CDKN2 Gene Research Articles

Genetic analysis of susceptibility to spontaneous and UV-induced carcinogenesis in Xiphophorus hybrid fish.

Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC(1)) hybrids produced from backcrossing F(1) hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC(1) hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC(1) hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC(1) hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianus CDKN2X alleles in BC(1) hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC(1) hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

Molecular and cytogenetic analysis of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary tumor occurring in the central nervous system of adults. Although progress has been made in clinical management of this tumor, little is known about the molecular defects underlying the initiation and progression of GBM. To address these issues, we have characterized five cases of GBM using cytogenetics, comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and direct sequencing. All of these tumors were observed to have clonal chromosome aberrations. Complicated chromosome translocations including der(18)t(2;4;12;18), der(X)t(X;10)(q27.1;p12.1) and der(10)t(10;15)(p11.23;q11.2), and der(1) (:1p31→1q44::7q11.3→7qter) were seen in three tumors. Loss of the CDKN2 gene was noted in four tumors. A gain of copy number of the Cathepsin L gene was seen in two tumors. Amplification of the CDK4, MDM2, and GLI/CHOP genes was noted in two tumors, and amplification of the PDGFR gene was detected in one tumor. Mutation of exon 5 of the TP53 gene was found in three tumors. No mutation of the BCL10 gene was detected in five cases of GBM analyzed, although deletion of chromosome 1p was seen in two tumors. These results provide information for further investigation of GBM.

Overexpression of a fish CDKN2 gene in a hereditary melanoma model.

The fish genus Xiphophorus provides a vertebrate model useful in etiological studies of cancer. Hybrid fish can spontaneously develop melanomas deriving from the inheritance of melanistic pigment patterns and the simultaneous absence of proper genetic regulation. A cyclin-dependent kinase inhibitor gene, termed CDKN2X, was mapped to a genomic region that is implicated in fish melanoma tumor suppression. The related human tumor suppressor locus CDKN2A (P16, INK4A, MTS1) is deleted, mutated or transcriptionally repressed through methylation of cytosine bases within the 5' CpG island in a variety of neoplasms, including melanoma. The fish CDKN2X locus harbors a CpG island within its promoter and first exon, analogous in location to CpG islands in human CDKN2A and CDKN2B loci. The methylation state of individual CpG dinucleotides was investigated in genomic DNA derived from control tissues and melanomas within the CDKN2X 5' CpG island. The studied genomic area was found to be virtually unmethylated in all tested tissues including melanomas. In addition, RNA expression studies of the fish CDKN2X locus revealed that it is significantly overexpressed in melanoma, in contrast to what has been reported for the human CDKN2A locus in melanoma. Such overexpression may be a consequence of the pronounced upregulation of the Xmrk-2 receptor tyrosine kinase oncogene reported in several Xiphophorus melanoma models.

The role of p16-cyclin d/CDK-pRb pathway in the tumorigenesis of endometrioid-type endometrial carcinoma.

We analysed p16 gene alteration and p16, cyclin-dependent kinase 4 (CDK4), CDK6, cyclin D1, cyclin D2, cyclin D3 and retinoblastoma protein (pRb) expression in ten normal endometriums (PE), 18 endometrial hyperplasias (EH) and 35 endometrial cancers (EC). Two of ten PE (20%), nine of 18 EH (50.0%) and 29 of 35 EC (82.9%) exhibited p16 nuclear staining. p16 expression was significantly higher in EC than EH (P = 0.0119). In the six p16 (–) EC, one was considered to have reduced gene dosage consistent with possible homozygous deletion of the CDKN2 gene and three had methylation in 5′CpG island in the promoter region of the p16 gene, whereas none showed such reduced gene dosage and four had methylation in the nine p16 (–) EH. Strong CDK4 staining was observed in 12 of 35 EC (34.3%) and one of 18 EH (5.6%). The strong expression of CDK4 was higher in EC than in EH (P = 0.0399). The expression of CDK4 was higher in EH than PE (P = 0.0054). The abnormalities of p16-cyclin D/CDK-pRb pathway were detected in 18 of 35 EC (51.4%). In conclusion, the expression of p16 and CDK4 may be an early event in the neoplastic transformation of endometrial cancer. © 2000 Cancer Research Campaign

Homozygous deletion of the p16/MTS-1/CDKN2 gene in malignant gliomas is infrequent among Japanese patients.

