Oscar Andriani

Hepatocellular carcinoma cells and their fibrotic microenvironment modulate bone marrow-derived mesenchymal stromal cell migration in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related death. Fibrogenesis is an active process characterized by the production of several proinflammatory cytokines, chemokines and growth factors. It involves the activation of hepatic stellate cells (HSCs) which accumulate at the site of injury and are the main source of the extracellular matrix deposits. There are no curative treatments for advanced HCC, thus, new therapies are urgently needed. Mesenchymal stromal cells (MSCs) have the ability to migrate to sites of injury or to remodeling tissues after in vivo administration; however, in several cancer models they demonstrated limited efficacy to eradicate experimental tumors partially due to poor engraftment. Thus, the aim of this work was to analyze the capacity of human MSCs (hMSCs) to migrate and anchor to HCC tumors. We observed that HCC and HSCs, but not nontumoral stroma, produce factors that induce hMSC migration in vitro. Conditioned media (CM) generated from established HCC cell lines were found to induce higher levels of hMSC migration than CM derived from fresh patient tumor samples. In addition, after exposure to CM from HCC cells or HSCs, hMSCs demonstrated adhesion and invasion capability to endothelial cells, type IV collagen and fibrinogen. Consistently, these cells were found to increase metalloproteinase-2 activity. In vivo studies with subcutaneous and orthotopic HCC models indicated that intravenously infused hMSCs migrated to lungs, spleen and liver. Seven days post-hMSC infusion cells were located also in the tumor in both models, but the signal intensity was significantly higher in orthotopic than in subcutaneous model. Interestingly, when orthotopic HCC tumors where established in noncirrhotic or cirrhotic livers, the amount of hMSCs localized in the liver was higher in comparison with healthy animals. A very low signal was found in lungs and spleens, indicating that liver tumors are able to recruit them at high efficiency. Taken together our results indicate that HCC and HSC cells produce factors that efficiently induce hMSC migration toward tumor microenvironment in vitro and in vivo and make MSCs candidates for cell-based therapeutic strategies to hepatocellular carcinoma associated with fibrosis.

Increased Migration of Human Mesenchymal Stromal Cells by Autocrine Motility Factor (AMF) Resulted in Enhanced Recruitment towards Hepatocellular Carcinoma

Background and Aims Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. Methods Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. Results AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. Conclusion AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.

Model for end-stage liver disease-based allocation system for liver transplantation in Argentina: does it work outside the United States?

BACKGROUND In July 2005, Argentina was the first country after the United States to adopt the MELD system. The purpose of the present study was to analyse the impact of this new system on the adult liver waiting list (WL). METHODS Between 2005 and 2009, 1773 adult patients were listed for liver transplantation: 150 emergencies and 1623 electives. Elective patients were categorized using the MELD system. A prospective database was used to analyse mortality and probability to be transplanted (PTBT) on the WL. RESULTS The waiting time increased inversely with the MELD score and PTBT positively correlated with MELD score. With scores >/= 18 the PTBT remained over 50%. However, the largest MELD subgroup with <10 points (n = 433) had the lower PTBT (3%). In contrast, patients with T(2) hepatocellular carcinoma benefited excessively with the highest PTBT (84.2%) and the lowest mortality rate (5.4%). The WL mortality increased after MELD adoption (10% vs. 14.8% vs. P < 0.01). Patients with <10 MELD points had >fourfold probability of dying on the WL than PTBT (14.3% vs. 3%; P < 0.0001). CONCLUSIONS After MELD implementation, WL mortality increased and most patients who died had a low MELD score. A comprehensive revision of the MELD system must be performed to include cultural and socio-economical variables that could affect each country individually.

Low Molecular Weight Hyaluronan-Pulsed Human Dendritic Cells Showed Increased Migration Capacity and Induced Resistance to Tumor Chemoattraction

We have shown that ex vivo pre-conditioning of bone marrow-derived dendritic cells (DC) with low molecular weight hyaluronan (LMW HA) induces antitumor immunity against colorectal carcinoma (CRC) in mice. In the present study we investigated the effects of LMW HA priming on human-tumor-pulsed monocytes-derived dendritic cells (DC/TL) obtained from healthy donors and patients with CRC. LMW HA treatment resulted in an improved maturation state of DC/TL and an enhanced mixed leucocyte reaction activity in vivo. Importantly, pre-conditioning of DC/TL with LMW HA increased their ability to migrate and reduced their attraction to human tumor derived supernatants. These effects were associated with increased CCR7 expression levels in DC. Indeed, a significant increase in migratory response toward CCL21 was observed in LMW HA primed tumor-pulsed monocyte-derived dendritic cells (DC/TL/LMW HA) when compared to LWM HA untreated cells (DC/TL). Moreover, LMW HA priming modulated other mechanisms implicated in DC migration toward lymph nodes such as the metalloproteinase activity. Furthermore, it also resulted in a significant reduction in DC migratory capacity toward tumor supernatant and IL8 in vitro. Consistently, LMW HA dramatically enhanced in vivo DC recruitment to tumor-regional lymph nodes and reduced DC migration toward tumor tissue. This study shows that LMW HA –a poorly immunogenic molecule- represents a promising candidate to improve human DC maturation protocols in the context of DC-based vaccines development, due to its ability to enhance their immunogenic properties as well as their migratory capacity toward lymph nodes instead of tumors.

