[Show abstract][Hide abstract]ABSTRACT: Background and aims:
Validation of non-invasive methods of liver fat quantification requires a reference standard. However, using standard histopathology assessment of liver biopsies is problematical because of poor repeatability. We aimed to assess a stereological method of measuring volumetric liver fat fraction (VLFF) in liver biopsies and to use the method to validate a magnetic resonance imaging method for measurement of VLFF.
VLFFs were measured in 59 subjects (1) by three independent analysts using a stereological point counting technique combined with the Delesse principle on liver biopsy histological sections and (2) by three independent analysts using the HepaFat-Scan® technique on magnetic resonance images of the liver. Bland Altman statistics and intraclass correlation (IC) were used to assess the repeatability of each method and the bias between the methods of liver fat fraction measurement.
Inter-analyst repeatability coefficients for the stereology and HepaFat-Scan® methods were 8.2 (95% CI 7.7-8.8)% and 2.4 (95% CI 2.2-2.5)% VLFF respectively. IC coefficients were 0.86 (95% CI 0.69-0.93) and 0.990 (95% CI 0.985-0.994) respectively. Small biases (≤3.4%) were observable between two pairs of analysts using stereology while no significant biases were observable between any of the three pairs of analysts using HepaFat-Scan®. A bias of 1.4±0.5% VLFF was observed between the HepaFat-Scan® method and the stereological method.
Repeatability of the stereological method is superior to the previously reported performance of assessment of hepatic steatosis by histopathologists and is a suitable reference standard for validating non-invasive methods of measurement of VLFF.
[Show abstract][Hide abstract]ABSTRACT: Chronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunological pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within MHC class-II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class-II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Treg after BMT also induced cGVHD, while adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class-II that is induced by aGVHD.
[Show abstract][Hide abstract]ABSTRACT: Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.
Full-text Article · Apr 2016 · The American Journal of Human Genetics
[Show abstract][Hide abstract]ABSTRACT: The incidence of esophageal adenocarcinoma (EAC) has risen significantly over recent decades. While survival has improved,
cure rates remain poor, with < 20% of patients surviving 5 years. This is the first study to explore methylome, transcriptome
and ENCODE data to characterize the role of methylation in EAC. We investigate the genome-wide methylation profile of 250
samples including 125 EAC, 19 Barrett’s esophagus (BE), 85 squamous esophagus and 21 normal stomach. Transcriptome data of
70 samples (48 EAC, 4 BE and 18 squamous esophagus) were used to identify changes in methylation associated with gene expression.
BE and EAC showed similar methylation profiles, which differed from squamous tissue. Hyper-methylated sites in EAC and BE
were mainly located in CpG-rich promoters. A total of 18,575 CpG sites associated with 5,538 genes were differentially methylated,
63% of these genes showed significant correlation between methylation and mRNA expression levels. Pathways involved in tumorigenesis
including cell adhesion, TGF and WNT signaling showed enrichment for genes aberrantly methylated. Genes involved in chromosomal
segregation and spindle formation were aberrantly methylated. Given the recent evidence that chromothripsis may be a driver
mechanism in EAC, the role of epigenetic perturbation of these pathways should be further investigated. The methylation profiles
revealed two EAC subtypes, one associated with widespread CpG island hyper-methylation overlapping H3K27me3 marks and binding
sites of the Polycomb proteins. These subtypes were supported by an independent set of 89 esophageal cancer samples. The most
hyper-methylated tumors showed worse patient survival.