We have analyzed the status of the p16/MST-1/CDKN2 gene in 63 brain tumors from Japanese patients. With quantitative multiplex polymerase chain reaction (PCR) assay using the exon 2 primers of the p16 gene and control chromosome 9qSTS primers, we found homozygous deletion of the p16 gene in 7 cases; in 1 out of 10 cases of anaplastic astrocytomas (WHO grade III), 6 out of 35 cases of glioblastoma multiformes (grade IV) but in none of the tumors of grade I or II. We also found mobility-shifted PCR products in 8 cases using the single-strand conformation polymorphism technique. DNA sequencing of the aberrantly migrated products revealed that 5 cases of glioblastoma multiforme had mutations which caused amino acid substitutions. We found one case with silent mutations and two cases with nucleotide changes in the non-coding region. The frequency of the alteration of the p16 gene, either homozygous deletion or mutation accompanied with amino acid substitutions, increased in malignant brain tumors (grade III and IV) compared with that in low grade tumors (grade I and II) (p=0.0275), suggesting possible role(s) of the gene in the progression of brain tumors. In addition, the low frequency of homozygous deletions shown in this study is quite different from previous reports that demonstrated frequently deleted p16 gene in malignant gliomas from Caucasian patients. We have also shown the presence of heterogeneous cell populations within the glioblastoma masses based on the variety of the mutated p16 sequences. The present study, therefore, suggests a possible racial difference in the mechanism of the tumorigenesis and a heterogeneity of malignant gliomas developed during the tumor progression.

Genetic abnormalities involved in the pathogenesis of gallbladder carcinoma.

While considerable progress has been made in the understanding of the genetic changes involved in the pathogenesis of several human neoplasms, there is limited information about the genetic changes involved in the development of gallbladder carcinoma. Several studies indicate that TP53 (17p13) and p16(Ink4)/CDKN2 (9p21-22) gene loci abnormalities are frequent and early events in the pathogenesis of this neoplasm, in some cases preceding the onset of histological changes of invasion. Preliminary data also suggest that deletions at other chromosomal regions (8p21 and DCC at 18q21 loci) may play an important role in the development of gallbladder carcinoma; however, they need to be further analyzed. K-ras gene mutations appear to be an infrequent event in this neoplasm, except in gallbladder carcinomas associated with congenital abnormalities of the biliary tract. Genetic studies confirm that the sequence dysplasia-carcinoma in situ (CIS) is the usual route for the development of gallbladder carcinoma, and our recent data strongly suggest that adenomas are not precursors of this neoplasm.

Partial allelotype of schistosomiasis-associated bladder cancer.

In Egypt and other regions of the Middle East where the trematode Schistosoma haematobium is endemic, bladder cancer is the most common adult cancer. Unlike bladder cancers in Western countries, which are predominantly transitional-cell carcinoma (TCC), these schistosomiasis-associated bladder cancers are predominantly squamous-cell carcinoma (SCC). Our aim was to assess a large series of schistosomiasis-associated bladder tumours for genetic alterations commonly found in TCC in the United Kingdom and the United States. We have carried out a partial allelotype of 70 tumours from patients with schistosomiasis. LOH was found on all chromosome arms studied (3p, 4p, 4q, 8p, 9p, 9q, 11p, 11q, 13q, 14q, 17p, 18q). The most frequent regions of LOH were 9p (65%), 17p (58%), 3p (40%), 9q (39%) and 8p (37%). LOH on 17p, where the TP53 gene is located, was more common in Egyptian TCC than in SCC. Similarly, 8p LOH was more common in TCC than SCC. The most striking difference between this group of tumours and TCCs from the United Kingdom and the United States was the high frequency of 9p LOH in the region of the CDKN2 gene (65%) and the relatively low frequency of 9q LOH (39%); 15 of 43 tumours with LOH of at least one marker on chromosome 9 showed LOH of 9p only. This suggests that a 9p gene, possibly CDKN2, may contribute to the development of the majority of schistosomiasis-associated bladder tumours but that genes on 9q play a much less important role.

