Comparing anti-tumor and anti-self immunity in a patient with melanoma receiving immune checkpoint blockade.

BACKGROUND: Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. METHODS: Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. RESULTS: While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression (CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1), including those in the COX-2/PGE2 pathway (IL1B, PTGER1/EP1 and PTGER4/EP4). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. CONCLUSIONS: This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.

Multi-Institutional Study of Pathologist Reading of the Programmed Cell Death Ligand-1 Combined Positive Score Immunohistochemistry Assay for Gastric or Gastroesophageal Junction Cancer.

The assessment of the expression of programmed cell death ligand-1 (PD-L1) using immunohistochemistry (IHC) has been controversial since its introduction. The methods of assessment and the range of assays and platforms contribute to confusion. Perhaps the most challenging aspect of PD-L1 IHC is the combined positive score (CPS) method of interpretation of IHC results. Although the CPS method is prescribed for more indications than any other PD-L1 scoring system, its reproducibility has never been rigorously assessed. In this study, we collected a series of 108 gastric or gastroesophageal junction cancer cases, stained them using the Food and Drug Administration-approved 22C3 assay, scanned them, and then circulated them to 14 pathologists at 13 institutions for the assessment of interpretative concordance for the CPS system. We found that higher cut points (10 or 20) performed better than a CPS of <1 or >1. We used the Observers Needed to Evaluate Subjective Tests algorithm to assess how the CPS system might perform in the real-world setting and found that the cut points of <1 or >1 showed an overall percent agreement of only 30% among the pathologist raters, with a plateau occurring at 8 raters. The raters performed better at higher cut points. However, the best cut point of <20 versus that of >20 was still disappointing, with a plateau at an overall percent agreement of 70% (at 7 raters). Although there is no ground truth for CPS, we compared the score with quantitative messenger RNA measurement and showed no relationship between the score (at any cut point) and messenger RNA amount. In summary, we showed that CPS shows high subjective variability among pathologist readers and is likely to perform poorly in the real-world setting. This system may be the root cause of the poor specificity and relatively low predictive value of IHC companion diagnostic tests for PD-1 axis therapies that use the CPS system.

High Interobserver Variability Among Pathologists Using Combined Positive Score to Evaluate PD-L1 Expression in Gastric, Gastroesophageal Junction, and Esophageal Adenocarcinoma.

Reliable, reproducible methods to interpret programmed death ligand-1 (PD-L1) expression on tumor cells (TC) and immune cells (IC) are needed for pathologists to inform decisions associated with checkpoint inhibitor therapies. Our international study compared interpathologist agreement of PD-L1 expression using the combined positive score (CPS) under standardized conditions on samples from patients with gastric/gastroesophageal junction/esophageal adenocarcinoma. Tissue sections from 100 adenocarcinoma pretreatment biopsies were stained in a single laboratory using the PD-L1 immunohistochemistry 28-8 and 22C3 (Agilent) pharmDx immunohistochemical assays. PD-L1 CPS was evaluated by 12 pathologists on scanned whole slide images of these biopsies before and after a 2-hour CPS training session by Agilent. Additionally, pathologists determined PD-L1-positive TC, IC, and total viable TC on a single tissue fragment from 35 of 100 biopsy samples. Scoring agreement among pathologists was assessed using the intraclass correlation coefficient (ICC). Interobserver variability for CPS for 100 biopsies was high, with only fair agreement among pathologists both pre- (range, 0.45-0.55) and posttraining (range, 0.56-0.57) for both assays. For the 35 single biopsy samples, poor/fair agreement was also observed for the total number of viable TC (ICC, 0.09), number of PD-L1-positive IC (ICC, 0.19), number of PD-L1-positive TC (ICC, 0.54), and calculated CPS (ICC, 0.14), whereas calculated TC score (positive TC/total TC) showed excellent agreement (ICC, 0.82). Retrospective histologic review of samples with the poorest interpathologist agreement revealed the following as possible confounding factors: (1) ambiguous identification of positively staining stromal cells, (2) faint or variable intensity of staining, (3) difficulty in distinguishing membranous from cytoplasmic tumor staining, and (4) cautery and crush artifacts. These results emphasize the need for objective techniques to standardize the interpretation of PD-L1 expression when using the CPS methodology on gastric/gastroesophageal junction cancer biopsies to accurately identify patients most likely to benefit from immune checkpoint inhibitor therapy.

