Gene signature distinguishes patients with chronic ulcerative colitis harboring remote neoplastic lesions

Abstract

Background: Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer. The standard method of surveillance for neoplasia in UC by colonoscopy is invasive and can miss flat lesions. We sought to identify a gene expression signature in nondysplastic mucosa without active inflammation that could serve as a marker for remote neoplastic lesions.

Methods: Gene expression was analyzed by complementary DNA microarray in 5 normal controls, 4 UC patients without dysplasia, and 11 UC patients harboring remote neoplasia. Common gene ontology pathways of significantly differentially expressed genes were identified. Expression of genes which were progressively and significantly upregulated from controls to UC without neoplasia, to UC with remote neoplasia were evaluated by real-time polymerase chain reaction. Several gene products were also examined by immunohistochemistry.

Results: Four hundred and sixty-eight genes were significantly upregulated, and 541 genes were significantly downregulated in UC patients with neoplasia compared with UC patients without neoplasia. Nine genes (ACSL1, BIRC3, CLC, CREM, ELTD1, FGG, S100A9, THBD, and TPD52L1) were progressively and significantly upregulated from controls to nondysplastic UC to UC with neoplasia. Immunostaining of proteins revealed increased expression of S100A9 and REG1α in UC-associated cancer and in nondysplastic tissue from UC patients harboring remote neoplasia compared with UC patients without neoplasia and controls.

Conclusions: Gene expression changes occurring as a field effect in the distal colon of patients with chronic UC identify patients harboring remote neoplastic lesions. These markers may lead to a more accurate and less invasive method of detection of neoplasia in patients with inflammatory bowel disease.

Figure 1

Heat map with genome wide analysis of gene expression. Using two-way unsupervised hierarchical clustering, patients clustered into phenotypic groups with the exception of one UC patient without dysplasia (S29) who had a gene expression profile more characteristic of those harboring remote neoplasia.

Figure 2

REG1α expression by real time PCR. Fold change in expression in UC patients without dysplasia and UC patients harboring remote neoplasia is compared to normal controls. Error bars are model based standard error estimates.

Figure 3

Genes clustered into 5 groups (C0-C4) based on expression patterns between Normal, UC, and UC harboring remote neoplasia. The number of genes in each group is listed at the top of each box. The blue line represents the expression pattern with standard deviation of the mean in red. The black boxes represent the average rank of gene expression levels by clinical condition (Normal – left, UC – middle, UCN – right).

Figure 4

Gene Ontology Pathways Common to Groups C3 (A) and C4 (B). Gene ontology categories were identified by Ontoexpress software; only biological processes with ≥4 genes and a p-value <0.05 are displayed. The numbers to the right of the column represent the number of unique genes in each pathway.

Figure 5

S100A9 Immunohistochemistry. Representative images from the sigmoid colon in (A) Normal colon (20x) (B) Quiescent UC (20x) and (C 10x, D 20x) non-dysplastic mucosa in a UC patient with a remote dysplastic lesion. Note increased stromal staining in (C, D) compared to (A, B). (E–H) IBD-cancer tissue array: Representative images from 2 sections with positive stromal staining (E, F) and 2 sections with glandular staining (G, H). Larger images are 20x magnification. Insets are at 40x magnification.

Figure 6

REG1α Immunohistochemistry. (A) Normal colon, (B, C) UC without dysplasia, and (D–F) UC harboring a remote dysplastic lesion. In patients with UC harboring remote dysplasia, there is increased crypt staining, primarily in base of crypts. (G–L) IBD-cancer tissue array (G) A cancer with no positive staining, (H) Cancer with diffuse weak positive staining (I) A cancer with focal strong positive staining (J–L) Cancers with diffuse strong glandular positivity. Images A–G at 10x magnification. Images G–L at 5x magnification.