Antonia D'Errico-Grigioni

Efficacy of selective transarterial chemoembolization in inducing tumor necrosis in small (<5 cm) hepatocellular carcinomas

Transarterial chemoembolization (TACE) is commonly used as a bridge therapy for patients awaiting liver transplantation (LT) and for downstaging patients initially not meeting the Milan criteria. The primary aim of this study was to analyze whether a difference exists between selective/superselective and lobar TACE in determining tumor necrosis by a pathological analysis of the whole lesion at the time of LT. The secondary aim was to investigate the relationship between the tumor size and the capacity of TACE to induce necrosis. Data were extracted from a prospective database of 67 consecutive patients who underwent LT for hepatocellular carcinoma and cirrhosis from 2003 to 2009 and were treated exclusively with TACE as a bridging (n = 53) or downstaging therapy (n = 14). We identified 122 nodules; 53.3% were treated with selective/superselective TACE. The mean histological necrosis level was 64.7%; complete tumor necrosis was obtained in 42.6% of the nodules. In comparison with lobar TACE, selective/superselective TACE led to significantly higher mean levels of necrosis (75.1% versus 52.8%, P = 0.002) and a higher rate of complete necrosis (53.8% versus 29.8%, P = 0.013). A significant direct relationship was observed between the tumor diameter and the mean tumor necrosis level (59.6% for lesions < 2 cm, 68.4% for lesions of 2.1‐3 cm, and 76.2% for lesions > 3 cm). Histological necrosis was maximal for tumors > 3 cm: 91.8% after selective/superselective TACE and 66.5% after lobar procedures. Independent predictors of complete tumor necrosis were selective/superselective TACE (P = 0.049) and the treatment of single nodules (P = 0.008). Repeat sessions were more frequently needed for nodules treated with lobar TACE (31.6% versus 59.3%, P = 0.049). Conclusion: Selective/superselective TACE was more successful than lobar procedures in achieving complete histological necrosis, and TACE was more effective in 3‐ to 5‐cm tumors than in smaller ones. (Hepatology 2011;)

Predictive Value of Biological Markers for Hepatocellular Carcinoma Patients Treated with Orthotopic Liver Transplantation

Purpose: To help stratify candidates with hepatocellular carcinoma (HCC) for orthotopic liver transplantation (OLT), biomarkers are needed that are capable of predicting recurrence of disease (ROD). We investigated the prognostic role in this setting of immunohistochemical markers reported previously to predict poor prognosis in HCC patients treated with resection. Experimental Design: Eighty-three patients with HCC who underwent OLT between 1987 and 2001 with a minimum clinical follow up of 12 months were included in this retrospective study. We analyzed immunohistochemical expression of the adhesion molecules E-cadherin and β-catenin (membrane/nuclear localization), MIB-1 proliferative index and the cyclin-dependent kinase inhibitor p27, alongside the main clinical-pathological variables. Results: At univariate analysis, vascular thrombosis, high MIB-1 index, lower membrane expression of E-cadherin and β-catenin, and nuclear β-catenin localization were associated with ROD. At multivariate analysis, only MIB-1 index, low equal E-cadherin (with respect to non-neoplastic surrounding tissue), and nuclear β-catenin appeared as independent predictors of ROD. The logistic regression analysis model indicated that detection of any one parameter was associated with at least 88% estimated risk of ROD (up to 99% for all three). Conclusions: We propose these three molecular parameters as an additional tool for rational selection of OLT candidates among HCC patients (stratification according to the risk of ROD might help provide a similar life expectancy for cirrhotic candidates with and without HCC).

Predictive value of frozen‐section analysis in the histological assessment of steatosis before liver transplantation

Histological quality assessment of donated livers is a key factor for extending the cadaveric donor pool for liver transplantation. We retrospectively compared frozen‐section analysis with routine histological permanent slides and the outcomes of grafts in liver biopsies from 294 candidate donors. The κ concordance coefficient of agreement between frozen‐section analysis and routine histological analysis was very good for macrosteatosis (κ = 0.934), microsteatosis (κ = 0.828), and total steatosis (κ = 0.814). The correlation between the mean amounts of macrosteatosis, microsteatosis, and total steatosis in frozen and permanent sections was also significant (P < 0.001, Spearmans test). Macrosteatosis and microsteatosis were overestimated to >30% in 4 of 32 cases (12.5%) and in 23 of 62 cases (37.1%), respectively. The only 2 histological parameters of frozen sections able to predict graft dysfunction within 7 days of transplantation were macrosteatosis and total steatosis (P = 0.018 and P = 0.015, respectively, Mann‐Whitney test). None of the other histopathological features evaluated in frozen sections, including portal inflammation, lobular necrosis, myointimal thickening, biliocyte regression, cholestasis, hepatocellular polymorphism, lipofuscin storage, and fibrous septa, were significantly correlated with the graft outcome. The frozen‐section histological evaluation of biopsies from cadaveric liver donors is an accurate, time‐effective, and predictive method for the assessment of graft suitability. Liver Transpl 15:1821–1825, 2009.

