p16INK4 gene 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 G1-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 mutational 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 p16INK4 gene mutations are relatively frequent and its inactivation might be important in ampullary carcinogenesis.     

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.     

Loss of p16/INK4A protein expression in non-Hodgkin's lymphomas is a frequent finding associated with tumor progression

The CDKN2A gene located on chromosome region 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16/INK4A, a negative cell cycle regulator. We analyzed p16/INK4A expression in different types of non-Hodgkin's lymphoma to determine whether the absence of this protein is involved in lymphomagenesis, while also trying to characterize the genetic events underlying this p16/INK4A loss. To this end, we investigated the levels of p16/INK4A protein using immunohistochemical techniques in 153 cases of non-Hodgkin's lymphoma, using as reference the levels found in reactive lymphoid tissue. The existence of gene mutation, CpG island methylation, and allelic loss were investigated in a subset of 26 cases, using single-strand conformational polymorphism and direct sequencing, Southern Blot, polymerase chain reaction, and microsatellite analysis, respectively. Loss of p16/INK4A expression was detected in 41 of the 112 non-Hodgkin's lymphomas studied (37%), all of which corresponded to high-grade tumors. This loss of p16/INK4A was found more frequently in cases showing tumor progression from mucosa-associated lymphoid tissue low-grade lymphomas (31 of 37) or follicular lymphomas (4 of 4) into diffuse large B-cell lymphomas. Analysis of the status of the p16/INK4A gene showed different genetic alterations (methylation of the 5'-CpG island of the p16/INK4A gene, 6 of 23 cases; allelic loss at 9p21, 3 of 16 cases; and nonsense mutation, 1 of 26 cases). In all cases, these events were associated with loss of the p16/INK4A protein. No case that preserved protein expression contained any genetic change. Our results demonstrate that p16/INK4A loss of expression contributes to tumor progression in lymphomas. The most frequent genetic alterations found were 5'-CpG island methylation and allelic loss.     

Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells

CDKN2/p16 inhibits the cyclin D/cyclin-dependent kinase complexes that phosphorylate pRb, thus blocking cell cycle progression. We previously reported that p16 levels are low to undetectable in normal human uroepithelial cells (HUCs) and in immortalized uroepithelial cells with functional pRb, whereas p16 levels are markedly elevated in immortal HUCs with altered pRb (T. Yeager et al., Cancer Res., 55: 493-497, 1995). We now report that elevation of p16 levels occurs at senescence in HUCs, including HUCs transformed by human papillomavirus 16 E7 or E6, whose oncoprotein products lead to functional loss of pRb and p53, respectively. We also report that six of six independently immortalized E7 HUCs show high levels of p16 similar to those observed at HUC senescence, whereas p16 is undetectable in five of five immortal E6 HUCs. Four of the five independent E6 HUCs that lost p16 at immortalization showed hemizygous deletion of the 9p21 region. However, no homozygous CDKN2 deletions were detected, and only one CDKN2 mutation was identified. For the first time, these data associate elevated p16 with senescence in human epithelial cells. These data also suggest that a component of immortalization may be abrogation, either by pRb inactivation (as in the E7-transformed HUCs) or by p16 inactivation (as in the E6-transformed HUCs), of a p16-mediated senescence cell cycle block.     

The 9p21 region in bladder cancer cell lines: large homozygous deletion inactivate the CDKN2, CDKN2B and MTAP genes

Homozygous and hemizygous deletions of 9p21 are the earliest and most common genetic alteration in bladder cancer. The identification of two cell cycle regulators, CDKN2 and CDKN2B, that map to the common region of deletion has prompted the hypothesis that they are critical tumor suppressor genes in this malignancy. However, controversy as to whether these genes are the only or even the most important target in bladder cancer oncogenesis remains. To more clearly determine the effect of these 9p21 alterations, we mapped the homozygous deletions and performed a detailed mutational and expression analysis for CDKN2, CDKN2B and a closely linked gene, methylthioadenoside phosphorylase (MTAP), in 16 established bladder cancer cell lines. Nine of the 16 lines exhibit large (30 to > 2000 kb) homozygous deletions on 9p21. All deletions include at least one exon of CDKN2, eight of nine include CDKN2B, and six of nine include MTAP. MTAP function correlates with the genomic deletions. SSCP and sequence analysis does not reveal any inactivating point mutations of CDKN2 or of CDKN2B in any of the cell lines without homozygous deletions, and all express the CDKN2 and the CDKN2B mRNA as well as the encoded p16 protein. The p16 protein levels vary widely and are correlated with absent pRb expression. We conclude that the 9p21 deletions in bladder cancer usually inactivate the CDKN2. CDKN2B, and MTAP genes but that CDKN2 is the most common target. Other mechanisms for inactivating this gene in bladder cancer appear to be uncommon.     

