Collagen and elastic fibres in acute and chronic liver injury.

The histological distinction between acute and chronic liver injury is a challenging aspect of liver histopathology. It is traditionally based on the interpretation of morphological changes to the extracellular matrix (ECM) at sites of hepatocyte loss using histochemical stains. Our aim was to investigate whether immunohistochemistry and multiplexing for collagen type (I & III) and elastic fibres and a modified Victoria blue method could be helpful. We studied 43 livers removed at transplantation for acute liver failure (ALF, 20 cases) or cirrhosis (23) plus 8 normal controls. In ALF the periportal ECM was normal in 2 cases, contained mainly collagen I associated with a ductular reaction in 6 cases, and delicate elastic strands in 11 cases. Periportal deposition of mainly collagen I and mature elastic fibres was observed in cirrhosis. In ALF the perisinusoidal ECM was intact in 4 cases, collapsed or condensed but of normal composition (predominantly collagen III) in 2 cases, and collapsed and condensed containing mostly collagen I in 17 cases (7 including delicate immature elastic strands). In contrast, bridging fibrous septa of cirrhosis contained abundant collagen 1 and bundles of mature elastin. We propose the use of a scale and the use of immunohistochemistry and multiplexing in additional to histochemical stains to characterise the ECM changes in acute and chronic liver injury.

Tissue curvature and apicobasal mechanical tension imbalance instruct cancer morphogenesis.

Tubular epithelia are a basic building block of organs and a common site of cancer occurrence1-4. During tumorigenesis, transformed cells overproliferate and epithelial architecture is disrupted. However, the biophysical parameters that underlie the adoption of abnormal tumour tissue shapes are unknown. Here we show in the pancreas of mice that the morphology of epithelial tumours is determined by the interplay of cytoskeletal changes in transformed cells and the existing tubular geometry. To analyse the morphological changes in tissue architecture during the initiation of cancer, we developed a three-dimensional whole-organ imaging technique that enables tissue analysis at single-cell resolution. Oncogenic transformation of pancreatic ducts led to two types of neoplastic growth: exophytic lesions that expanded outwards from the duct and endophytic lesions that grew inwards to the ductal lumen. Myosin activity was higher apically than basally in wild-type cells, but upon transformation this gradient was lost in both lesion types. Three-dimensional vertex model simulations and a continuum theory of epithelial mechanics, which incorporate the cytoskeletal changes observed in transformed cells, indicated that the diameter of the source epithelium instructs the morphology of growing tumours. Three-dimensional imaging revealed that-consistent with theory predictions-small pancreatic ducts produced exophytic growth, whereas large ducts deformed endophytically. Similar patterns of lesion growth were observed in tubular epithelia of the liver and lung; this finding identifies tension imbalance and tissue curvature as fundamental determinants of epithelial tumorigenesis.

Irreversible electroporation in borderline resectable pancreatic adenocarcinoma for margin accentuation.

Achieving clear microscopic resection margins following pancreaticoduodenectomy (PD) is challenging particularly in borderline resectable pancreatic carcinoma (BRPC). Positive resection margins has been identified as a major independent prognostic factor. Irreversible electroporation (IRE) has emerged as a promising non-thermal ablative method that could be used in the treatment of pancreatic cancer as an adjunct to chemotherapy and surgery. This case report describes the successful simultaneous intraoperative IRE and PD in a patient with BRPC, achieving clear microscopic resection margins. Technical aspects and histology showing the effect of IRE are presented. The role of IRE in the treatment of pancreatic adenocarcinoma should be further evaluated in prospective studies.

Tumour diameter is not reliable for management of non-secreting pancreatic neuroendocrine tumours.

