Multiplex Gene Profiling of Cell-Free DNA in Patients With Metastatic Melanoma for Monitoring Disease.

PURPOSE: Hotspot blood cell-free DNA (cfDNA) biomarker assays have limited utility in profiling tumor heterogeneity and burden and in capturing regional metastasis with low disease burden in patients with melanoma. We investigated the utility of a sensitive 54-cancer gene digital next-generation sequencing approach targeting blood cfDNA single nucleotide variants (SNVs) and copy number amplification for monitoring disease in patients with melanoma with regional or distant organ metastasis (DOM). PATIENTS AND METHODS: A total of 142 blood samples were evaluated by digital next-generation sequencing across two patient cohorts. Cohort 1 contained 44 patients with stage II, III, or IV disease with matched tumor DNA at the time of surgery or DOM. Cohort 2 consisted of 12 overlapping patients who were longitudinally monitored after complete lymph node dissection to DOM. RESULTS: In cohort 1, cfDNA SNVs were detected in 75% of patients. Tumor-cfDNA somatic SNV concordance was 85% at a variant allele fraction of ≥ 0.5%. An SNV load (number of unique SNVs detected) of greater than two SNVs and an SNV burden (total cumulative SNV VAF) of > 0.5% were significantly associated with worse overall survival (P < .05) in stage IV patients. In cohort 2, 98 longitudinal blood samples along with matched regional and distant metastases from 12 stage III patients were analyzed before complete lymph node dissection and throughout disease progression. cfDNA SNV levels correlated with tumor burden (P = .019), enabled earlier detection of recurrence compared with radiologic imaging (P < .01), captured tumor heterogeneity, and identified increasing SNVs levels before recurrence. CONCLUSION: This study demonstrates significant utility for cfDNA profiling in patients with melanoma with regional and/or distant metastasis for earlier detection of recurrence and progression and in capturing tumor evolution and heterogeneity, thus impacting how patients with melanoma are monitored.

Liquid biopsy utility for the surveillance of cutaneous malignant melanoma patients.

Cutaneous melanoma is one of the highest incident-rate cancers with increasing prevalence in Western societies. Despite the advent of new approved therapeutics, the 5-year overall survival rate of stage IV melanoma patients remains below 15%. Current treatments for late stage disease have shown higher efficacy when treated at a lower disease burden. Thus, blood-based biomarkers capable of detecting melanoma prior to clinically evident distant metastasis, will improve the treatment and outcomes for melanoma patients. To that end, effective treatment of melanoma necessitates identification of patients at risk for developing distant metastases. Furthermore, employing blood biomarkers that monitor cancer progression over the course of treatment is a promising solution to post-treatment drug resistance often developed in melanoma patients. Non-invasive blood biomarker assays allow for regular dynamic monitoring of disease. "Liquid Biopsy" of blood, which exploits circulating tumor cells (CTCs), cell-free circulating tumor DNA (ctDNA) and cell-free circulating microRNA (cmiRNA), has been shown to detect prognostic factors for relapse in AJCC stage III and stage IV melanoma patients. Moreover, molecular characterization of CTC and analysis of various forms of ctDNA present promising potential in development of individualized therapy for melanoma patients. New approaches such as massive parallel sequencing (MPS) provide a comprehensive view of the disease progression, allowing for the selection of therapeutic options for individual patients. With advancements of improving molecular assays, liquid biopsy analysis as a powerful, routine clinical assay for melanoma patients, is highly promising prospective.

In Situ Sodium Bisulfite Modification of Genomic DNA from Microdissected Melanoma Paraffin-Embedded Archival Tissues.

DNA methylation analysis of paraffin-embedded archival tumor tissues (PEAT) is important in clinical and translational research studies. Efficient identification and isolation of homogeneous cell population, optimal DNA extraction, and sodium bisulfite modification (SBM) are essential, particularly in small tumor lesions. Laser capture microdissection (LCM) coupled with an in situ SBM improves the specificity, through histopathology accuracy, and the amount of genomic DNA modified for downstream methylation assays.