Deletions and loss of expression of p16INK4a and p21Waf1 genes are associated with aggressive variants of mantle cell lymphomas

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1 gene overexpression. Some aggressive variants of MCL have been described with blastic or large cell morphology, higher proliferative activity, and shorter survival. The cyclin-dependent kinase inhibitors (CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of p21Waf1 and p16INK4a gene alterations in MCLs, we examined the expression, deletions, and mutations of these genes in a series of 24 MCLs, 18 typical, and 6 aggressive variants. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes were detected in 4 (67%) aggressive MCLs but in none of the typical variants. Two aggressive MCLs showed a loss of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1 expression at both protein and mRNA levels was detected in an additional aggressive MCL. No p21Waf1 gene deletions or mutations were found in this case. The p21Waf1 expression in MCLs was independent of p53 mutations. The two cases with p53 mutations showed p21Waf1 and p16INK4a expression whereas the 4 aggressive MCLs with p16INK4a and p21Waf1 gene alterations had a wild-type p53. p21Waf1 and p16INK4a were expressed at mRNA and protein levels in all typical MCLs examined. No gene deletions or point mutations were found in typical variants. Two typical MCLs showed an anomalous single-stranded conformation polymorphism corresponding to the known polymorphisms at codon 148 of p16INK4a gene and codon 31 of p21Waf1 gene. These findings indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and deletions of p16INK4a gene are associated with aggressive variants of MCLs, and they occur in a subset of tumors with a wild-type p53 gene.     

High frequency of p16INK4A gene alterations in hepatocellular carcinoma

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. In order to investigate the role of p16 gene in the tumorigenesis of hepatocellular carcinoma (HCC), 48 cases of HCC were analysed for p16 alterations by: methylation-specific PCR (MSP) to determine the methylation status of the p16 promoter region; comparative multiplex PCR to detect homozygous deletion; PCR-SSCP and DNA sequencing analysis to identify mutation of the p16 gene. We found high frequency of hypermethylation of the 5' CpG island of the p16 gene in 30 of 48 cases (62.5%) of HCC tumors. Moreover, homozygous deletion at p16 region were present in five of 48 cases (10.4%); and missense mutation were detected in three of 48 cases (6.3%). The overall frequency of p16 alterations, including homozygous deletion, mutation and hypermethylation, in HCC tumors was 70.8% (34 of 48 cases). These findings suggest that: (a) the inactivation of the p16 is a frequent event in HCC; (b) the p16 gene is inactivated by multiple mechanisms including homozygous deletion, promoter hypermethylation and point mutation; (c) the most common somatic alteration of the p16 gene in HCC is de novo hypermethylation of the 5' CpG island; and (d) in contrast to other studies, high frequency of genomic alterations are not uncommon in the 9p21 of the p16 gene. Our results strongly suggest that the p16 gene plays an important role in the pathogenesis of HCC.     

Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in primary non-small cell lung cancers (NSCLC)

Homozygous deletions of the tumor suppressor gene p16INK4A and deficiency of methylthioadenosine phosphorylase (MTAP), both located on chromosome 9p21, have been independently reported in non-small cell lung cancer (NSCLC). To determine the frequency of co-deletion of these two genes, we investigated 50 samples of primary NSCLC using a quantitative PCR-ELISA. All specimens were fixed in formalin, paraffin embedded and stored until assayed. Histologic subtypes included 25 adenocarcinomas (50%), 21 squamous cell carcinomas (42%) and four large cell carcinomas (8%). Homozygous deletions of MTAP exon 8 could be detected in 19 of 50 NSCLC samples (38%). Adenocarcinoma (11 of 25, 44%) showed a higher deletion frequency than squamous cell carcinoma (six of 21, 29%). In contrast, homozygous p16INK4A deletions were detected in only nine of 50 (18%) samples using specific primers for p16INK4A exon 1alpha. No difference between the histological subtypes and p16INK4A deletion frequency was observed. We further investigated the ten samples with MTAP deletions but intact p16INK4A exon 1alpha with primers specific for p16INK4A exon 3, the exon nearest to MTAP exon 8. Interestingly, none of the ten samples had deletion of the p16INK4A exon 3 coding region. Fine mapping analysis performed in ten samples showed a frequent breakpoint between MTAP exon 4 and exon 5. In addition, p16 protein expression could not be detected in five out of six samples with intact p16 but deleted MTAP locus. These data show a high frequency of homozygous MTAP deletions in NSCLC which is associated with detectable co-deletion of p16INK4A in only half of the cases. This result suggests the existence either of another tumor suppressor gene telomeric of p16INK4A or of deletions involving 3'-untranslated (3'-UTR) regulatory regions of p16INK4A that can interfere with its expression or function.     