Small non-functioning pancreatic NETs (pNETs) ≤2 cm can pose a management dilemma in terms of surveillance or resection. There is evidence to suggest that a surveillance approach can be considered since there are no significant radiological changes observed in lesions during long-term follow-up. However, other studies have suggested loco-regional spread can be present in ≤2 cm pNETs. The aim of this study was to characterise the prevalence of malignant features and identify any useful predictive variables in a surgically resected cohort of pNETs. 418 patients with pNETs were identified from 5 NET centres. Of these 227 were included for main analysis of tumour characteristics. Mean age of patients was 57 years, 47% were female. The median follow-up was 48.2 months. Malignant features were identified in 38% of ≤2 cm pNETs. ROC analysis showed that the current cut-off of 20 mm had a sensitivity of 84% for malignancy. The rate of malignant features is in keeping with other surgical series and challenges the belief that small pNETs have a low malignant potential. This study does not support a 20 mm size cut-off as being a solitary safe parameter to exclude malignancy in pNETs.

AST-to-platelet ratio index in non-invasive assessment of long-term graft fibrosis following pediatric liver transplantation.

Long-term graft fibrosis occurs in the majority of pediatric liver transplant recipients. Serial biopsies to monitor graft health are impractical and invasive. The APRI has been evaluated in pediatric liver disease, but not in the context of post-transplantation fibrosis. We aimed to investigate the validity of APRI as a predictor of long-term graft fibrosis in pediatric liver transplant recipients. This was a retrospective, observational study of a cohort of children who underwent liver transplantation at King's College Hospital between 1989 and 2003, with a relevant dataset available. Protocol liver biopsies were performed at 10-yr follow-up and fibrosis was graded using the Ishak scoring system, with S3-6 denoting "significant fibrosis." APRI was calculated concurrently with biopsy. A total of 39 asymptomatic patients (20 males; median age at transplant, 1.43 yr) underwent protocol liver biopsies at a median of 10.39 yr post-transplantation. APRI was associated with significant fibrosis (p = 0.012). AUROC for APRI as a predictor of significant fibrosis was 0.74 (p = 0.013). The optimal cutoff APRI value for significant fibrosis was 0.45 (sensitivity = 0.67; specificity = 0.79; PPV = 0.67; NPV = 0.79). APRI appears to be a useful non-invasive adjunct in the assessment of significant graft fibrosis in the long-term follow-up of pediatric liver transplant survivors.

The effect of anterograde persufflation on energy charge and hepatocyte function in donation after cardiac death livers unsuitable for transplant.

Donation after cardiac death (DCD) livers are considered to be marginal organs for solid organ and cell transplantation. Low energy charge (EC) and low purine quantity within the liver parenchyma has been associated with poor outcome after liver transplantation. The aim of this work was to assess the effect of anterograde persufflation (A-PSF) using an electrochemical concentrator on DCD liver energy status and hepatocyte function. Organs utilized for research were DCD livers considered not suitable for transplant. Each liver was formally split, and the control non-persufflated (non-PSF) section was stored in University of Wisconsin (UW) solution at 4°C. The A-PSF liver section was immersed in UW solution on ice, and A-PSF was performed via the portal vein with 40% oxygen. Tissue samples were taken 2 hours after A-PSF from the A-PSF and control non-PSF liver sections for snap freezing. Purine analysis was performed with photodiode array detection. Hepatocytes were isolated from A-PSF and control non-PSF liver sections using a standard organs utilized for research were DCD livers considered not suitable for transplant collagenase perfusion technique. Hepatocyte function was assessed using mitochondrial dehydrogenase activity {3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl tetrazolium bromide (MTT)} and the sulforhodamine B (SRB) assay for cell attachment. In DCD livers with <30% steatosis (n = 6), A-PSF increased EC from 0.197 ± 0.025 to 0.23 ± 0.035 (P = 0.04). In DCD livers with >30% steatosis (n = 4), A-PSF had no beneficial effect. After isolation (n=4, <30% steatosis), A-PSF was found to increase MTT from 0.92 ± 0.045 to 1.19 ± 0.55 (P < 0.001) and SRB from 2.53 ± 0.12 to 3.2 ± 0.95 (P < 0.001). In conclusion, A-PSF can improve the EC and function of isolated hepatocytes from DCD livers with <30% steatosis.