Digital imaging software versus the "eyeball" method in quantifying steatosis in a liver biopsy.

Steatotic livers represent a potentially underutilized resource to increase the donor graft pool; however, 1 barrier to the increased utilization of such grafts is the heterogeneity in the definition and the measurement of macrovesicular steatosis (MaS). Digital imaging software (DIS) may better standardize definitions to study posttransplant outcomes. Using HALO, a DIS, we analyzed 63 liver biopsies, from 3 transplant centers, transplanted between 2016 and 2018, and compared macrovesicular steatosis percentage (%MaS) as estimated by transplant center, donor hospital, and DIS. We also quantified the relationship between DIS characteristics and posttransplant outcomes using log-linear regression for peak aspartate aminotransferase, peak alanine aminotransferase, and total bilirubin on postoperative day 7, as well as logistic regression for early allograft dysfunction. Transplant centers and donor hospitals overestimated %MaS compared with DIS, with better agreement at lower %MaS and less agreement for higher %MaS. No DIS analyzed liver biopsies were calculated to be >20% %MaS; however, 40% of liver biopsies read by transplant center pathologists were read to be >30%. Percent MaS read by HALO was positively associated with peak aspartate aminotransferase (regression coefficient= 1.04 1.08 1.12 , p <0.001), peak alanine aminotransferase (regression coefficient = 1.04 1.08 1.12 , p <0.001), and early allograft dysfunction (OR= 1.10 1.40 1.78 , p =0.006). There was no association between HALO %MaS and total bilirubin on postoperative day 7 (regression coefficient = 0.99 1.01 1.04 , p =0.3). DIS provides reproducible quantification of steatosis that could standardize MaS definitions and identify phenotypes associated with good clinical outcomes to increase the utilization of steatite livers.

Induction of innate lymphoid cell-derived interleukin-22 by the transcription factor STAT3 mediates protection against intestinal infection.

Inhibitors of the transcription factor STAT3 target STAT3-dependent tumorigenesis but patients often develop diarrhea from unknown mechanisms. Here we showed that STAT3 deficiency increased morbidity and mortality after Citrobacter rodentium infection with decreased secretion of cytokines including IL-17 and IL-22 associated with the transcription factor RORγt. Administration of the cytokine IL-22 was sufficient to rescue STAT3-deficient mice from lethal infection. Although STAT3 was required for IL-22 production in both innate and adaptive arms, by using conditional gene-deficient mice, we observed that STAT3 expression in RORγt(+) innate lymphoid cells (ILC3s), but not T cells, was essential for the protection. However, STAT3 was required for RORγt expression in T helper cells, but not in ILC3s. Activated STAT3 could directly bind to the Il22 locus. Thus, cancer therapies that utilize STAT3 inhibitors increase the risk for pathogen-mediated diarrhea through direct suppression of IL-22 from gut ILCs.

NCCN Guidelines® Insights: Biliary Tract Cancers, Version 2.2023.

In 2023, the NCCN Guidelines for Hepatobiliary Cancers were divided into 2 separate guidelines: Hepatocellular Carcinoma and Biliary Tract Cancers. The NCCN Guidelines for Biliary Tract Cancers provide recommendations for the evaluation and comprehensive care of patients with gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma. The multidisciplinary panel of experts meets at least on an annual basis to review requests from internal and external entities as well as to evaluate new data on current and emerging therapies. These Guidelines Insights focus on some of the recent updates to the NCCN Guidelines for Biliary Tract Cancers as well as the newly published section on principles of molecular testing.

Balancing Safety and Efficacy to Determine the Most Suitable Size of Imaging-Visible Embolic Microspheres for Bariatric Arterial Embolization in a Preclinical Model.