Mutations in the p16INK4/MTS1/CDKN2, p15INK4B/MTS2, and p18 genes in primary and metastatic lung cancer

We examined the genomic status of cyclin-dependent kinase-4 and -6 inhibitors, p16INK4,p15INK4B, and p18, in 40 primary lung cancers and 31 metastatic lung cancers. Alterations of the p16INK4 gene were detected in 6 (2 insertions and 4 homozygous deletions) of 22 metastatic non-small cell lung cancers (NSCLCs; 27%), but none were detected in 25 primary NSCLCs, 15 primary small cell lung cancers (SCLCs), or 9 metastatic SCLCs, indicating that mutation in the p16INK4 gene is a late event in NSCLC carcinogenesis. Although three intragenic mutations of the p15INK4B gene were detected in 25 primary NSCLCs (12%) and five homozygous deletions of the p15INK4B gene were detected in 22 NSCLCs (23%), no genetic alterations of the p15INK4B gene were found in primary and metastatic SCLCs. The p18 gene was wild type in these 71 lung cancers, except 1 metastatic NSCLC which showed loss of heterozygosity. We also examined alterations of these three genes and expression of p16INK4 in 21 human lung cancer cell lines. Alterations of the p16INK4 and p15INK4B genes were detected in 71% of the NSCLC cell lines (n = 14) and 50% of the NSCLC cell lines (n = 14), respectively, but there were none in the 7 SCLC cell lines studied. No p18 mutations were detected in these 21 cell lines. These results indicate that both p16INK4 and p15INK4B gene mutations are associated with tumor progression of a subset of NSCLC, but not of SCLC, and that p15INK4B mutations might also be an early event in the molecular pathogenesis of a subset of NSCLC.     

Early acquisition of homozygous deletions of p16/p19 during squamous cell carcinogenesis and genetic mosaicism in bladder cancer

We looked for p16/p19 deletion and p16 promoter methylation, as well as loss of 9p21 heterozygosity in pure squamous cell carcinomas (SCC), and in transitional cell carcinomas (TCC) of the bladder with SCC components. Homozygous deletion of p16/p19 was detected in 11 of 21 (52%) cases of pure SCCs and in three of ten (30%) cases of TCC with SCC. Three cases of TCC with SCC had p16/p19 deletion, hypermethylation of the p16 promoter, or LOH on 9p21 only in the SCC components, suggesting that these molecular alterations occurred preferentially in SCC. Interestingly, homozygous deletion of p16/p19 was observed in squamous metaplasia from bladder cancer patients (five of 11, 45%), showing that this change occurred in preneoplastic cells. On the other hand, p16/p19 deletions were not found in squamous metaplasias from non cancerous patients. Hypermethylation of the p16 promoter was observed in two of 14 tumors (14%) and none of seven metaplasias examined. These data suggest that: (a) p16/p19 deletion is associated with early carcinogenesis of SCC of the bladder, and squamous metaplasia of the bladder cancer patient has already sustained genetic changes found in cancer, and (b) genetic mosaicism occurs in cases of TCC with SCC, with the SCC component showing more frequent 9p21 alterations than the TCC component.     

Loss of heterozygosity and mutation analysis of the p16 (9p21) and p53 (17p13) genes in squamous cell carcinoma of the head and neck

We analyzed allelic loss at the p53 gene (17p13) and at chromosome region 9p21 in 35 primary head and neck squamous cell carcinomas. Loss of heterozygosity (LOH) at p53 and 9p21 was found in 50 and 75% of informative cases, respectively. LOH at the p53 gene did not increase significantly with tumor stage, but was more frequent in moderately and poorly differentiated tumors than in well-differentiated tumors. LOH plus mutation or homozygous deletion of p53 was limited to advanced stage and poorly differentiated tumors. Allelic loss at 9p21 is frequent in early stage head and neck squamous cell carcinoma and is not significantly associated with LOH at p53. The second exon of the p16/MTS1/CDKN2 gene was found to be homozygously deleted in 1 of 19 cases showing LOH at 9p21, but direct sequencing did not show mutations in the remaining 18 cases. This suggests that p16 plays a limited role in the development of head and neck squamous cell carcinoma.     