Studies on immunoproteasome in human liver. Part I: Absence in fetuses, presence in normal subjects, and increased levels in chronic active hepatitis and cirrhosis

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive (alpha4 and beta1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28alphabeta regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical data have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28alphabeta. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention.

Antiinflammatory effect of phytosterols in experimental murine colitis model: prevention, induction, remission study.

Phytosterols, besides hypocholesterolemic effect, present anti-inflammatory properties. Little information is available about their efficacy in Inflammatory Bowel Disease (IBD). Therefore, we have evaluated the effect of a mixture of phytosterols on prevention/induction/remission in a murine experimental model of colitis. Phytosterols were administered x os before, during and after colitis induction with Dextran Sodium Sulfate (DSS) in mice. Disease Activity Index (DAI), colon length, histopathology score, 18F-FDG microPET, oxidative stress in the intestinal tissue (ileum and colon) and gallbladder ileum and colon spontaneous and carbachol (CCh) induced motility, plasma lipids and plasma, liver and biliary bile acids (BA) were evaluated. A similar longitudinal study was performed in a DSS colitis control group. Mice treated with DSS developed severe colitis as shown by DAI, colon length, histopathology score, 18F-FDG microPET, oxidative stress. Both spontaneous and induced ileal and colonic motility were severely disturbed. The same was observed with gallbladder. DSS colitis resulted in an increase in plasma cholesterol, and a modification of the BA pattern. Phytosterols feeding did not prevent colitis onset but significantly reduced the severity of the disease and improved clinical and histological remission. It had strong antioxidant effects, almost restored colon, ileal and gallbladder motility. Plasmatic levels of cholesterol were also reduced. DSS induced a modification in the BA pattern consistent with an increase in the intestinal BA deconjugating bacteria, prevented by phytosterols. Phytosterols seem a potential nutraceutical tool for gastrointestinal inflammatory diseases, combining metabolic systematic and local anti-inflammatory effects.

Increased Plin2 Expression in Human Skeletal Muscle Is Associated with Sarcopenia and Muscle Weakness

Human aging is associated with a progressive loss of muscle mass and strength and a concomitant fat accumulation in form of inter-muscular adipose tissue, causing skeletal muscle function decline and immobilization. Fat accumulation can also occur as intra-muscular triglycerides (IMTG) deposition in lipid droplets, which are associated with perilipin proteins, such as Perilipin2 (Plin2). It is not known whether Plin2 expression changes with age and if this has consequences on muscle mass and strength. We studied the expression of Plin2 in the vastus lateralis (VL) muscle of both healthy subjects and patients affected by lower limb mobility limitation of different age. We found that Plin2 expression increases with age, this phenomenon being particularly evident in patients. Moreover, Plin2 expression is inversely correlated with quadriceps strength and VL thickness. To investigate the molecular mechanisms underpinning this phenomenon, we focused on IGF-1/p53 network/signalling pathway, involved in muscle physiology. We found that Plin2 expression strongly correlates with increased p53 activation and reduced IGF-1 expression. To confirm these observations made on humans, we studied mice overexpressing muscle-specific IGF-1, which are protected from sarcopenia. These mice resulted almost negative for the expression of Plin2 and p53 at two years of age. We conclude that fat deposition within skeletal muscle in form of Plin2-coated lipid droplets increases with age and is associated with decreased muscle strength and thickness, likely through an IGF-1- and p53-dependent mechanism. The data also suggest that excessive intramuscular fat accumulation could be the initial trigger for p53 activation and consequent loss of muscle mass and strength.

Blood Monitoring of Granzyme B and Perforin Expression After Intestinal Transplantation : Considerations on Clinical Relevance

