RESUMO
OBJECTIVE: To identify and characterise DNA methylation subtypes in oesophageal adenocarcinoma (EAC) and its precursor Barrett's oesophagus (BE). DESIGN: We performed genome-wide DNA methylation profiling on samples of non-dysplastic BE from cancer-free patients (n=59), EAC (n=23), normal squamous oesophagus (n=33) and normal fundus (n=9), and identified methylation subtypes using a recursively partitioned mixture model. We assessed genomic alterations for 9 BE and 22 EAC samples with massively parallel sequencing of 243 EAC-associated genes, and we conducted integrative analyses with transcriptome data to identify epigenetically repressed genes. We also carried out in vitro experiments treating EAC cell lines with 5-Aza-2'-Deoxycytidine (5-Aza-dC), short hairpin RNA knockdown and anticancer therapies. RESULTS: We identified and validated four methylation subtypes of EAC and BE. The high methylator subtype (HM) of EAC had the greatest number of activating events in ERBB2 (p<0.05, Student's t-test) and the highest global mutation load (p<0.05, Fisher's exact test). PTPN13 was silenced by aberrant methylation in the HM subtype preferentially and in 57% of EACs overall. In EAC cell lines, 5-Aza-dC treatment restored PTPN13 expression and significantly decreased its promoter methylation in HM cell lines (p<0.05, Welch's t-test). Inhibition of PTPN13 expression in the SK-GT-4 EAC cell line promoted proliferation, colony formation and migration, and increased phosphorylation in ERBB2/EGFR/Src kinase pathways. Finally, EAC cell lines showed subtype-specific responses to topotecan, SN-38 and palbociclib treatment. CONCLUSIONS: We identified and characterised methylator subtypes in BE and EAC. We further demonstrated the biological and clinical relevance of EAC methylator subtypes, which may ultimately help guide clinical management of patients with EAC.
Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Metilação de DNA , Neoplasias Esofágicas/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Esôfago de Barrett/tratamento farmacológico , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , DNA de Neoplasias/genética , Receptores ErbB/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Estudo de Associação Genômica Ampla/métodos , Humanos , Mutação , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 13/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 13/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais/genéticaRESUMO
Droplet digital (ddPCR) is a recent advance in PCR technology that enables the precise detection and absolute quantification of nucleic acid target sequences and that has a range of applications for both research and clinical diagnostic studies. Here, we discuss the parameters important in the design and performance of ddPCR for the detection and quantification of methylated DNA. We provide explicit instructions for conducting methylation specific ddPCR (aka MethyLight ddPCR). We also present an example that demonstrates the sensitivity and precision of the method for detecting methylated DNA in the promoter region of mir342/EVL, a potential DNA methylation biomarker for colon cancer risk. Common technical problems and troubleshooting for conducting successful MethyLight ddPCR assays are also discussed.
Assuntos
Biomarcadores Tumorais/isolamento & purificação , Neoplasias do Colo/diagnóstico , Metilação de DNA/genética , DNA de Neoplasias/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Biomarcadores Tumorais/genética , Moléculas de Adesão Celular/genética , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA de Neoplasias/genética , Humanos , MicroRNAs/genética , Reação em Cadeia da Polimerase/instrumentação , Regiões Promotoras Genéticas/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The CpG island methylator phenotype (CIMP) in stage III colon cancer (CRC) has been associated with improved survival after treatment with adjuvant irinotecan-based chemotherapy. In this analysis, we determine whether CIMP status in the primary CRC is concordant with the CIMP status of matched metastases in order to determine if assessment of CIMP status in the primary tumor can be used to predict CIMP status of metastatic disease, which is relevant for patient management as well as for understanding the biology of CIMP CRCs. METHODS: We assessed the CIMP status of 70 pairs of primary CRC and matched metastases using a CRC-specific panel of five markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) where CIMP positive was defined as 3/5 positive markers at a percent methylated reference threshold of ≥10%. Concordance was compared using the Fisher's exact test and P < 0.05 was considered significant. RESULTS: Sixty-nine of the pairs (98.6%) showed concordant CIMP status in the primary tumor and matched metastasis; five (7.0%) of the pairs were concordantly CIMP positive. Only one pair (1.4%) had divergent CIMP status, demonstrating CIMP positivity (4/5 markers positive) in the primary tumor, while the matched metastasis was CIMP negative (0 markers positive). CONCLUSIONS: CIMP status is generally concordant between primary CRCs and matched metastases. Thus, CIMP status in the primary tumor is maintained in matched metastases and can be used to inform CIMP-based therapy options for the metastases.