RASSF8 regulates progression of cutaneous melanoma through nuclear factor-κb.

Our group previously demonstrated that the RASSF1 gene has a significant tumor suppressor role in cutaneous melanoma. The RASSF8 gene is a member of the N-terminal RASSF gene family. Previously, we identified RASSF8 (HOJ1, NCBI Gene ID:11228) expression in cutaneous melanoma; however the functional role of RASSF8 in melanoma is not known. RASSF8 expression was assessed in melanoma cell lines and tumors of different AJCC stages. Results indicated that RASSF8 expression was low in metastatic melanoma lines and decreased with melanoma progression. We then explored the mechanism of RASSF8 downregulation in melanoma by assessing methylation of RASSF8 and demonstrated that methylation of RASSF8 gene promoter was higher in advanced than in early stages melanomas. Functional activity of RASSF8 in melanoma lines by knockdown and overexpression of RASSF8 demonstrated that RASSF8 expression significantly inhibited cell growth, cell migration and invasion, whereas knockdown of RASSF8 expression significantly increased cell growth, cell migration and invasion of melanoma cells by increasing expression of P65 and its downstream target IL-6. Moreover RASSF8 was found to induce apoptosis in melanoma cells by activating the P53-P21 pathway, and also in vivo studies demonstrated that inhibiting RASSF8 increases the tumorigenic properties of human melanoma xenografts. These results suggest that RASSF8 plays a significant role in suppressing the progression of cutaneous melanoma.

MicroRNA-93 activates c-Met/PI3K/Akt pathway activity in hepatocellular carcinoma by directly inhibiting PTEN and CDKN1A.

To assess the role of microRNAs (miR) in hepatocellular carcinoma (HCC), we performed comprehensive microRNA expression profiling using HCC cell lines and identified miR-93 as a novel target associated with HCC. We further verified miR-93 expression levels in advanced HCC tumors (n=47) by a direct PCR assay and found that elevated miR-93 expression level is significantly correlated with poor prognosis. Elevated miR-93 expression significantly stimulated in vitro cell proliferation, migration and invasion, and additionally inhibited apoptosis. We confirmed that miR-93 directly bound with the 3' untranslated regions of the tumor-suppressor genes PTEN and CDKN1A, respectively,and inhibited their expression. As a result of this inhibition, the c-Met/PI3K/Akt pathway activity was enhanced. IHC analysis of HCC tumors showed significant correlation between c-Met protein expression levels and miR-93 expression levels. Knockdown of c-Met inhibited the activation of the c-Met/PI3K/Akt pathway regardless of hepatocyte growth factor (HGF) treatment, and furthermore reduced the expression of miR-93 in these HCC cells. miR-93 also rendered cells to be more sensitive to sorafenib and tivantinib treatment. We concluded that miR-93 stimulated cell proliferation, migration, and invasion through the oncogenic c-Met/PI3K/Akt pathway and also inhibited apoptosis by directly inhibiting PTEN and CDKN1A expression in human HCC.

Patient-specific driver gene prediction and risk assessment through integrated network analysis of cancer omics profiles.

Extensive and multi-dimensional data sets generated from recent cancer omics profiling projects have presented new challenges and opportunities for unraveling the complexity of cancer genome landscapes. In particular, distinguishing the unique complement of genes that drive tumorigenesis in each patient from a sea of passenger mutations is necessary for translating the full benefit of cancer genome sequencing into the clinic. We address this need by presenting a data integration framework (OncoIMPACT) to nominate patient-specific driver genes based on their phenotypic impact. Extensive in silico and in vitro validation helped establish OncoIMPACT's robustness, improved precision over competing approaches and verifiable patient and cell line specific predictions (2/2 and 6/7 true positives and negatives, respectively). In particular, we computationally predicted and experimentally validated the gene TRIM24 as a putative novel amplified driver in a melanoma patient. Applying OncoIMPACT to more than 1000 tumor samples, we generated patient-specific driver gene lists in five different cancer types to identify modes of synergistic action. We also provide the first demonstration that computationally derived driver mutation signatures can be overall superior to single gene and gene expression based signatures in enabling patient stratification and prognostication. Source code and executables for OncoIMPACT are freely available from http://sourceforge.net/projects/oncoimpact.

