RNAscape: geometric mapping and customizable visualization of RNA structure.

Analyzing and visualizing the tertiary structure and complex interactions of RNA is essential for being able to mechanistically decipher their molecular functions in vivo. Secondary structure visualization software can portray many aspects of RNA; however, these layouts are often unable to preserve topological correspondence since they do not consider tertiary interactions between different regions of an RNA molecule. Likewise, quaternary interactions between two or more interacting RNA molecules are not considered in secondary structure visualization tools. The RNAscape webserver produces visualizations that can preserve topological correspondence while remaining both visually intuitive and structurally insightful. RNAscape achieves this by designing a mathematical structural mapping algorithm which prioritizes the helical segments, reflecting their tertiary organization. Non-helical segments are mapped in a way that minimizes structural clutter. RNAscape runs a plotting script that is designed to generate publication-quality images. RNAscape natively supports non-standard nucleotides, multiple base-pairing annotation styles and requires no programming experience. RNAscape can also be used to analyze RNA/DNA hybrid structures and DNA topologies, including G-quadruplexes. Users can upload their own three-dimensional structures or enter a Protein Data Bank (PDB) ID of an existing structure. The RNAscape webserver allows users to customize visualizations through various settings as desired. URL: https://rnascape.usc.edu/.

Imaging Delay Following Liver-Directed Therapy Increases Progression Risk in Early- to Intermediate-Stage Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related deaths in the world. Patients with early-stage HCC are treated with liver-directed therapies to bridge or downstage for liver transplantation (LT). In this study, the impact of HCC care delay on HCC progression among early-stage patients was investigated. Early-stage HCC patients undergoing their first cycle of liver-directed therapy (LDT) for bridge/downstaging to LT between 04/2016 and 04/2022 were retrospectively analyzed. Baseline variables were analyzed for risk of disease progression and time to progression (TTP). HCC care delay was determined by the number of rescheduled appointments related to HCC care. The study cohort consisted of 316 patients who received first-cycle LDT. The HCC care no-show rate was associated with TTP (p = 0.004), while the overall no-show rate was not (p = 0.242). The HCC care no-show rate and HCC care delay were further expanded as no-show rates and rescheduled appointments for imaging, laboratory, and office visits, respectively. More than 60% of patients experienced HCC care delay for imaging and laboratory appointments compared to just 8% for office visits. Multivariate analysis revealed that HCC-specific no-show rates and HCC care delay for imaging (p < 0.001) were both independently associated with TTP, highlighting the importance of minimizing delays in early-stage HCC imaging surveillance to reduce disease progression risk.

Pediatric Patients Discharged After Transfer to a Pediatric Emergency Department: Opportunities for Telehealth?

STUDY OBJECTIVE: Interemergency department pediatric transfers can be costly, involve risk, and may be disruptive to patients and families. Telehealth could be a way to safely reduce the number of transfers. We made an estimate of the proportion of transfers of pediatric patients to our emergency department (ED) that may have been avoidable using telehealth. METHODS: This was a retrospective analysis of electronic health record data of all pediatric patients (younger than 19 years) who were transferred to a single urban, academic medical center pediatric emergency department (PED) (annual pediatric volume approximately 15,000) between June 1, 2016, and December 29, 2021. We defined transfers as potentially avoidable with telehealth (the primary outcome) when the encounter at the receiving ED resulted in ED discharge and 1) met our definition of low-resource intensity (had no laboratory tests, diagnostic imaging, procedures, or consultations) or 2) could have used initial ED resources with telehealth guidance. RESULTS: Among 4,446 PED patients received in transfer during the study period, 406 (9%) were low-resource intensity. Of the non-low-resource intensity encounters, as many as another 1,103 (24.8%) potentially could have been avoided depending on available telehealth and initial ED resources, ranging from 210 (4.7%) with only telehealth specialty consultation to 538 (7.4%) with imaging and telehealth specialty consultation, and up to 1,034 (23.3%) with laboratory, imaging, and telehealth specialty consultation. CONCLUSION: Our results suggest that depending on available telehealth and initial ED resources, between 9% and 33% of pediatric inter-ED transfers may have been avoidable. This information may guide health system design and PED operations when considering implementing pediatric telehealth.

Lineage-specific regulation of PD-1 expression in early-stage hepatocellular carcinoma following 90yttrium transarterial radioembolization - Implications in treatment outcomes.

