Colorectal metastases in the liver grow according to three histological patterns: a pushing pattern, a replacement pattern, and a desmoplastic pattern. The objective of the current study was to ...explore the prognostic significance of these three growth patterns for survival. The study included 217 consecutive patients, liver resected between 2007 and 2011 due to hepatic metastases from colorectal adenocarcinoma. The growth patterns were assessed on archival hematoxylin and eosin-stained tissue sections. In 150 metastases, the density of the immune cell infiltrate at the tumor periphery was judged by a semi-quantitative method. The prevalence of the pushing-type, the desmoplastic-type, and the replacement-type was 33%, 32%, and 11%, respectively; 24% of the metastases displayed a mixed pattern. Kaplan–Meier analysis and Cox regression demonstrated a prognostic significance of the growth patterns (P=0.0006, log-rank test), as the replacement pattern appeared as an independent predictor of poor overall survival. For patients with replacement growth, the hazard of death was 2–2.5 times higher than for patients with pushing growth (P=0.004, cox regression) or mixed growth (P=0.01), and nearly four times higher than for patients with desmoplastic growth (P<0.0001). The negative prognostic effect of the replacement growth pattern was even more pronounced after adjusting for tumor size. Desmoplastic growth corresponded with small tumor size, dense lymphocytic infiltration and a more favorable prognosis. Eventually, the growth patterns may contribute to a histology-based prognostic biomarker for patients with colorectal liver metastases.
Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it ...difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient's tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine.
Aim: The present study describes the ability of a newly developed N‐terminal pro‐peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme‐linked immunosorbent assay (ELISA) for describing ...liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100% homologous in the human, rat, and mouse species.
Methods: Monoclonal antibodies were raised against selected P4NP 7S specific sequences. Antibodies were screened and a competitive ELISA assay was developed using a selected antibody. The assay was evaluated in relation to technical performance, and in two preclinical liver fibrosis models; the bile duct ligation model (BDL) and the carbon tetrachloride model (CCL4) both performed in rats.
Results: A technically robust P4NP 7S ELISA assay using a monoclonal antibody was produced. In the BDL and CCL4 liver fibrosis models it was observed that the P4NP 7S levels were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P < 0.001) and correlated to the amount of hepatic type IV collagen expression in BDL rats (r = 0.49, P < 0.05) in contrast to sham (r = −0.12). P4NP 7S also correlated to total collagen in CCL4 treated livers (P < 0.001, r = 0.67), however, not in controls (r = 0.04).
Conclusions: This newly developed serum assay specific for P4NP 7S was highly related to liver fibrosis and correlated to extent of hepatic fibrosis. This assay may improve fibrosis quantification.
Histologic scoring systems specific for primary sclerosing cholangitis (PSC) are not validated. We recently determined the applicability and prognostic value of three histological scoring systems in ...a single PSC cohort. The aim of this study was to validate their prognostic use and reproducibility across a multicenter PSC cohort. Liver biopsies from PSC patients were collected from seven European institutions. Histologic scoring was performed using the Nakanuma, Ishak, and Ludwig scoring systems. Biopsies were independently scored by six liver pathologists for interobserver agreement. The prognostic value of clinical, biochemical, and all three histologic scoring systems on predicting composite endpoints 1 (PSC‐related death and liver transplantation), 2 (liver transplantation), and 3 (liver‐related events), was assessed using univariable and multivariable Cox proportional hazards modeling. A total of 119 PSC patients were identified, and the median follow‐up was 142 months. During follow‐up, 31 patients died (20 PSC‐related deaths), 31 patients underwent liver transplantation, and 35 patients experienced one or more liver‐related events. All three staging systems were independent predictors of endpoints 2 and 3 (Nakanuma system: hazard ratio HR, 3.16 95% confidence interval (CI), 1.49‐6.68 for endpoint 2 and HR, 2.05 95% CI, 1.17‐3.57 for endpoint 3; Ishak system: HR, 1.55 95% CI, 1.10‐2.18 for endpoint 2 and HR, 1.43 95% CI, 1.10‐1.85 for endpoint 3; Ludwig system: HR, 2.62 95% CI, 1.19‐5.80 for endpoint 2 and HR, 2.06 95% CI, 1.09‐3.89 for endpoint 3). Only the Nakanuma staging system was independently associated with endpoint 1: HR, 2.14 (95% CI, 1.22‐3.77). Interobserver agreement was moderate for Nakanuma stage (κ = 0.56) and substantial for Nakanuma component fibrosis (κ = 0.67), Ishak stage (κ = 0.64), and Ludwig stage (κ = 0.62). Conclusion: We confirm the independent prognostic value and demonstrate for the first time the reproducibility of staging disease progression in PSC using the Nakanuma, Ishak, and Ludwig staging systems. The Nakanuma staging system—incorporating features of chronic biliary disease—again showed the strongest predictive value. (Hepatology 2017;65:907‐919).
