Serum selenium levels predict survival after breast cancer.

BACKGROUND: Studies have reported an inverse relationship between serum selenium levels and cancer incidence, but the impact of low serum selenium status on survival after a diagnosis of breast cancer has not been established. METHODS: We obtained a blood sample from 546 women diagnosed with a first primary invasive breast cancer between 2008 and 2015 in the region of Szczecin, Poland. Blood was collected after diagnosis, but prior to treatment. Serum selenium was quantified by mass spectroscopy and each patient was assigned to one of four categories (quartiles) based on the distribution in the entire cohort. Patients were followed from diagnosis to death over a mean follow-up of 3.8 years. Vital status was obtained by linkage to the Polish National Death Registry. RESULTS: The 5-year overall actuarial survival was 68.1% for women in the lowest (< 64.4 µg/L) and 82.5% for those in the highest (> 81.0 µg/L) quartile of serum selenium. In an adjusted analysis, the hazard ratio for death was 2.49 (95%CI 1.53-4.04; P = 0.0002) for patients in the lowest quartile of serum selenium, compared to those in all other quartiles. The effect of low selenium on breast cancer-specific mortality was stronger for women who were past smokers (HR 6.03; 95%CI 1.96-18.6; P = 0.0002). CONCLUSIONS: This study suggests that a selenium level in excess of 64.4 µg//L might be beneficial for women undergoing treatment for breast cancer and that selenium supplementation to achieve this level may favorably impact the outcome. Further studies are needed to confirm this association and to evaluate the impact of selenium supplementation on breast cancer survival among women with low post-diagnostic selenium levels.

The impact of an expanded genetic testing program and selective oophorectomy on the incidence of ovarian cancer in West Pomerania.

The aim of the study was to evaluate the impact of a regional population-based genetic testing program on the incidence of ovarian cancer in West Pomerania. Between 1999 and 2010, a total of 37,552 women ages 35 to 70 were tested for three BRCA1 founder mutations at the outpatient genetics clinic of the Pomeranian Medical University in Szczecin, Poland. A total of 641 women were found to carry a mutation (1.7%) and of these, 220 had a prophylactic oophorectomy (34.3%). A total of 12 women had an occult cancer diagnosed at the time of prophylactic oophorectomy (5.5%). We estimate that 26 more ovarian cancers would have been diagnosed by January 2015 in the absence of these oophorectomies and that an additional 25 cancers will be prevented in the future (total 51). During this period, 1611 ovarian cancers were diagnosed in the region; therefore we estimate that approximately 1.6% of ovarian cancers were prevented between 1999 and 2015 by our genetic testing program. We conclude that the prophylactic oophorectomies performed between 1999 and 2010 as a result of widespread BRCA1 mutation testing have reduced the incidence of ovarian cancer in Pomerania by a small amount (about 1.6%), and that the impact of genetic testing will increase in the coming years.

Cumulative effects of genetic markers and the detection of advanced colorectal neoplasias by population screening.

Genetic markers associated with colorectal cancer may be used in population screening for the early identification of patients at elevated risk of disease. We genotyped 3059 individuals with no cancer family history for eight markers previously associated with colorectal cancer. After colonoscopy, the genetic profile of cases with advanced colorectal neoplasia (213) was compared with the rest (2846). rs2066847 and rs6983267 were significantly associated with the risk of advanced colorectal neoplasia but with limited effect on their own [odds ratio (OR) 1.59; 95% confidence interval (CI) 1.02-2.41; p = 0.033 and OR 1.45; 95% CI 1.02-2.12; p = 0.044, respectively]. Cumulative effects, in contrast, were associated with high risk: the combination of rs2066847, rs6983267, rs4779584, rs3802842 and rs4939827 minimized the number of markers considered, while maximizing the relative size of the carrier group and the risk associated to it, for example, for at least two cumulated risk markers, OR is 2.57 (95% CI 1.50-4.71; corrected p-value 0.0079) and for three or more, OR is 3.57 (95% CI 1.91-6.96; corrected p-value 0.00074). The identification of cumulative models of - otherwise - low-risk markers could be valuable in defining risk groups, within an otherwise low-risk population (no cancer family history).

Mutations predisposing to breast cancer in 12 candidate genes in breast cancer patients from Poland.

