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Cancer investigators frequently conduct studies to examine tumor samples from pairs of apparently independent primary tumors with a view to determine whether they share a "clonal" origin. The genetic fingerprints of the tumors are compared using a panel of markers, often representing loss of heterozygosity (LOH) at distinct genetic loci. In this article we evaluate candidate significance tests for this purpose. The relevant information is derived from the observed correlation of the tumors with respect to the occurrence of LOH at individual loci, a phenomenon that can be evaluated using Fisher's exact test. Information is also available from the extent to which losses at the same locus occur on the same parental allele. Data from these combined sources of information can be evaluated using a simple adaptation of Fisher's exact test. The test statistic is the total number of loci at which concordant mutations occur on the same parental allele, with higher values providing more evidence in favor of a clonal origin for the two tumors. The test is shown to have high power for detecting clonality for plausible models of the alternative (clonal) hypothesis, and for reasonable numbers of informative loci, preferably located on distinct chromosomal arms. The method is illustrated using studies to identify clonality in contralateral breast cancer. Interpretation of the results of these tests requires caution due to simplifying assumptions regarding the possible variability in mutation probabilities between loci, and possible imbalances in the mutation probabilities between parental alleles. Nonetheless, we conclude that the method represents a simple, powerful strategy for distinguishing independent tumors from those of clonal origin.

Cyclin-dependent kinase inhibitor type 2A (CDKN2A) has been identified as a major melanoma susceptibility gene based on the presence of germline mutations in high-risk melanoma families. In this study, we sought to identify and characterize the spectrum of CDKN2A mutations affecting p16 inhibitor of cyclin-dependent kinase type 4 (INK4a) in individuals with melanoma using a population-based study design. DNA samples from 1189 individuals with incident multiple primary melanoma (MPM) and 2424 with incident single primary melanoma unselected for family history of melanoma were available for screening of CDKN2A (p16INK4a) mutations. Variants were classified for functional impact based on intragenic position, existing functional data, sequence, and structural analysis. The impact of individual mutations and functional groupings was assessed by comparing frequencies in cases of MPM versus cases with a single first primary melanoma, and by comparing the reported incidence rates in first-degree relatives. Our results show that mutations occur infrequently in these high-risk groups, and that they occur mainly in exons 1alpha and 2. Rare coding variants with putative functional impact are observed to increase substantially the risk of melanoma. With the exception of the variant in position -34 of CDKN2A of known functional consequence, the remaining rare variants in the non-coding region have no apparent impact on risk.

Objective: Sun exposure is the main cause of melanoma in populations of European origin. No previous study has examined the effect of sun exposure on risk of multiple primary melanomas compared with people who have one melanoma., Methods: We identified and enrolled 2,023 people with a first primary melanoma (controls) and 1,125 with multiple primary melanomas (cases) in seven centers in four countries, recorded their residential history to assign ambient UV and interviewed them about their sun exposure., Results: Risk of multiple primary melanomas increased significantly (P<0.05) to OR=2.10 for the highest exposure quarter of ambient UV irradiance at birth and 10 years of age, to OR=1.38 for lifetime recreational sun exposure, to OR=1.85 for beach and waterside activities, to OR=1.57 for vacations in a sunnier climate, to OR=1.50 for sunburns. Occupational sun exposure did not increase risk (OR=1.03 for highest exposure). Recreational exposure at any age increased risk and appeared to add to risk from ambient UV in early life., Conclusions: People who have had a melanoma can expect to reduce their risk of a further melanoma by reducing recreational sun exposure whatever their age. The same is probably true for a person who has never had a melanoma.

Natural variation in the coding region of the melanocortin-1 receptor (MC1R) gene is associated with constitutive pigmentation phenotypes and development of melanoma and nonmelanoma skin cancers. We investigated the effect of MC1R variants on melanoma using a large, international population-based study design with complete determination of all MC1R coding region variants. Direct sequencing was completed for 2,202 subjects with a single primary melanoma (controls) and 1,099 subjects with second or higher-order primary melanomas (cases) from Australia, the United States, Canada, and Italy. We observed 85 different MC1R variants, 10 of which occurred at a frequency >1%. Compared with controls, cases were more likely to carry two previously identified red hair ("R") variants [D84E, R151C, R160W, and D294H; odds ratio (OR), 1.6; 95% confidence interval (95% CI), 1.1-2.2]. This effect was similar among individuals carrying one R variant and one r variant (defined as any non-R MC1R variant; OR, 1.6; 95% CI, 1.3-2.2) and among those carrying only one R variant (OR, 1.5; 95% CI, 1.1-1.9). There was no statistically significant association among those carrying only one or two r variants. Effects were similar across geographic regions and categories of pigmentation characteristics or number of moles. Our results confirm that MC1R is a low-penetrance susceptibility locus for melanoma, show that pigmentation characteristics may not modify the relationship of MC1R variants and melanoma risk, and suggest that associations may be smaller than previously reported in part due to the study design.

