that homozygous deletion is a rare event in the group of tumours analysed. Loss of heterozygosity (LOH) analysis was performed by polymerase chain reaction (PCR) using two microsatellite markers (IFNA and D9S171) from the 9p21 region. Taking into account only the informative cases, 17 of 32 tumours (53%) showed LOH for at least one of the markers analysed. The methylation status of the CpG sites in the exon 1 of the p16 gene was analysed using methylation-sensitive restriction enzymes and PCR amplification. Hypermethylation was observed in 22 (47%) of the head and neck tumours analysed. In our series of head and neck tumours, evidence for inactivation of both p16 alleles was observed in 13 cases with hypermethylation and LOH, two cases with hypermethylation and mutation, four cases with mutation and LOH and one case with homozygous deletion. These findings provide further evidence that genetic alterations, especially hypermethylation and LOH, leading to the inactivation of the p16 tumour suppressor gene are common in primary head and neck tumours.     

p16 mutations/deletions are not frequent events in prostate cancer.

Cyclin-dependent kinase-4 inhibitor gene (p16INK4) has recently been mapped to chromosome 9p21. Homozygous deletions of this gene have been found at high frequency in cell lines derived from different types of tumours. These findings suggested therefore, that p16INK4 is a tumour-suppressor gene involved in a wide variety of human cancers. To investigate the frequency of p16INK mutations/deletions in prostate cancer, we screened 20 primary prostate tumours and four established cell lines by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis for exon 1 and exon 2. In contrast to most previous reports, no homozygous deletions were found in prostate cancer cell lines, but one cell line (DU145) has revealed to a mutation at codon 76. Only two SSCP shifts were detected in primary tumours: one of them corresponds to a mutation at codon 55 and the other one probably corresponds to a polymorphism. These data suggest that mutation of the p16INK4 gene is not a frequent genetic alteration implicated in prostate cancer development.     

CDKN2A mutation and deletion status in thin and thick primary melanoma.

Human melanoma cell lines and tumor tissue from familial and sporadic melanomas have frequent, nonrandom chromosomal breaks and deletions on chromosome 9p21, a region that includes the tumor suppressor gene CDKN2A/p16INK4A. Germ-line mutations within this gene have been observed in some familial melanoma kindreds, but somatic mutation in sporadic primary melanoma is infrequent. Thirty-nine archival, paraffin-embedded, sporadic, primary cutaneous malignant melanomas (20 >3-mm-thick and 19 <0.75-mm-thick cases) were examined for mutations of the CDKN2A gene using single-strand conformational polymorphism analysis and direct sequencing. No mutations were detected. Loss of heterozygosity for the 9p21 microsatellite marker D9S942 was detected in 6 of 17 informative thick lesions (35%) but 0 of 18 thin lesions (P = 0.006). These results support other studies indicating that intragenic mutation is an infrequent mechanism of CDKN2A inactivation in primary melanoma. The finding of loss of heterozygosity for the 9p21 microsatellite D9S942 in thick but not thin primary melanoma suggests that deletion or inactivation of CDKN2A or other tumor suppressor gene(s) at this locus is involved in the progression rather than initiation of sporadic malignant 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.     

肺癌组织9p21区域杂合性缺失和p16基因异常的分析

目的：研究肺癌组织９ｐ２１杂合性缺失和ｐ１６基因的变化。方法：４９例癌组织和相应的１６例转移性肺门淋巴结ＲＮＡ进行ＲＴ－ＰＣＲ和ＳＳＣＰ分析，对其中３８例肺癌组织ＤＮＡ进行杂合性缺失分析（９不７１和ＩＦＮＡ两个位点）。结果：９ｐ２１区域杂合笥缺失频率：４７．３％（１８／３８）。Ｄ９Ｓ１７１和ＩＦＮＡ位点杂合性缺失频率分别为：２６．３％（１０／３８）和３６．８％（１４／３８）。原发灶和转移性林巴结果     

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.     

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.     

Somatic INK4a-ARF locus mutations: a significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16(INK4a) (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14(ARF) tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G(1) arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14(ARF) genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1alpha, 1beta, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1alpha. No mutations were found in exon 1beta. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14(ARF) proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild-type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29- to 38-fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14(ARF) proteins, seems to be a prevalent event in the development of SCCHN. Mol. Carcinog. 30:26-36, 2001.     