[Show abstract][Hide abstract]ABSTRACT: The interplay between the inflammatory infiltrate and tissue resident cell populations invokes fibrogenesis. However, the temporal and mechanistic contributions of these cells to fibrosis are obscure. To address this issue, liver inflammation, ductular reaction (DR), and fibrosis were induced in C57BL/6 mice by thioacetamide administration for up to 12 weeks. Thioacetamide treatment induced two phases of liver fibrosis. A rapid pericentral inflammatory infiltrate enriched in F4/80(+) monocytes co-localized with SMA(+) myofibroblasts resulted in early collagen deposition, marking the start of an initial fibrotic phase (1 to 6 weeks). An expansion of bone marrow-derived macrophages preceded a second phase, characterized by accelerated progression of fibrosis (>6 weeks) after DR migration from the portal tracts to the centrilobular site of injury, in association with an increase in DR/macrophage interactions. Although chemokine (C-C motif) ligand 2 (CCL2) mRNA was induced rapidly in response to thioacetamide, CCL2 deficiency only partially abrogated fibrosis. In contrast, colony-stimulating factor 1 receptor blockade diminished C-C chemokine receptor type 2 [CCR2(neg) (Ly6C(lo))] monocytes, attenuated the DR, and significantly reduced fibrosis, illustrating the critical role of colony-stimulating factor 1-dependent monocyte/macrophage differentiation and linking the two phases of injury. In response to liver injury, colony-stimulating factor 1 drives early monocyte-mediated myofibroblast activation and collagen deposition, subsequent macrophage differentiation, and their association with the advancing DR, the formation of fibrotic septa, and the progression of liver fibrosis to cirrhosis.
Article · Jan 2016 · American Journal Of Pathology
[Show abstract][Hide abstract]ABSTRACT: Background:
Macrophages play critical roles in liver regeneration, fibrosis development and resolution. They are among the first responders to liver injury and are implicated in orchestrating the fibrogenic response via multiple mechanisms. Macrophages are also intimately associated with the activated hepatic progenitor cell (HPC) niche or ductular reaction that develops in parallel with fibrosis. Among the many macrophage-derived mediators implicated in liver disease progression, a key role for macrophage-derived Wnt proteins in driving pro-regenerative HPC activation towards a hepatocellular fate has been suggested. Wnt proteins, in general, however, have been associated with both pro- and anti-fibrogenic activities in the liver and other organs. We investigated the role of macrophage-derived Wnt proteins in fibrogenesis and HPC activation in murine models of chronic liver disease by conditionally deleting Wntless expression, which encodes a chaperone essential for Wnt protein secretion, in LysM-Cre-expressing myeloid cells (LysM-Wls mice).
Fibrosis and HPC activation were exacerbated in LysM-Wls mice compared to littermate controls, in the absence of an apparent increase in myofibroblast activation or interstitial collagen mRNA expression, in both the TAA and CDE models of chronic liver disease. Increased Epcam mRNA levels paralleled the increased HPC activation and more mature ductular reactions, in LysM-Wls mice. Increased Epcam expression in LysM-Wls HPC was also observed, consistent with a more cholangiocytic phenotype. No differences in the mRNA expression levels of key pro-inflammatory and pro-fibrotic cytokines or the macrophage-derived HPC mitogen, Tweak, were observed. LysM-Wls mice exhibited increased expression of Timp1, encoding the key Mmp inhibitor Timp1 that blocks interstitial collagen degradation, and, in the TAA model, reduced expression of the anti-fibrotic matrix metalloproteinases, Mmp12 and Mmp13, suggesting a role for macrophage-derived Wnt proteins in restraining fibrogenesis during ongoing liver injury.
In summary, these data suggest that macrophage-derived Wnt proteins possess anti-fibrogenic potential in chronic liver disease, which may be able to be manipulated for therapeutic benefit.
Full-text Article · Dec 2015 · Fibrogenesis & Tissue Repair
[Show abstract][Hide abstract]ABSTRACT: Objective:
Sessile serrated adenomas (SSAs) are the precursors of at least 15% of colorectal carcinomas, but their biology is incompletely understood. We performed a clinicopathological and molecular analysis of a large number of the rarely observed SSAs with dysplasia/carcinoma to better define their features and the pathways by which they progress to carcinoma.
A cross-sectional analysis of 137 SSAs containing regions of dysplasia/carcinoma prospectively collected at a community GI pathology practice was conducted. Samples were examined for BRAF and KRAS mutations, the CpG island methylator phenotype (CIMP) and immunostained for MLH1, p53, p16, β-catenin and 0-6-methylguanine DNA methyltransferase (MGMT).