Contact allergy in children

We examined the genomic status of the p16INK4A (inhibitor of cyclin-dependent kinase 4 A) and cyclin-dependent kinase 4 (CDK4) genes in 62 human hepatocellular carcinomas (HCCs), 5 cholangiocellular carcinomas and 6 cell lines derived from human liver cancers. Although no samples showed the homozygous deletion of the p16INK4A gene, we detected intragenic mutations of the p16INK4A gene in 3 HCCs and one HCC cell line, which led to an amino-acid substitution or a frameshift. In 2 HCC samples with mis-sense mutations of the p16INK4A gene, loss of heterozygosity on 9p22 was also detected, suggesting that the loss of function of p16 was induced during hepatocarcinogenesis. On the other hand, amplification or rearrangement of the CDK4 gene was not detected in any samples examined in this study. These results indicated that the mutations or deletions of the p16INK4A gene are not frequent, but may play a role in a sub-set of human HCC.     

Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis

It is still unclear whether the sporadic form of dysplastic nevi (SDN) represents a premalignant lesion of malignant melanoma and whether genetic alterations are involved in the development of SDN. To determine whether p16INK4a and p53 genetic abnormalities could be associated with development of SDN, nevus cell nests were procured selectively from H & E-stained slide sections by using a modified microdissection technique and were screened for the presence of mutations and loss of heterozygosity (LOH) of p16INK4a and p53 genes using a polymerase chain reaction-based LOH, single-strand conformation polymorphism, and direct DNA sequencing analyses. Hemizygous deletion was detected in 9 of 12 informative cases (75%) for 9p21-22 (p16INK4a) at one or more loci and 60% (6/10) for 17p13 (p53). As for mutation, we found 3 missense mutations and 1 mutation in the first intron in p16INK4a and 2 missense mutations in p53. Among these mutations in p16INK4a and p53, 5 of 6 mutations were of the C:G to T:A transitional type; this is known to be related to ultraviolet radiation as previously confirmed in other skin cancers. This indicates that p16INK4a and p53 genetic alterations may play an important role in the evolution of SDN and may represent an early event in the development of malignant melanoma. Furthermore, ultraviolet radiation might be the predominant etiologic agent in the development of SDN.     

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 (p16INK4A, MTS1) 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 sequence. 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.     

p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas

The molecular mechanisms underlying the pathogenesis of aggressive lymphomas and the histological transformation of indolent variants are not well known. To determine the role of p16(INK4a) gene alterations in the pathogenesis of non-Hodgkin's lymphomas (NHLs) and the histological progression of indolent variants, we have analyzed the expression, deletions, and mutations of this gene in a series of 112 NHLs. Hypermethylation of the gene was also examined in a subset of tumors with lack of protein expression but without mutations or deletions of the gene. p16(INK4a) gene alterations were detected in 3 out of 64 (5%) indolent lymphomas but in 16 out of 48 (33%) primary or transformed aggressive variants. In the low-grade tumors, p16(INK4a) alterations were detected in 1 (4%) chronic lymphocytic leukemia (hemizygous missense mutation), 1 (6%) follicular lymphoma (homozygous deletion), and 1 (5%) typical mantle cell lymphoma (homozygous deletion). The two later cases followed an aggressive clinical evolution. In the aggressive tumors, p16(INK4a) gene alterations were observed in 2 (29%) Richter's syndromes (2 homozygous deletions), 3 (33%) transformed follicular lymphomas (1 homozygous deletion and 2 nonsense mutations), 3 (43%) blastoid mantle cell lymphomas (2 homozygous and 1 hemizygous deletions), 5 (28%) de novo large-cell lymphomas (1 homozygous deletion and 4 hypermethylations), 2 lymphoblastic lymphomas (2 homozygous deletions), and 1 of 2 anaplastic large cell lymphomas (hypermethylation). Protein expression was lost in all tumors with p16(INK4a) alterations except in the typical chronic lymphocytic leukemia (CLL) with hemizygous point mutation. Sequential samples of the indolent and transformed phase of three cases showed the presence of p16(INK4a) deletions in the Richter's syndrome but not in the CLL component of two cases, whereas in a follicular lymphoma the deletion was present in both the follicular tumor and in the diffuse large-cell lymphoma. In conclusion, these findings indicate that p16(INK4a) gene alterations are a relatively infrequent phenomenon in NHLs. However, deletions, mutations, and hypermethylation of the gene with loss of protein expression are associated with aggressive tumors and they may also participate in the histological progression of indolent lymphomas.