During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, ...increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis.
Mass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)-treated rats.
Intra- and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl(4)-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R(2) = 0.58, p<0.0001) in CCl(4)- treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003).
This novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.
Cytoglobin (Cygb) is a member of the hemoglobin family and is thought to protect against cellular hypoxia and oxidative stress. These functions may be particularly important in inflammation-induced ...cancer, e.g., in patients with ulcerative colitis (UC). In this study, we investigated the development of inflammation and tumors in a murine model of inflammation-induced colorectal cancer using a combined treatment of azoxymethane and dextran sulfate sodium. A bioinformatics analysis of genome-wide expression data revealed increased colonic inflammation at the molecular level accompanied by enhanced macroscopic tumor development in Cygb-deficient mice. Moreover, the expression of the UC-associated gene neurexophilin and PC-esterase domain family member 4 (Nxpe4) depended on the presence of Cygb in the inflamed colonic mucosa. Compared to wild type mice, RT-qPCR confirmed a 14-fold (p = 0.0003) decrease in Nxpe4 expression in the inflamed colonic mucosa from Cygb-deficient mice. An analysis of Cygb protein expression suggested that Cygb is expressed in fibroblast-like cells surrounding the colonic crypts. Histological examinations of early induced lesions suggested that the effect of Cygb is primarily at the level of tumor promotion. In conclusion, in this model, Cygb primarily seemed to inhibit the development of established microadenomas.
One of the most serious complications of ulcerative colitis is the development of colorectal cancer. Screening patients with ulcerative colitis by standard histological examination of random ...intestinal biopsy samples might be inefficient as a method of cancer surveillance. This Review focuses on the current understanding of the pathogenesis of ulcerative colitis-associated colorectal cancer and how this knowledge can be transferred into patient management to assist clinicians and pathologists in identifying patients with ulcerative colitis who have an increased risk of colorectal cancer. Inflammation-driven mechanisms of DNA damage, including the generation and effects of reactive oxygen species, microsatellite instability, telomere shortening and chromosomal instability, are reviewed, as are the molecular responses to genomic stress. We also discuss how these mechanisms can be translated into usable biomarkers. Although progress has been made in the understanding of inflammation-driven carcinogenesis, markers based on these findings possess insufficient sensitivity or specificity to be usable as reliable biomarkers for risk of colorectal cancer development in patients with ulcerative colitis. However, screening for mutations in p53 could be relevant in the surveillance of patients with ulcerative colitis. Several other new biomarkers, including senescence markers and α-methylacyl-CoA-racemase, might be future candidates for preneoplastic markers in ulcerative colitis.