OBJECTIVES: To identify the most suitable size of imaging-visible embolic agents with balanced safety and efficacy for bariatric arterial embolization (BAE) in a preclinical model. MATERIALS AND METHODS: Twenty-seven pigs were divided into 3 cohorts. In Cohort I, 16 pigs were randomized to receive (n = 4 each) 40-100-µm microspheres in 1 or 2 fundal arteries, 70-340-µm radiopaque microspheres in 2 fundal arteries, or saline. In Cohort II, 3 pigs underwent renal arterial embolization with either custom-made 100-200-µm, 200-250-µm, 200-300-µm, or 300-400-µm radiopaque microspheres or Bead Block 300-500 µm with microsphere distribution assessed histologically. In Cohort III, 8 pigs underwent BAE in 2 fundal arteries with tailored 100-200-µm radiopaque microspheres (n = 5) or saline (n = 3). RESULTS: In Cohort I, no significant differences in weight or ghrelin expression were observed between BAE and control animals. Moderate-to-severe gastric ulcerations were noted in all BAE animals. In Cohort II, renal embolization with 100-200-µm microspheres occluded vessels with a mean diameter of 139 µm ± 31, which is within the lower range of actual diameters of Bead Block 300-500 µm. In Cohort III, BAE with 100-200-µm microspheres resulted in significantly lower weight gain (42.3% ± 5.7% vs 51.6% ± 2.9% at 8 weeks; P = .04), fundal ghrelin cell density (16.1 ± 6.7 vs 23.6 ± 12.6; P = .045), and plasma ghrelin levels (1,709 pg/mL ± 172 vs 4,343 pg/mL ± 1,555; P < .01) compared with controls and superficial gastric ulcers (5/5). CONCLUSIONS: In this preclinical model, tailored 100-200-µm microspheres were shown to be most suitable for BAE in terms of safety and efficacy.

ß-Catenin destruction complex-independent regulation of Hippo-YAP signaling by APC in intestinal tumorigenesis.

Mutations in Adenomatous polyposis coli (APC) underlie familial adenomatous polyposis (FAP), an inherited cancer syndrome characterized by the widespread development of colorectal polyps. APC is best known as a scaffold protein in the ß-catenin destruction complex, whose activity is antagonized by canonical Wnt signaling. Whether other effector pathways mediate APC's tumor suppressor function is less clear. Here we report that activation of YAP, the downstream effector of the Hippo signaling pathway, is a general hallmark of tubular adenomas from FAP patients. We show that APC functions as a scaffold protein that facilitates the Hippo kinase cascade by interacting with Sav1 and Lats1. Consistent with the molecular link between APC and the Hippo signaling pathway, genetic analysis reveals that YAP is absolutely required for the development of APC-deficient adenomas. These findings establish Hippo-YAP signaling as a critical effector pathway downstream from APC, independent from its involvement in the ß-catenin destruction complex.

Homeostatic control of Hippo signaling activity revealed by an endogenous activating mutation in YAP.

The Hippo signaling pathway converges on YAP to regulate growth, differentiation, and regeneration. Previous studies with overexpressed proteins have shown that YAP is phosphorylated by its upstream kinase, Lats1/2, on multiple sites, including an evolutionarily conserved 14-3-3-binding site whose phosphorylation is believed to inhibit YAP by excluding it from the nucleus. Indeed, nuclear localization of YAP or decreased YAP phosphorylation at this site (S168 in Drosophila, S127 in humans, and S112 in mice) is widely used in current literature as a surrogate of YAP activation even though the physiological importance of this phosphorylation event in regulating endogenous YAP activity has not been defined. Here we address this question by introducing a Yap(S112A) knock-in mutation in the endogenous Yap locus. The Yap(S112A) mice are surprisingly normal despite nuclear localization of the mutant YAP protein in vivo and profound defects in cytoplasmic translocation in vitro. Interestingly, the mutant Yap(S112A) mice show a compensatory decrease in YAP protein levels due to increased phosphorylation at a mammalian-specific phosphodegron site on YAP. These findings reveal a robust homeostatic mechanism that maintains physiological levels of YAP activity and caution against the assumptive use of YAP localization alone as a surrogate of YAP activity.