Epigenetic changes of EGFR have an important role in BRAF inhibitor-resistant cutaneous melanomas.

BRAF mutations are frequent in cutaneous melanomas, and BRAF inhibitors (BRAFi) have shown remarkable clinical efficacy in BRAF mutant melanoma patients. However, acquired drug resistance can occur rapidly and tumor(s) often progresses thereafter. Various mechanisms of BRAFi resistance have recently been described; however, the mechanism of resistance remains controversial. In this study, we developed BRAFi-resistant melanoma cell lines and found that metastasis-related epithelial to mesenchymal transition properties of BRAFi-resistant cells were enhanced significantly. Upregulation of EGFR was observed in BRAFi-resistant cell lines and patient tumors because of demethylation of EGFR regulatory DNA elements. EGFR induced PI3K/AKT pathway activation in BRAFi-resistant cells through epigenetic regulation. Treatment of EGFR inhibitor was effective in BRAFi-resistant melanoma cell lines. The study demonstrates that EGFR epigenetic activation has important implications in BRAFi resistance in melanoma.

Genome-wide hypomethylation and specific tumor-related gene hypermethylation are associated with esophageal squamous cell carcinoma outcome.

INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) is a cancer of variable outcomes with limited effective treatments resulting in poor overall survival (OS). Epigenetic alterations contributing to this deadly cancer type that can be used as novel therapeutic or diagnostic targets are still poorly understood. METHODS: We explored genome-wide DNA methylation data from The Cancer Genome Atlas project and identified a panel of tumor-related genes hypermethylated in ESCC. The methylation statuses of RASSF1, RARB, CDKN2A (p16INK4a, p14ARF), APC, and RUNX3 genes and long interspersed nucleotide element-1 (LINE-1) were validated in a large cohort (n = 140) of clinically well-annotated ESCC specimens and esophageal normal mucosa (n = 28) using a quantitative methylation-specific polymerase chain reaction. RESULTS: Hypermethylation of RARB, p16INK4a, RASSF1, APC, RUNX3, and p14ARF were observed in 55%, 24%, 20%, 19%, 14%, and 8% of specimens, respectively. Hypermethylation of APC was significantly associated with tumor depth (p = 0.02) and American Joint Committee on Cancer stage (p = 0.03). Global DNA methylation level, assessed by LINE-1, was significantly lower in ESCC than in normal mucosa (p < 0.0001), and lower in greater than or equal to T2 (n = 69) than T1 tumors (n = 45; p = 0.03). There was a significant inverse correlation between LINE-1 and RARB methylation (p = 0.008). Importantly, hypermethylation of RASSF1 and APC genes was significantly associated with overall survival (OS; p = 0.006 and p = 0.007, respectively). In addition, patients with tumors containing a higher number of methylated genes (greater than two genes) presented worse OS (p = 0.003). CONCLUSIONS: This study demonstrates that epigenetic alterations of a panel of tumor-related genes and the noncoding region LINE-1 can be used as prognostic indicators and help in clinical management of ESCC patients.

Genome-wide characterization of circulating tumor cells identifies novel prognostic genomic alterations in systemic melanoma metastasis.

