Characterization of the neoplastic phenotype in the familial atypical multiple-mole melanoma-pancreatic carcinoma syndrome

BACKGROUND: Previous studies suggest that the familial atypical multiple-mole melanoma (FAMMM) syndrome may predispose affected families to nonmelanoma carcinomas, including adenocarcinoma of the pancreas. It has been found that many of these families harbor mutations in the CDKN2A gene on chromosome 9p21. The phenotypic expression of CDKN2A mutations in these families has not been characterized fully. METHODS: The authors studied eight families that appeared to inherit multiple nevi, cutaneous melanomas, and pancreatic carcinomas in association with a CDKN2A germline mutation. The expression of disease within these families was examined, and segregation ratios were estimated to assess the patterns of inheritance according to various definitions of phenotype. RESULTS: Either multiple nevi or pancreatic carcinoma was diagnosed in 53% of first-degree relatives of the probands. The offspring of parents affected with multiple nevi, melanoma, or pancreatic carcinoma were significantly more likely to be affected themselves compared with the offspring of unaffected parents (48.9% vs. 16.7%; P = 0.004). CONCLUSIONS: The current results provide additional evidence that multiple nevi, melanoma, or pancreatic carcinoma may be inherited as autosomal-dominant traits in families known to harbor CDKN2A mutations. Other malignancies may be a part of the phenotype in these families, although this hypothesis requires additional study.     

Definition of the role of chromosome 9p21 in sporadic melanoma through genetic analysis of primary tumours and their metastases. The Melanoma Cooperative Group

Malignant melanoma (MM) is thought to arise by sequential accumulation of genetic alterations in normal melanocytes. Previous cytogenetic and molecular studies indicated the 9p21 as the chromosomal region involved in MM pathogenesis. In addition to the CDKN genes (p16/CDKN2A, p15/CDKN2B and p19(ARF), frequently inactivated in familial MM), widely reported data suggested the presence within this region of other melanoma susceptibility gene(s). To clearly assess the role of the 9p21 region in sporadic melanoma, we evaluated the presence of microsatellite instability (MSI) and loss of heterozygosity (LOH) in primary tumours as well as in synchronous or asynchronous metastases obtained from the same MM patients, using 9 polymorphic markers from a 17-cM region at 9p21. LOH and MSI were found in 27 (41%) and 11 (17%), respectively, out of 66 primary tumours analysed. In corresponding 58 metastases, MSI was found at higher rate (22; 38%), whereas a quite identical pattern of allelic deletions with 27 (47%) LOH+ cases were observed. Although the CDKN locus was mostly affected by LOH, an additional region of common allelic deletion corresponding to marker D9S171 was also identified. No significant statistical correlation between any 9p21 genetic alteration (LOH, MSI or both) and clinicopathological parameters was observed.     

Germline mutation and large deletion analysis of the CDKN2A and ARF genes in families with multiple melanoma or an aggregation of malignant melanoma and breast cancer

The CDKN2A/ARF genes have been associated with increased risk of malignant melanoma (MM) in families with multiple members affected by disease and in families characterized by the constellation of breast cancer and MM. The exact contribution of CDKN2A/ARF to disease risk remains poorly characterized, especially in diverse populations. In this report, the contribution of CDKN2A/ARF germline mutations and large rearrangements to disease in Polish familial MM (FMM) and aggregations of breast cancer and MM were assessed using a strategy that included genomic sequencing, restriction fragment length polymorphism and multiplex ligation-dependent probe amplification. We examined 16 FMM cases (group 1), 44 MM probands with a cancer family aggregation (CFA) that included at least one breast cancer (group 2) and 22 breast cancer probands with CFA and MM (group 3). The results revealed a paucity of mutations in CDKN2A/ARF, suggesting that in the Polish population this gene does not contribute significantly to either FMM or MM within the context of CFA.     