Genetic aberrations in glioblastoma multiforme: translocation of chromosome 10 in an O-2A-like cell line.

We have examined the genetic aberrations in two near-diploid glioblastoma multiforme cell lines that appear to have arisen from different glial lineages. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This line generates, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. In Hu-O-2A/Gb1 the sole homologue of chromosome 10 is disrupted at band 10p11–12.1 by translocation with chromosomes X and 15. The translocation breakpoint is localized between genetic markers D10S2103 and [D10S637, D10S1962, D10S355]. Other aberrations include a 5;14 translocation, deletion of the long and short arms of chromosome 16 and loss of one copy of the CDKN2 gene. IN1434 cells share some cytogenetic abnormalities with Hu-O-2A/Gb1 cells, despite their apparent derivation from a different biological origin, but also have translocations involving the long and short arms of chromosome 1 and the long arm of chromosome 7, and deletion of chromosome 13 at bands 13q12–21. © 1999 Cancer Research Campaign

P53 and p16/CDKN2 gene mutations in esophageal tumors from a high-incidence area in South Africa.

Squamous cell carcinoma of the esophagus has an uneven geographic distribution, with a high incidence in the Transkei region of Eastern Cape Province, South Africa. The precise molecular events associated with tumorigenesis of esophageal cancer in this region have not been characterized. DNA from human esophageal squamous cell carcinomas (n = 76), as well as adjacent tissue samples (n = 9) and blood (n = 50) from the same patients from the Transkei region were screened for somatic mutations. Exons 5-8 of the p53 gene and exons 1-2 of the p16/CDKN2 gene were examined for mutations using PCR-SSCP procedures and DNA sequence analysis. Results show that 17% of the tumors contained small deletions, insertions and point mutations, resulting in frameshifts or amino acid changes in the p53 gene. Among the mutations in the structural p16/CDKN2 gene, 9 were point mutations, 4 were deletions and 3 were insertions. A novel C to T mutation, 25 bp upstream from the ATG start site of p16/CDKN2, which sometimes occurs together with other structural gene variations, was found. The mutations described here are somatic in origin since none of the DNA samples from the adjacent control tissues or blood samples from the same patients had them.

Allelotype analysis of oesophageal adenocarcinoma: loss of heterozygosity occurs at multiple sites.

Deletions of tumour-suppressor genes can be detected by loss of heterozygosity (LOH) studies, which were performed on 23 cases of adenocarcinoma of the oesophagus, using 120 microsatellite primers covering all non-acrocentric autosomal chromosome arms. The chromosomal arms most frequently demonstrating LOH were 3p (64% of tumours), 5q (45%), 9p (52%), 11p (61%), 13q (50%), 17p (96%), 17q (55%) and 18q (70%). LOH on 3p, 9p, 13q, 17p and 18q occurred mainly within the loci of the VHL, CDKN2, Rb, TP53 and DCC tumour-suppressor genes respectively. LOH on 5q occurred at the sites of the MSH3 mismatch repair gene and the APC tumour-suppressor gene. 11p15.5 and 17q25-qter represented areas of greatest LOH on chromosomes 11p and 17q, and are putative sites of novel tumour-suppressor genes. LOH on 9p was significantly associated with LOH on 5q, and tumours demonstrating LOH at both the CDKN2 (9p21) and MSH3 (5q11-q12) genes had a significantly higher fractional allele loss than those retaining heterozygosity at these sites. Six of nine carcinomas displaying microsatellite alterations also demonstrated LOH at CDKN2, which may be associated with widespread genomic instability. Overall, there are nine sites of LOH associated with oesophageal adenocarcinoma.

Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor

Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor

Localization of aCDKN2 gene in linkage group V ofXiphophorus fishes defines it as a candidate for theDIFF tumor suppressor

The Xiphophorus hybrid melanoma model represents one of the earliest reported cases of genetically regulated tumor susceptibility. Melanoma formation in Xiphophorus hybrids may be explained by the inheritance of two genes: a sex-linked oncogene, Xmrk, and a putative tumor suppressor locus, termed DIFF, located in Linkage Group V (LG V). Several genetic mapping procedures were used to produce a new Xiphophorus LG V map with 20 loci. All markers, particularly a recently cloned Xiphophorus CDKN2 gene family member, called CDKN2X, were tested for associations of genotype with degree of macromelanophore pigment pattern modification and susceptibility to melanoma formation in backcross hybrids of seven genetic types, involving 1,110 fish and three pigment patterns. Highly significant associations of CDKN2X genotypes with such phenotypic effects suggests that this gene is a strong candidate for the classically defined DIFF tumor suppressor gene. Because published results have documented the involvement of the CDKN2A (p16, MTS1, and INK4A) tumor suppressor gene in human melanoma formation, the possibility of CDKN2 genes acting as tumor suppressors in both man and Xiphophorus is likely.

Mutational analysis of the P16-binding domain of cyclin-dependent kinase 4 in tumors in the head region of the pancreas.

Alterations in genes involved in cell cycle regulation are common in many tumor types. In pancreatic adenocarcinomas, inactivating mutations in the CDKN2 gene, encoding the cyclin-dependent kinase inhibitor p16, are frequently observed. CDKN2 mutations have also been identified in the germline of 50% of patients with hereditary melanoma. Interestingly, such patients also have an increased risk for pancreatic cancers. In melanoma-prone kindreds with CDKN2 wild-type status, a mutation in one of the targets of p16, cyclin-dependent kinase 4 (CDK4) was reported, which abolishes p16 inhibition. To test the possible involvement of CDK4 mutations in pancreatic carcinoma, we analyzed sequence alterations in the p16-binding domain of CDK4 in DNA isolated from 32 tumors in the head region of the pancreas. Alterations in the CDK4 region between codon 1 and codon 56 were not observed in any of the tumors. Our results do not support disruption of the p16 pathway through CDK4 mutation as an oncogenic mechanism in pancreatic head tumorigenesis.

Inactivation of the von Hippel–Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: Evidence for aVHL-independent pathway in clear cell renal tumourigenesis

Inactivation of tumour suppressor gene(s) (TSGs) on 3p appears to be a critical event in the pathogenesis of clear cell renal cell carcinoma (CC-RCC). Analysis of loss of heterozygosity (LOH) in sporadic RCC samples has implicated roles for TSGs in three specific regions of 3p in RCC development: (1) 3p12-p14, which includes the breakpoint of the familial t(3;8) constitutional translocation involved in hereditary RCC development and a recently cloned putative TSG, the FHIT gene: (2) 3p21.2-p21.3, a common region of deletion in many cancers including lung; and (3) 3p25-p26, which contains the von Hippel-Lindau (VHL) disease TSG. We and others have shown that most primary sporadic CC-RCCs contain somatic VHL gene mutations, clearly implicating inactivation of the VHL gene in the pathogenesis of CC-RCC. It is not known if CC-RCC without VHL gene mutations have alternative mechanisms of VHL gene inactivation or result from an alternative non-VHL pathway to RCC, e.g., inactivation of TSGs in 3p12-p21. We and others have reported hypermethylation and silencing of the VHL TSG in RCC from patients with VHL disease and in CC-RCC cell lines. However, the incidence and specificity of VHL methylation in primary sporadic RCC has not been defined. Therefore, we analysed methylation of the VHL, CDKN2, MYC, and H19 genes in primary RCC samples. Hypermethylation of the VHL promoter region was detected in 11% (11/99) of the primary RCCs analysed. In 10 of these tumours, there was no evidence of concomitant VHL gene mutation. VHL methylation was specific to CC-RCC (15%, 7/45) but was not detected in any non-CC tumours (n = 16). None of the 11 RCCs methylated at VHL had evidence of methylation at either CDKN2 or MYC (methylation at CDKN2 was, however, detected in 3%, or 1/33, of RCCs without VHL methylation). A normal methylation pattern at H19 was demonstrated in the three RCCs with methylated VHL analysed. Previous studies have suggested that, in addition to VHL, other 3p TSGs at 3p12-p14 and 3p21 may be involved in CC-RCC tumourigenesis. However, the interpretation of these studies has been difficult because information on VHL gene status has not been available for these data sets. Therefore, we investigated a subset of 55 sporadic RCCs (of known VHL gene methylation and mutation status) for LOH at polymorphic markers close to candidate TSG loci in the 3p14.2 and 3p21.2-p21.3 regions. Among tumours with LOH at one or more 3p markers, the incidence of 3p25 allele loss was higher in tumours with VHL alterations (mutation or methylation) than in those without. For tumours without detectable VHL alterations, the frequency of 3p14-p21 LOH was significantly higher than the frequency of 3p25-p26 LOH (93%, 13/14 vs. 43%, 6/14; P = 0.013), whereas, in RCC samples with VHL methylation or mutation, the frequency of 3p14-p21 LOH did not differ from that of sp25-p26 (72%, 18/25 vs. 59%, 13/22; P = 0.376). None of the 11 RCCs with 3p25 allele loss that were informative at 3p21 and 3p14 showed LOH at 3p25 only. These findings suggest that (1) VHL methylation is a specific and important event in the pathogenesis of CC-RCC; (2) in CC-RCC with 3p LOH but without VHL inactivation, mutations in TSGs at 3p14-p21 appear to have a primary role in tumourigenesis; and (3) inactivation of other 3p TSGs in addition to VHL may also be required for malignant transformation in tumours with VHL gene inactivation.

Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: evidence for a VHL-independent pathway in clear cell renal tumourigenesis.

Inactivation of tumour suppressor gene(s) (TSGs) on 3p appears to be a critical event in the pathogenesis of clear cell renal cell carcinoma (CC-RCC). Analysis of loss of heterozygosity (LOH) in sporadic RCC samples has implicated roles for TSGs in three specific regions of 3p in RCC development: (1) 3p12-p14, which includes the breakpoint of the familial t(3;8) constitutional translocation involved in hereditary RCC development and a recently cloned putative TSG, the FHIT gene: (2) 3p21.2-p21.3, a common region of deletion in many cancers including lung; and (3) 3p25-p26, which contains the von Hippel-Lindau (VHL) disease TSG. We and others have shown that most primary sporadic CC-RCCs contain somatic VHL gene mutations, clearly implicating inactivation of the VHL gene in the pathogenesis of CC-RCC. It is not known if CC-RCC without VHL gene mutations have alternative mechanisms of VHL gene inactivation or result from an alternative non-VHL pathway to RCC, e.g., inactivation of TSGs in 3p12-p21. We and others have reported hypermethylation and silencing of the VHL TSG in RCC from patients with VHL disease and in CC-RCC cell lines. However, the incidence and specificity of VHL methylation in primary sporadic RCC has not been defined. Therefore, we analysed methylation of the VHL, CDKN2, MYC, and H19 genes in primary RCC samples. Hypermethylation of the VHL promoter region was detected in 11% (11/99) of the primary RCCs analysed. In 10 of these tumours, there was no evidence of concomitant VHL gene mutation. VHL methylation was specific to CC-RCC (15%, 7/45) but was not detected in any non-CC tumours (n = 16). None of the 11 RCCs methylated at VHL had evidence of methylation at either CDKN2 or MYC (methylation at CDKN2 was, however, detected in 3%, or 1/33, of RCCs without VHL methylation). A normal methylation pattern at H19 was demonstrated in the three RCCs with methylated VHL analysed. Previous studies have suggested that, in addition to VHL, other 3p TSGs at 3p12-p14 and 3p21 may be involved in CC-RCC tumourigenesis. However, the interpretation of these studies has been difficult because information on VHL gene status has not been available for these data sets. Therefore, we investigated a subset of 55 sporadic RCCs (of known VHL gene methylation and mutation status) for LOH at polymorphic markers close to candidate TSG loci in the 3p14.2 and 3p21.2-p21.3 regions. Among tumours with LOH at one or more 3p markers, the incidence of 3p25 allele loss was higher in tumours with VHL alterations (mutation or methylation) than in those without. For tumours without detectable VHL alterations, the frequency of 3p14-p21 LOH was significantly higher than the frequency of 3p25-p26 LOH (93%, 13/14 vs. 43%, 6/14; P = 0.013), whereas, in RCC samples with VHL methylation or mutation, the frequency of 3p14-p21 LOH did not differ from that of sp25-p26 (72%, 18/25 vs. 59%, 13/22; P = 0.376). None of the 11 RCCs with 3p25 allele loss that were informative at 3p21 and 3p14 showed LOH at 3p25 only. These findings suggest that (1) VHL methylation is a specific and important event in the pathogenesis of CC-RCC; (2) in CC-RCC with 3p LOH but without VHL inactivation, mutations in TSGs at 3p14-p21 appear to have a primary role in tumourigenesis; and (3) inactivation of other 3p TSGs in addition to VHL may also be required for malignant transformation in tumours with VHL gene inactivation.