BACKGROUND: Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related deaths in the world. Liver-directed therapies, including 90Yttrium (90Y) radioembolization, play an integral role in the management of HCC with excellent response rates. This has led to clinical trials of immunotherapy in combination with 90Y. Elevated PD-1 expression and lymphopenia were recently shown as risk factors for disease progression in early-stage HCC treated with liver-directed therapies. The aim of this study was to investigate PD-1 expression dynamics in bridge/downstage to transplant in HCC patients receiving first-cycle 90Y and evaluate the impact of these changes on response rates and time-to-progression (TTP). METHODS: Patients with HCC receiving first-cycle 90Y as a bridge to liver transplantation (n = 99) were prospectively enrolled. Blood specimens were collected before 90Y and again during routine imagining follow-up to analyze PD-1 expression via flow cytometry. Complete and objective response rates (CR and ORR) were determined using mRECIST. RESULTS: In 84/88 patients with available follow-up imaging, 83% had a localized ORR with 63% having localized CR. For overall response, 71% and 54% experienced ORR and CR, respectively. Post-90Y PD-1 upregulation in CD8 + associated with HCC progression and decreased TTP. Treatment with 90Y was associated with an anticipated significant post-treatment drop in lymphocytes (P < 0.001) that was independent of PD-1 expression for either CD4+ or CD8+ T cells (P = 0.751 and P = 0.375) and not associated with TTP risk. The change in lymphocytes was not correlated with PD-1 expression following treatment nor TTP. CONCLUSIONS: Elevated PD-1 expression on peripheral T cells is associated with increased risk of HCC progression and shorter time to progression in bridging/downstaging to transplant HCC patients undergoing first-cycle 90Y. Treatment-induced lymphopenia was not associated with treatment response, or increased progression risk, suggesting this anticipated adverse event does not impact short-term HCC outcomes.

A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis.

Introduction: Extracellular vesicles could serve as a non-invasive biomarker for early cancer detection. However, limited methods to quantitate cancer-derived vesicles in the native state remain a significant barrier to clinical translation. Aim: This research aims to develop a rapid, one-step immunoaffinity approach to quantify HCC exosomes directly from a small serum volume. Methods: HCC-derived exosomes in the serum were captured using fluorescent phycoerythrin (PE)-conjugated antibodies targeted to GPC3 and alpha-fetoprotein (AFP). Total and HCC-specific exosomes were then quantified in culture supernatant or patient-derived serums using fluorescence nanoparticle tracking analysis (F-NTA). The performance of HCC exosome quantification in the serum was compared with the tumor size determined by MRI. Results: Initially we tested the detection limits of the F-NTA using synthetic fluorescent and non-fluorescent beads. The assay showed an acceptable sensitivity with a detection range of 104-108 particles/mL. Additionally, the combination of immunocapture followed by size-exclusion column purification allows the isolation of smaller-size EVs and quantification by F-NTA. Our assay demonstrated that HCC cell culture releases a significantly higher quantity of GPC3 or GPC3+AFP positive EVs (100-200 particles/cell) compared to non-HCC culture (10-40 particles/cell) (p<0.01 and p<0.05 respectively). The F-NTA enables absolute counting of HCC-specific exosomes in the clinical samples with preserved biological immunoreactivity. The performance of F-NTA was clinically validated in serum from patients ± cirrhosis and with confirmed HCC. F-NTA quantification data show selective enrichment of AFP and GPC3 positive EVs in HCC serum compared to malignancy-free cirrhosis (AUC values for GPC3, AFP, and GPC3/AFP were found 0.79, 0.71, and 0.72 respectively). The MRI-confirmed patient cohort indicated that there was a positive correlation between total tumor size and GPC3-positive exosome concentration (r:0.78 and p<0.001). Conclusion: We developed an immunocapture assay that can be used for simultaneous isolation and quantification of HCC-derived exosomes from a small serum volume with high accuracy.

The number of hepatocellular carcinoma foci as predictor of poor response to tumor-directed therapies in patients awaiting liver transplantation: a prospective cohort study.