A number of genes other than BRCA1 and BRCA2 have been associated with breast cancer predisposition, and extended genetic testing panels have been proposed. It is of interest to establish the full spectrum of deleterious mutations in women with familial breast cancer.We performed whole-exome sequencing of 144 women with familial breast cancer and negative for 11 Polish founder mutations in BRCA1, CHEK2 and NBS1, and we evaluated the sequences of 12 known breast cancer susceptibility genes. A truncating mutation in a breast cancer gene was detected in 24 of 144 women (17%) with familial breast cancer. A BRCA2 mutation was detected in 12 cases, a (non-founder) BRCA1 mutation was detected in 5 cases, a PALB2 mutation was detected in 4 cases and an ATM mutation was detected in 2 cases. Polish women with familial breast cancer who are negative for founder mutations in BRCA1, CHEK2 and NBS1 should be fully screened for mutations in BRCA1, BRCA2 and PALB2. The PALB2 founder mutation c.509_519delGA should be included in the panel of Polish founder mutations.

Recurrent mutations of BRCA1 and BRCA2 in Poland: an update.

Three founder alleles of BRCA1 (C61G, 4153delA, 5382insC) were reported in Poland in 2000, and these three mutations have comprised the standard testing panel used throughout the country. However, since 2000, other recurrent mutations of BRCA1 and BRCA2 have been reported. To establish if the inclusion of one or more of these mutations will increase the sensitivity of the standard test panel, we studied 1164 Polish women with unselected breast cancer diagnosed at age of 50 or below. All women were genotyped for 12 recurrent mutations of BRCA1 and BRCA2. We identified a mutation in 83 of 1164 patients (7.1%) including 61 women with one of the original three mutations (C61G, 4153delA, 5382insC) and 22 women with a different mutation (1.9%). Three new mutations (3819del5, 185delAG and 5370C>T) were seen in multiple families. By including these three mutations in the extended panel, the mutant frequency increased from 5.2 to 6.7%. Polish women with breast cancer diagnosed at age of 50 or below should be screened with a panel of six founder mutations of BRCA1 (C61G, 4153delA, 5382insC, 3819del5, 185delAG and 5370C>T).

Common variants of ZNF750, RPTOR and TRAF3IP2 genes and psoriasis risk.

Psoriasis vulgaris is a genetically heterogenous disease with unclear molecular background. We assessed the association of psoriasis and its main clinical phenotypes with common variants of three potential psoriasis susceptibility genes: ZNF750, RPTOR and TRAF31P2. We genotyped 10 common variants in a cohort of 1,034 case-control individuals using Taqman genotyping assays and sequencing. Minor alleles of all four TRAF3IP2 variants were more frequent among cases. The strongest, significant association was observed for rs33980500 (OR = 2.5, p = 0.01790). Minor allele of this SNP was always present in two haplotypes found to be associated with increased psoriasis risk: rs13196377_G + rs13190932_G + rs33980500_T + rs13210247_A (OR = 2.7, p = 0.0054) and rs13196377_A + rs13190932_A + rs33980500_T + rs13210247_G (OR = 1.8, p = 0.0008). Analyses of clinically relevant phenotypes revealed association of rs33980500 with pustular psoriasis (OR = 1.2, p = 0.0109). We observed significant connection of severity of cutaneous disease with variation at rs13190932 and suggestive with three remaining TRAF3IP2 SNPs. Another positive associations were found between age of onset and familial aggregation of disease: smoking and younger age of onset, smoking and occurrence of pustular psoriasis, nail involvement and arthropatic psoriasis, nail involvement and more severe course of psoriasis. We found no statistically significant differences in the prevalence of the examined variants of RPTOR and ZNF750 genes among our cases and controls. We have replicated the association of TRAF3IP2-_rs33980500 variant with the susceptibility to psoriasis. We have found new associations with clinically relevant subphenotypes such as pustular psoriasis or moderate-to-severe cases. We ascertain no connection of RPTOR and ZNF750 variants with psoriasis or its subphenotypes.

Common variants of the EPDR1 gene and the risk of Dupuytren's disease.

