Faculty Bibliography

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

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need to develop more effective treatments. We screened 2,000 compounds from the Spectrum Library in human melanoma cell lines to identify compounds that enhanced the cytotoxic effect of temozolomide, a drug used to treat metastatic melanoma. Screening was done with the temozolomide-resistant melanoma cell line SK-MEL-19, and six compounds were identified that had little or no inherent cytotoxicity but significantly enhanced growth-inhibition by temozolomide. These compounds were tested in five additional melanoma cell lines. Cell proliferation and death assays were used to compare the efficacy of single agent temozolomide versus combination treatments. Effects of combination treatment on levels of DNA double-strand breaks, the DNA repair protein O(6)-methylguanine-DNA-methyltransferase, apoptosis [measured by cleaved caspase-3 and poly(ADP-ribose) polymerase], and cell cycle were examined. Pyrimethamine, an antiparasitic, sensitized melanoma cells to temozolomide. Temozolomide combined with Pyrimethamine synergistically inhibited cell proliferation in melanoma cells with combination index values of 0.7 or less. In addition, combination treatment induced cell cycle arrest and increased both DNA damage and apoptosis. The increase in cell death due to combination treatment was rescued by leucovorin. Other folate antagonists were also effective enhancers of temozolomide-induced cytotoxicity, and the effects of antifolates were also evident in gliomas. Our screening approach led to the identification of Pyrimethamine, an orally available drug that efficiently crosses the blood-brain barrier, as a potent enhancer of the efficacy of temozolomide as an antineoplastic agent via inhibition of folate metabolism

African American (AA) men with prostate cancer (PCa) have worse disease, with a higher incidence, younger age and more advanced disease at diagnosis, and a worse prognosis, compared to Caucasian (CA) men. In addition to socioeconomic factors and lifestyle differences, molecular alterations contribute to this discrepancy. In this review, we summarize molecular genetics research results interrelated with the biology of PCa racial disparity. Androgen and androgen receptor (AR) pathways have long been associated with prostate growth. Racial differences have also been found among variants of the genes of the enzymes involved in androgen biosynthesis and metabolism, such as SRD5A2, CYP17, and CYP3A4. The levels of expression and CAG repeat length of AR also show racial divergence and may be critical molecular alterations for racial disparity. Growth factors and their receptors, which promote cancer cell growth, are another potential cause of the disparity; both EGFR and EPHB2, two of the most studied receptors, show interethnic differences. Differences have also been found among genes regulating cell apoptosis, such as BCL2, which is increased in PCa in the AA population. Recent developments in genetics, proteomics, and genomics, among other molecular biotechnologies, will greatly aid the advancement of translational research on PCa racial disparity, hopefully culminating in the discovery of novel mechanisms of disease, in addition to prognostic markers and novel therapeutic approaches

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

PURPOSE: In certain cancers, MDM2 SNP309 has been associated with early tumor onset in women. In melanoma, incidence rates are higher in women than in men among individuals less than 40 years of age, but among those older than 50 years of age, melanoma is more frequent in men than in women. To investigate this difference, we examined the association among MDM2 SNP309, age at diagnosis, and gender among melanoma patients. EXPERIMENTAL DESIGN: Prospectively enrolled melanoma patients (N = 227) were evaluated for MDM2 SNP309 and the related polymorphism, p53 Arg72Pro. DNA was isolated from patient blood samples, and genotypes were analyzed by PCR-restriction fragment length polymorphism. Associations among MDM2 SNP309, p53 Arg72Pro, age at diagnosis, and clinicopathologic features of melanoma were analyzed. RESULTS: The median age at diagnosis was 13 years earlier among women with a SNP309 GG genotype (46 years) compared with women with TG+TT genotypes (59 years; P = 0.19). Analyses using age dichotomized at each decade indicated that women with a GG genotype had significantly higher risks of being diagnosed with melanoma at ages <50 years compared with women >or=50 years, but not when the comparison was made between women <60 and >or=60 years. At ages <50 years, women with a GG genotype had a 3.89 times greater chance of being diagnosed compared with women with TG+TT genotypes (P = 0.01). Similar observations were not seen among men. CONCLUSIONS: Our data suggest that MDM2 may play an important role in the development of melanoma in women. The MDM2 SNP309 genotype may help identify women at risk of developing melanoma at a young age

BACKGROUND:: Despite the lack of an established survival benefit of sentinel lymph node (SLN) biopsy, this technique has been increasingly applied in the staging of thin (</=1 mm) melanoma patients, without clear evidence to support this recommendation. The authors performed a meta-analysis to estimate the risk, potential predictors, and outcome of SLN positivity in this group of patients. METHODS:: MEDLINE, EMBASE, and Cochrane databases were searched for rates of SLN positivity in patients with thin melanoma. The methodologic quality of included studies was assessed using the Methodological Index for Non-Randomized Studies criteria. Heterogeneity was assessed using the Cochran Q statistic, and publication bias was examined through funnel plot and the Begg and Mazumdar method. Overall SLN positivity in thin melanoma patients was estimated using the DerSimonial-Laird random effect method. RESULTS:: Thirty-four studies comprising 3651 patients met inclusion criteria. The pooled SLN positivity rate was 5.6%. Significant heterogeneity among studies was detected (P = .005). There was no statistical evidence of publication bias (P = .21). Eighteen studies reported select clinical and histopathologic data limited to SLN-positive patients (n = 113). Among the tumors from these patients, 6.1% were ulcerated, 31.5% demonstrated regression, and 47.5% were Clark level IV/V. Only 4 melanoma-related deaths were reported. CONCLUSIONS:: Relatively few patients with thin melanoma have a positive SLN. To the authors' knowledge, there are no clinical or histopathologic criteria that can reliably identify thin melanoma patients who might benefit from this intervention. Given the increasing diagnosis of thin melanoma, in addition to the cost and potential morbidity of this procedure, alternative strategies to identify patients at risk for lymph node disease are needed. Cancer 2009. (c) 2008 American Cancer Society

The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31-34) frequently amplified in melanoma, is commonly up-regulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmia-associated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182's proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy

PURPOSE: The tumor microenvironment significantly influences prostate cancer progression. Androgen receptor exerts its effect through downstream target genes to regulate prostate cancer cell proliferation. The c-FLIP gene was recently shown to be an androgen receptor target gene. c-FLIP is an inactive homologue of caspase-8 and, thus, it inhibits the death receptor mediated apoptosis pathway. c-FLIP over expression was shown to accelerate the progression of prostate cancer cells to androgen independence. We evaluated the role of c-FLIP expression in stromal cells in prostate cancer development. MATERIALS AND METHODS: We examined c-FLIP expression in 53 androgen dependent and 21 androgen independent prostate cancer stromal cells by immunohistochemical analysis. The effects of c-FLIP over expression in stromal cells on the growth and invasion of LNCaP and PC3 prostate cancer cells were determined in indirect coculture systems. RESULTS: At the androgen dependent stage the stromal c-FLIP level was increased in prostate cancer tissue. The expression level of stromal c-FLIP was associated with tumor differentiation. However, stromal c-FLIP expression was not increased in androgen independent human prostate cancer. c-FLIP over expression in stromal cells stimulated the growth and invasion of prostate cancer, including LNCaP and PC3 cells in vitro. CONCLUSIONS: These results indicate the over expression of stromal c-FLIP and its function for promoting prostate cancer growth and invasion