Background. The use of biomarkers for rejection monitoring represents a major goal in intestinal transplantation. We analyzed the blood expression of Granzyme B (GB) and Perforin (PF) in the following pathological conditions after intestinal transplantation: acute rejection (AR), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection, and posttransplant lymphoproliferative disease (PTLD). The diagnostic accuracy and the clinical utility of these tests are finally discussed. Methods. GB and PF levels were measured by real time polymerase chain reaction on peripheral blood samples from 32 intestinal recipients. Blood samples (n=494) after comparison of clinical, histological, and microbiological data were assigned to the following groups: normal (n=307), AR (n=30), EBV infection (n=107), CMV infection (n=25), and PTLD (n=25). Results. Mean levels of GB and PF in the AR (GB=279.7; PF=256.7), PTLD (GB=199; PF=185.9), EBV (GB=133.2; PF=143.7), and CMV (GB=151.3; PF=144) groups were significantly higher than in the normal group (GB=100.1; PF=101.1) (all P<0.05, except for PF in CMV infection). The best accuracy was obtained for the diagnosis of AR with sensitivity and specificity of 80% and 79% for GB and 70% and 79% for PF, respectively. The area under the receiver-operator characteristics curve was 0.87 for GB and 0.82 for PF. Conclusions. GB and PF are diagnostic molecular markers of AR. GB and PF blood levels are also increased in case of viral infections or PTLD. Serial blood testing for GB and PF might be predictive of early intestinal graft dysfunction and should be interpreted in the context of the histological and virological analyses.

Tissue hepatitis C virus RNA quantification and protein expression help identify early hepatitis C virus recurrence after liver transplantation

We compared tissue hepatitis C virus (HCV) RNA polymerase chain reaction quantification and HCV immunohistochemistry (IHC) to histology in biopsy tissues in order to differentiate between acute rejection and HCV hepatitis recurrence early after orthotopic liver transplantation (OLT). We analyzed the first biopsy performed because of alteration of serum aminotransferases in 65 consecutive OLT patients with HCV genotype 1b. In the histological analysis, we quantified the portal tracts, Councilman bodies, Councilman body/portal tract (CP) ratio, steatosis, and Knodell and Ishak scores. The 52 patients (80%) with histological HCV recurrence [recurrence‐positive (Rec+)] were separated from the 6 (9%) with acute rejection and the 7 (11%) with undetermined pathological features [recurrence‐negative (Rec−)]. HCV RNA strongly correlated with HCV IHC, regardless of the histological diagnosis (P < 0.001). Both HCV RNA and HCV IHC were significantly associated with CP ratio (P = 0.041 and P = 0.008). No statistical correlation was found between HCV RNA, HCV IHC, and the other histopathologic features or the hepatitis scores. HCV RNA, HCV IHC, and CP ratio were the only variables able to discriminate between Rec+ and Rec− patients (Mann‐Whitney test P < 0.001, P < 0.001, P = 0.014). In conclusion, a combined evaluation of histology, tissue HCV RNA, and HCV IHC significantly discriminated between OLT patients with or without HCV recurrence. Liver Transpl 14:313–320, 2008.

The role of 3D microenvironmental organization in MCF-7 epithelial-mesenchymal transition after 7 culture days.

We present a multi-technique study on in vitro epithelial-mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(l-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, β-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, β-catenin and vimentin) and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and β-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-β2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold.

Nestin and WT1 expression in atheromathous plaque neovessels: association with vulnerability.

INTRODUCTION Neoangiogenesis is crucial for the progression and vulnerability of atheromasic lesions. Since adult vasa vasorum, which represent the neoangiogenetic burden of healthy arteries, constitutively express Nestin and Wilms Tumor (WT1), the aims of the present study are: i) to describe and quantify Nestin and WT1 in plaque neovessels; ii) to investigate the relationship between neovessel phenotype and plaque instability. METHODS We prospectively evaluated 49 consecutive carotid endarterectomy specimens. Histopathological characteristics were separately collected, particularly the intraplaque histological complications. Immunohistochemistry was carried out for CD34, Nestin and WT1; the density of positivity was evaluated for each marker. RT-PCR was performed to assess Nestin and WT1 mRNA levels on the first 10 plaques and on 10 control arteries. RESULTS Six (12.2%) plaques showed no neoangiogenesis. In the others, the mean immunohistochemical densities of CD34, Nestin, and WT1-positive structures were 41.88, 28.84 and 17.68/mm2. Among the CD34+ neovessels, 68% and 42% expressed Nestin and WT1 respectively, i.e., nearly 36% of the neovessels resulted to be Nestin+/WT1-. Furthermore, complicated plaques (n=30) showed significantly more CD34 and Nestin-positive vessels than uncomplicated plaques (n=13; P=0.045 and P=0.009), while WT1 was not increased (P=0.139). RT-PCR confirmed that WT1 gene expression was 3-fold lower than Nestin gene in plaques (p=0.001). CONCLUSIONS Plaque neoangiogenesis shows both a Nestin+/WT1- and a Nestin+/WT1+ phenotype. The Nestin+/WT1- neovessels are significantly more abundant in complicated (vulnerable) plaques. The identification of new transcription factors in plaque neoangiogenesis, and their possible regulation, can open new perspectives in the therapy of vulnerable plaques.