The median polyp size was 9 mm and 86.5% were proximal. Most were BRAF mutated (92.7%) and 94.0% showed CIMP. Mismatch repair deficiency, evidenced by loss of MLH1 (74.5%) is associated with older age (76.7 versus 71.0; p<0.0029), female gender (70% versus 36%; p<0.0008), proximal location (91% versus 72%; p<0.02), CIMP (98% versus 80%; p<0.02) and lack of aberrant p53 (7% versus 34%; p<0.001) when compared with the mismatch repair-proficient cases. Loss of p16 (43.1%) and gain of nuclear β-catenin (55.5%) were common in areas of dysplasia/cancer, irrespective of mismatch repair status.
SSAs containing dysplasia/carcinoma are predominantly small (<10 mm) and proximal. The mismatch repair status separates these lesions into distinct clinicopathological subgroups, although WNT activation and p16 silencing are common to both. Cases with dysplasia occur at a similar age to cases with carcinoma. This, together with the rarity of these 'caught in the act' lesions, suggests a rapid transition to malignancy following a long dwell time as an SSA without dysplasia.
[Show abstract][Hide abstract]ABSTRACT: Importance
Laparoscopic procedures are generally thought to have better outcomes than open procedures. Because of anatomical constraints, laparoscopic rectal resection may not be better because of limitations in performing an adequate cancer resection.Objective
To determine whether laparoscopic resection is noninferior to open rectal cancer resection for adequacy of cancer clearance.Design, Setting, and Participants
Randomized, noninferiority, phase 3 trial (Australasian Laparoscopic Cancer of the Rectum; ALaCaRT) conducted between March 2010 and November 2014. Twenty-six accredited surgeons from 24 sites in Australia and New Zealand randomized 475 patients with T1-T3 rectal adenocarcinoma less than 15 cm from the anal verge.Interventions
Open laparotomy and rectal resection (n = 237) or laparoscopic rectal resection (n = 238).Main Outcomes and Measures
The primary end point was a composite of oncological factors indicating an adequate surgical resection, with a noninferiority boundary of Δ = −8%. Successful resection was defined as meeting all the following criteria: (1) complete total mesorectal excision, (2) a clear circumferential margin (≥1 mm), and (3) a clear distal resection margin (≥1 mm). Pathologists used standardized reporting and were blinded to the method of surgery.Results
A successful resection was achieved in 194 patients (82%) in the laparoscopic surgery group and 208 patients (89%) in the open surgery group (risk difference of −7.0% [95% CI, −12.4% to ∞]; P = .38 for noninferiority). The circumferential resection margin was clear in 222 patients (93%) in the laparoscopic surgery group and in 228 patients (97%) in the open surgery group (risk difference of −3.7% [95% CI, −7.6% to 0.1%]; P = .06), the distal margin was clear in 236 patients (99%) in the laparoscopic surgery group and in 234 patients (99%) in the open surgery group (risk difference of −0.4% [95% CI, −1.8% to 1.0%]; P = .67), and total mesorectal excision was complete in 206 patients (87%) in the laparoscopic surgery group and 216 patients (92%) in the open surgery group (risk difference of −5.4% [95% CI, −10.9% to 0.2%]; P = .06). The conversion rate from laparoscopic to open surgery was 9%.Conclusions and Relevance
Among patients with T1-T3 rectal tumors, noninferiority of laparoscopic surgery compared with open surgery for successful resection was not established. Although the overall quality of surgery was high, these findings do not provide sufficient evidence for the routine use of laparoscopic surgery. Longer follow-up of recurrence and survival is currently being acquired.Trial Registration
anzctr.org Identifier: ACTRN12609000663257
Article · Oct 2015 · JAMA The Journal of the American Medical Association
[Show abstract][Hide abstract]ABSTRACT: Most colorectal polyps are readily classified, but a subset of tubulovillous adenomas (TVA) with prominent serrated architecture cause diagnostic confusion. We aimed to 1) identify histological features that separate serrated TVAs from both conventional TVAs and traditional serrated adenomas (TSA) and 2) perform a clinicopathological and molecular analysis to determine if the serrated TVA has unique features.