The object of this study was the investigation of 3 common variants of single nucleotide polymorphisms of the ependymin-related gene 1 and its association with the occurrence of Dupuytren's disease. DNA samples were obtained from the peripheral blood of 508 consecutive patients. The control group comprised 515 healthy adults who were age-matched with the Dupuytren's patients. 3 common variants were analysed using TaqMan® genotyping assays and sequencing. The differences in the frequencies of variants of single nucleotide polymorphisms in patients and the control group were statistically tested. Additionally, haplotype frequency and linkage disequilibrium were analysed for these variants. A statistically significant association was noted between rs16879765_CT, rs16879765_TT and rs13240429_AA variants and Dupuytren's disease. 2 haplotypes: rs2722280_C+rs13240429_A+rs16879765_C and rs2722280_C+rs13240429_G+rs16879765_T were found to be statistically significantly associated with Dupuytren's disease. Moreover, we found that rs13240429 and rs16879765 variants were in strong linkage disequilibrium, while rs2722280 was only in moderate linkage disequilibrium. No significant differences were found in the frequencies of the variants of the gene between the groups with a positive and negative familial history of Dupuytren's disease. In conclusion, results of this study suggest that EPDR1 gene can be added to a growing list of genes associated with Dupuytren's disease development.

A personalised approach to prostate cancer screening based on genotyping of risk founder alleles.

BACKGROUND: To evaluate whether genotyping for 18 prostate cancer founder variants is helpful in identifying high-risk individuals and for determining optimal screening regimens. METHODS: A serum PSA level was measured and a digital rectal examination (DRE) was performed on 2907 unaffected men aged 40-90. Three hundred and twenty-three men with an elevated PSA (≥4 ng ml⁻¹) or an abnormal DRE underwent a prostate biopsy. All men were genotyped for three founder alleles in BRCA1 (5382insC, 4153delA and C61G), for four alleles in CHEK2 (1100delC, IVS2+1G>A, del5395 and I157T), for one allele in NBS1 (657del5), for one allele in HOXB13 (G84E), and for nine low-risk single-nucleotide polymorphisms (SNPs). RESULTS: On the basis of an elevated PSA or an abnormal DRE, prostate cancer was diagnosed in 135 of 2907 men (4.6%). In men with a CHEK2 missense mutation I157T, the cancer detection rate among men with an elevated PSA or an abnormal DRE was much higher (10.2%, P=0.0008). The cancer detection rate rose with the number of SNP risk genotypes observed from 1.2% for men with no variant to 8.6% for men who carried six or more variants (P=0.04). No single variant was helpful on its own in predicting the presence of prostate cancer, however, the combination of all rare mutations and SNPs improved predictive power (area under the curve=0.59; P=0.03). CONCLUSION: These results suggest that testing for germline CHEK2 mutations improves the ability to predict the presence of prostate cancer in screened men, however, the clinical utility of incorporating DNA variants in the screening process is marginal.

Xeroderma pigmentosum genes and melanoma risk.

Xeroderma pigmentosum is a rare autosomal recessive disease that is associated with a severe deficiency in nucleotide excision repair. The presence of a distinct the nucleotide excision repair (NER) mutation signature in melanoma suggests that perturbations in this critical repair process are likely to be involved with disease risk. We hypothesized that persons with polymorphic NER gene(s) are likely to have reduced NER activity and are consequently at an increased risk of melanoma development. We assessed the association between 94 SNPs within seven XP genes (XPA-XPG) and the melanoma risk in the Polish population. We genotyped 714 unselected melanoma patients and 1,841 healthy adults to determine if there were any polymorphisms differentially represented in the disease group. We found that a significantly decreased risk of melanoma was associated with the Xeroderma pigmentosum complementation (XPC) rs2228000_CT genotype (odds ratio [OR] = 0.15; p < 0.001) and the rs2228000_TT genotype (OR = 0.11; p < 0.001) compared to the reference genotype. Haplotype analysis within XPC revealed the rs2228001_A + G1475A_G + G2061A_A + rs2228000_T + rs3731062_C haplotype (OR = 0.26; p < 0.05) was associated with a significantly decreased disease risk. The haplotype analysis within the Xeroderma pigmentosum group D (XPD) showed a modest association between two haplotypes and a decrease in melanoma risk. There were no major differences between the prevalence of the XP polymorphisms among young or older patients with melanoma. Linkage disequilibrium of XPC: rs2228001, G1475A, G2061A, rs2228000 and rs3731062 was found. The data from our study support the notion that only XPC and XPD genes are associated with melanoma susceptibility.