BACKGROUND: Circulating tumor cells (CTC) have been found in patients with metastatic melanoma and are associated with advanced melanoma stage and poor patient outcome. We hypothesize that CTC harbor genomic changes critical in the development of distant systemic metastasis. Here, we present the first genome-wide copy-number aberration (CNA) and loss of heterozygosity (LOH)-based characterization of melanoma CTC. METHODS: CTC were isolated from peripheral blood monocytes of 13 melanoma patients with regional metastasis stage IIIB/C using antibodies against melanoma-associated cell surface gangliosides. RESULTS: We characterized 251 CNA in CTC. Comparative analysis demonstrated >90% concordance in single-nucleotide polymorphism profiles between paired CTC and tumor metastases. In particular, there were notable recurring CNA across patients. In exploratory studies, the presence of several top CTC-associated CNA was verified in distant metastasis (stage IV) from 27 patients, suggesting that certain genomic changes are propagated from regional metastasis to CTC and to distant systemic metastases. Lastly, an exploratory biomarker panel derived from 5 CTC-associated CNA [CSMD2 (CUB and Sushi multiple domains 2), 1p35.1; CNTNAP5 (contactin associated protein-like 5), 2q14.3; NRDE2 (NRDE-2, necessary for RNA interference, domain containing), 14q32.11; ADAM6 (ADAM metallopeptidase domain 6, pseudogene), 14q32.33; and TRPM2 (transient receptor potential cation channel, subfamily m, member 2), 21q22.3] conferred prognostic utility for melanoma recurrence [hazard ratio (HR), 1.14; CI, 1.00-1.44; P = 0.0471] and death (HR, 2.86; CI, 1.23-14.42; P = 0.0014) in 35 patients with stage IIIB/C melanoma, with a 5-year disease-free survival of 13% vs 69% (P = 0.0006) and overall survival of 28% vs 94% between high-risk and low-risk groups defined by the biomarker panel, respectively. CONCLUSIONS: This study provides the first detailed CNA-based profile of melanoma CTC and illustrates how CTC may be used as a novel approach for identification of systemic metastasis.

Epigenome-wide DNA methylation landscape of melanoma progression to brain metastasis reveals aberrations on homeobox D cluster associated with prognosis.

Melanoma brain metastasis (MBM) represents a frequent complication of cutaneous melanoma. Despite aggressive multi-modality therapy, patients with MBM often have a survival rate of <1 year. Alteration in DNA methylation is a major hallmark of tumor progression and metastasis; however, it remains largely unexplored in MBM. In this study, we generated a comprehensive DNA methylation landscape through the use of genome-wide copy number, DNA methylation and gene expression data integrative analysis of melanoma progression to MBM. A progressive genome-wide demethylation in low CpG density and an increase in methylation level of CpG islands according to melanoma progression were observed. MBM-specific partially methylated domains (PMDs) affecting key brain developmental processes were identified. Differentially methylated CpG sites between MBM and lymph node metastasis (LNM) from patients with good prognosis were identified. Among the most significantly affected genes were the HOX family members. DNA methylation of HOXD9 gene promoter affected transcript and protein expression and was significantly higher in MBM than that in early stages. A MBM-specific PMD was identified in this region. Low methylation level of this region was associated with active HOXD9 expression, open chromatin and histone modifications associated with active transcription. Demethylating agent induced HOXD9 expression in melanoma cell lines. The clinical relevance of this finding was verified in an independent large cohort of melanomas (n = 145). Patients with HOXD9 hypermethylation in LNM had poorer disease-free and overall survival. This epigenome-wide study identified novel methylated genes with functional and clinical implications for MBM patients.

Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma.

Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2'-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients.

B7-H3 associated with tumor progression and epigenetic regulatory activity in cutaneous melanoma.

B7-H3, a cell surface transmembrane glycoprotein, was assessed for its functional and prognostic role in cutaneous melanoma progression. B7-H3 expression in melanoma cells was shown to be related to specific downstream signal transduction events as well as associated with functional epigenetic activity. B7-H3 expression and prognostic utility were shown by reverse transcription and real-time PCR and immunohistochemistry analysis on individual melanoma specimens and then verified in clinically annotated melanoma stage III and stage IV metastasis tissue microarrays in a double-blind study. B7-H3 messenger RNA expression was shown to be significantly increased with stage of melanoma (P<0.0001) and significantly associated with melanoma-specific survival in both stage III (P<0.0001) and stage IV (P<0.012) melanoma patients. B7-H3 expression was related to migration and invasion; overexpression of B7-H3 increased migration and invasion, whereas knockdown of B7-H3 reduced cell migration and invasion. MiR-29c expression was shown to inversely regulate B7-H3 expression. Furthermore, we demonstrated that melanoma B7-H3 expression was correlated to phosphorylated signal transducer and activator of transcription-3 activity level in melanoma tissues and cell lines. These studies demonstrate that B7-H3 is a significant factor in melanoma progression and events of metastasis.