Erythropoietic effects of vadadustat in patients with anemia associated with chronic kidney disease.

Patients with chronic kidney disease (CKD) develop anemia largely because of inappropriately low erythropoietin (EPO) production and insufficient iron available to erythroid precursors. In four phase 3, randomized, open-label, clinical trials in dialysis-dependent and non-dialysis-dependent patients with CKD and anemia, the hypoxia-inducible factor prolyl hydroxylase inhibitor, vadadustat, was noninferior to the erythropoiesis-stimulating agent, darbepoetin alfa, in increasing and maintaining target hemoglobin concentrations. In these trials, vadadustat increased the concentrations of serum EPO, the numbers of circulating erythrocytes, and the numbers of circulating reticulocytes. Achieved hemoglobin concentrations were similar in patients treated with either vadadustat or darbepoetin alfa, but compared with patients receiving darbepoetin alfa, those receiving vadadustat had erythrocytes with increased mean corpuscular volume and mean corpuscular hemoglobin, while the red cell distribution width was decreased. Increased serum transferrin concentrations, as measured by total iron-binding capacity, combined with stable serum iron concentrations, resulted in decreased transferrin saturation in patients randomized to vadadustat compared with patients randomized to darbepoetin alfa. The decreases in transferrin saturation were associated with relatively greater declines in serum hepcidin and ferritin in patients receiving vadadustat compared with those receiving darbepoetin alfa. These results for serum transferrin saturation, hepcidin, ferritin, and erythrocyte indices were consistent with improved iron availability in the patients receiving vadadustat. Thus, overall, vadadustat had beneficial effects on three aspects of erythropoiesis in patients with anemia associated with CKD: increased endogenous EPO production, improved iron availability to erythroid cells, and increased reticulocytes in the circulation.

Identifying the Ideal Target Vessel Size for Bariatric Embolization: Histologic Analysis of Swine and Human Gastric Fundi.

This study aimed to identify the ideal arteriole size to target in bariatric embolization, with the goal of maximizing weight loss efficacy while maintaining patient safety. Although all published clinical trials of bariatric embolization have used embolic microspheres that were at least 300 µm in diameter, optimal weight loss outcomes have been achieved safely in swine using 50-µm embolics. Human fundal remnants from bariatric surgery were compared with swine fundal sections after bariatric embolization with 50-µm embolic microspheres to assess the ideal fundal vessel size for bariatric embolization. In swine, the 50-µm embolic microspheres deposited in the luminal half of the submucosa with a mean arteriole size of 49 µm ± 30. The mean arteriole diameter in the corresponding submucosal layer of the human gastric fundi was 40 µm ± 30. These measurements may inform future clinical trials and direct the development of embolic agents for bariatric embolization.

Nuclear PTEN and p53 suppress stress-induced liver cancer through distinct mechanisms.

PTEN and p53 are highly mutated in many cancers. These two tumor suppressors have critical functions in the nucleus, such as DNA repair, cell cycle progression, and genome maintenance. However, the in vivo functional relationship of nuclear PTEN and p53 is unknown. Here, we analyzed the liver of mice in which nuclear PTEN and p53 are individually or simultaneously depleted. We found that nuclear PTEN loss greatly upregulates p53 expression upon oxidative stress, while the loss of p53 potentiates stress-induced accumulation of PTEN in the nucleus. Next, we examined oxidative stress-induced DNA damage in hepatocytes, and found that nuclear PTEN loss aggravated the damage while p53 loss did not. Notably, mice lacking nuclear PTEN had increased hepatocellular carcinoma under oxidative stress, while mice lacking p53 in hepatocytes had accelerated hepatocellular carcinoma and intrahepatic cholangiocarcinoma. The formation of cholangiocarcinoma appears to involve the transformation of hepatocytes into cholangiocarcinoma. Simultaneous loss of nuclear PTEN and p53 exacerbated both types of liver cancers. These data suggest that nuclear PTEN and p53 suppress liver cancers through distinct mechanisms.

