Quantification and Characterization of CTCs and Clusters in Pancreatic Cancer by Means of the Hough Transform Algorithm.

Circulating Tumor Cells (CTCs) are considered a prognostic marker in pancreatic cancer. In this study we present a new approach for counting CTCs and CTC clusters in patients with pancreatic cancer using the IsofluxTM System with the Hough transform algorithm (Hough-IsofluxTM). The Hough-IsofluxTM approach is based on the counting of an array of pixels with a nucleus and cytokeratin expression excluding the CD45 signal. Total CTCs including free and CTC clusters were evaluated in healthy donor samples mixed with pancreatic cancer cells (PCCs) and in samples from patients with pancreatic ductal adenocarcinoma (PDAC). The IsofluxTM System with manual counting was used in a blinded manner by three technicians who used Manual-IsofluxTM as a reference. The accuracy of the Hough-IsofluxTM approach for detecting PCC based on counted events was 91.00% [84.50, 93.50] with a PCC recovery rate of 80.75 ± 16.41%. A high correlation between the Hough-IsofluxTM and Manual-IsofluxTM was observed for both free CTCs and for clusters in experimental PCC (R2 = 0.993 and R2 = 0.902 respectively). However, the correlation rate was better for free CTCs than for clusters in PDAC patient samples (R2 = 0.974 and R2 = 0.790 respectively). In conclusion, the Hough-IsofluxTM approach showed high accuracy for the detection of circulating pancreatic cancer cells. A better correlation rate was observed between Hough-IsofluxTM approach and with the Manual-IsofluxTM for isolated CTCs than for clusters in PDAC patient samples.

Pancreatobiliary maljunction: association with gallbladder cancer.

BACKGROUND: Pancreatobiliary maljunction is a rare disease characterized by the junction of the pancreatic and biliary ducts outside of the duodenal wall, which normally results in a large common duct. As a result, there is a greater risk of acute pancreatitis and cancer of the gallbladder and biliary tract. CASE REPORT: We present the case of a 43-year-old female diagnosed with a pancreatobiliary maljunction and an associated stenosis of the bile duct, secondary to an episode of acute pancreatitis. She underwent several endoscopic retrograde cholangiopancreatography procedures over the course of three years, without improvement of the stenosis, and therefore a surgical approach was taken. Prior to the surgical intervention, magnetic resonance imaging showed the presence of an 11-mm polyp in the gallbladder. A histological study of the surgical sample identified intramucosal adenocarcinoma over a tubular adenoma of the gallbladder. DISCUSSION: Pancreatobiliary maljunction can be considered as a premalignant entity due to the risk of developing cancer of the biliary tree and gallbladder. Therefore, these patients should undergo a prophylactic intervention, despite being asymptomatic.

Hepatocellular carcinoma in patients without advanced fibrosis after HCV eradication antiviral treatment.

From the last few years, hepatitis C virus and the new direct antiviral treatments are being more and more important. In consequence, case studies like the one we present, the appearance of hepatocelullar carcinoma after its eradication with fibrosis grade 2, are getting special interest. In 2007, our patient was treated with pegylated interferon α-2a and ribavirin, having a sustained virological response after it. In this way, liver fibrosis grade 2 was confirmed by a biopsy. Finally, after corroborating a good liver functioning, the patient was discharged (as, according to the guidebooks, an ultrasound scan of screeing every 6 months was not required). In 2014, the patient came to hospital because of a pain at right hypochondrium and he was diagnosed with hepatocelullar carcinoma. A hepatectomy was done objectifying the surgical piece, liver fibrosis grade 2, one more time. Subsequently, a tumour relapse through an abdominal CT scan, a tumour relapse was found and despite the Sorafenib treatment, the patient died on January 2015. This case study provokes curiosity and uncertainty about the attitude which should be taken respect to the monitoring, and hepatocelullar carcinoma screening overall, in patients with a sustained virological response after eradicator treatment and without advanced fibrosis. Nowadays, with the benefits of the new treatments, the amount of patients in this situation is increasing significantly.

Post-transplant lymphoproliferative disease in liver transplant recipients.