BACKGROUND AND AIMS: Tumor-directed therapies (TDTs) are a constitutive part of hepatocellular carcinoma (HCC) treatment in patients awaiting liver transplantation (LT). While most patients benefit from TDTs as a bridge to LT, some patients drop out from the waiting list due to tumor progression. The study aimed to determine the risk factors for poor treatment outcome following TDTs among patients with HCC awaiting LT. METHODS: A total of 123 patients with HCC were evaluated with 92 patients meeting Milan Criteria enrolled in the prospective cohort study. Tumor response was evaluated using the modified Response Evaluation Criteria for Solid Tumors for HCC 1 month after the procedure. The risk factors for progressive disease (PD) and dropout were evaluated. RESULTS: After TDT, 55 patients (59.8%) achieved complete or partial response (44.6% and 15.2% respectively), 17 patients (18.5%) had stable disease, and 20 patients (21.7%) were assessed as PD. Multivariate analysis revealed a significant and independent association between the number of HCC foci and PD ( P  = 0.03, OR = 2.68). There was no statistically significant association between treatment response and demographics, MELDNa score, pre-and post-treatment alpha-fetoprotein (AFP), cumulative tumor burden the largest tumor size, or TDT modality. PD was the major cause of dropout in our cohort. Pre-treatment AFP levels ≥200 ng/ml had a strong association with dropout after TDTs ( P  = 0.0005). CONCLUSION: This study demonstrated the presence of multifocal HCC is the sole prognostic factor for PD following TDTs in HCC patients awaiting LT. We recommend prioritizing patients with multifocal HCC within Milan criteria by exception points for LT to improve the dropout rate.

Normothermic Machine Perfusion of Donor Livers for Transplantation in the United States: A Randomized Controlled Trial.

OBJECTIVE: To compare conventional low-temperature storage of transplant donor livers [static cold storage (SCS)] with storage of the organs at physiological body temperature [normothermic machine perfusion (NMP)]. BACKGROUND: The high success rate of liver transplantation is constrained by the shortage of transplantable organs (eg, waiting list mortality >20% in many centers). NMP maintains the liver in a functioning state to improve preservation quality and enable testing of the organ before transplantation. This is of greatest potential value with organs from brain-dead donor organs (DBD) with risk factors (age and comorbidities), and those from donors declared dead by cardiovascular criteria (donation after circulatory death). METHODS: Three hundred eighty-three donor organs were randomized by 15 US liver transplant centers to undergo NMP (n = 192) or SCS (n = 191). Two hundred sixty-six donor livers proceeded to transplantation (NMP: n = 136; SCS: n = 130). The primary endpoint of the study was "early allograft dysfunction" (EAD), a marker of early posttransplant liver injury and function. RESULTS: The difference in the incidence of EAD did not achieve significance, with 20.6% (NMP) versus 23.7% (SCS). Using exploratory, "as-treated" rather than "intent-to-treat," subgroup analyses, there was a greater effect size in donation after circulatory death donor livers (22.8% NMP vs 44.6% SCS) and in organs in the highest risk quartile by donor risk (19.2% NMP vs 33.3% SCS). The incidence of acute cardiovascular decompensation at organ reperfusion, "postreperfusion syndrome," as a secondary outcome was reduced in the NMP arm (5.9% vs 14.6%). CONCLUSIONS: NMP did not lower EAD, perhaps related to the inclusion of lower-risk liver donors, as higher-risk donor livers seemed to benefit more. The technology is safe in standard organ recovery and seems to have the greatest benefit for marginal donors.

Timing of Transport of Patients with COVID-19.

RATIONALE: The objective of this study was to evaluate the risk of mortality or ECMO cannulation for patients with confirmed or suspected COVID-19 transferred from sending hospitals to receiving tertiary care centers as a function of the duration of time at the sending hospital. OBJECTIVE: To determine outcomes of critically ill patients with COVID-19 who were transferred to tertiary or quarternary care medical centers. MATERIALS AND METHODS: Retrospective cohort study of critical care transports of patients to one of seven consortium tertiary care centers from March 1, 2020, through September 4, 2020. Age 14 years and older with confirmed or suspected COVID-19 transported from a sending hospital to a receiving tertiary care center by the critical care transport organization. RESULTS: Patients transported with confirmed or suspected COVID-19 to tertiary care centers had a mortality rate of 38.0%. Neither the number of days admitted, nor the number of days intubated at the sending hospital correlated with mortality (correlation coefficient 0.051 and -0.007, respectively). Similarly, neither the number of days admitted, nor number of days intubated at the sending hospital correlated with ECMO cannulation (correlation coefficient 0.008 and -0.036, respectively). CONCLUSION: It may be reasonable to transfer a critically ill COVID-19 patient to a tertiary care center even if they have been admitted at the sending hospital for several days.

