The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis

Background Prolonged exposure of the lip to sunlight may cause actinic cheilitis (AC) and squamous cell carcinoma (SCC). Maspin is a serpin with tumor suppressor functions. This work analyzed the presence and distribution of maspin in AC and lip SCC. Methods Sections from 36 cases diagnosed as AC (18 cases with mild epithelial dysplasia, 11 with moderate and 7 with severe), 18 cases diagnosed as lip SCC and 7 specimens containing normal lip vermillion epithelium were submitted for immunohistochemical analysis to detect maspin. Results All AC cases with mild and two cases with moderate dysplasia were scored 3. The remaining nine cases with moderate dysplasia were identified as score 2, whereas all cases with severe dysplasia were scored 1. Positive staining for maspin decreased from the basal layer to the surface. Among the 18 lip SCCs studied, 15 cases showed abundant staining for maspin. Epithelium adjacent to the SCCs also showed intense positive staining in all cells. Conclusions Our results suggest that the loss of maspin expression occurs from the basal layer to the surface. Lip SCCs related to solar radiation show an intense presence of maspin protein in almost all tumor cells as well as the neighboring epithelium. Fontes A, Sousa SM, Santos E, Martins MT. The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis.

Morphological Study of Myoneural Interaction between the Levator Labii Superioris Muscle and its Motor Nerve in Rats

The progress of science in search of new techniques of the nerve regeneration and the functional repair in reinnervated muscle has been the target of many researchers around the world. Consequently, nerves and muscles in different body segments asked for more enlightenment of their morphology, their interrelation with other anatomic structures and their peculiarities. One of the most significant areas that need deeper studies is the region of the head and neck, since they are often affected by important pathologies. In order to offer the researcher`s community a morphological myoneural interaction model, this study elected the levator labii superioris muscle and its motor nerve, the buccal branch of the facial nerve (VII pair) not only for its special characteristics, but also its value on the facial expression. The rat was chosen for this investigation for being easy to obtain, to keep, to manipulate and to compare this experiment with many others studies previously published. The techniques used were Mesoscopic (dissection), histoenzymologic and morphometric ones. In the results the muscle proved to have a predominance of fast twich fibers (FG and FOG) and superficial location, with a proximal bone and a distal cutaneous insertion. Its motor nerve, the buccal branch of the facial nerve (VII pair), breaks through the muscle belly into its deep face, and comprised a heterogeneous group of myelinic nerve fibers disposed in a regular form in all fascicle. Near the motor point, the nerve showed to be composed of two fascicles with different sizes. Due to the small nerve dimensions, the nerve fibers have a smaller diameter if compared to the motor nerve of pectineus muscle of the cat. Further studies with neural tracers have already had a start in order to provide more information about the distribution and the architecture of these fibers.

Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or indeterminate 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition. (C) 2001 Wiley-Liss, Inc.

Heritability and gene-environment interactions for melanocytic nevus density examined in a U.K. adolescent twin study

Risk factors for melanoma include environmental (particularly ultraviolet exposure) and genetic factors. In rare families, susceptibility to melanoma is determined by high penetrance mutations in the genes CDKN2A or CDK4, with more common, less penetrant genes also postulated. A further, potent risk factor for melanoma is the presence of large numbers of melanocytic nevi so that genes controlling nevus phenotype could be such melanoma susceptibility genes. A large Australian study involving twins aged 12 y of predominantly U.K. ancestry showed strong evidence for genetic influence on nevus number and density. We carried out essentially the same study in the U.K. to gain insight into gene-environment interactions for nevi. One hundred and three monozygous (MZ) and 118 dizygous (DZ) twin pairs aged 10-18 y were examined in Yorkshire and Surrey, U.K. Nevus counts were, on average, higher in boys (mean = 98.6) than girls (83.8) (p = 0.009) and higher in Australia (110.4) than in the U.K. (79.2, adjusted to age 12 y, p < 0.0001), and nevus densities were higher on sun-exposed sites (92 per m(2)) than sun-protected sites (58 per m(2)) (p < 0.0001). Correlations in sex and age adjusted nevus density were higher in MZ pairs (0.94, 95% CI 0.92-0.96) than in DZ pairs (0.61, 95%CI 0.49-0.72), were notably similar to those of the Australian study (MZ = 0.94, DZ = 0.60), and were consistent with high heritability (65% in the U.K., 68% in Australia). We conclude that emergence of nevi in adolescents is under strong genetic control, whereas environmental exposures affect the mean number of nevi.

