James C. Hardwick

Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4–β-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.

Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes

Gastric cancer, a leading cause of cancer-related deaths, is a heterogeneous disease. We aim to establish clinically relevant molecular subtypes that would encompass this heterogeneity and provide useful clinical information. We use gene expression data to describe four molecular subtypes linked to distinct patterns of molecular alterations, disease progression and prognosis. The mesenchymal-like type includes diffuse-subtype tumors with the worst prognosis, the tendency to occur at an earlier age and the highest recurrence frequency (63%) of the four subtypes. Microsatellite-unstable tumors are hyper-mutated intestinal-subtype tumors occurring in the antrum; these have the best overall prognosis and the lowest frequency of recurrence (22%) of the four subtypes. The tumor protein 53 (TP53)-active and TP53-inactive types include patients with intermediate prognosis and recurrence rates (with respect to the other two subtypes), with the TP53-active group showing better prognosis. We describe key molecular alterations in each of the four subtypes using targeted sequencing and genome-wide copy number microarrays. We validate these subtypes in independent cohorts in order to provide a consistent and unified framework for further clinical and preclinical translational research.

Endoscopic tri-modal imaging for surveillance in ulcerative colitis: randomised comparison of high-resolution endoscopy and autofluorescence imaging for neoplasia detection; and evaluation of narrow-band imaging for classification of lesions

Background: Endoscopic tri-modal imaging (ETMI) incorporates white light endoscopy (WLE), autofluorescence imaging (AFI) and narrow-band imaging (NBI). Aims: To assess the value of ETMI for the detection and classification of neoplasia in patients with longstanding ulcerative colitis. Design: Randomised comparative trial of tandem colonoscopies. Setting: Academic Medical Centre Amsterdam, Netherlands. Patients and methods: Fifty patients with ulcerative colitis underwent surveillance colonoscopy with ETMI. Each colonic segment was inspected twice, once with AFI and once with WLE, in random order. All detected lesions were inspected by NBI for Kudo pit pattern analysis and additional random biopsies were taken. Main outcome measures: Neoplasia miss-rates of AFI and WLE, and accuracy of the Kudo classification by NBI. Results: Among patients assigned to inspection with AFI first (n = 25), 10 neoplastic lesions were primarily detected. Subsequent WLE detected no additional neoplasia. Among patients examined with WLE first (n = 25), three neoplastic lesions were detected; subsequent inspection with AFI added three neoplastic lesions. Neoplasia miss-rates for AFI and WLE were 0% and 50% (p = 0.036). The Kudo classification by NBI had a sensitivity and specificity of 75% and 81%; however, all neoplasia was coloured purple on AFI (sensitivity 100%). No additional patients with neoplasia were detected by random biopsies. Conclusion: Autofluorescence imaging improves the detection of neoplasia in patients with ulcerative colitis and decreases the yield of random biopsies. Pit pattern analysis by NBI has a moderate accuracy for the prediction of histology, whereas AFI colour appears valuable in excluding the presence of neoplasia. Trial registration number: ISRCTN05272746

One to 2-year surveillance intervals reduce risk of colorectal cancer in families with Lynch syndrome

BACKGROUND & AIMS Two percent to 4% of all cases of colorectal cancer (CRC) are associated with Lynch syndrome. Dominant clustering of CRC (non-Lynch syndrome) accounts for 1%-3% of the cases. Because carcinogenesis is accelerated in Lynch syndrome, an intensive colonoscopic surveillance program has been recommended since 1995. The aim of the study was to evaluate the effectiveness of this program. METHODS The study included 205 Lynch syndrome families with identified mutations in one of the mismatch repair genes (745 mutation carriers). We also analyzed data from non-Lynch syndrome families (46 families, 344 relatives). Patients were observed from January 1, 1995, until January 1, 2009. End points of the study were CRC or date of the last colonoscopy. RESULTS After a mean follow-up of 7.2 years, 33 patients developed CRC under surveillance. The cumulative risk of CRC was 6% after the 10-year follow-up period. The risk of CRC was higher in carriers older than 40 years and in carriers of MLH1 and MSH2 mutations. After a mean follow-up of 7.0 years, 6 cases of CRC were detected among non-Lynch syndrome families. The risk of CRC was significantly higher among families with Lynch syndrome, compared with those without. CONCLUSIONS With surveillance intervals of 1-2 years, members of families with Lynch syndrome have a lower risk of developing CRC than with surveillance intervals of 2-3 years. Because of the low risk of CRC in non-Lynch syndrome families, a less intensive surveillance protocol can be recommended.