Detection of p16 gene alteration in cervical cancer using tissue microdissection and LOH study

The p16 gene was identified as cyclin-dependent kinase inhibitor (CDKI) and this may negatively regulate the cell cycle by acting as a tumor suppressor. Using tissue microdissection, the molecular changes at p16 and Rb genes were analysed in the spectrum of disease from dysplasia to invasive cancer of the uterine cervix. Six of 27 (22%) cases informative for D9S171 and IFNA of 9p21-22 marker (p16INK4a) showed loss of one or both alleles in at least one of these loci. LOH of pRb was detected in 29% (5/17). Gene alterations at p16 and pRb loci were only detectable in some cases of HPV-16/18 DNA positive cervical cancer. Three cases demonstrated mutational changes of p16INK4a, and the alterations were determined to be G to T shift, suggesting transitional missense mutation. In summary, the inactivation of the p16/cdk-cyclin/Rb cascade may play an additional role during the malignant progression in HPV-16/18 positive cervical cancers.     

Deletion in p16INK4a and loss of p16 expression in human skin primary and metastatic melanoma cells

p16INK4a gene mapped at chromosome 9p21 region encodes a tumor suppressor protein p16 which is frequently inactivated in human cancers, including skin melanoma. In order to clarify the importance of p16 alterations in melanoma, we examined the deletions of p16INK4a and expression of p16 protein in eight unselected primary and metastatic melanoma cell lines from human skin melanomas. Normal skin melanocytes were used as controls. Deletions of entire exons in the p16INK4a gene were detected by PCR technique and expression of the p16 protein was examined by Western blotting and immunocytochemistry. Results showed that the fragments from exons 2A, 2C and 3 in p16INK4a gene were totally deleted in the metastatic melanoma cell line, FM28.7 and the fragment from exon 3 was deleted in the metastatic melanoma cell line, FM55M2. P16 protein was strongly expressed in two of the primary melanomas cell lines (FM55P and RaH3). The p16 protein was weakly expressed in one of the metastatic melanoma cell lines (FM55M1) and negative in the other metastasis (FM55M2) as compared to their matched primary melanoma cells (FM55P). The p16 protein was strongly expressed in normal skin melanocytes. Immunocytochemistry showed that p16 protein was mainly localized in the nuclei of the melanoma cells and normal melanocytes, if it was expressed. Deletions of p16INK4a gene was uncommon and loss of p16 protein expression was common event in melanoma, especially in the later stages of melanoma.     

The contribution of large genomic deletions at the CDKN2A locus to the burden of familial melanoma

Mutations in two genes encoding cell cycle regulatory proteins have been shown to cause familial cutaneous malignant melanoma (CMM). About 20% of melanoma-prone families bear a point mutation in the CDKN2A locus at 9p21, which encodes two unrelated proteins, p16(INK4a) and p14(ARF). Rare mutations in CDK4 have also been linked to the disease. Although the CDKN2A gene has been shown to be the major melanoma predisposing gene, there remains a significant proportion of melanoma kindreds linked to 9p21 in which germline mutations of CDKN2A have not been identified through direct exon sequencing. The purpose of this study was to assess the contribution of large rearrangements in CDKN2A to the disease in melanoma-prone families using multiplex ligation-dependent probe amplification. We examined 214 patients from independent pedigrees with at least two CMM cases. All had been tested for CDKN2A and CDK4 point mutation, and 47 were found positive. Among the remaining 167 negative patients, one carried a novel genomic deletion of CDKN2A exon 2. Overall, genomic deletions represented 2.1% of total mutations in this series (1 of 48), confirming that they explain a very small proportion of CMM susceptibility. In addition, we excluded a new gene on 9p21, KLHL9, as being a major CMM gene.     

Inactivation of the p16INK4A gene by methylation is not a frequent event in sporadic ovarian carcinoma

The p16INK4A tumour suppressor gene (p16) encodes for a cyclin-dependant kinase inhibitor which plays a role in the phosphorylation of the retinoblastoma protein (pRb) during regulation of the G1-S phase of the cell cycle. Loss of heterozygosity at 9p21, the chromosomal location of the p16 gene, has been reported in a broad range of tumours and this is usually indicative of the presence of a tumour suppressor gene, the second allele being frequently inactivated by mutation or deletion. The p16 gene, however, is often found not be mutated or deleted and it has been suggested that hypermethylation of the CpG islands of the gene may be an alternative mechanism of gene inactivation. We sought to determine the levels of p16 abnormality in a series of epithelial ovarian carcinomas in an attempt to clarify the presently conflicting evidence of whether or not hypermethylation of the p16 gene plays a role in ovarian carcinogenesis. We analysed 57 primary ovarian tumours and their corresponding blood DNA using SSCP analysis, sequencing and the methylation specific PCR (MSP) technique. We found low levels of mutation (6.7% of malignant tumours) and no evidence of methylation in any of our samples, suggesting that neither mutation or hypermethylation of the CpG islands of the p16 gene play an important role in ovarian carcinogenesis.     