INTRODUCTION: Post-transplant lymphoproliferative syndrome (PTLD) is a rare and potentially life-threatening complication after liver transplantation. The aim of this study was to analyze the clinicopathologic features related to PTLD in a single institution after liver transplantation. METHODS: Observational study where we have retrospectively analyzed 851 cases who underwent liver transplantation. Ten cases have developed PTLD. Their clinical-pathological characteristics and the treatment received have been analyzed. RESULTS: PTLD incidence was 1.2% (10/851). The mean time from liver transplantation to PTLD diagnosis was 36 months (range 1.2 to 144 months). PTLD localization was extranodal in all cases, the most frequent location being intestinal. Seven cases showed a monomorphic lymphoma which in all cases was differentiated B cell lymphomas. Fifty per cent of the series were seropositive for Epstein-Barr virus. Five patients were alive at the time of the review. Among these patients, we observed three cases of complete remission and two cases of disease stabilization. The death rate was higher in the first year after diagnosis of PTLD. CONCLUSION: PTLD is a rare complication after liver transplantation, but it may pose a threat to the life of a liver transplant recipient. It is essential to identify patients at risk, to establish an early diagnosis and treatment that can change the outcome of the disease.

Cryopreserved nanostructured fibrin-agarose hydrogels are efficient and safe hemostatic agents.

Uncontrolled bleeding during surgery is associated with high mortality and prolonged hospital stay, necessitating the use of hemostatic agents. Fibrin sealant patches offer an efficient solution to achieve hemostasis and improve patient outcomes in liver resection surgery. We have previously demonstrated the efficacy of a nanostructured fibrin-agarose hydrogel (NFAH). However, for the widespread distribution and commercialization of the product, it is necessary to develop an optimal preservation method that allows for prolonged stability and facilitates storage and distribution. We investigated cryopreservation as a potential method for preserving NFAH using trehalose. Structural changes in cryopreserved NFAH (Cryo-NFAH) were investigated and comparative in vitro and in vivo efficacy and safety studies were performed with freshly prepared NFAH. We also examined the long-term safety of Cryo-NFAH versus TachoSil in a rat partial hepatectomy model, including time to hemostasis, intra-abdominal adhesion, hepatic hematoma, inflammatory factors, histopathological variables, temperature and body weight, hemocompatibility and cytotoxicity. Structural analyses demonstrated that Cryo-NFAH retained most of its macro- and microscopic properties after cryopreservation. Likewise, hemostatic efficacy assays showed no significant differences with fresh NFAH. Safety evaluations indicated that Cryo-NFAH had a similar overall profile to TachoSil up to 40 days post-surgery in rats. In addition, Cryo-NFAH demonstrated superior hemostatic efficacy compared with TachoSil while also demonstrating lower levels of erythrolysis and cytotoxicity than both TachoSil and other commercially available hemostatic agents. These results indicate that Cryo-NFAH is highly effective hemostatic patch with a favorable safety and tolerability profile, supporting its potential for clinical use.

GEMA-Na and MELD 3.0 severity scores to address sex disparities for accessing liver transplantation: a nationwide retrospective cohort study.

Background: The Gender-Equity Model for liver Allocation corrected by serum sodium (GEMA-Na) and the Model for End-stage Liver Disease 3.0 (MELD 3.0) could amend sex disparities for accessing liver transplantation (LT). We aimed to assess these inequities in Spain and to compare the performance of GEMA-Na and MELD 3.0. Methods: Nationwide cohort study including adult patients listed for a first elective LT (January 2016-December 2021). The primary outcome was mortality or delisting for sickness within the first 90 days. Independent predictors of the primary outcome were evaluated using multivariate Cox's regression with adjusted relative risks (RR) and 95% confidence intervals (95% CI). The discrimination of GEMA-Na and MELD 3.0was assessed using Harrell c-statistics (Hc). Findings: The study included 6071 patients (4697 men and 1374 women). Mortality or delisting for clinical deterioration occurred in 286 patients at 90 days (4.7%). Women had reduced access to LT (83.7% vs. 85.9%; p = 0.037) and increased risk of mortality or delisting for sickness at 90 days (adjusted RR = 1.57 [95% CI 1.09-2.28]; p = 0.017). Female sex remained as an independent risk factor when using MELD or MELD-Na but lost its significance in the presence of GEMA-Na or MELD 3.0. Among patients included for reasons other than tumours (n = 3606; 59.4%), GEMA-Na had Hc = 0.753 (95% CI 0.715-0.792), which was higher than MELD 3.0 (Hc = 0.726 [95% CI 0.686-0.767; p = 0.001), showing both models adequate calibration. Interpretation: GEMA-Na and MELD 3.0 might correct sex disparities for accessing LT, but GEMA-Na provides more accurate predictions of waiting list outcomes and could be considered the standard of care for waiting list prioritization. Funding: Instituto de Salud Carlos III, Agencia Estatal de Investigación (Spain), and European Union.

TARTESSUS: A Customized Electrospun Drug Delivery System Loaded with Irinotecan for Local and Sustained Chemotherapy Release in Pancreatic Cancer.