Germ-line mutations of CDKN2A have been identified as strong risk factors for melanoma in studies of multiple-case families. However, an assessment of their relative risk for melanoma in the general population has been difficult because they occur infrequently. We addressed this issue using a novel population-based case-control study design in which "cases" have incident second- or higher-order melanomas [multiple primary melanoma (MPM)] and "controls" have incident first primary melanoma [single primary melanoma (SPM)]. Participants were ascertained from nine geographic regions in Australia, Canada, Italy, and United States. In the 1,189 MPM cases and 2,424 SPM controls who were eligible and available for analysis, the relative risk of a subsequent melanoma among patients with functional mutations who have an existing diagnosis of melanoma, after adjustments for age, sex, center, and known phenotypic risk factors, is estimated to be 4.3 (95% confidence interval, 2.3-7.7). The odds ratio varied significantly depending on the type of mutation involved. The results suggest that the relative risk of mutation carriers in the population may be lower than currently believed and that different mutations on the CDKN2A gene may confer substantially different risks of melanoma.

Many endometrial adenocarcinomas, particularly those of endometrioid type, express estrogen receptors (ERs), progesterone receptors (PRs), and vimentin. This typical immunophenotype is frequently considered a standard against which others are compared when immunohistochemistry is used for differential diagnosis. We tested large numbers of endometrial cancers, enriched for high-grade tumors, to determine whether this reported immunophenotype was valid and whether expression differences between types of endometrial carcinoma could be exploited for diagnostic purposes. Immunohistochemical stains were performed on the following types of endometrial cancers using established methodology: International Federation of Gynecology and Obstetrics (FIGO) grades 1 and 2 endometrioid-42; FIGO grade 3 endometrioid-40; serous-24; clear cell-11; carcinosarcoma-9. In total, 92% of serous carcinomas expressed p16 strongly compared to weak-to-moderate expression of p16 in 7-67% of other tumors (FIGO grades 1 and 2 carcinoma and carcinosarcoma, respectively). A total of 84% of FIGO grades 1 and 2 carcinomas expressed ER compared to 9-54% of other tumors (clear cell and serous carcinomas respectively); 83% of FIGO grades 1 and 2 expressed PR compared to 11-54% of other carcinomas (carcinosarcoma and serous carcinoma, respectively). Most carcinomas were negative for monoclonal carcinoembryonic antigen (mCEA), and those that were positive showed mostly only focal membrane expression. Vimentin was expressed in nearly every tumor. Most tumors were diffusely vimentin positive, but a large range of expression patterns, from focal to diffuse and from weak to strong, was noted. Only 70% of FIGO grades 1 and 2 endometrioid carcinomas and 26% of grade 3 endometrioid carcinomas possessed the reportedly characteristic endometrial cancer immunophenotype p16 (-), ER (+), PR (+), mCEA (-), and vimentin (+). Endometrial cancers demonstrate substantial immunophenotypic diversity that remained apparent even within groups of similar histologic subtype and grade. ER, PR, and p16 expression was more illustrative of tumor type and degree of differentiation than they were of endometrial origin. In contrast, the vimentin-positive/CEA-negative phenotype remained the most constant among all endometrial cancers.

Background: The population-based case-control study is not suited to the evaluation of rare genetic (or environmental) factors. The use of a novel case-control design in which cases have second primaries and controls are cancer survivors has been proposed for this purpose., Methods: We report results from an international study of melanoma that involved population-based ascertainment of incident cases of second or subsequent primary melanoma as the 'case' group and incident cases of first primary melanoma as the 'control' group. We evaluate the validity of the study design by comparing the results obtained for phenotypic factors that have been shown consistently to be associated with melanoma in previous conventional studies with the results from a conventional case-control study conducted in Connecticut and from literature reviews., Results: All but one of the known risk factors for melanoma were shown to be significantly associated with melanoma in our study, though the individual odds ratios appear to be somewhat attenuated relative to the magnitudes typically observed in the literature., Conclusions: Patients with a second or subsequent primary cancer of a single type represent a potentially valuable and under-utilized resource for the study of cancer aetiology.