Parental Mental Health and Childhood Respiratory Outcomes in a Severe Bronchiolitis Cohort.

Parental mental health conditions appear to contribute to the development of childhood respiratory illness. We investigated the relationship between parental mental health and childhood respiratory illness using data from a 17-center prospective cohort study of infants hospitalized with bronchiolitis between 2011 and 2014 (n = 921). Among 779 (84.6%) participants with self-reported parental mental health history data, 184 (23.6%) had parental history of depression and 155 (19.9%) had anxiety. Multivariable analyses found that both parental history of depression (hazard ratio [HR] 1.41, 95% confidence interval [CI] 1.01-1.99) and anxiety (HR 1.28, 95% CI 1.08-1.52) were associated with an increased risk of recurrent wheezing by age 3 years. However, only parental history of anxiety was associated with asthma by age 5 years (odds ratio 1.79, 95% CI 1.25-2.55). Further research on exposure severity, other early life stressors, and effective methods of parental psychosocial support is needed to develop targeted risk factor prevention strategies to reduce the burden of childhood respiratory illness.

Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles.

Background and Aim: HCC development in liver cirrhosis is associated with impaired autophagy leading to increased production of extracellular vesicles (EVs) including exosomes and microvesicles. The goal of the study is to determine which of these particles is primarily involved in releasing of HCC-specific biomarker glypican-3 (GPC3) when autophagy is impaired. Methods: Streptavidin-coated magnetic beads were coupled with either biotinylated CD63 or Annexin A1 antibodies. Coupled beads were incubated with EVs isolated from either HCC culture or serum. EVs captured by immuno-magnetic beads were then stained with FITC or PE fluorescent-conjugated antibodies targeting exosomes (CD81), and microvesicles (ARF6). The percentage of GPC3 enrichment in the microvesicles and exosomes was quantified by flow cytometry. The impact of autophagy modulation on GPC3 enrichment in exosomes and microvesicles was assessed by treating cells with Torin 1 and Bafilomycin A1. For clinical validation, GPC3 content was quantified in microvesicles, and exosomes were isolated from the serum of patients with a recent HCC diagnosis. Results: The immune-magnetic bead assay distinguishes membrane-derived microvesicles from endosome-derived exosomes. The GPC3 expression was only seen in the CD63 beads group but not in the Annexin A1 beads group, confirming that in HCC, GPC3 is preferentially released through exosomes. Furthermore, we found that autophagy induction by Torin1 decreased GPC3-positive exosome secretion and decreased microvesicle release. Conversely, autophagy inhibition by Bafilomycin A1 increased the secretion of GPC3-positive exosomes. Serum analysis showed CD81+ve EVs were detected in exosomes and ARF6+ve vesicles were detected in microvesicles, suggesting that immunoaffinity assay is specific. The exosomal GPC3 enrichment was confirmed in isolated EVs from the serum of patients with HCC. The frequency of GPC3-positive exosomes was higher in patients with HCC (12.4%) compared to exosomes isolated from non-cirrhotic and healthy controls (3.7% and 1.3% respectively, p<0.001). Conclusion: Our results show that GPC3 is enriched in the endolysosomal compartment and released in exosome fractions when autophagy is impaired.

Distinct Gene Expression Profiles in Viable Hepatocellular Carcinoma Treated With Liver-Directed Therapy.

Background: Hepatocellular carcinoma is a heterogeneous tumor that accumulates a mutational burden and dysregulated signaling pathways that differ from early to advanced stages. Liver transplant candidates with early-stage hepatocellular carcinoma (HCC) undergo liver-directed therapy (LDT) to delay disease progression and serve as a bridge to liver transplantation (LT). Unfortunately, >80% of LDT-treated patients have viable HCC in the explant liver, dramatically increasing recurrence risk. Understanding the effect of LDT on early-stage HCC could help identify therapeutic targets to promote complete pathologic necrosis and improve recurrence-free survival. In this study, transcriptomic data from viable HCC in LDT-treated bridged to transplant patients were investigated to understand how treatment may affect tumor signaling pathways. Methods: Multiplex transcriptomic gene analysis was performed with mRNA extracted from viable tumors of HCC patients bridged to transplant using LDT. The NanoString nCounter® Tumor Signaling 360 panel was used that contained 780 genes from 48 pathways involved in tumor biology within the microenvironment as well as antitumoral immune responses. Results: Hierarchical clustering separated tumors into three subtypes (HCC-1, HCC-2, and HCC-3) each with distinct differences in anti-tumoral signaling and immune infiltration within the tumor microenvironment. Immune infiltration (neutrophils, T cells, and macrophages) were all lowest in subtype HCC-3. The tumor inflammatory signature consisting of 18 genes associated with PD-1/PD-L1 inhibition, antigen presentation, chemokine secretion, and adaptive immune responses was highest in subtype HCC-1 and lowest in HCC-3. History of decompensation and etiology were associated with HCC subtype favoring downregulations in inflammation and immune infiltration with upregulation of lipid metabolism. Gene expression among intrahepatic lesions was remarkably similar with >85% of genes expressed in both lesions. Genes differentially expressed (<8 genes per patient) in multifocal disease were all upregulated in LDT-treated tumors from pathways involving epithelial mesenchymal transition, extracellular matrix remodeling, and/or inflammation potentially implicating intrahepatic metastases. Conclusion: Incomplete response to LDT may drive expression patterns that inhibit an effective anti-tumoral response through immune exclusion and induce intrahepatic spread.