Common variants of the ALDH2 and DHDH genes and the risk of Dupuytren's disease.

The object of this study was the investigation of four common variants of single nucleotide polymorphisms of the aldehyde dehydrogenase H2 gene and dihydrodiol dehydrogenase gene and their association with the occurrence of Dupuytren's disease. DNA samples were obtained from the peripheral blood of 300 consecutive patients. The control group comprised 300 healthy adults who were age matched with the Dupuytren's patients. All four common variants were analysed using TaqMan® genotyping assays and sequencing. The differences in the frequencies of variants of single nucleotide polymorphisms in patients and the control group were statistically tested. No significant differences were found in the frequencies of the variants of the aldehyde dehydrogenase H2 and dihydrodiol dehydrogenase genes between the groups. Likewise, no significant differences were found in the frequencies of the variants of the genes between men and women. We noted a statistically significantly higher prevalence of the variants of the dihydrodiol dehydrogenase gene (rs2270941 and rs11666105) in the group with a positive familial history of Dupuytren's disease, in comparison with the group with a negative familial history.

An inherited NBN mutation is associated with poor prognosis prostate cancer.

BACKGROUND: To establish the contribution of eight founder alleles in three DNA damage repair genes (BRCA1, CHEK2 and NBS1) to prostate cancer in Poland, and to measure the impact of these variants on survival among patients. METHODS: Three thousand seven hundred fifty men with prostate cancer and 3956 cancer-free controls were genotyped for three founder alleles in BRCA1 (5382insC, 4153delA, C61G), four alleles in CHEK2 (1100delC, IVS2+1G>A, del5395, I157T), and one allele in NBS1 (657del5). RESULTS: The NBS1 mutation was detected in 53 of 3750 unselected cases compared with 23 of 3956 (0.6%) controls (odds ratio (OR)=2.5; P=0.0003). A CHEK2 mutation was seen in 383 (10.2%) unselected cases and in 228 (5.8%) controls (OR=1.9; P<0.0001). Mutation of BRCA1 (three mutations combined) was not associated with the risk of prostate cancer (OR=0.9; P=0.8). In a subgroup analysis, the 4153delA mutation was associated with early-onset (age ≤ 60 years) prostate cancer (OR=20.3, P=0.004). The mean follow-up was 54 months. Mortality was significantly worse for carriers of a NBS1 mutation than for non-carriers (HR=1.85; P=0.008). The 5-year survival for men with an NBS1 mutation was 49%, compared with 72% for mutation-negative cases. CONCLUSION: A mutation in NBS1 predisposes to aggressive prostate cancer. These data are relevant to the prospect of adapting personalised medicine to prostate cancer prevention and treatment.

The contribution of founder mutations in BRCA1 to breast cancer in Belarus.

Mutations in the BRCA1 gene increase susceptibility to both breast and ovarian cancer. In some countries, including several in Eastern Europe, founder mutations in the BRCA1 gene are responsible for a significant proportion of breast cancer cases. To estimate the hereditary proportion of breast cancer in Belarus, we sought the presence of any of three founder mutations in BRCA1 (4153delA, 5382insC and C61G) in 500 unselected cases of breast cancer. These mutations have previously been identified in breast/ovarian cancer families from Belarus and from other Slavic countries, including Poland and Russia. One of the three founder mutations in BRCA1 was present in 38 of 500 unselected cases of breast cancer (7.6%). A mutation was found in 12.6% of women diagnosed before age 50 and 5.6% of women diagnosed after age 50. A mutation was identified in 2 of 251 newborn controls (0.8%). The hereditary proportion of breast cancers in Belarus is among the highest of any countries studied to date.

Vitamin D receptor variants and the malignant melanoma risk: a population-based study.

