Faculty Bibliography

Background: Most skin cancer-related deaths are due to malignant melanoma. Risk assessment criteria for melanoma currently include skin phenotype, family and sun exposure history, factors that are subject to observer and recall bias. Genetic markers of susceptibility have been identified in association studies; however little progress has been made in developing them to improve screening and identification of individuals at risk of melanoma. Tyrosinase (TYR), a known susceptibility gene and a determinant of skin pigmentation, was thus investigated further to characterize its association with melanoma susceptibility and to identify markers which can be used in a risk assessment model. Methods: The cohort consisted of 326 individuals diagnosed with melanoma and 400 control subjects. TYR was interrogated using fifteen tag single nucleotide polymorphisms (SNPs) spanning the gene and statistical association tests performed. Additionally, ancestry informative markers were utilized to correct for population genetic sub-structure. Haplotype analysis was performed to determine if specific regions of the gene contributed more significantly to susceptibility. Coding regions of the gene are currently being sequenced and identified variants will be tested for impact on enzymatic function. Results: Of the 15 SNPs, 8 were associated with melanoma; 4 with decreased risk (Odds ratios 0.41-0.71) and 4 with increased risk (Odds ratios 1.43-1.96). SNPs localized to 2 regions of the gene (spanning exon 1 to intron 2 and intron 3 to 4) with markers of increased as well as decreased susceptibility present in both areas. With the exception of one coding region variant, SNPs were localized to introns. Conclusions: SNPs localized to TYR may serve as useful biomarkers for determining susceptibility to melanoma. We are currently sequencing the gene in our population in order to identify additional and potentially more potent markers of melanoma susceptibility. Coding region variants are being characterized for their effect on protein stability and enzyme activity such that functional active variants (most likely to affect susceptibility to melanoma) can be identified and assessed for their utility in melanoma risk assessment

Background: Several studies have provided evidence that solid tumors are polyclonal malignancies, an observation which may contribute to difficulties in achieving durable treatment responses. In some patients, molecularly targeted therapies may be compromised due to heterogeneity among tumor subclones. In this study we compared conventional DNA sequencing with a fluorescent-based mutant-specific PCR (MS-PCR) assay to detect the BRAF hotspot mutation V600E in a large panel of patient tumors, including paired primary and metastatic tumors from individual patients. Methods: BRAF MS-PCR and conventional sequencing were performed on DNA from 304 tumors (112 melanoma, 110 ovarian, 82 prostate) to determine the presence of the BRAFV600E hot-spot mutation. Among the melanomas were 18 matched primary and metastatic specimens, and 40 metastatic specimens from 19 patients, each of whom had 2 or more metastases. Results: DNA sequencing detected mutations in 5/110 (4.5%) ovarian tumors, 1/82 (1.2%) prostate tumors, and 36/112 (32%) melanomas. In contrast, the MS-PCR assay detected mutations in 12/110 (11%) ovarian tumors, 15/82 (18%) prostate tumors and 85/112 (76%) melanomas. The presence of contaminating normal tissue was scored for each melanoma sample, but excess normal tissue did not influence the results using either methodology. In all cases mutations detected by sequencing were also detected by MSPCR. Among 18 patients with matched primary and metastatic melanoma, 8/18 (44%) had discordant results including 2 patients with mutant primary tumors and wild-type metastases; among the 19 patients with multiple metastases 5/19 (26%) had discordant (both wild-type and mutant) tumors. Conclusions: Using a highly sensitive BRAF mutation detection method, we observed substantial evidence for heterogeneity within clinical tumor specimens. This was especially true in melanoma samples, where multiple specimens from individual patients differed with respect to the presence of the mutant BRAF allele. These results suggest that failures of molecularly targeted therapies, such as those directed against mutant BRAF, may be due in part to a lack of clonality among the tumors under treatment

PURPOSE: Both phosphatidylinositol 3-kinase/AKT and RAS/mitogen-activated protein kinase signal transduction pathways mediate 4E-BP1 phosphorylation, releasing 4E-BP1 from the mRNA cap and permitting translation initiation. Given the prevalence of PTEN and BRAF mutations in melanoma, we first examined translation initiation, as measured by phosphorylated 4E-BP1 (p-4E-BP1), in metastatic melanoma tissues and cell lines. We then tested the association between amounts of total and p-4E-BP1 and patient survival. EXPERIMENTAL DESIGN: Seven human metastatic melanoma cells lines and 72 metastatic melanoma patients with accessible metastatic tumor tissues and extended follow-up information were studied. Expression of 4E-BP1 transcript, total 4E-BP1 protein, and p-4E-BP1 was examined. The relationship between 4E-BP1 transcript and protein expression was assessed in a subset of patient tumors (n = 41). The association between total and p-4E-BP1 levels and survival was examined in the larger cohort of patients (n = 72). RESULTS: 4E-BP1 was hyperphosphorylated in 4 of 7 melanoma cell lines harboring both BRAF and PTEN mutations compared with untransformed melanocytes or RAS/RAF/PTEN wild-type melanoma cells. 4E-BP1 transcript correlated with 4E-BP1 total protein levels as measured by the semiquantitative reverse-phase protein array (P = 0.012). High levels of p-4E-BP1 were associated with worse overall and post-recurrence survival (P = 0.02 and 0.0003, respectively). CONCLUSION: Our data show that translation initiation is a common event in human metastatic melanoma and correlates with worse prognosis. Therefore, effective inhibition of the pathways responsible for 4E-BP1 phosphorylation should be considered to improve the treatment outcome of metastatic melanoma patients