We collected 48 serrated TVAs, 50 conventional TVAs and 66 BRAF wild-type TSAs for analysis. For each polyp we performed a clinicopathological assessment, BRAF and KRAS mutation profiling, CpG island methylator phenotype status, MGMT methylation and immunohistochemical assessment of seven markers (MLH1, p16, p53, β-catenin, Ki67, CK7 and CK20).
We found that serrated TVAs can be reliably diagnosed and have features distinct from both conventional TVAs and TSAs. Compared to conventional TVAs, serrated TVAs are larger, more often proximal, more histologically advanced, show more CpG island methylation and more frequent KRAS mutation. Compared to TSAs, they are more often proximal, show less CpG island methylation, more frequent MGMT methylation and more frequent nuclear staining for β-catenin.
The serrated TVA can be reliably diagnosed and has unique features. It represents a precursor of KRAS mutated, microsatellite stable colorectal carcinoma. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract]ABSTRACT: Current tools for risk stratification of chronic liver disease subjects are limited. We aimed to determine whether the serum-based ELF (Enhanced Liver Fibrosis) test predicted liver-related clinical outcomes, or progression to advanced liver disease, and to compare the performance of ELF to liver biopsy and non-invasive algorithms.
300 patients with ELF scores assayed at the time of liver biopsy were followed up (median 6.1 years) for liver-related clinical outcomes (n=16) and evidence of progression to advanced fibrosis (n=18), by review of medical records and clinical data.
Fourteen of 73 (19.2%) patients with ELF score indicative of advanced fibrosis (≥9.8, the manufacturer's cut-off) had a liver-related clinical outcome, compared to only 2 of 227 (<1%) patients with ELF score <9.8. By contrast, the simple scores APRI and FIB-4 would only have predicted subsequent decompensation in 6 and 4 patients, respectively. A unit increase in ELF score was associated with a 2.53-fold increased risk of a liver-related event (adjusted for age and stage of fibrosis). In patients without advanced fibrosis on biopsy at recruitment, 61% (11/18) with an ELF score ≥9.8 showed evidence of progression to advanced fibrosis (after an average 6 years), whereas only 13.5% of those with an ELF score <9.8 (28/207) progressed (average 14 years). In these subjects, a unit increase in ELF score was associated with a 4.34-fold increased risk of progression. Conclusions The ELF score is a valuable tool for risk stratification of patients with chronic liver disease. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Full-text Article · Jun 2015 · Liver international: official journal of the International Association for the Study of the Liver
[Show abstract][Hide abstract]ABSTRACT: Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus–cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver.
[Show abstract][Hide abstract]ABSTRACT: Barrett's esophagus (BE), a common condition, is the only known precursor to esophageal adenocarcinoma (EAC). There is uncertainty about the best way to manage BE, since most people with BE never develop EAC and most patients diagnosed with EAC have no preceding diagnosis of BE. Moreover, there have been recent advances in knowledge and practice about the management of BE and early EAC. To aid clinical decision-making in this rapidly moving field, Cancer Council Australia convened an expert working party to identify pertinent clinical questions. The questions covered a wide range of topics including endoscopic and histologic definitions of BE and early EAC; prevalence, incidence, natural history and risk factors for BE; and methods for managing BE and early EAC. The latter considered modification of lifestyle factors; screening and surveillance strategies; and medical, endoscopic and surgical interventions. To answer each question, the working party systematically reviewed the literature and developed a set of recommendations through consensus. Evidence underpinning each recommendation was rated according to quality and applicability.
This article is protected by copyright. All rights reserved.
Article · Jan 2015 · Journal of Gastroenterology and Hepatology