Post-surgical chemotherapy in pancreatic cancer has notorious side effects due to the high dose required. Multiple devices have been designed to tackle this aspect and achieve a delayed drug release. This study aimed to explore the controlled and sustained local delivery of a reduced drug dose from an irinotecan-loaded electrospun nanofiber membrane (named TARTESSUS) that can be placed on the patients' tissue after tumor resection surgery. The drug delivery system formulation was made of polycaprolactone (PCL). The mechanical properties and the release kinetics of the drug were adjusted by the electrospinning parameters and by the polymer ratio between 10 w.t.% and 14 w.t.% of PCL in formic acid:acetic acid:chloroform (47.5:47.5:5). The irinotecan release analysis was performed and three different release periods were obtained, depending on the concentration of the polymer in the dissolution. The TARTESSUS device was tested in 2D and 3D cell cultures and it demonstrated a decrease in cell viability in 2D culture between 72 h and day 7 from the start of treatment. In 3D culture, a decrease in viability was seen between 72 h, day 7 (p < 0.001), day 10 (p < 0.001), 14 (p < 0.001), and day 17 (p = 0.003) as well as a decrease in proliferation between 72 h and day 10 (p = 0.030) and a reduction in spheroid size during days 10 (p = 0.001), 14 (p < 0.001), and 17 (p < 0.001). In conclusion, TARTESSUS showed a successful encapsulation of a chemotherapeutic drug and a sustained and delayed release with an adjustable releasing period to optimize the therapeutic effect in pancreatic cancer treatment.

Development of a liver graft assessment expert machine-learning system: when the artificial intelligence helps liver transplant surgeons.

Background: The complex process of liver graft assessment is one point for improvement in liver transplantation. The main objective of this study is to develop a tool that supports the surgeon who is responsible for liver donation in the decision-making process whether to accept a graft or not using the initial variables available to it. Material and method: Liver graft samples candidate for liver transplantation after donor brain death were studied. All of them were evaluated "in situ" for transplantation, and those discarded after the "in situ" evaluation were considered as no transplantable liver grafts, while those grafts transplanted after "in situ" evaluation were considered as transplantable liver grafts. First, a single-center, retrospective and cohort study identifying the risk factors associated with the no transplantable group was performed. Then, a prediction model decision support system based on machine learning, and using a tree ensemble boosting classifier that is capable of helping to decide whether to accept or decline a donor liver graft, was developed. Results: A total of 350 liver grafts that were evaluated for liver transplantation were studied. Steatosis was the most frequent reason for classifying grafts as no transplantable, and the main risk factors identified in the univariant study were age, dyslipidemia, personal medical history, personal surgical history, bilirubinemia, and the result of previous liver ultrasound (p < 0.05). When studying the developed model, we observe that the best performance reordering in terms of accuracy corresponds to 76.29% with an area under the curve of 0.79. Furthermore, the model provides a classification together with a confidence index of reliability, for most cases in our data, with the probability of success in the prediction being above 0.85. Conclusion: The tool presented in this study obtains a high accuracy in predicting whether a liver graft will be transplanted or deemed non-transplantable based on the initial variables assigned to it. The inherent capacity for improvement in the system causes the rate of correct predictions to increase as new data are entered. Therefore, we believe it is a tool that can help optimize the graft pool for liver transplantation.

Impact of a Donor Age >75 Years on the Survival of Liver Transplant Recipients.

BACKGROUND: An organ shortage is the reason why it is necessary to expand the pool of donors, which can be achieved by using elderly donors. The main goal of this study is to analyze the outcomes of liver transplant (LT) when it is performed with donors older than 75 years. METHODS: We carried out a retrospective case-control study (N = 212) that included LTs with donors older than 75 years (group A, n = 106 cases) that were performed in our center between the years 2010 and 2020. This cohort has been paired off with a similar control group (group B, n = 106) whose donors were significantly younger. A survival analysis using the Kaplan-Meier model was performed. RESULTS: Average (SD) age of donors in group A was statistically greater than group B (A, 79.1 [3.0] years vs B, 54.4 [15.3], P < .001). There were no differences either in the average age of the recipients or in the Model for End-Stage Liver Disease score of both groups. Indications for LT were distributed equally in both groups: the most common was cellular hepatocarcinoma followed by alcohol-related cirrhosis. Survival rates for group A were 81%, 78%, and 67%, in 1, 3, and 5 years, respectively, while in group B they were 85%, 76%, and 71%, respectively, without differences found between the groups (P = .57). CONCLUSIONS: Using elderly liver donors is safe, achieving good outcomes in terms of short- and midterm rates of survival.