BACKGROUND: There is continuing interest in identifying low-penetrance genes which are associated with an increased susceptibility to common types of cancer, including malignant melanoma. METHODS: We sought to examine the association between four VDR common variants (rs1544410, rs731236, rs10735810, rs4516035) and the risk of melanoma in the Polish population. We also determined the prevalence of compound carriers of VDR and known MM genetic risk factors MC1R and CDKN2A (A148T) variants. We examined 763 unselected melanoma cases, 763 healthy adults matched for sex and age with the melanoma cases and 777 newborns. RESULTS: None of the VDR variants alone or as compound carriers of two or more of the VDR genotypes were associated with MM risk. There were no major differences between the prevalences of the examined variants among patients with MM on UV-exposed and UV-non exposed skin areas, as well as among early-onset and late-onset cases. We found no association between VDR and MC1R or between VDR and CDKN2A common variants. A statistically significant over-representation of one VDR haplotype: rs731236_A+rs1544410_T (OR=3.2, p=0.02) was detected. Linkage disequilibrium of rs1544410 and rs731236 was confirmed. CONCLUSION: To answer the question, whether VDR can be regarded as melanoma susceptibility gene, additional, large multi-center association studies have to be performed.

Cancer risks in first-degree relatives of CHEK2 mutation carriers: effects of mutation type and cancer site in proband.

It is important to have accurate knowledge of the range of cancers associated with various CHEK2 mutations, and of the lifetime risks of cancer associated with each. We wished to establish the relationship between family history, mutation type and cancer risk in families with a CHEK2 mutation. We obtained a blood sample and pedigree information from 2012 unselected women with breast cancer, from 2007 men with prostate cancer and from 1934 patients with colon cancer, from hospitals throughout Poland. Genetic testing was carried out for four founder CHEK2 mutations on all 5953 specimens and 533 carriers were identified. We estimated the risk to age 75 for any cancer in the 2544 first-degree relatives to be 22.3%. After adjusting for mutation type, the risk of breast cancer was much higher among relatives of probands with breast cancer than among relatives of patients with prostate or colon cancer (HR=3.6; 95% CI=2.1-6.2; P=0.0001). Similarly, the risk of prostate cancer was higher among relatives of probands with prostate cancer than among relatives of patients with breast or colon cancer (HR=4.4; 95% CI=2.2-8.7; P=0.0001) and the risk of colon cancer was higher among relatives of probands with colon cancer than among relatives of patients with prostate or breast cancer (HR=4.2; 95% CI=2.4-7.8; P=0.0001). These analyses suggest that the risk of cancer in a carrier of a CHEK2 mutation is dependent on the family history of cancer.

Effect of CHEK2 missense variant I157T on the risk of breast cancer in carriers of other CHEK2 or BRCA1 mutations.

BACKGROUND: Carriers of heterozygous mutations in CHEK2 or BRCA1 are at increased risk of breast cancer. These mutations are rare and a very small number of women in a population will carry two mutations. However, it is of interest to estimate the breast cancer risks associated with carrying two mutations because this information may be informative for genetic counsellors and may provide clues to the carcinogenic process. METHODS: We genotyped 7782 Polish breast cancer patients and 6233 controls for seven founder mutations in BRCA1 and CHEK2. Odds ratios (OR) and 95% confidence intervals (CI) were estimated for the mutations, singly and in combination. RESULTS: Of the 7782 women with breast cancer, 1091 had one mutation (14.0%) and 37 had two mutations (0.5%). Compared to controls, the odds ratio for a BRCA1 mutation in isolation was 13.1 (95% CI 8.2 to 21). The odds ratio was smaller for BRCA1 mutation carriers who also carried a CHEK2 mutation (OR 6.6, 95% CI 1.5 to 29), but the difference was not statistically significant. In contrast, the odds ratio for women who carried two CHEK2 mutations (OR 3.9, 95% CI 1.5 to 10) was greater than that for women who carried one CHEK2 mutation (OR 1.9, 95% CI 1.6 to 2.1). The odds ratio for women who carried both a truncating mutation and the missense mutation in CHEK2 was 7.0 (95% CI 0.9 to 56) and was greater than for women who carried the truncating mutation alone (OR 3.3, 95% CI 2.4 to 4.3) or the missense mutation alone (OR 1.6, 95% CI 1.4 to 1.9), but the difference was not statistically significant. CONCLUSION: Our study suggests that the risk of breast cancer in carriers of a deleterious CHEK2 mutation is increased if the second allele is the I157T missense variant. However, the presence of a CHEK2 mutation in women with a BRCA1 mutation may not increase their risk beyond that of the BRCA1 mutation alone. These suggestive findings need to be verified in other studies.

Vitamin D receptor variants and breast cancer risk in the Polish population.

