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DCLK1 as a Marker/Indicator of Stem Cell Response in Barrett’s Esophagus/Esophageal Adenocarcinoma

ABSTRACT: The hypotheses are: 1) the intestinal stem cell marker, DCLK1, which is increased in both the epithelium and stroma in colon cancer is also increased in BE (Barrett’s esophagus) with HGD (high grade dysplasia) and in EAC (esophageal adenocarcinoma), 2) this expression correlates with disease progression towards EAC and 3) eradication of cells expressing stem cell markers occurs after therapy of EMR (endoscopic mucosal resection) or RFA (radiofrequency ablation) to eradicate BE with HGD and intramucosal adenocarcinoma and esophagectomy for EAC. We will test our hypotheses with the following aims: 1) To characterize the cell specific expression patterns of intestinal stem cell biomarkers in BE patients and correlate them with serum expression and disease progression, 2) To examine prospectively the effects of EMR, RFA or esophagectomy on the expression of stem cell biomarkers and the progression to EAC.

DISEASE(S): Adenocarcinoma,Esophageal Neoplasms,Barrett's Esophagus,Esophageal Adenocarcinoma,Barrett Esophagus

PROVIDER: 2273018 | ecrin-mdr-crc |

REPOSITORIES: ECRIN MDR

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Project description:Introduction: The identification of biomarkers in Barrett’s oesophagus (BO) that stratify groups at risk of progressing to oesophageal adenocarcinoma (OAC) would allow tailored surveillance strategies. We have applied a novel high throughput RNA sequencing analysis characterizing the BO mRNA transcriptome across the metaplasia-dysplasia sequence to identify potential markers of progression in an unbiased fashion and functionally validated these at the protein level. Methods: Matched biopsy samples for histology and RNA extraction were taken from BO patients of known histological grade. RNA was extracted from matched samples and sequenced to 60bp length (paired-end).21 samples were sequenced (HGD,7; LGD, 7 and SIM, 7). Reads obtained were mapped to NCBI build37.2 using TopHat. Read count generation, normalisation and differential expression (DE) analysis was performed using the HTSeq-DESeq pipeline. Significantly DE genes (>2 fold change in expression with B-H adjusted p-value <0.1) were further assessed for network and biological relevance using Ingenuity Pathway analysis. Candidate genes were selected and validated in a larger cohort (n=64) using RT-PCR. Targets were further validated (by ELISA and immunohistochemistry) in independent cohorts of patients using serum and tissue microarrays. Results: DE analysis was performed in 3 groups with 2 conditions at a time using the lower grade cohort as control and the higher grade as comparator: SIM vs. LGD (demonstrated 218 DE genes, 131 up-regulated in LGD, 87 down-regulated compared to SIM), SIM vs. HGD (49 DE, 27 up, 22 down) and LGD vs. HGD (317 DE, 81 up, 216 down). Six network-central candidate genes (FOSB, IL-1B, SERPINA3, KLK7, GSTM5 &SCUBE2) were selected for RT-PCR validation following network and functional analysis. Circulating IL-1β and SERPINA-3 demonstrated progressive significant increases in expression across the dysplasia sequence to OAC (p<0.005)). This was confirmed at the tissue level showing significant differences between SIM and dysplastic BO (p<0.05). Conclusion: The use of RNA-sequencing as a detailed and unbiased analysis method identifies IL-1β and SERPINA-3 as novel candidates differentially expressed along the metaplasia-dysplasia-cancer sequence in BO.

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DCLK1 as a Marker/Indicator of Stem Cell Response in Barrett’s Esophagus/Esophageal Adenocarcinoma

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