Germline splicing mutations of CDKN2A predispose to melanoma

Coding mutations of the CDKN2A gene on chromosome 9p21 cosegregate with 25-60% of familial melanoma cases, but there remains a number of 9p21-linked kindreds that lack germline coding mutations of CDKN2A. We sequenced CDKN2A exons 1alpha, 2, 3, and the adjacent intronic regions in 167 melanoma-prone families (at least two affected first-degree relatives), and detected four splice site variations, three of which cosegregate with the disease. RT-PCR experiments verified that these three variants, including an AGgt to ATgt mutation that demonstrates a founder effect, do affect splicing. While an exon 1alpha splice donor site mutation incompletely abolishes splicing, the correctly spliced mRNA yields a protein (Q50P) that cannot effectively interact with CDK4. We also performed RT-PCR on mRNA from 16 melanoma-prone kindreds to search for cryptic splice sites deep within introns, but identified no splice variants. Meanwhile, we screened 139 affected families using allele-specific PCR for the recently discovered IVS2-105A>G mutation, but found only one family that possesses this alteration. We conclude that splice site mutations do predispose to disease in a subset of melanoma-prone kindreds. Characterization of additional splice site variants and other noncoding alterations of CDKN2A should allow us to detect a wider range of mutations in at-risk patients.     

Associations of 9p21 variants with cutaneous malignant melanoma, nevi, and pigmentation phenotypes in melanoma-prone families with and without CDKN2A mutations

Chromosome 9p21 has been implicated in the pathogenesis of cutaneous malignant melanoma (CMM). In addition to CDKN2A, the major known high-risk susceptibility gene for CMM, recent studies suggest that other 9p21 genes may be involved in melanoma/nevi development. To identify 9p21 variants that influence susceptibility to CMM and number of nevi in CMM-prone families with and without CDKN2A mutations, we analyzed 562 individuals (183 CMM) from 53 families (23 CDKN2A+, 30 CDKN2A?) for 233 tagging SNPs in 21 genes at 9p21. Single SNP- and gene-based regression analyses were used to assess the risk of CMM, nevi count, skin complexion, and tanning ability associated with these SNPs and genes. We found that SNP rs7023329 in the MTAP gene was associated with number of nevi (P _trend = 0.003) confirming a recent finding by a genome-wide association study. In addition, three SNPs in the ACO1 gene, rs7855483 (P _trend = 0.002), rs17288067 (P _trend = 0.0009), and rs10813813 (P _trend = 0.005), showed the strongest associations with CMM risk. None of the examined 9p21 SNPs was associated with skin complexion, whereas two SNPs, rs10964862 in IFNW1 (P _trend = 0.003), and rs13290968 in TUSC1 (P _trend = 0.0006), were associated with tanning ability. Gene-based analyses suggested that the ACO1 gene was significantly associated with CMM (P = 0.0004); genes IFNW1 (P = 0.002) and ACO1 (P = 0.0002) were significantly associated with tanning ability. Our findings are consistent with recent proposals that additional 9p21 genes may contribute to CMM susceptibility in CMM-prone families. These genetic variants may, at least partially, exert their effects through nevi and tanning ability.     

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.     

High- and low-penetrance cutaneous melanoma susceptibility genes

The aim of this review is to report the current understanding of the molecular genetics of melanoma predisposition. To date, two high-penetrance melanoma susceptibility genes, cyclin-dependent kinas inhibitor (CDKN)2A on chromosome 9p21 and cyclin-dependent kinase (CDK4) on 12q13, have been identified. Germline inactivating mutations of the CDKN2A gene are the most common cause of inherited susceptibility to melanoma. Worldwide, a few families have been found to harbor CDK4 mutations. However, predisposing alterations to familial melanoma are still unknown in a large proportion of kindreds. Other melanoma susceptibility loci have been mapped through genome-wide linkage analysis, although the putative causal genes at these loci have yet to be identified. Much ongoing research is being focused on the identification of low-penetrance melanoma susceptibility genes that confer a lower melanoma risk with more frequent variations. Specific variants of the MC1R gene have been demonstrated to confer an increase in melanoma risk. In addition, conflicting data are available on other potential low-penetrance genes encoding proteins involved in pigmentation, cell growth and differentiation, DNA repair or detoxifying of metabolites.     