Bone morphogenetic protein signalling in colorectal cancer

Much of the current understanding of colorectal cancer stems from the study of rare, inherited colorectal cancer syndromes. Mutations in the bone morphogenetic protein (BMP) pathway have been found in juvenile polyposis, an inherited polyposis syndrome that predisposes to colorectal cancer. The hamartomas that develop in these patients and in BMP pathway mutant mice have a remarkable mesenchymal component. Further evidence in mice suggests a primary role for mesenchymal loss of BMP signalling in hamartoma development. Here, we examine this evidence and question its relevance to sporadic colorectal carcinogenesis.

ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response

Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER) stress and activity of the unfolded protein response (UPR) are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

Effect of aspirin on the Wnt/beta-catenin pathway is mediated via protein phosphatase 2A.

Nonsteroidal anti-inflammatory drugs show chemopreventive efficacy in colon cancer, but the mechanism behind this remains unclear. Elucidating this mechanism is seen as vital to the development of new chemopreventive agents. We studied the effects of aspirin on the oncogenic Wnt/β-catenin pathway activity in colorectal cancer cell lines and observed that aspirin dose-dependently decreased the activity of this pathway, as judged by TCF-driven luciferase activity, reduced Wnt target gene expression and increased phosphorylation of β-catenin by immunoblotting. Furthermore, the ubiquitination and cytoplasmic levels of β-catenin were assessed by immunoblotting, and also the localization of β-catenin was shown by green fluorescent protein-tagged β-catenin and time-lapse fluorescent imaging. Importantly, aspirin treatment caused increased phosphorylation of protein phosphatase 2A (PP2A), an event associated with inhibition of PP2A enzymatic activity, which was confirmed by a reduction in enzymatic PP2A activity. Moreover, this inhibition of PP2A enzymatic activity was essential for the effects of aspirin on the Wnt/β-catenin pathway as shown by transient transfection with PP2A constructs. The findings in this article provide a molecular explanation for the efficacy of aspirin in chemoprevention of colorectal cancer and shows biochemical evidence that PP2A is an important regulator of Wnt/β-catenin pathway activity in these cells.

NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps

The factors that govern the progression from colonic adenomatous polyp to colon cancer are poorly understood. The observation that NSAIDs act as chemopreventative agents and reduce the size of colonic polyps suggests the involvement of inflammatory signalling, but inflammatory signalling in colonic polyps has not been studied. We investigated the expression of the active forms of NF-κB, JNK and p38 MAPK using immunohistochemistry with activation specific antibodies in human colonic adenomas. We show that active NF-κB is seen in stromal macrophages that also express COX-2 and TNF-α, active JNK is seen in stromal and intraepithelial T-lymphocytes and periendothelial cells of new blood vessels, and active p38 MAPK is most highly expressed in macrophages and other stromal cells. These results demonstrate the presence of active inflammatory signal transduction in colonic polyps and that these are predominantly in the stroma. In the case of NF-κB this coincides with the cellular localisation of COX-2. These results support evidence that NSAIDs may act through effects on stromal cells rather than epithelial cells.

Clinical evaluation of endoscopic trimodal imaging for the detection and differentiation of colonic polyps.

BACKGROUND & AIMS Endoscopic trimodal imaging (ETMI) incorporates high-resolution endoscopy (HRE) and autofluorescence imaging (AFI) for adenoma detection, and narrow-band imaging (NBI) for differentiation of adenomas from nonneoplastic polyps. The aim of this study was to compare AFI with HRE for adenoma detection and to assess the diagnostic accuracy of NBI for differentiation of polyps. This was a randomized trial of tandem colonoscopies. The study was performed at the Academic Medical Center in Amsterdam. METHODS One hundred patients underwent colonoscopy with ETMI. Each colonic segment was examined twice for polyps, once with HRE and once with AFI, in random order per patient. All detected polyps were assessed with NBI for pit pattern and with AFI for color, and subsequently removed. Histopathology served as the gold standard for diagnosis. The main outcome measures of this study were adenoma miss-rates of AFI and HRE, and diagnostic accuracy of NBI and AFI for differentiating adenomas from nonneoplastic polyps. RESULTS Among 50 patients examined with AFI first, 32 adenomas were detected initially. Subsequent inspection with HRE identified 8 additional adenomas. Among 50 patients examined with HRE first, 35 adenomas were detected initially. Successive AFI yielded 14 additional adenomas. The adenoma miss-rates of AFI and HRE therefore were 20% and 29%, respectively (P = .351). The sensitivity, specificity, and overall accuracy of NBI for differentiation were 90%, 70%, and 79%, respectively; corresponding figures for AFI were 99%, 35%, and 63%, respectively. CONCLUSIONS The overall adenoma miss-rate was 25%; AFI did not significantly reduce the adenoma miss-rate compared with HRE. Both NBI and AFI had a disappointing diagnostic accuracy for polyp differentiation, although AFI had a high sensitivity.