The aim of the study was to determine whether four VDR gene single nucleotide polymorphisms (SNPs: rs1544410, rs731236, rs10735810 and rs4516035) are associated with breast cancer risk in Polish population. Two independent series of female patients were employed: 960 consecutive breast cancer cases, and 800 unselected early onset cases diagnosed under the age of 51. The control group for the consecutive breast cancer cases consisted of 960 healthy, age-matched women with a negative cancer family history. 550 healthy women, aged 51 or less, with negative cancer family history were selected as the independent controls for the early onset breast cancer cases. The frequencies of the VDR polymorphisms in the unselected cases when compared to the respective control population failed to reveal any association between the individual SNPs and disease. Examination of the group of early-onset patients, revealed an association between rs10735810 and increased breast cancer risk. Heterozygous carriers for the change had an OR = 1.73 (95% CI 1.33-2.26, P < 0.0001) and homozygous carriers OR = 2.34 (95% CI 1.71-3.21, P < 0.0001). The remaining three examined SNPs failed to show any association with disease risk. In summary, this study has identified an association between the VDR gene and early onset breast cancer risk in the Polish population.

Estrogen receptor status in CHEK2-positive breast cancers: implications for chemoprevention.

To investigate the relationship between CHEK2 mutation status and estrogen receptor (ER) status in unselected cases of early-onset breast cancer from Poland, we screened 4441 women diagnosed with breast cancer younger than 51 years and 7217 controls for three inherited mutations in CHEK2 (1100delC, IVS2+1G>A, del5395). ER status was compared between CHEK2-positive and CHEK2-negative breast cancer cases. A truncating mutation in CHEK2 was seen in 140 of 4441 cases and in 70 of 7217 controls [odds ratio (OR) = 3.3; 95% CI = 2.5-4.4; p < 0.0001]. ER status was available for 92 of 140 mutation carriers and for 3001 of 4301 non-carriers with breast cancer. The OR was higher for ER-positive cancers (OR = 3.9; 95% CI = 2.7-5.4; p < 0.0001) than for ER-negative cancers (OR = 2.1; 95% CI = 1.3-3.3; p = 0.002). Sixty-six of the 92 breast cancers in carriers of CHEK2 truncating mutations were ER positive compared with 1742 of the 3001 breast cancers in non-carriers (72% vs 58%; p = 0.01). Women with a CHEK2 mutation face a fourfold increase in the risk of ER-positive breast cancer and might be candidates for tamoxifen chemoprevention.

An evaluation of the polymorphisms Ins16bp and Arg72Pro in p53 as breast cancer risk modifiers in BRCA1 and BRCA2 mutation carriers.

The close functional relationship between p53 and the breast cancer susceptibility genes BRCA1 and BRCA2 has promoted the investigation of various polymorphisms in the p53 gene as possible risk modifiers in BRCA1/2 mutation carriers. Specifically, two polymorphisms in p53, c.97-147ins16bp and p.Arg72Pro have been analysed as putative breast cancer susceptibility variants, and it has been recently reported that a p53 haplotype combining the absence of the 16-bp insertion and the presence of proline at codon 72 (No Ins-72Pro) was associated with an earlier age at the onset of the first primary tumour in BRCA2 mutation carriers in the Spanish population. In this study, we have evaluated this association in a series of 2932 BRCA1/2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2.

Common variants of DNA repair genes and malignant melanoma.

In the current study, we evaluated the possible associations of seven common variants of the DNA repair and cell cycle control genes BRCA2 and CHEK2 with malignant melanoma (MM). We genotyped 630 unselected MM patients and over 3700 controls (newborns, age- and sex-matched healthy adults with negative cancer family histories, and the adults selected at random by family doctors) for the prevalence of three common variants of the BRCA2 (T1915M, N991D and N372H) and four common variants of the CHEK2 (1100delC, VS2+1G --> A, I157T and del5395). Our study strongly suggests that the common variant of the BRCA2 gene -- the N991D variant is associated with malignant melanoma risk (OR=1.8, p=0.002 after Bonferroni correction). Patients homozygote for the N991D variant were present in 0.32% of cases and only 0.13% of controls. The other variants studied were not over-represented among MM patients when compared to the general population. In conclusion, we report an increased melanoma risk among carriers of the N991D change of the BRCA2 and no association of the CHEK2 changes with malignant melanoma.