Heterogeneous epigenetic regulation of TIMP3 in prostate cancer.

Tissue inhibitor of metalloproteinase-3 (TIMP3) is a tumor suppressor gene frequently downregulated in prostate cancer. The mechanisms involved in TIMP3 transcriptional repression are not fully understood, but evidence suggests that promoter hypermethylation may not be the predominant epigenetic alteration in prostate cancer. To clarify this issue, we examined the contribution of both CpG site promoter methylation and histone modifications on TIMP3 downregulation. Using publicly available data sets, we confirmed that TIMP3 mRNA expression is decreased in prostate tumors relative to normal glands. Immunohistochemical analysis also showed decreased TIMP3 levels in high-grade primary tumors, but promoter hypermethylation was only detected in 6 of 28 (21%) high-grade specimens. Similarly, in prostate cancer cells, TIMP3 hypermethylation was only observed in DU145 cells. Treatment of DU145 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored TIMP3 expression, and this was significantly amplified by co-treating the cells with the HDAC inhibitor trichostatin A (TSA). Alternatively, in cells that did not exhibit aberrant TIMP3 methylation (LNCaP and PC3), TIMP3 expression could be upregulated by the combination of histone methylation inhibitor 3-Deazaneplanocin A (DZNep) and TSA. This reversal of transcriptional repression was associated with decreased H3K27me3 and increased H3K9ac histone marks at the TIMP3 promoter, as demonstrated by chromatin immunoprecipitation. Collectively, these results indicate that histone modifications can contribute to TIMP3 repression in the absence of promoter hypermethylation, and suggest that the combination of histone modifying agents could restore TIMP3 expression in prostate tumors harboring aberrant histone modifications at the TIMP3 promoter.

AIM1 and LINE-1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome.

Aberrations in the methylation status of noncoding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in long interspersed nucleotide element-1 (LINE-1) and absent in melanoma-1 (AIM1; 6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissue (PEAT; n = 133) and in melanoma patients' serum (n = 56). LINE-1 U-Index (hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma (n = 100) was significantly higher than that of normal skin (n = 14) and nevi (n = 12; P = 0.0004). LINE-1 U-Index level was elevated with increasing American Joint Committee on Cancer (AJCC) stage (P<0.0001). AIM1 promoter hypermethylation was found in higher frequency (P = 0.005) in metastatic melanoma (65%) than in primary melanomas (38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival (DFS) and overall survival (OS) in stage I/II patients (P = 0.017 and 0.027, respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS (P = 0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III (n = 20) and stage IV (n = 36) patients compared with healthy donors (n = 14; P = 0.022). Circulating methylated AIM1 was detected in patients' serum and was predictive of OS in stage IV patients (P = 0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients' tumors and serum.

LC/MS-based quantitative proteomic analysis of paraffin-embedded archival melanomas reveals potential proteomic biomarkers associated with metastasis.

BACKGROUND: Melanoma metastasis status is highly associated with the overall survival of patients; yet, little is known about proteomic changes during melanoma tumor progression. To better understand the changes in protein expression involved in melanoma progression and metastasis, and to identify potential biomarkers, we conducted a global quantitative proteomic analysis on archival metastatic and primary melanomas. METHODOLOGY AND FINDINGS: A total of 16 metastatic and 8 primary cutaneous melanomas were assessed. Proteins were extracted from laser captured microdissected formalin fixed paraffin-embedded archival tissues by liquefying tissue cells. These preparations were analyzed by a LC/MS-based label-free protein quantification method. More than 1500 proteins were identified in the tissue lysates with a peptide ID confidence level of >75%. This approach identified 120 significant changes in protein levels. These proteins were identified from multiple peptides with high confidence identification and were expressed at significantly different levels in metastases as compared with primary melanomas (q-Value<0.05). CONCLUSIONS AND SIGNIFICANCE: The differentially expressed proteins were classified by biological process or mapped into biological system networks, and several proteins were implicated by these analyses as cancer- or metastasis-related. These proteins represent potential biomarkers for tumor progression. The study successfully identified proteins that are differentially expressed in formalin fixed paraffin-embedded specimens of metastatic and primary melanoma.