Model for end-stage liver disease exceptions committee activity in Argentina: does it provide justice and equity among adult patients waiting for a liver transplant?

BACKGROUND In 2005, the model of end-stage liver disease (MELD)-based allocation system was adopted to assess potential liver transplant (LT) recipients in Argentina. The aim of the present study was to revise the activity of the MELD Exception Experts Committee. METHODS Between 2005 and 2009, 1623 patients were listed for LT. Regulation provides extra-MELD points for amyloidosis, hepatopulmonary syndrome (HPS) and T(2) hepatocellular carcinoma (T(2) HCC). Centres could also request priority for other situations. Using a prospective database, we identified patients in whom priority points were requested. Pathology reports of explanted livers were analysed for patients with T(2) HCC. RESULTS From 234 out of 1623 (14.4%) requests, the overall approval rate was 60.2% including: 2 amyloidosis, 6 HPS, 111 T(2) HCC and 22 non-regulated situations. Of the 111 patients with T(2) HCC, 6 died (5.4%), 8 had tumour progression (7.2%), 94 were transplanted (84.2%) and 3 are still waiting. An explants correlation showed that presumed diagnosis of T(2) HCC was incorrect in 20/94 (22%) and was correct in only 41/94 (43%) cases being T(1) HCC in 9 and T(3) HCC in 23. CONCLUSIONS MELD exceptions are frequently requested in Argentina. Unfortunately, most receiving priority points for T(2) HCC benefited by medical error or imaging limitations. An intense review process is urgently needed to maintain equity and justice in the allocation system.

IL-8, GRO and MCP-1 produced by hepatocellular carcinoma microenvironment determine the migratory capacity of human bone marrow-derived mesenchymal stromal cells without affecting tumor aggressiveness

New therapies are needed for advanced hepatocellular carcinoma (HCC) and the use of mesenchymal stromal cells (MSCs) carrying therapeutic genes is a promising strategy. HCC produce cytokines recruiting MSCs to the tumor milieu and modifying its biological properties. Our aim was to study changes generated on human MSCs exposed to conditioned media (CM) derived from human HCC fresh samples and xenografts. All CM shared similar cytokines expression pattern including CXCL1-2-3/GRO, CCL2/MCP-1 and CXCL8/IL-8 being the latter with the highest concentration. Neutralizing and knockdown experiments of CCL2/MCP-1, CXCL8/IL-8, CXCR1 and CXCR2 reduced in vitro MSC migration of ≥20%. Simultaneous CXCR1 and CXCR2 neutralization resulted in 50% of MSC migration inhibition. MSC stimulated with CM (sMSC) from HuH7 or HC-PT-5 showed a 2-fold increase of migration towards the CM compared with unstimulated MSC (usMSC). Gene expression profile of sMSC showed ~500 genes differentially expressed compared with usMSC, being 46 genes related with cell migration and invasion. sMSC increased fibroblasts and endothelial cells chemotaxis. Finally, sMSC with HuH7 CM and then inoculated in HCC tumor bearing-mice did not modify tumor growth. In this work we characterized factors produced by HCC responsible for the changes in MSC chemotactic capacity with would have an impact on therapeutic use of MSCs for human HCC.

Screening for liver cancer during transplant waiting list: a multicenter study from South America.

Background and aims Surveillance during liver transplantation (LT) waiting list has scarcely been reported in South America. We aimed to describe hepatocellular carcinoma (HCC) surveillance during the LT waiting list in the daily practice. Patients and methods A multicenter retrospective analysis in cirrhotic patients was carried out. All patients underwent an ultrasound (US) every 6 months and the last pre-LT US was compared with explanted liver findings. A false-negative case was considered when incidentally found HCC (iHCC) was detected, whereas a false-positive case was considered when HCC diagnosed before LT (cHCC) was not confirmed in the explanted liver. US performance was assessed after excluding cHCC patients referred to transplant evaluation. Results Of 643 patients, 129 had HCC, of whom 92 had cHCC (71.3%) and 37 had iHCC (28.7%). Five patients (5.4%) had nonconfirmed cHCC (n=3 regenerative nodules, n=1 biliary hamartoma, and n=1 cholangiocarcinoma). Patients with iHCC had a higher MELD score (23±10 vs. 15±10; P<0.0001), and were more frequently Child–Pugh C (62.2 vs. 36.6%; P=0.006) compared with patients with cHCC. The number of US performed during waiting list was 1.7±1.6 (median 1.0). During transplant waiting list, the sensitivity and specificity of US were 33 and 99%, with positive and negative predictive values of 0.89 and 0.93, respectively. Multivariate analysis showed that the strongest variable related to iHCC finding was pre-LT Child–Pugh C status (OR 3.5; P=0.004). Conclusion Screening for liver cancer remains an important issue during transplant waiting list. However, the US screening method should be reviewed particularly for Child–Pugh C patients.