Hereditary cutaneous melanoma

Members of some kindreds have a hereditary predisposition for development of cutaneous melanoma. Cytogenetic and linkage studies implicated chromosomes 1p and 9p as possible locations for genetic alterations predisposing for melanoma. Germline mutations in the CDKN2A gene on chromosome 9p21 have been identified in hereditary melanoma, but are present in only approximately 40% of kindreds with linkage to 9p21, indicating that changes in other gene(s) at this location may also predispose to melanoma. In a few families, germline mutations in the CDK4 gene are present. The genetic alterations underlying disease predisposition in a large proportion of melanoma families remain unknown.     

Loh and mutation analysis of CDKN2 in primary human ovarian cancers

The CDKN2 gene encodes a cell cycle regulatory protein and is located on chromosome 9p21, a region deleted in a wide variety of primary tumours. While mutations in the CDKN2 gene itself are frequently observed in tumour cell lines, they are less common in primary tumours. We have investigated the role of the CDKN2 gene in ovarian cancer by analysis for allelic loss of 9p21 and single-strand conformational polymorphism analysis of exons 1 and 2 of CDKN2 in 67 primary ovarian tumours. Loss of heterozygosity on 9p21 was frequently observed (24/50 informative tumours) and was common in early-stage tumours, suggesting that it is an early event in ovarian tumorigenesis. Homozygous deletion of the CDKN2 gene was detected in only 1 tumour. No somatic or germline mutations were observed in CDKN2, though a codon 140 polymorphism was detected in 2 cases. This suggests that CDKN2 is not involved in ovarian tumorigenesis and that another gene(s) may be the target of the frequent 9p allelic losses observed.     

CDKN2 (MTS1/p16INK4A) Gene Alterations in Hematological Malignancies

Cyclin dependent kinases (CDKs) make complexes with cyclins, and regulate cell cycle progression by their serine/threonine kinase activities. CDK inhibitors (CDKIs) arrest the inappropriate progression of the cell cycle by combining with CDKs. Because the functional loss of CDKIs may permit unlimited cell growth, their disruptions are thought to be associated with tumorigenesis. Recently, one CDKI, p16, was found, and its gene, CDKN2 (MTS1/p16^INK4A), was identified on chromosome 9p21. Intensive investigations of the CDKN2 gene in various tumors have shown that alterations frequently occur in this gene, thus suggesting that the CDKN2 gene is a tumor suppressor gene. In hematological malignancies, CDKN2 gene alterations may be limited to lymphoid malignancies, especially T-cell type acute lymphocytic leukemias, in which frequent chromosomal abnormalities in the 9p21 region have been reported. The CDKN2 gene is also inactivated in some patients with non-Hodgkin's lymphomas, adult T-cell leukemias, and lymphoid blastic crisis of chronic myelogenous leukemias. The main mechanism of CDKN2 gene inactivation is thought to be homozygous deletion, but point mutations may also inactivate it in some cases. The CDKN2 gene appears to be the major tumor suppressor gene on chromosome 9p21, and it is thought to be involved in the tumorigenesis of various lymphoid malignancies.     