PDA-Based Glyconanomicelles for Hepatocellular Carcinoma Cells Active Targeting Via Mannose and Asialoglycoprotein Receptors.

Hepatocellular carcinoma (HCC) is the sixth most common neoplasia and the fourth most common cause of cancer-related mortality worldwide. Sorafenib is the first-line molecular therapy for patients in an advanced stage of HCC. However, the recommended clinical dose of Sorafenib is associated with several complications, which derive from its lack of cell specificity and its very low water solubility. To circumvent these drawbacks, in the present study we developed two sugar-coated polydiacetylene-based nanomicelles-Sorafenib carriers targeting mannose and asialoglycoprotein receptors (MR and ASGPR, respectively). The strategies allowed the inducement of apoptosis and reduction of cell proliferation at a nanomolar, instead of micromolar, range in liver cancer cells. The study showed that, contrary to literature data, Sorafenib included into the pMicMan (Man = mannose) vector (targeting MR) is more efficient than pMicGal (Gal = galactose) (targeting ASGPR). Indeed, pMicMan increased the endosomal incorporation with an increased intracellular Sorafenib concentration that induced apoptosis and reduced cell proliferation at a low concentration range (10-20 nM).

Circulating Tumor Cells Enumeration from the Portal Vein for Risk Stratification in Early Pancreatic Cancer Patients.

BACKGROUND: Effective biomarkers are needed to enable personalized medicine for pancreatic cancer patients. This study analyzes the prognostic value, in early pancreatic cancer, of single circulating tumor cell (CTC) and CTC clusters from the central venous catheter (CVC) and portal blood (PV). METHODS: In total, 7 mL of PV and CVC blood from 35 patients with early pancreatic cancer were analyzed. CTC were isolated using a positive immunomagnetic selection. The detection and identification of CTC were performed by immunocytochemistry (ICC) and were analyzed by Epi-fluorescence and confocal microscopy. RESULTS: CTC and the clusters were detected both in PV and CVC. In both samples, the CTC number per cluster was higher in patients with grade three or poorly differentiated tumors (G3) than in patients with well (G1) or moderately (G2) differentiated. Patients with fewer than 185 CTC in PV exhibited a longer OS than patients with more than 185 CTC (24.5 vs. 10.0 months; p = 0.018). Similarly, patients with fewer than 15 clusters in PV showed a longer OS than patients with more than 15 clusters (19 vs. 10 months; p = 0.004). These significant correlations were not observed in CVC analyses. CONCLUSIONS: CTC presence in PV could be an important prognostic factor to predict poor prognosis in early pancreatic cancer. In addition, the number of clustered-CTC correlate to a tumor negative differentiation degree and, therefore, could be used as a diagnostic biomarker for pancreatic cancer.

Risk Factors for No Valid Liver Graft. Multivariate Study Based on the Variables Included in the Donation Protocol of the National Trasplant Organisation. / Factores de riesgo para injertos hepáticos no válidos. Estudio multivariante a partir de las variables recogidas en el protocolo de donación de la Organización Nacional de Trasplantes.

INTRODUCTION: Among the strategies designed to optimize the number of existing liver grafts for transplantation, the implementation of the graft assessment process is one of the least explored. The main objective is to identify the risk factors presented by liver donors for «NO validity¼. Secondly, we analyzed the coincidence between the surgeon's assessment and that of the anatomo-pathologist in the invalid donors. MATERIAL AND METHOD: Retrospective study conducted from a prospective database that analyzes 190 liver donors, 95 valid and 95 NOT valid. The variables of each of them corresponding to the donation protocol of the National Transplant Organization are studied. Through a multivariate study we determine the independent risk factors of NO validity. We checked the causes of NO validity argued with the histopathological findings of these grafts. RESULTS: The independent risk factors of non-validity in the multivariate study (P < .05) were: dyslipidemia, personal medical history other than cardiovascular and abdominal surgical risk factors, GGT, BrT, and the result of previous liver ultrasound. The 3 most frequent causes of NO validity were: steatosis, fibrosis and macroscopic appearance of the organ. 78% of the biopsies confirmed the NO validity of the graft (in 57.9% of the cases the histological findings coincided with those described by the surgeon). The 22.1% of the biopsies hadnt pathological findings. CONCLUSIONS: The determination of the risk factors of NO validity will contribute to the design of future assessment scores that are useful tools in the process of liver graft assessment.).

Body Mass Index as a Prognostic Factor in Liver Transplantation.