SLAMF1 is expressed and secreted by hepatocytes and the liver in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disease in the United States and worldwide. Nonalcoholic steatohepatitis (NASH), the most advanced form of NAFLD, is characterized by hepatic steatosis associated with inflammation and hepatocyte death. No treatments are currently available for NASH other than lifestyle changes, and the disease lacks specific biomarkers. The signaling lymphocytic activation molecule family 1 (SLAMF1) protein is a self-ligand receptor that plays a role in orchestrating an immune response to some pathogens and cancers. We found that livers from humans and mice with NASH showed a more prominent immunohistochemistry staining for SLAMF1 than non-NASH controls. Furthermore, SLAMF1 levels are significantly increased in NASH plasma samples from mice and humans compared with their respective controls. In mice, the levels of SLAMF1 correlated significantly with the severity of the NASH phenotype. To test whether SLAMF 1 is expressed by hepatocytes, HepG2 cells and primary murine hepatocytes were treated with palmitic acid (PA) to induce a state of lipotoxicity mimicking NASH. We found that PA treatments of HepG2 cells and primary hepatocytes lead to significant increases in SLAMF1 levels. The downregulation of SLAMF1 in HepG2 cells improved the cell viability and reduced cytotoxicity. The in vivo data using mouse and human NASH samples suggests a potential role for this protein as a noninvasive biomarker for NASH. The in vitro data suggest a role for SLAMF1 as a potential therapeutic target to prevent hepatocyte death in response to lipotoxicity.NEW & NOTEWORTHY This study identified for the first time SLAMF1 as a mediator of hepatocyte death in nonalcoholic fatty liver disease (NASH) and as a marker of NASH in humans. There are no pharmacological treatments available for NASH, and diagnostic tools are limited to invasive liver biopsies. Therefore, since SLAMF1 levels correlate with disease progression and SLAMF1 mediates cytotoxic effects, this protein can be used as a therapeutic target and a clinical biomarker of NASH.

COVID-19 and transplantation-Data censoring.

During the early wave of the COVID-19 pandemic, the Scientific Registry of Transplant Recipients (SRTR) designated a "black out" period between March 12, 2020, and June 12, 2020, for transplant outcomes reporting. We discuss the implications and potential bias it has introduced as it may selectively favor the outcomes for certain regions and harm other regions due to varied effects of different waves of COVID-19 infections across the United States.

Hypoalbuminemia Is a Hepatocellular Carcinoma Independent Risk Factor for Tumor Progression in Low-Risk Bridge to Transplant Candidates.

Due to active hepatocellular carcinoma (HCC) surveillance, many patients are diagnosed with early-stage disease and are usually amendable to curative treatments. These patients lack poor prognostic factors associated with Milan Criteria and alpha fetoprotein (AFP) biomarker levels. There are currently limited strategies to assess prognosis in the patients who remain at risk of post-treatment HCC progression. In a cohort of liver transplant (LT) candidates with HCC, this study seeks to identify factors prior to liver-directed therapy (LDT) associated with time to progression (TTP). This is a retrospective analysis of prospectively collected data from LT candidates with recently diagnosed HCC and receiving LDT as a bridge to LT at three interventional oncology programs within a single system (n = 373). Demographics, clinical hepatology and serology, and factors related to HCC burden were extracted and analyzed for associations with TTP risk. Albumin level below the cohort median (3.4 g/dL) emerged as an independent risk factor for TTP controlling for AFP > 20 ng/mL as well as Milan, T-stage, and Barcelona Clinic Liver Cancer (BCLC) stage individually. In modality-specific subgroup survival analysis, albumin-based TTP stratification was restricted to patients receiving first cycle microwave ablation (p = 0.007). In n = 162 patients matching all low-risk criteria for Milan, T-stage, BCLC stage, and AFP, the effect of albumin < 3.4 g/dL remained significant for TTP (p = 0.004) with 2-year TTP rates of 68% (<3.4 g/dL) compared to 95% (≥3.4 g/dL). In optimal bridge to LT candidates with small HCC and low AFP biomarker levels, albumin level at treatment baseline provides an HCC-independent positive prognostic factor for risk of HCC progression prior to LT.