MC1R genotype modifies risk of melanoma in families segregating CDKN2A mutations

Mutations in the exons of the cyclin-dependent kinase inhibitor gene CDKN2A are melanoma-predisposition alleles which have high penetrance, although they have low population frequencies. In contrast, variants of the melanocortin-1 receptor gene, MC1R, confer much lower melanoma risk but are common in European populations. Fifteen Australian CDKN2A mutation-carrying melanoma pedigrees were assessed for MC1R genotype, to test for possible modifier effects on melanoma risk. A CDKN2A mutation in the presence of a homozygous consensus MC1R genotype had a raw penetrance of 50%, with a mean age at onset of 58.1 years. When an MC1R variant allele was also present, the raw penetrance of the CDKN2A mutation increased to 84%, with a mean age at onset of 37.8 years (P=0.1). The presence of a CDKN2A mutation gave a hazard ratio of 13.35, and the hazard ratio of 3.72 for MC1R variant alleles was also significant. The impact of MC1R variants on risk of melanoma was mediated largely through the action of three common alleles, Arg151Cys, Arg160Trp, and Asp294His, that have previously been associated with red hair, fair skin, and skin sensitivity to ultraviolet light.

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 90 involved in the development of melanoma, Although LOH at 90 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 90. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations hy 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 at least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

No evidence of a role for activating CDK2 mutations in melanoma

Inactivation of p16(INK4a) and/or activation of cyclin-dependent kinase-4 (CDK4) are strongly associated with both susceptibility and progression in melanoma. Activating CDK4 mutations prevent the binding and inhibition of CDK4 by p16(INK4a). A second, more indirect role for CDK4 is in late G(1), where It may sequester the inhibitors p27(KIP1) or p21(CIP1) away from CDK2, and in doing so upregulate the CDK2 activity necessary for cells to proceed completely through G(1) into S phase. As the pivotal residues around the most predominant R24C activating CDK4 mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27(KIP1) or p21(CIP1). However, except for a silent polymorphism, we detected no variants within this region of the CDK2 gene in 60 melanoma cell lines. Thus, if CDK2 activity is dysregulated in melanoma it is likely to occur by a means other than mutations causing loss of direct inhibition. We also examined the expression of the CDK2 gene in melanoma cell lines, to assess its possible co-regulation with the gene for the melanocyte-lineage antigen pmel17, which maps less than 1 kb away in head to head orientation with CDK2 and may be transcribed off the same bidirectional promoter. However, expression of the genes is not co-regulated. (C) 2001 Lippincott Williams & Wilkins.

Reproduction in the squirrel glider, Petaurus norfolcensis (Petauridae) in south-east Queensland

Reproduction was studied across a three-year period in two south-east Queensland populations of the squirrel glider, Petaurus norfolcensis, by measuring morphological (body weight, scrotal diameter, cutaneous scent-gland activity, condition index) and physiological (plasma steroid levels) variables. Reproduction showed a seasonal pattern, with peak numbers of pouch young recorded in late autumn and winter. Declines in oestrogen concentrations outside the breeding period indicate that females are anoestrous in the summer months. Most (83%) reproductive females captured during the study were 2-3 years old, but all individuals over one year of age were found to have bred. Average litter size was 1.73 +/- 0.01 (n = 23). Scrotal diameter and testosterone concentrations showed no significant seasonal variation. It is suggested that this is due to the presence of both socially dominant and subordinate males in the data set. Maximum testosterone concentrations did coincide with periods of mating. The condition index showed no relationship with reproductive variables, but it is likely that this results from the manner in which the index was generated.