Alterations of CDKN2 (MTS1/p16INK4A) gene in paraffin-embedded tumor tissues of human stomach, lung, cervix and liver cancers.

The CDKN2 (MTS1/p16INK4A) gene, encoding cyclin dependent kinase inhibitor, was found to be homozygously deleted at a high frequency in cell lines from many different types of cancer and some primary cancers. To determine the frequency of CDKN2 mutations in most common human cancers in Korea, PCR and PCR-SSCP analyses for the exon 2 of CDKN2 were performed on each set of 20 formalin-fixed and paraffin-embedded tumor tissues of stomach adenocarcinomas, lung cancers, cervix cancers and hepatocellular carcinomas. No mutations in exon 2 of CDKN2 were found in 20 stomach adenocarcinomas. In contrast to rare mutations in stomach adenocarcinomas, a high frequency of CDKN2 mutations was identified in other 3 cancers, 11 of 20 (55%) lung cancers (7 of 10 NSCLCs and 4 of 10 SCLCs), 14 of 20 (70%) cervix cancers and 11 of 20 (55%) hepatocellular carcinomas. These results suggest that mutations of the CDKN2 gene might be an important genetic change in NSCLCs, cervix cancers and hepatocellular carcinomas.

Codeletion of CDKN2 and MTAP genes in a subset of non‐Hodgkin's lymphoma may be associated with histologic transformation from low‐grade to diffuse large‐cell lymphoma

Identifying the various genetic alterations that contribute to lymphomagenesis is key to our improved understanding of the biological behavior of the disease. Recently, we and others have defined a tumor suppressor region on the short arm of chromosome 9 harboring a cluster of genes, including MTAP, CDKN2A(p16INK4a), and CDKN2B(p15INK4B), which is frequently deleted in a variety of tumor types. To determine whether this region is involved in a particular subset of malignant lymphomas, we have examined 16 cases of diffuse large-cell lymphoma (DLCL) (including three cases that evolved from low-grade non-Hodgkin lymphoma (NHL) (transformed DLCL)), and nine cases of low-grade NHL that had subpopulations of large cells with a diffuse growth pattern (seven follicular NHL, one chronic lymphocytic leukemia, one mycosis fungoides). Interphase fluorescence in situ hybridization was performed on these samples using a 250-kb cosmid contig (COSp16), which encompasses MTAP, CDKN2A, and CDKN2B. Six of the 16 DLCLs and one of nine low-grade NHLs had deletions of COSp16. COSp16 was homozygously deleted in four cases; two cases had hemizygous deletions, and one case had a partial homozygous deletion of the cosmid contig. Three of 13 cases of de novo DLCL, all three transformed DLCLs, and one of nine low-grade NHL had COSp16 deletions. Although the numbers are small, COSp16 deletion was associated with transformed DLCL in contrast to de novo DLCL (P < 0.04, Fisher's exact test) or low-grade NHL (P < 0.02). The COSp16 deletion was mostly submicroscopic and was not observed in association with any specific recurring cytogenetic abnormalities. These results suggest that targeted deletion of the CDKN2A region occurs in a subset of non-Hodgkin's lymphomas, and may be associated with transformed lymphomas. Genes Chromosomes Cancer 22:72–78, 1998. © 1998 Wiley-Liss, Inc.