PD-1 expression in hepatocellular carcinoma predicts liver-directed therapy response and bridge-to-transplant survival.

BACKGROUND: Hepatocellular carcinoma (HCC) patients undergo liver-directed therapy (LDT) to control tumor burden while awaiting liver transplantation with response impacting waitlist survival. In this study, we investigate the link between absolute lymphocyte count (ALC) and PD-1 expression with response to LDT and bridge-to-transplant survival. METHODS: Treatment-naïve HCC patients (n = 86) undergoing LDT were enrolled at a single center from August 2016-March 2020. Response to LDT was determined using mRECIST. Blood samples were collected on the day of LDT and at follow-up. Cells were analyzed for phenotype by flow cytometry. Outcomes were liver transplantation or tumor progression. RESULTS: Incomplete response to initial LDT was associated with tumor progression precluding liver transplantation (OR: 7.6, 1.7 - 33.3, P < 0.001). Univariate analysis of baseline T cell phenotypes revealed ALC (OR: 0.44, 0.24-0.85, P = 0.009) as well as intermediate expression of PD-1 on CD4 (OR: 3.3, 1.03-10.3, P = 0.034) and CD8 T cells (OR: 3.0, 0.99-8.8 P = 0.043) associated with incomplete response to LDT. Elevations in PD-1 expression were associated with increased risk of bridge-to-transplant tumor progression (HR: 3.2, 1.2-9.4). In patients successfully bridged to liver transplantation, pre-treatment peripheral PD-1 profile was associated with advanced tumor staging (P < 0.005) with 2/4 of patients with elevations in PD-1 having T3-T4 TNM staging compared to 0 with low PD-1 expression. CONCLUSION: Low lymphocyte count or elevated expression of the PD-1 checkpoint inhibitor is associated with incomplete response to LDT and increased risk of bridge-to-transplant tumor progression. Patients with impaired T cell homeostasis may benefit from PD-1 immunotherapy to improve response to LDT and improve bridge-to-transplant outcomes.

Experimental Validation of Novel Glypican 3 Exosomes for the Detection of Hepatocellular Carcinoma in Liver Cirrhosis.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) developing in the context of preexisting cirrhosis is characterized by impaired autophagy that results in increased exosome release. This study was conducted to determine whether circulating exosomes expressing glypican 3 (GPC3) could be utilized as a biomarker for HCC detection and treatment response in patients with cirrhosis. METHODS: Immunohistochemistry was performed to assess p62 and GPC3 expression in the lesion and adjacent tissue from cirrhosis with HCC. GPC3-enriched exosomes were captured by an enzyme-linked immunosorbent assay (ELISA). The diagnostic specificity of serum exosome-derived GPC3 (eGPC3) was determined using samples obtained from malignancy-free controls, malignancy-free cirrhotics, cirrhotics with confirmed HCC, and patients with a non-HCC malignancy. The performance of eGPC3 was validated using serum samples of HCC patients received chemotherapy. RESULTS: We found that the expression of p62 and GPC3 was significantly increased in HCC tissues compared to adjacent cirrhotic liver. Impaired autophagy and exosome shedding were confirmed in HCC cell lines. Mass spectroscopic analysis revealed that GPC3 was enriched in exosomes isolated from HCC cell lines. An affinity ELISA assay was developed that specifically captures GPC3 positive exosomes in the serum. Total exosome concentration and eGPC3 were significantly elevated in cirrhotic patients with HCC as compared to the reference control groups. Furthermore, decreases in post-treatment exosome concentration and eGPC3 levels were more closely correlated with response to locoregional chemotherapy compared to change in serum AFP in HCC patients awaiting liver transplantation. CONCLUSION: We developed an affinity exosome capture assay to detect GPC3 enriched exosomes. Our preliminary assessment shows that GPC3 positive exosomes can be used for HCC detection and prediction of treatment outcomes.

