Molecular Characterization of Perineural Invasion in Pancreatic Ductal Adenocarcinoma: Proteomic Analysis and In Vitro Modelling.

PhDPancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and the 5th most common cause of cancer death in the UK. One of the peculiarities of this malignancy is its ability to invade nerves, a process called perineural invasion (PNI). PNI is found in almost 100% of PDAC, and is associated with poor prognosis, tumour recurrence and generation of pain. However, the molecular bases of PNI remain largely unknown. We investigated the molecular alterations underlying the neuro-epithelial interactions in PNI using one and two dimensional liquid chromatography – mass spectrometry (1D and 2D LC-MS) of laser microdissected PNI and non-PNI cancer from formalin fixed, paraffin embedded PDAC tissues. We also performed 1D LC-MS analysis of invaded and non-invaded nerves from the same cases. In addition, we developed an in vitro model of PNI using a co-culture system comprising PC12 cells, a rat pheochromocytoma cell line, as the neuronal element and PDAC cell lines. The overall proteomic profiles of PNI and non-PNI cancer appeared largely similar; of very few deregulated proteins, we have validated the up-regulation of antiapoptotic protein Olfactomedin 4 in PNI cancer using immunohistochemistry. In contrast, nerve samples demonstrated widespread molecular alterations characteristic of neuronal plasticity upon invasion by cancer cells. Immunohistochemistry confirmed the up-regulation of VGF in nerves from PDAC and chronic pancreatitis (CP) specimens compared to normal pancreas, as well as in invaded compared to non-invaded nerves in PDAC tissues. Furthermore, VGF expression strongly correlated with pain in CP; similar analysis in PDAC cases is still pending. Using the in vitro co-culture model, several PDAC cell lines were able to induce PC12 cells neuronal plasticity including survival, neurite extension as well as VGF expression, recapitulating thus the changes observed in human tissues. PDAC-induced PC12 plasticity was not mediated via NGF, a neurotrophin acting upstream of VGF and thought to be involved in the neuro-epithelial interactions. The induction of VGF expression was shown not to be necessary for PC12 cell survival, however, it contributed to the neurite extension induced by PDAC cell lines. In summary, based on proteomics analysis and in vitro modelling, we show the complex and intricate involvement and crosstalk of both tumoral and neural elements that are activated during perineural invasion in pancreatic cance

Development and Validation of a Comprehensive Model to Estimate Early Allograft Failure Among Patients Requiring Early Liver Retransplant

Importance: Expansion of donor acceptance criteria for liver transplant increased the risk for early allograft failure (EAF), and although EAF prediction is pivotal to optimize transplant outcomes, there is no consensus on specific EAF indicators or timing to evaluate EAF. Recently, the Liver Graft Assessment Following Transplantation (L-GrAFT) algorithm, based on aspartate transaminase, bilirubin, platelet, and international normalized ratio kinetics, was developed from a single-center database gathered from 2002 to 2015. Objective: To develop and validate a simplified comprehensive model estimating at day 10 after liver transplant the EAF risk at day 90 (the Early Allograft Failure Simplified Estimation [EASE] score) and, secondarily, to identify early those patients with unsustainable EAF risk who are suitable for retransplant. Design, setting, and participants: This multicenter cohort study was designed to develop a score capturing a continuum from normal graft function to nonfunction after transplant. Both parenchymal and vascular factors, which provide an indication to list for retransplant, were included among the EAF determinants. The L-GrAFT kinetic approach was adopted and modified with fewer data entries and novel variables. The population included 1609 patients in Italy for the derivation set and 538 patients in the UK for the validation set; all were patients who underwent transplant in 2016 and 2017. Main outcomes and measures: Early allograft failure was defined as graft failure (codified by retransplant or death) for any reason within 90 days after transplant. Results: At day 90 after transplant, the incidence of EAF was 110 of 1609 patients (6.8%) in the derivation set and 41 of 538 patients (7.6%) in the external validation set. Median (interquartile range) ages were 57 (51-62) years in the derivation data set and 56 (49-62) years in the validation data set. The EASE score was developed through 17 entries derived from 8 variables, including the Model for End-stage Liver Disease score, blood transfusion, early thrombosis of hepatic vessels, and kinetic parameters of transaminases, platelet count, and bilirubin. Donor parameters (age, donation after cardiac death, and machine perfusion) were not associated with EAF risk. Results were adjusted for transplant center volume. In receiver operating characteristic curve analyses, the EASE score outperformed L-GrAFT, Model for Early Allograft Function, Early Allograft Dysfunction, Eurotransplant Donor Risk Index, donor age 7 Model for End-stage Liver Disease, and Donor Risk Index scores, estimating day 90 EAF in 87% (95% CI, 83%-91%) of cases in both the derivation data set and the internal validation data set. Patients could be stratified in 5 classes, with those in the highest class exhibiting unsustainable EAF risk. Conclusions and relevance: This study found that the developed EASE score reliably estimated EAF risk. Knowledge of contributing factors may help clinicians to mitigate risk factors and guide them through the challenging clinical decision to allocate patients to early liver retransplant. The EASE score may be used in translational research across transplant centers