RESUMO
PURPOSE: Wnt pathways control key biological processes that potentially impact on tumor progression and patient survival. We aimed to evaluate DNA methylation at promoter CpG islands (CGI) of Wnt pathway genes in ovarian tumors at presentation and identify biomarkers of patient progression-free survival (PFS). EXPERIMENTAL DESIGN: Epithelial ovarian tumors (screening study n = 120, validation study n = 61), prospectively collected through a cohort study, were analyzed by differential methylation hybridization at 302 loci spanning 189 promoter CGIs at 137 genes in Wnt pathways. The association of methylation and PFS was examined by Cox proportional hazards model. RESULTS: DNA methylation is associated with PFS at 20 of 302 loci (P < 0.05, n = 111), with 5 loci significant at false discovery rate (FDR) less than 10%. A total of 11 of 20 loci retain significance in an independent validation cohort (n = 48, P ≤ 0.05, FDR ≤ 10%), and 7 of these loci, at FZD4, DVL1, NFATC3, ROCK1, LRP5, AXIN1, and NKD1 genes, are independent from clinical parameters (adjusted P < 0.05). Increased methylation at these loci associates with increased hazard of disease progression. A multivariate Cox model incorporates only NKD1 and DVL1, identifying two groups differing in PFS [HR = 2.09; 95% CI (1.39-3.15); permutation test P < 0.005]. Methylation at DVL1 and NFATC3 show significant association with response. Consistent with their epigenetic regulation, reduced expression of FZD4, DVL1, and ROCK1 is an indicator of early-disease relapse in an independent ovarian tumor cohort (n = 311, adjusted P < 0.05). CONCLUSION: The data highlight the importance of epigenetic regulation of multiple promoter CGIs of Wnt pathway genes in ovarian cancer and identify methylation at NKD1 and DVL1 as independent predictors of PFS.
Assuntos
Biomarcadores Tumorais/metabolismo , Ilhas de CpG/genética , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/fisiopatologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/fisiopatologia , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação ao Cálcio , Carcinoma Epitelial do Ovário , Proteínas de Transporte/metabolismo , Estudos de Coortes , Intervalo Livre de Doença , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Ovarianas/mortalidade , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Proteínas Wnt/genéticaRESUMO
The isolation of fully human monoclonal antibodies (MAb) against tumor targets has to date relied largely on combinatorial library-based antibody display techniques, which generally require lengthy antigen selection procedures due to a low frequency of clones expressing compatible heavy (VH) and light chain (VL) variable genes. Here we describe a method to directly isolate immunoglobulin sequences in situ from antibody-producing cells infiltrating human tumor tissue. Single B cells and plasma cells infiltrating cervical cancer were microdissected from tissue sections using laser-assisted microscopy, and VH and VL expressed by each individual cell amplified using nested reverse transcriptase- polymerase chain reaction (RT-PCR), thus retaining the native VH and VL pairing. Sequencing analysis determined that the isolated cells expressed functional immunoglobulin variable genes, consistent with an antitumor antibody response. The immunoglobulin sequences can be reassembled as Fab or scFv fragments using conventional recombinant antibody expression plasmids. This method will allow a more direct assessment of the humoral immune response to cancer, and the potential identification of novel human therapeutic cancer antibodies.