Baseline Alpha-Fetoprotein, Alpha-Fetoprotein-L3, and Des-Gamma-Carboxy Prothrombin Biomarker Status in Bridge to Liver Transplant Outcomes for Hepatocellular Carcinoma.

The biomarkers α-fetoprotein (AFP), Lens culinaris agglutinin-reactive AFP fraction (AFP-L3), and des-γ-carboxy prothrombin (DCP) have emerging implications in hepatocellular carcinoma (HCC) surveillance, overall prognosis, and post-surgical recurrence risk. This retrospective study investigated treatment and bridge to liver transplant (LT) prognosis associated with AFP, AFP-L3%, and DCP biomarker profiles prior to liver-directed therapy (LDT). In a 140-patient cohort, each biomarker was associated with HCC progression risk using the established thresholds of AFP > 20 ng/mL, AFP-L3 > 15%, and DCP > 7.5 ng/mL. Over 60% of the cohort expressed at least one biomarker at baseline. Although most biomarker-positive patients expressed the clinical standard AFP (57/87), only 32% were positive for AFP alone. Biomarker accumulation increased HCC progression risk but was not associated with demographic factors or preserved liver function. Biomarker triple negative patients had smaller index HCC (p = 0.003), decreased multifocal burden (p = 0.010), and a higher objective response rate (ORR, 62% compared to 46%, p = 0.011). Expressing all three biomarkers at baseline was associated with dismal first-line ORR (12%) with a median time to progression (TTP) of only 181 days post-LDT. Patients with triple negative status for the HCC biomarkers AFP, AFP-L3%, and DCP have the highest first-line ORR with < 5% HCC progression 1-year post-LDT. Biomarker profiling can establish baseline prognosis for identifying optimal bridge to LT and downstaging to LT candidates with triple negative biomarker status and providing an ideal post-LDT target as a compliment to radiographic response.

Critical Care Transport of Patients With COVID-19.

PURPOSE: Critical care transport is associated with a high rate of adverse events, and the risks and outcomes of transporting critically ill patients during the COVID-19 pandemic have not been previously described. MATERIALS AND METHODS: We performed a retrospective review of transports of subjects with suspected or confirmed COVID-19 from sending hospitals to tertiary care hospitals in Boston. Follow-up data were obtained for patients transported between March 1st and April 20th, 2020. RESULTS: Of 254 charts identified, 250 patients were transported. Nine patients (3.5%) had cardiac arrest prior to transport. Twenty-nine (11.6%) had hypotension, 22 (8.8%) had a critical desaturation, and 4 (1.6%) had both en route. Hospital follow-up data were available for 189 patients. Of those intubated during their hospitalization, 44 (25.0%) had died, 59 (33.5%) had been extubated, and 13 (17.6%) had been discharged alive. For the subgroup with prior cardiac arrest, follow-up data available for 6. Of these 6, 2 died and 4 (66.7%) have been discharged alive. CONCLUSIONS: Few patients with COVID-19 had an adverse event in transport. The in-hospital mortality rate was 25%, with a 33.5% extubation rate. Patients resuscitated from cardiac arrest prior to transport had a 66.7% discharge rate among those transported to consortium hospitals.

Hepatitis C-associated focal proliferative glomerulonephritis in an aviremic recipient of a hepatitis C-positive antibody donor liver.

Shortage of organs for liver transplantation (LT) and the availability of highly efficient pan-genotypic direct-acting antivirals (DAAs) against hepatitis C virus (HCV) have allowed the use of livers from HCV-positive antibody/negative nucleic acid test donors (dHCV Ab+/NAT-) into aviremic HCV recipients over the last few years. We report the case of a patient who received an LT from an HCV Ab+/NAT- donor and, after HCV viremic conversion, developed a nephrotic syndrome due to a focal proliferative glomerulonephritis early after LT. Patient's renal function and proteinuria resolved after successful treatment with DAAs. Renal and hepatic function remain normal over 24 months of follow-up. This case restates the success of LT using livers from dHCV Ab+/NAT- in aviremic recipients in the context of DAAs while illustrating the risk for potential complications associated with the HCV transmission and reinforcing the importance of early initiation of anti-HCV therapy.