Sun exposure and interaction with family history in risk of melanoma, Queensland, Australia

Sun exposure is the main environmental risk factor for melanoma, but the timing of exposure during life that confers increased risk is controversial. Here we provide the first report of the association between lifetime and age-specific cumulative ultraviolet exposure and cutaneous melanoma in Queensland, Australia, an area of high solar radiation, and examine the association separately for families at high, intermediate and low familial melanoma risk. Subjects were a population-based sample of melanoma cases diagnosed and registered in Queensland between 1982 and 1990 and their relatives. The analysis included 1,263 cases and relatives with confirmed cutaneous melanoma and 3,111 first-degree relatives without melanoma as controls. Data an lifetime residence and sun exposure, family history and other melanoma risk factors were collected by a mailed questionnaire. Using conditional multiple logistic regression with stratification by family, cumulative sun exposure in childhood and in adulthood after age 20 was significantly associated with melanoma, with estimated relative risks of 1.15 per 5,000 minimal erythemal doses (MEDs) from age 5 to 12 years, and 1.52 per 5 MEDs/day from age 20. There was no association with sun exposure in families at high familial melanoma risk. History of nonmelanoma skin cancer (relative risk [RR] = 1.26) and multiple sunburns (RR = 1.31) were significant risk factors. These findings indicate that sun exposure in childhood and in adulthood are important determinants of melanoma but not in those rare families with high melanoma susceptibility, in which genetic factors are likely to be more important. (C) 2002 Wiley-Liss, Inc.

Melanoma in adolescents: A case-control study of risk factors in Queensland, Australia

The incidence of melanoma increases markedly in the second decade of life but almost nothing is known of the causes of melanoma in this age group. We report on the first population-based case-control study of risk factors for melanoma in adolescents (15-19 years). Data were collected through personal interviews with cases, controls and parents. A single examiner conducted full-body nevus counts and blood samples were collected from cases for analysis of the CDKN2A melanoma predisposition gene. A total of 201 (80%) of the 250 adolescents with melanoma diagnosed between 1987 and 1994 and registered with the Queensland Cancer Registry and 205 (79%) of 258 age-, gender- and location-matched controls who were contacted agreed to participate. The strongest risk factor associated with melanoma in adolescents in a multivariate model was the presence of more than 100 nevi 2 mm or more in diameter (odds ratio [OR] = 46.5, 95% confidence interval [Cl] = 11.4-190.8). Other risk factors were red hair (OR = 5.4, 95%Cl = 1.0-28.4); blue eyes (OR = 4.5, 95%Cl = 1.5- 13.6); inability to tan after prolonged sun exposure (OR = 4.7, 95%Cl = 0.9-24.6); heavy facial freckling (OR = 3.2, 95% Cl = 0.9-12.3); and family history of melanoma (OR = 4.0, 95%Cl = 0.8-18.9). Only 2 of 147 cases tested had germline variants or mutations in CDKN2A. There was no association with sunscreen use overall, however, never/rare use of sunscreen at home under the age of 5 years was associated with increased risk (OR = 2.2, 95%Cl = 0.7-7.1). There was no difference between cases and controls in cumulative sun exposure in this high-exposure environment. Factors indicating genetic susceptibility to melanoma, in particular, the propensity to develop nevi and freckles, red hair, blue eyes, inability to tan and a family history of the disease are the primary determinants of melanoma among adolescents in this high solar radiation environment. Lack of association with reported sun exposure is consistent with the high genetic susceptibility in this group. (C) 2002 Wiley-Liss, Inc.