OBJECTIVE: Obesity is one of the main growing epidemics of the last century and is responsible for many deaths worldwide. The aim of this study is to analyze the impact of the body mass index (BMI) of the recipient on survival and morbidity after liver transplantation (LT). MATERIAL AND METHODS: We conducted a retrospective cohort study of all transplanted recipients in a third-level hospital between 2006 and 2018. The following donor variables were analyzed: age, sex, weight, BMI, cause of death. Additionally, the following recipient variables were analyzed: age, sex, weight, height, BMI, procedure indication. Finally, outcome variables were analyzed: postoperative complications, early mortality, graft loss, and overall survival. This study strictly complies with the Helsinki Congress and the Istanbul Declaration regarding donor source. RESULTS: We analyzed 825 of 837 LTs from January 2006 to December 2018. These were grouped by BMI categories: 271 (29%) normal, 322 (34.3%) overweight, and 228 (24.3%) obesity. The overall survival at 5 years was 83% in the normal group, 76% in the overweight group, and 71% in the obesity group. These differences were statistically significant (P = .027). The early mortality rate was 4.42% in the normal weight group, 6.5% in the overweight group, and 5.26% in the obesity group. No differences were found between groups in terms of postoperative complications: hemorrhagic, vascular, biliary, respiratory, hemodynamic, digestive, renal, neurologic, rebel ascites, and infections. No differences were found regarding the need for re-operation. CONCLUSIONS: In this study, overall survival in LT decreases as the BMI of recipient increases; but overweight and obesity do not constitute a risk factor for early morbidity and mortality in LT.

Nanostructured fibrin agarose hydrogel as a novel haemostatic agent.

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin-agarose hydrogel patch, with (c-NFAH) or without cells (a-NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post-operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a-NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a-NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a-NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures.

Incidence and survival rate of de novo tumors in liver transplants. / Incidencia y supervivencia de los tumores de novo en el trasplante hepático.

INTRODUCTION: The greater survival of transplanted patients is accompanied by an increase in the rate of de novo malignancies (NM), which are the most frequent late-onset complication. We can distinguish between non-melanoma skin cancers (NMSC), post-transplant lymphoproliferative disorders (PTLD) and solid organ cancers (SOC). Our objective is to determine the incidence of the different types of NM, the time elapsed until diagnosis and survival rates in our setting. METHODS: We conducted a retrospective study of 1071 liver transplant patients from 1990 to 2015 at our center. We analyzed the demographic variables, incidence of NM and survival. RESULTS: 184 NM developed in 1071 transplant patients (17%), specifically 19% of the males and 13% of the females (P=.004). The most frequent NM were NMSC (29%), lung (18%), head and neck (16%), PTLD (10%) and gastrointestinal (8%). The median time of diagnosis was 7.9 years in NMSC, 3.9 years in PTLD and 9.8 years in SOC. Patients with NMSC had significantly better survival than those with PTLD or SOC. The incidence of de novo tumors (excluding NMSC) was 1889/100,000 transplants/year. By gender, lung cancer was the most common TOS in men and breast cancer in women. CONCLUSION: In our setting, excluding NMSC, the incidence is 8.8 times greater than estimations for the general population, with a high rate of lung cancer, so we should implement preventive and diagnostic strategies.

Fibrinogen-thrombin collagen patch reinforcement of high-risk colonic anastomoses in rats.

AIM: To evaluate the effectiveness of human fibrinogen-thrombin collagen patch (TachoSil®) in the reinforcement of high-risk colon anastomoses. METHODS: A quasi-experimental study was conducted in Wistar rats (n = 56) that all underwent high-risk anastomoses (anastomosis with only two sutures) after colectomies. The rats were divided into two randomized groups: Control group (24 rats) and treatment group (24 rats). In the treatment group, high-risk anastomosis was reinforced with TachoSil® (a piece of TachoSil® was applied over this high-risk anastomosis, covering the gap). Leak incidence, overall survival, intra-abdominal adhesions, and histologic healing of anastomoses were analyzed. Survivors were divided into two subgroups and euthanized at 15 and 30 d after intervention in order to analyze the adhesions and histologic changes. RESULTS: Overall survival was 71.4% and 57.14% in the TachoSil® group and control group, respectively (P = 0.29); four rats died from other causes and six rats in the treatment group and 10 in the control group experienced colonic leakage (P > 0.05). The intra-abdominal adhesion score was similar in both groups, with no differences between subgroups. We found non-significant differences in the healing process according to the histologic score used in both groups (P = 0.066). CONCLUSION: In our study, the use of TachoSil® was associated with a non-statistically significant reduction in the rate of leakage in high-risk anastomoses. TachoSil® has been shown to be a safe product because it does not affect the histologic healing process or increase intra-abdominal adhesions.