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.     

Alterations of the p16(ink4a) gene in resected nonsmall cell lung tumors and exfoliated cells within sputum

Recently, we reported that p16 protein expression was nondetectable in 49.5% of 107 resected nonsmall cell lung cancers (NSCLCs), suggesting that the p16(INK4a) gene is frequently inactivated in primary NSCLC. To identify the molecular basis for this p16 immunohistochemical negativity further, we performed a genetic and epigenetic study of p16(INK4a) status in a series of 115 NSCLC samples parallel to the clinicopathologic and prognostic analyses. Microdissected tumor DNA samples were screened for homozygous deletion using comparative multiplex-polymerase chain reaction (PCR), for intragenic mutation using direct sequencing and for loss of heterozygosity (LOH) using an intragenic microsatellite marker, D9S942. Of these samples, 67 were further analyzed by SmaI-based PCR methylation assay to evaluate aberrant methylation at the gene. To examine the correlation of aberrant methylation in tumor and sputum samples, sputum samples from 12 matched patients were assessed for this change. We found that methylation of the p16(INK4a) gene was present in 38 of the 67 (56.7%) tumors and was significantly associated with negative p16 protein expression (p = 0.029). A 92% (11/12) concordance of sputum samples with matched resected tumors was found. The survival rates among adenocarcinoma patients with p16(INK4a) methylation were lower, but at a level of borderline significance compared with those patients without methylation (p = 0.071). In addition, 29.4% of the informative cases were found to harbor LOH at D9S942. None of the 115 microdissected tumors exhibited homozygous deletion in the p16(INK4a) gene. Only 1 patient exhibited a complex mutation at the fourth ankyrin repeat consensus sequence and concordantly demonstrated p16 immunohistochemical negativity. Overall, 69% (79/115) of NSCLC tumors had at least 1 type of p16(INK4a) alteration. Our data provide compelling evidence that p16(INK4a) alterations are involved in NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in p16(INK4a) deregulation.     

High frequency of homozygous deletion and methylation of p16(INK4A) gene in oral squamous cell carcinomas

p16(INK4A) inactivation was analyzed in ten squamous cell carcinoma (SCC) cell lines and 32 primary SCCs, using the polymerase chain reaction (PCR), PCR-single-strand conformation polymorphism, methylation-specific PCR, and cycle sequencing. In the study of cell lines, we detected three deletions in exon 1alpha and exon 2, and detected two methylations. Among tumor samples, we detected the homozygous deletions (HDs) of 43.8% in exon 1alpha 34.4% in exon 2, and methylation was found in 50.0%. The lack of p16(INK4A) with immunohistochemistry was detected in 71.9% and matched the alteration of p16(INK4A) gene. These results suggest that p16(INK4A) inactivation is predominantly caused by HD and methylation, and immunohistochemical evaluation of p16(INK4A) is a useful method.     

A melanoma-associated germline mutation in exon 1beta inactivates p14ARF

The INK4a/ARF locus encodes the cyclin dependent kinase inhibitor, p16(INK4a) and the p53 activator, p14ARF. These two proteins have an independent first exon (exon 1alpha and exon 1beta, respectively) but share exons 2 and 3 and are translated in different reading frames. Germline mutations in this locus are associated with melanoma susceptibility in 20-40% of multiple case melanoma families. Although most of these mutations specifically inactivate p16(INK4a), more than 40% of the INK4a/ARF alterations located in exon 2, affect both p16(INK4a) and p14ARF. We now report a 16 base pair exon 1beta germline insertion specifically altering p14ARF, but not p16(INK4a), in an individual with multiple primary melanomas. This mutant p14ARF, 60ins16, was restricted to the cytoplasm, did not stabilize p53 and was unable to arrest the growth of a p53 expressing melanoma cell line. This is the first example of an exon 1beta mutation that inactivates p14ARF, and thus implicates a role for this tumour suppressor in melanoma predisposition.