Background: Germline mutations in the CDKN2A gene have been linked to melanoma incidence in many families with multiple cases of the disease. Previous studies of multiple-case families have indicated that the lifetime risk (i.e., penetrance) of melanoma in CDKN2A mutation carriers is very high, ranging from 58% in Europe to 91% in Australia by age 80 years. In this study, we examined lifetime melanoma risk among CDKN2A mutation carriers using carriers who were identified in a population-based study of melanoma., Methods: Probands for the study were incident case patients with either first or subsequent melanoma who were identified in nine geographic regions in Australia, Canada, the United States, and Italy. A total of 3626 probands (53% participation rate) with adequate DNA for analysis were recruited and genotyped for CDKN2A mutations. From the 3550 probands whose DNA could be amplified by polymerase chain reaction of CDKN2A exons 1alpha, 2, and 3 and surrounding regions, 65 mutation carriers were identified. Melanoma histories in first-degree relatives of these probands were used to calculate the lifetime risk in CDKN2A mutation carriers using the kin-cohort method., Results: The risk of melanoma in CDKN2A mutation carriers was approximately 14% (95% CI = 8% to 22%) by age 50 years, 24% (95% CI = 15% to 34%) by age 70 years, and 28% (95% CI = 18% to 40%) by age 80 years. Eighteen probands had three or more first-degree relatives with melanoma, but only one was a carrier of a CDKN2A mutation., Conclusions: CDKN2A mutation carriers in the general population have a much lower risk of melanoma than that suggested by estimates obtained from multiple-case families. The preponderance of familial clustering of melanoma occurs in families without identifiable mutations in CDKN2A.

Endometrial stromal sarcomas are low-grade malignant tumors that may pose a diagnostic challenge, especially when they are present in an extrauterine site. Owing to the presence of an arborizing vasculature and cells with an undifferentiated appearance, endometrial stromal sarcomas can be confused with several soft-tissue neoplasms. We studied 17 endometrial stromal sarcomas, eight hemangiopericytomas, 14 solitary fibrous tumors, and 16 synovial sarcomas immunohistochemically, detecting the following antigens: CD10, estrogen receptor, progesterone receptor, bcl-2, CD34, smooth muscle antigen, epithelial membrane antigen and cytokeratin (AE1/AE3). Most endometrial stromal sarcomas stained positively for CD10 (16/17), estrogen receptor (17/17), progesterone receptor (15/17), and bcl-2 (17/17). Staining with antismooth muscle antigen was seen in 11 of 17 cases of endometrial stromal sarcoma, with more intense staining seen in areas showing smooth muscle differentiation. Staining with AE1/3 was seen in four of 17 endometrial stromal sarcomas, with two of the positive cases containing epithelioid cells. None of the endometrial stromal sarcomas expressed epithelial membrane antigen or CD34. More than half of the hemangiopericytomas (4/8) and solitary fibrous tumors (9/14) cases demonstrated CD10 expression either focally or in a patchy cytoplasmic and membranous pattern. Hemangiopericytomas, solitary fibrous tumors, and synovial sarcomas did not express estrogen receptor. Four of eight hemangiopericytomas and seven of 14 solitary fibrous tumors also showed patchy progesterone receptor expression. CD34 expression was identified in six of eight hemangiopericytomas and 13 of 14 solitary fibrous tumors, but we did not find expression of CD34 in synovial sarcoma. Differences between endometrial stromal sarcoma and other soft-tissue tumors were detected for all of the immunohistochemical markers (P<0.05), except anti-bcl-2 and AE1/3. Antibodies against CD10 mark a substantial number of hemangiopericytomas and solitary fibrous tumors (albeit not diffusely) and should always be combined with antiestrogen receptor and CD34 when the differential diagnosis includes endometrial stromal sarcoma. Unlike estrogen receptor antibodies, progesterone receptor antibodies show at least focal nuclear staining in most hemangiopericytomas, solitary fibrous tumors and rare synovial sarcomas, and are not useful for this differential diagnosis. All endometrial stromal sarcomas expressed bcl-2, mostly in a diffuse pattern, but this did not distinguish between endometrial stromal sarcoma and mimics. We therefore recommend the use of a small antibody panel comprising anti-CD10, anti-estrogen receptor, and anti-CD34 to distinguish endometrial stromal sarcomas from tumors with a predominant hemangiopericytomatous growth pattern.