Loss of p16 expression is associated with histological features of melanoma invasion

While mutations of CDKN2A are associated with melanoma predisposition, the precise role of its gene product p16 in the development of sporadic melanoma is less clearly understood. We sought to determine the prevalence of p16 expression using immunohistochemical analysis in a population-based sample of melanoma tumours, and also to identify histological, phenotypic and environmental factors associated with the presence or absence of p16 expression. We conducted face-to-face interviews with 108 patients newly diagnosed with melanoma to ascertain their history of sun exposure, and recorded various phenotypic parameters. Paraffin sections of tumours from these patients were stained with an anti-p16 monoclonal antibody following antigen retrieval. Overall, 52 (48%) tumours expressed p16; nodular melanomas had significantly lower levels of p16 immunoreactivity than superficial spreading melanomas (P = 0.015). While no association was found between p16 expression and host phenotype, loss of p16 staining was associated with thicker lesions (p = 0.084) and a high mitotic index (P = 0.013). Taken together, these findings are consistent with loss of p16 being a late event in the progression of sporadic primary melanomas, being associated with tumours of a more aggressive nature. (C) 2002 Lippincott Williams Wilkins.

Pathways to Melanoma Development: Lessons from the Mouse

Because of subtle differences between mouse and human skin, mice have traditionally not been an ideal model to study melanoma development. Understanding of the molecular mechanisms of melanoma predisposition, however, has been greatly improved by modeling various pathway defects in the mouse. This review analyzes the latest developments in mouse models of melanoma, and summarizes what these may indicate about the development of this neoplasm in humans. Mutations of genes involved in human melanoma have been recapitulated with some unexpected results, particularly with respect to the role of the two transcripts (Ink4a and Arf) encoded by the Cdkn2a locus. Both the Ink4a/pRb and Arf/p53 pathways are involved in melanoma development in mice, and possible mechanisms of cross-talk between the two pathways are discussed. We also know from mouse models that Ras/mitogen-activated protein kinase pathway activation is very important in melanoma development, either through direct activation of Ras (e.g., Hras G12V), or via activation of Ras-effector pathways by other oncogenes (e.g., Ret, Hgf/Sf). Ras can cooperate with the Arf/p53 pathway, and probably the Ink4a/Rb pathway, to induce melanoma. These three growth regulation pathways (Ink4a/pRb, Arf/p53, and Ras/mitogen-activated protein kinase) seem to represent three major axes of melanoma development in mice. Finally, we summarize experiments using genetically modified mice that have given indications of the intensity and timing of ultraviolet radiation exposure that may be most responsible for melanoma development.

Geographical variation in the penetrance of CDKN2A mutations for melanoma

Background: Germline mutations in the CDKN2A gene, which encodes two proteins (p16INK4A and p14ARF), are the most common cause of inherited susceptibility to melanoma. We examined the penetrance of such mutations using data from eight groups from Europe, Australia and the United States that are part of The Melanoma Genetics Consortium Methods: We analyzed 80 families with documented CDKN2A mutations and multiple cases of cutaneous melanoma. We modeled penetrance for melanoma using a logistic regression model incorporating survival analysis. Hypothesis testing was based on likelihood ratio tests. Covariates included gender, alterations in p14APF protein, and population melanoma incidence rates. All statistical tests were two-sided. Results: The 80 analyzed families contained 402 melanoma patients, 320 of whom were tested for mutations and 291 were mutation carriers. We also tested 713 unaffected family members for mutations and 194 were carriers. Overall, CDKN2A mutation penetrance was estimated to be 0.30 (95% confidence interval (CI) = 0.12 to 0.62) by age 50 years and 0.67 (95% CI = 0.31 to 0.96) by age 80 years. Penetrance was not statistically significantly modified by gender or by whether the CDKN2A mutation altered p14ARF protein. However, there was a statistically significant effect of residing in a location with a high population incidence rate of melanoma (P = .003). By age 50 years CDKN2A mutation penetrance reached 0.13 in Europe, 0.50 in the United States, and 0.32 in Australia; by age 80 years it was 0.58 in Europe, 0.76 in the United States, and 0.91 in Australia. Conclusions: This study, which gives the most informed estimates of CDKN2A mutation penetrance available, indicates that the penetrance varies with melanoma population incidence rates. Thus, the same factors that affect population incidence of melanoma may also mediate CDKN2A penetrance.