Search for germline alterations in CDKN2A/ARF and CDK4 of 42 Jewish melanoma families with or without neural system tumours

To gain insight into the molecular mechanisms involved in the inherited predisposition to melanoma and associated neural system tumours, 42 Jewish, mainly Ashkenazi, melanoma families with or without neural system tumours were genotyped for germline point mutations and genomic deletions at the CDKN2A/ARF and CDK4 loci. CDKN2A/ARF deletion detection was performed using D9S1748, an intragenic microsatellite marker. Allele dosage at the p14ARF locus was analysed by quantitative real-time PCR employing a TaqMan probe that anneals specifically to exon 1beta of the p14ARF gene. For detecting point mutations, dHPLC and direct sequencing of the coding sequences of CDKN2A/ARF and CDK4 was used. No germline alterations in any of the tested genes were detected among the families under study. We conclude that in the majority of Ashkenazi Jewish families, the genes tested are unlikely to be implicated in the predisposition to melanoma and associated neural system tumours.     

Genomic profiling of malignant melanoma using tiling-resolution arrayCGH

Malignant melanoma is an aggressive, heterogeneous disease where new biomarkers for diagnosis and clinical outcome are needed. We searched for chromosomal aberrations that characterize its pathogenesis using 47 different melanoma cell lines and tiling-resolution bacterial artificial chromosome-arrays for comparative genomic hybridization. Major melanoma genes, including BRAF, NRAS, CDKN2A, TP53, CTNNB1, CDK4 and PTEN, were examined for mutations. Distinct copy number alterations were detected, including loss or gain of whole chromosomes but also minute amplifications and homozygous deletions. Most common overlapping regions with losses were mapped to 9p24.3-q13, 10 and 11q14.1-qter, whereas copy number gains were most frequent on chromosomes 1q, 7, 17q and 20q. Amplifications were delineated to oncogenes such as MITF (3p14), CCND1 (11q13), MDM2 (12q15), CCNE1 (19q12) and NOTCH2 (1p12). Frequent findings of homozygous deletions on 9p21 and 10q23 confirmed the importance of CDKN2A and PTEN. Pair-wise comparisons revealed distinct sets of alterations, for example, mutually exclusive mutations in BRAF and NRAS, mutual mutations in BRAF and PTEN, concomitant chromosome 7 gain and 10 loss and concomitant chromosome 15q22.2-q26.3 gain and 20 gain. Moreover, alterations of the various melanoma genes were associated with distinct chromosomal imbalances suggestive of specific genomic programs in melanoma development.     

CDKN2A gene inactivation in epithelial sporadic ovarian cancer.

The tumour suppressor gene CDKN2A, located on chromosome 9p21, encodes the cell cycle regulatory protein p16. Inactivation of the CDKN2A gene could lead to uncontrolled cell growth. In order to determine the role of CDKN2A in the development of sporadic ovarian cancer, loss of heterozygosity at 9p21-22, homozygous deletion, mutation and methylation status of the CDKN2A gene as well as CDKN2A expression were examined in a panel of serous papillary ovarian cancer. The frequency of loss of heterozygosity (LOH) for one or more informative markers at 9p21-22 was 65% (15/23). The most common deleted region was located between interferon (IFN)-alpha and D9S171. Homozygous deletions and mutations of the CDKN2A gene were not found. There was no evidence of methylation in exon 1, but methylation in exon 2 of CDKN2A gene was found in 26% (6/23). Absence of CDKN2A gene expression was shown in 27% (6/22) at mRNA level and 21% (4/19) at protein level. These data suggest that the CDKN2A gene is involved in the tumorigenesis of ovarian cancer, but the mechanisms of CDKN2A gene inactivation in serous papillary ovarian cancer remains unclear.     

Somatic mutations of TRAIL-receptor 1 and TRAIL-receptor 2 genes in non-Hodgkin's lymphoma

Tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIL-R1) and tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) are cell-surface receptors involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell-death signaling. TRAIL-R1 and TRAIL-R2 genes have recently been mapped to chromosome 8p21-22, which is a frequent site of allelic deletions in many types of human tumors, including non-Hodgkin's lymphoma (NHL). Because TRAIL/TRAIL receptor system plays an important role in lymphocyte homeostasis, we hypothesized that the mutations of TRAIL-R1 and TRAIL-R2 may be involved in the development of NHL and that such mutations may be responsible for the allelic losses of 8p21-22 in NHL. In this study, we analysed the entire coding region of TRAIL-R2 gene and the death domain region of TRAIL-R1 gene for the detection of the somatic mutations in a series of 117 human NHLs using polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis. Overall, eight tumors (6.8%) were found to have two TRAIL-R1 gene mutations or six TRAIL-R2 gene mutations. Interestingly, of the eight mutations, six missense mutations (two TRAIL-R1 and four TRAIL-R2) were detected in the death domains and one nonsense mutation of TRAIL-R2 was detected just before the death domain. Our data suggest that somatic mutations of TRAIL-R1 and TRAIL-R2 genes may play a role in the pathogenesis of some NHLs and that TRAIL-R1 and TRAIL-R2 genes might be the relevant genes to the frequent loss of chromosome 8p21-22 in human NHL.     

High- and low-penetrance cutaneous melanoma susceptibility genes

The aim of this review is to report the current understanding of the molecular genetics of melanoma predisposition. To date, two high-penetrance melanoma susceptibility genes, cyclin-dependent kinas inhibitor (CDKN)2A on chromosome 9p21 and cyclin-dependent kinase (CDK4) on 12q13, have been identified. Germline inactivating mutations of the CDKN2A gene are the most common cause of inherited susceptibility to melanoma. Worldwide, a few families have been found to harbor CDK4 mutations. However, predisposing alterations to familial melanoma are still unknown in a large proportion of kindreds. Other melanoma susceptibility loci have been mapped through genome-wide linkage analysis, although the putative causal genes at these loci have yet to be identified. Much ongoing research is being focused on the identification of low-penetrance melanoma susceptibility genes that confer a lower melanoma risk with more frequent variations. Specific variants of the MC1R gene have been demonstrated to confer an increase in melanoma risk. In addition, conflicting data are available on other potential low-penetrance genes encoding proteins involved in pigmentation, cell growth and differentiation, DNA repair or detoxifying of metabolites.     

Screening of germline mutations in the CDK4, CDKN2C and TP53 genes in familial melanoma: A clinic-based population study

Germline mutations within the CDKN2A gene, coding for the cyclin-dependent kinase inhibitor p16, have been detected by screening in 8% of Swedish families with an inheritance of cutaneous melanoma (FMM) and dysplastic nevus syndrome (DNS). Contrastingly, the closely related gene CDKN2B had no disease-related mutations in these families. A majority of Swedish families with hereditary melanoma predisposition thus lack germline mutations in these cell cycle G₁ checkpoint-regulating genes. Additional genes with the potential to contribute to increased melanoma risk may code for related components of the cell cycle-regulating machinery. The gene for cyclin-dependent kinase 4, CDK4, has been found in mutated form in the germline from individuals belonging to 2 melanoma kindreds in the United States. The CDKN2C gene coding for the cyclin-dependent kinase inhibitor p18 is localized on 1p32, a region frequently involved in chromosomal changes in melanomas and other tumors. The TP53 suppressor gene, involved in cell cycle regulation and maintenance of genetic stability, is found mutated in the germline of patients with hereditary Li-Fraumeni syndrome, leading to early onset of several human cancers, including melanoma. The present investigation reports the results of screening the 100 Swedish melanoma families for germline mutations in the CDK4, CDKN2C and TP53 genes. No disease-related mutations were detected in the coding regions. A direct contribution of these genes to the hereditary risk for melanoma in members of Swedish melanoma kindreds therefore appears unlikely. Int. J. Cancer 78:13–15, 1998.© 1998 Wiley-Liss, Inc.     

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.     

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 9p involved in the development of melanoma. Although LOH at 9p has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 9p. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations by single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele. Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748 the markers closest to CDKN2A. Of the remaining 11 tumors with LOH 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion. This report supports the conclusions of previous studies that a least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.