Neurological events after liver transplantation: a single-center experience

The aim of this study was to identify potential risk factors linked to neurologic events (NE) occurring after liver transplantation (LT) and use them to construct a model to predict such events. From odds ratios (OR) of risk factors, a scoring system was assessed using multivariate regression analysis. Forty‐one of 307 LT patients presented NE (13.3%), with prolonged hospital stay and decreased post‐LT survival. On multivariate analysis, factors associated with NE included: severe pre‐LT ascites OR 3.9 (1.80–8.41; P = 0.001), delta sodium ≥12 mEq/l OR 3.5 (1.36–8.67; P = 0.01), and post‐LT hypomagnesemia OR 2.9 (1.37–5.98; P = 0.005). Points were assigned depending on ORs as follows: ascites 4 points, and hypomagnesemia and delta sodium ≥12 mEq/l, 3 points each (score range = 0–10 points). ROC curve analysis suggested good discriminative power for the model, with a c‐statistic of 0.72 (CI 0.62–0.81; P < 0.0001), best performance for a cutoff value >3 points (71% sensitivity, 60% specificity). NE risk increased progressively from 6.4%, to 10.3%, 12.8%, 31.5% and 71.0% as scores rose from 0 to 3, 4, 6–7 and 10 cumulative points, respectively. The score described helps to identify patients potentially at risk for neurologic events, and its prevention would decrease morbidity and mortality after LT.

Identifying patients at higher risk of hepatocellular carcinoma recurrence after liver transplantation in a multicenter cohort study from Argentina.

Background and aim The Up-to-7 criteria on the basis of the explanted liver features categorize patients at higher risk of hepatocellular carcinoma (HCC) recurrence after liver transplantation (LT). The aim of this study was to propose a novel pretransplant scoring system to predict recurrence including pre-LT data. Patients and methods From 763 consecutive adult patients who underwent transplantation in four LT centers from Argentina, 124 patients with HCC were included. A scoring system was developed in 87 patients from pre-LT risk factors for recurrence as determined by hazard ratios (HRs) from a multivariate Cox regression analysis. Results Overall survival and recurrence rates at 5 years were 63.3 and 13.7%, respectively, during a follow-up period of 3.5±2.2 years. Variables associated with HCC recurrence on multivariate analysis were &agr;-fetoprotein more than 100 ng/ml (HR=5.6, P=0.001) and tumor beyond Up-to-7 imaging criteria (HR=6.3, P=0.001). Bootstrap validation showed that overfitting was negligible. Scoring points were assigned as follows (0–2 points): pre-LT &agr;-fetoprotein more than 100 ng/ml (presence=1 point, absence=0 point), and tumor beyond Up-to-7 imaging criteria (presence=1 point, absence=0 point). AUROC curve indicated a c-statistic of 0.74 (0.58–0.88, P=0.003). Two distinct subgroups of patients were identified with a cut-off more than or equal to 1 point (62% sensitivity and 82% specificity): low risk (0 point) and high risk (1–2 points). The 5-year recurrence rate was 9.4 and 44.5% (P=0.0001) and the 5-year overall survival was 78.1 and 34.8% (P=0.0001) in the low-risk and high-risk groups, respectively. Conclusion This scoring model may be a useful additional tool for HCC recurrence risk stratification before LT. Prospective studies are needed to evaluate our model.

Predicting early discharge from hospital after liver transplantation (ERDALT) at a single center: a new model.

BACKGROUND & RATIONALE Limited information related to Liver Transplantation (LT) costs in South America exists. Additionally, costs analysis from developed countries may not provide comparable models for those in emerging economies. We sought to evaluate a predictive model of Early Discharge from Hospital after LT (ERDALT = length of hospital stay ≤ 8 days). A predictive model was assessed based on the odds ratios (OR) from a multivariate regression analysis in a cohort of consecutively transplanted adult patients in a single center from Argentina and internally validated with bootstrapping technique. RESULTS ERDALT was applicable in 34 of 289 patients (11.8%). Variables independently associated with ERDALT were MELD exception points OR 1.9 (P = 0.04), surgery time < 4 h OR 3.8 (P = 0.013), < 5 units of blood products consumption (BPC) OR 3.5 (P = 0.001) and early weaning from mechanical intubation OR 6.3 (P = 0.006). Points in the predictive scoring model were allocated as follows: MELD exception points (absence = 0 points, presence = 1 point), surgery time < 4 h (0-2 points), < 5 units of BPC (0-2 points), and early weaning (0-3 points). Final scores ranged from 0 to 8 points with a c-statistic of 0.83 (95% CI 0.77-0.90; P < 0.0001). Transplant costs were significantly lower in patients with ERDALT (median