Abstract Interleukin-33 (IL-33) is a novel member of the IL-1 cytokine family. It has been shown to elicit a Th2-like cytokine response in immunocompetent cells through binding and activation of the ...T1/ST2 receptor. IL-33 has recently been associated with immune responses to helminthic intestinal infections, airway inflammation and arthritis in animal models. We now report IL-33 to be produced by colonic epithelial cells in humans and it is highly upregulated in ulcerative colitis (UC). Little mRNA expression was found in control subjects ( N = 9), whereas patients with UC in remission ( N = 7) and active UC ( N = 9) had a 3-fold ( p < 0.006) and 13-fold ( p < 0.0002) increased expression, respectively. On the protein level, IL-33 in its uncleaved form was overexpressed in active UC compared to controls ( p < 0.006) and inactive UC ( p < 0.03). Immunohistochemistry of IL-33 confirmed expression in active UC in colonic epithelial cells, whereas no detectable epithelial expression was seen in control specimens. Caspase 1, which is known to activate IL-33, was expressed in colonocytes, albeit at just detectable levels when the activated p20 caspase 1 was measured. Since IL-33 recently has been shown to be biologically active in its pro-form, and cleavage seems to inactivate IL-33, IL-33 is suggested to be active in UC. We found no IL-33 expression in Caco2 cells, regardless of stimulation by pro-inflammatory cytokines. In contrast to the IL-33 expression data, we could not show any difference in the production of another member of the IL-1 cytokine family, IL-1β. This is the first study to describe that IL-33 is upregulated in UC. If IL-33 is driving a Th2-like cytokine response in UC, inhibition of the IL-33 T1/ST2 receptor pathway could be a future therapeutic option in UC.
Hepatocellular adenomas (HCA) are rare, benign liver tumours that often occur in women of reproductive age. HCA has been associated with the use of oral contraceptives, but the increased incidence of ...the tumour in older women and in men has linked the tumour type to other diseases, including the metabolic syndrome. Genotypical classification of the adenomas has led to the identification of four subgroups that correlate genotype with phenotype: human hepatocyte nuclear factor‐1 alpha (HNF1α) inactivating HCA, β‐catenin activating HCA, inflammatory HCA and unclassified HCA. HNF1α inactivating HCA is associated with bi‐allelic mutations in the TCF1 gene and morphologically has marked steatosis. β‐catenin activating HCA has increased activity of the Wnt/β‐catenin pathway and is associated with possible malignant transformation. Inflammatory HCA is characterized by an oncogene‐induced inflammation due to alterations in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. In the diagnostic setting, sub classification of HCA is based primarily on immunohistochemical analyzes, and has had an increasing impact on choice of treatment and individual prognostic assessment. This review offers an overview of the reported gene mutations associated with hepatocellular adenomas together with a discussion of the diagnostic and prognostic value.
The treatment of patients with colorectal liver metastasis has improved significantly and first line therapy is often combined chemotherapy and bevacizumab, although it is unknown who responds to ...this regimen. Colorectal liver metastases grow in different histological growth patterns showing differences in angiogenesis. To identify possible response markers, histological markers of angiogenesis were assessed. Patients who underwent resection of colorectal liver metastasis at Rigshospitalet, Copenhagen, Denmark from 2007 to 2011 were included (n = 254) including untreated and patients treated with chemotherapy or chemotherapy plus bevacizumab. The resected liver metastases were characterised with respect to growth pattern, endothelial and tumour cell proliferation as well as microvessel density and tumour regression. Tumour regression grade of liver metastases differed significantly between untreated/chemotherapy treated patients in comparison to chemotherapy plus bevacizumab treated patients (both p < 0.0001). Microvessel density was decreased in liver metastases from patients treated with bevacizumab in comparison to those from untreated/chemotherapy‐treated patients (p = 0.006/p = 0.002). Tumour cell proliferation assessed by Ki67 expression correlated to a shorter recurrence free survival in the total patient cohort. In conclusion, liver metastases from patients treated with neo‐adjuvant chemotherapy and bevacizumab had significantly lower microvessel densities and tumour regression grades when compared to liver metastases from untreated or chemotherapy treated patients. This may indicate that bevacizumab treatment results in altered vascular biology and tumour viability, with possible tumour reducing effect.
What's new?
A first‐line therapy for colorectal liver metastasis is chemotherapy combined with vascular endothelial growth factor (VEGF) inhibitor bevacizumab. Patient response markers are however lacking. In this first study on the effect of bevacizumab on angiogenesis in colorectal liver metastases, the authors evaluate endothelial cell proliferation and microvessel density in resected liver metastases from patients who were either untreated or received chemotherapy alone or in combination with bevacizumab. Microvessel density is significantly lower and the degree of tumor regression is highest in liver metastases from patients treated with chemotherapy and bevacizumab. These findings may help develop response markers for bevacizumab treatment.
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