Excess Cancer Mortality in Six Dutch Pedigrees with the Familial Atypical Multiple Mole-Melanoma Syndrome from 1830 to 1994

An increased incidence of systemic cancers has been described in some reports of familial atypical multiple mole-melanoma kindreds. If the gene defect underlying the familial atypical multiple mole-melanoma syndrome is not only important for the development of melanoma of the skin, the impact of the defect on life expectancy may be much higher than previously thought. We investigated all-cause mortality from 1830 to the present and causes of death from 1941 to 1994 in proven, obligate, and potential CDKN2 mutation carriers to obtain an estimate of the impact of a hereditary defect of the CDKN2 gene on mortality. From 1830 to 1994 there were 65 deaths, although only 42 deaths were expected [standardized mortality ratio (SMR) 1.6, 95% confidence interval (CI) 1.2-2.0] and the SMR doubled with calendar time. Excess mortality was shown in most of the families, but was confined to ages 35-70 y (SMR 2.1, 95%CI 1.5-2.9). Excess mortality could be fully attributed to cancer mortality, especially to pancreatic carcinoma and melanoma of the skin. There appeared to be some heterogeneity among the families, especially due to the specific cancer pattern within a family. The impact of the defect of the CDKN2 gene is rising over calendar time, mainly because the mortality in the general population has been falling. Excess mortality was not only due to melanoma, but also to pancreatic carcinoma. Therefore, follow-up programs of affected family members should not be confined to a regular check of the atypical nevi.

Disruption of the RB pathway and cell-proliferative activity in non-small-cell lung cancers.

The pathway consisting of retinoblastoma protein (pRB), cyclin D1 and p16 (RB pathway) which is involved in the phosphorylation of pRB plays an important role in G1/S progression. The disruption of this RB pathway has been reported in several types of human neoplasm. An immunohistochemical study of 101 non-small-cell lung cancers (NSCLCs) showed loss of p16 is in 47 tumors (46.5%) and loss of pRB in 42 tumors (41.6%). In 79 of 101 NSCLCs (78.2%), the expression of p16 and pRB was complementary (p < 0.0001). Methylation of the cdkn2 gene was detected in 50% of p16-negative tumors and in 11% of p16-positive tumors. Aberrant expression of cyclin D1 was found in 45 tumors (44.5%). The cyclin-D1-positive tumors had significantly higher Ki-67 indices than the cyclin-D1-negative tumors irrespective of the tumor p16 or pRB expression. Thus, 91 (90%) of 101 NSCLCs showed disturbed expression of at least 1 of the 3 components of the RB pathway. Our results suggest that the disruption of the RB pathway plays an important role in tumorigenesis in NSCLCs and that increased cyclin-D1 expression leads to strong proliferative activity which may over-ride the suppressive effect of p16 and pRB.

An AC-repeat adjacent to mouse Cdkn2B allows the detection of specific allelic losses in the p15INK4b and p16INK4a tumor suppressor genes.

The cyclin-dependent kinase inhibitors p15INK4b and p16INK4a are involved in the development of a wide range of human and murine tumors. These tumor suppressor genes are inactivated by deletions frequently associated to point mutations in the coding regions or hypermethylation of their promoters. In this work, we describe a simple-sequence length polymorphism located in mouse Chromosome (Chr) 4, between the Cdkn2B (p15INK4b) and Cdkn2A (p16INK4a) genes, only 700 bp downstream of the Cdkn2B locus. This DNA region was analyzed in different inbred strains showing a variable AC-repetitive DNA sequence. We used this microsatellite to detect loss of heterozygosity of the Cdkn2A and Cdkn2B loci in gamma-irradiation-induced thymic lymphomas of C57BL/6J x RF/J F1 hybrids. Using this specific marker, we were able to locate additional allelic losses not detected by other microsatellites. Since the allelic losses can be detected by a simple PCR amplification, this AC-repetitive sequence is specially useful as a genetic marker for these Cdkn2 genes and specifically for the p15INK4b cell cycle inhibitor.