Histological aspects of post-TACE hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer in men and the seventh in women and is the third most common cause of death from cancer worldwide [http://globocan.iarc.fr]. The overall incidence of HCC remains high in developing countries and is steadily rising in most industrialized countries [Shariff MI et al., 2009]. A variety of therapeutic modalities is available for treating hepatocellular carcinoma, but orthotopic liver transplantation (OLT) represents a curative option. Due to the shortage of donor organs and the increasing need for liver transplantation in the last decade, local ablation therapy (LAT) has been increasingly used in many centers as a bridge to transplant [Majno PE et al., 1997; Decaens T et al., 2005; Herber S et al., 2005; Bharat A et al., 2006; Obed A et al., 2007; Otto G et al., 2007]. We retrieved from the archive in the Histopathology Laboratory, Institute of Liver Studies, King's College Hospital, London, UK, 28 cases of HCC, which underwent treatment with TACE (Doxorubicin 40 mg/m²) as a bridge to transplantation, between 2008 and 2010. We also analyzed 14 additional post-TACE tumors, classified according to the architectural patterns published by Morisco F et al. (2008), for quantification of necrosis. Extensive tumor necrosis was observed in 12 (42.85%) of the patients. Viable hepatocellular carcinoma showed a wide range of differentiation, from well to poorly differentiated. The phenotype of the tumors was mostly hepatocelluar, but 14% showed a mixed phenotype, including glandular/pseudoglandular formation and cholangiocellular components. The percentage of necrosis ranged between 0% and 100%, with an average of 50.6%. There was no statistical correlation between the total size of the nodules and the surface of necrosis in our series (p=0.125). In conclusion, the systematic pathological assessment of post-TACE resected HCC can help in investigating the biology of treated tumors but needs to incorporate sampling protocols, digital image analysis, phenotypic classification by immunohistochemistry and enzymatic function.

Immunohistochemical phenotyping of the inflammatory infiltrate in de novo autoimmune hepatitis after liver transplantation in children.

We have investigated the inflammatory infiltrate in post-transplant dn-AIH, a form of late insidious graft rejection, focusing on transcription factors defining effector and T-regs, using an antigen retrieval immunohistochemical method on archived liver tissue, and compared it with ACR and classical AIH. Paraffin-embedded liver biopsies from pediatric patients with dn-AIH (n = 10), ACR (n = 10), and AIH (n = 13) were selected randomly and stained using antibodies directed to CD4, CD8, T-bet (marker of Th1 polarization), GATA-3 (marker of Th2 polarization), FOXP3 (marker for T regulatory cells), IL-17, CD56 (NK cells), and perforin. Portal and lobular lymphocytic infiltrate was assessed semi-quantitatively. Prominent CD4, CD8, and T-bet positivity were present in both the lobular and portal infiltrate of all three conditions. Overall T-bet score of lobular inflammation in the dn-AIH group was lower than in the ACR and AIH groups (p = 0.02). In contrast, most samples showed absent or minimal GATA-3 positivity. FOXP3, CD56, IL-17, and perforin staining of mild to moderate severity were present in all three groups in both the portal and lobular infiltrate. A Th1 polarization of the inflammatory infiltrate characterizes dn-AIH, but also ACR and AIH.

Whole-genome DASL gene expression profiling of hepatocellular carcinoma sub-populations isolated by laser microdissection on formalin-fixed and paraffin-embedded liver tissue samples.