Proteomic profiling of primary breast cancer predicts axillary lymph node metastasis.

To determine if protein expression in primary breast cancers can predict axillary lymph node (ALN) metastasis, we assessed differences in protein expression between primary breast cancers with and without ALN metastasis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Laser capture microdissection was performed on invasive breast cancer frozen sections from 65 patients undergoing resection with sentinel lymph node (SLN) or level I and II ALN dissection. Isolated proteins from these tumors were applied to immobilized metal affinity capture (IMAC-3) ProteinChip arrays and analyzed by SELDI-TOF-MS to generate unique protein profiles. Correlations between unique protein peaks and histologically confirmed ALN status and other known clinicopathologic factors were examined using ANOVA and multivariate logistic regression. Two metal-binding polypeptides at 4,871 and 8,596 Da were identified as significant risk factors for nodal metastasis (P = 0.034 and 0.015, respectively) in a multivariate analysis. Lymphovascular invasion (LVI) was the only clinicopathologic factor predictive of ALN metastasis (P = 0.0038). In a logistic regression model combining the 4,871 and 8,596 Da peaks with LVI, the area under the receiver operating characteristic curve was 0.87. Compared with patients with negative ALN, those with > or =2 positive ALN or non-SLN metastases were significantly more likely to have an increased peak at 4,871 Da (P = 0.016 and 0.0083, respectively). ProteinChip array analysis identified differential protein peaks in primary breast cancers that predict the presence and number of ALN metastases and non-SLN status.

Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients.

Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hypermethylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-beta2 (RAR-beta2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methylatransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n=20) compared to metastatic melanomas. However, hypermethylation of RAR-beta2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had > or =1 gene(s) and 59% had > or =2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5'-aza 2'-deoxycytidine (5Aza-dC). Analysis of melanoma patients' plasma (preoperative blood; n=31) demonstrated circulating hypermethylated MGMT, RAR-beta2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.

Induction of a systemic immune response by a polyvalent melanoma-associated antigen DNA vaccine for prevention and treatment of malignant melanoma.

Studies have demonstrated that active-specific immunotherapy has potential for controlling melanoma progression. We developed a polyvalent melanoma gene vaccine using a plasmid vector to deliver the immunogenic human melanoma-associated antigens (MAAs) gp100 and TRP-2. The MAA-containing plasmids were delivered individually in vivo using the hemagglutinating virus Japan (HVJ)-anionic liposome delivery system. C57BL/6 mice were immunized weekly by intramuscular (i.m.) injection or intranasal (i.n.) inoculation for 3 weeks. Although both i.m. and i.n. immunization induced Th1 (T helper) and Th2 cell responses to gp100 and TRP2, the i.m. route induced a better Th1 response. MAA-specific IgG2a, IgG1, and delayed-type hypersensitivity (DTH) responses were induced against both MAAs by i.m. immunization. We assessed the vaccine for its prophylactic and therapeutic effect against the murine B16 F10 melanoma. Animals vaccinated and subsequently challenged with a lethal dose of B16 cells were significantly (P<0.01) protected against tumor progression and had significantly (P<0.01) enhanced survival compared with treatment using control plasmid. We also developed a therapeutic model in which mice were given B16 cells and subsequently immunized with the vaccine or treated with control plasmid. In animals treated with the vaccine, tumor growth was significantly (P<0.01) controlled, and survival was prolonged compared with controls. These studies demonstrate that the polyvalent DNA vaccine induces an effective systemic Th response.