In the last ten years, a multitude of studies focusing on gene expression profiling have attempted to shed light on the molecular and genomic mechanisms leading to hepatocarcinogenesis. One of the downsides of the technology available until recently was that it was limited to RNA extracted from fresh/frozen tissue or cell cultures. Recent advances have made it possible to obtain good quality RNA from formalin-fixed paraffin-embedded (FFPE) tissue, allowing access to a virtually limitless archival resource to be available for retrospective and long-term prospective clinico-pathological studies. Laser-capture microdissection allows the isolation of specific cell populations or of specific microscopic areas of interest from tissue samples. This allows the selective evaluation of gene expression of targeted cell clusters, especially in a very heterogeneous environment as the malignant tissue. In our study, we demonstrated that by laser microdissecting the areas of interest from FFPE tissue we could obtain gene expression signals by running the purified RNA through the Whole Genome DASL assay. A large number of genes were expressed in both subpopulations of hepatocellular carcinoma (classical HCC and cholangiocellular differentiation) as well as in the cirrhotic and non-cirrhotic liver background.

Histological and ultrastructural analysis of the seminiferous tubule wall in ageing testis.

The authors evaluated possible morphological changes of basement membrane (BM) and lamina propria (LP) of seminiferous tubule wall (ST) related to ageing. Surgical samples of testicular tissue from 28 cases with orchiectomy for prostate adenocarcinoma were processed for light microscopy and transmission electron microscopy (TEM) examination. Seven age groups (AgGr) between 50 and 80 years were designed. Tissue samples were immunomarked for collagen IV and smooth muscle actin. Images were acquired and measured with a specialized software. Thirty ST were randomly selected, with ×40-objective, for each case. Five random determinations for each ST and each parameter were performed. Mean values/tubule, case and AgGr were calculated for each parameter. Regression line (RL), slope and significance test for slope were determined for each parameter correlation with ageing. BM mean value was around 0.5 µm, with narrow limits of ranging in AgGr but more extended individual limits. RL showed discrete decreasing trend with ageing but without an obvious statistical correlation. LP mean value was around 6 µm, also with narrow limits of ranging in AgGr and more extended individual limits. RL decreased discretely with ageing but without an obvious statistical correlation. TEM showed more prominent BM material and more collagen fibers and less fibroblasts in LP of older AgGr and higher fibroblasts density in LP of younger AgGr. Our results showed that BM thickness is apparently decreasing with ageing whereas LP presents extremely variable degenerative changes, with a "mosaic", focal distribution and no tendency to advance with ageing.

Correlations between intralobular interstitial morphological changes and epithelial changes in ageing testis.

The study focuses on the possible influences of intra (I) lobular (L) stromal compounds [intertubular spaces and seminiferous (S) tubule (T) wall (W)] morphologic changes on S epithelium (E) during ageing process. The material consisted of surgical samples of testicular tissue from 192 patients with orchidectomy for prostate carcinoma. Seven age groups were designed, from 50 to 80 years. Tissue samples were fixed in neutral buffered formalin, embedded in paraffin stained with HE, Goldner and Gömöri and immunomarked (in a subgroup of 28 cases) for smooth muscle actin, collagen IV, and CD34. SE had an uneven involution, both individually and inter-individually, but with normal spermatogenesis in many of ST. E degenerative changes were seen mainly in L periphery. Different stages of maturation arresting were more frequent in older patients. IL septae had changes with extremely variable intensity, dispersed mainly in L periphery, without significant spread and without extensive trend with ageing. Leydig cells showed focal hyperplasia without extensive trend related with ageing. STW presented strictly in the internal layer of lamina propria (apposed to basement membrane of ES) a focal sclerosis, with variable extension concerning its presence, thickness and T circumference (T without sclerosis, with focal sclerosis and with fibro-hyaline "collar" - FHyC) but not related with ageing. IL arteriolae showed focal areas of degeneration with a wide individual and inter-individual range of intensity and extension, but not related with age. Capillary network (CN), with both its peri-T and intramural segments, was present in all age groups, with no quantitative endothelial changes and decreasing only in very old cases. FHyC was often associated with E atrophy. STW focal sclerosis could explain focal degeneration of SE in senescence, although CN undergoes no significant changes.