Hepatobiliary Cancers, Version 2.2021, NCCN Clinical Practice Guidelines in Oncology.

The NCCN Guidelines for Hepatobiliary Cancers focus on the screening, diagnosis, staging, treatment, and management of hepatocellular carcinoma (HCC), gallbladder cancer, and cancer of the bile ducts (intrahepatic and extrahepatic cholangiocarcinoma). Due to the multiple modalities that can be used to treat the disease and the complications that can arise from comorbid liver dysfunction, a multidisciplinary evaluation is essential for determining an optimal treatment strategy. A multidisciplinary team should include hepatologists, diagnostic radiologists, interventional radiologists, surgeons, medical oncologists, and pathologists with hepatobiliary cancer expertise. In addition to surgery, transplant, and intra-arterial therapies, there have been great advances in the systemic treatment of HCC. Until recently, sorafenib was the only systemic therapy option for patients with advanced HCC. In 2020, the combination of atezolizumab and bevacizumab became the first regimen to show superior survival to sorafenib, gaining it FDA approval as a new frontline standard regimen for unresectable or metastatic HCC. This article discusses the NCCN Guidelines recommendations for HCC.

Development, Practice Patterns, and Early Clinical Outcomes of a Multidisciplinary Liver Cancer Clinic.

Multidisciplinary care has been associated with improved survival in patients with primary liver cancers. We report the practice patterns and real world clinical outcomes for patients presenting to the Johns Hopkins Hospital (JHH) multidisciplinary liver clinic (MDLC). We analyzed hepatocellular carcinoma (HCC, n = 100) and biliary tract cancer (BTC, n = 76) patients evaluated at the JHH MDLC in 2019. We describe the conduct of the clinic, consensus decisions for patient management based on stage categories, and describe treatment approaches and outcomes based on these categories. We describe subclassification of BCLC stage C into 2 parts, and subclassification of cholangiocarcinoma into 4 stages. A treatment consensus was finalized on the day of MDLC for the majority of patients (89% in HCC, 87% in BTC), with high adherence to MDLC recommendations (91% in HCC, 100% in BTC). Among patients presenting for a second opinion regarding management, 28% of HCC and 31% of BTC patients were given new therapeutic recommendations. For HCC patients, at a median follow up of 11.7 months (0.7-19.4 months), median OS was not reached in BCLC A and B patients. In BTC patients, at a median follow up of 14.2 months (0.9-21.1 months) the median OS was not reached in patients with resectable or borderline resectable disease, and was 11.9 months in patients with unresectable or metastatic disease. Coordinated expert multidisciplinary care is feasible for primary liver cancers with high adherence to recommendations and a change in treatment for a sizeable minority of patients.

Phase 2 study of vismodegib, a hedgehog inhibitor, combined with gemcitabine and nab-paclitaxel in patients with untreated metastatic pancreatic adenocarcinoma.

BACKGROUND: The Hedgehog (Hh) signalling pathway is overexpressed in pancreatic ductal adenocarcinoma (PDA). Preclinical studies have shown that Hh inhibitors reduce pancreatic cancer stem cells (pCSC), stroma and Hh signalling. METHODS: Patients with previously untreated metastatic PDA were treated with gemcitabine and nab-paclitaxel. Vismodegib was added starting on the second cycle. The primary endpoint was progression-free survival (PFS) as compared with historical controls. Tumour biopsies to assess pCSC, stroma and Hh signalling were obtained before treatment and after cycle 1 (gemcitabine and nab-paclitaxel) or after cycle 2 (gemcitabine and nab-paclitaxel plus vismodegib). RESULTS: Seventy-one patients were enrolled. Median PFS and overall survival (OS) were 5.42 months (95% confidence interval [CI]: 4.37-6.97) and 9.79 months (95% CI: 7.85-10.97), respectively. Of the 67 patients evaluable for response, 27 (40%) had a response: 26 (38.8%) partial responses and 1 complete response. In the tumour samples, there were no significant changes in ALDH + pCSC following treatment. CONCLUSIONS: Adding vismodegib to chemotherapy did not improve efficacy as compared with historical rates observed with chemotherapy alone in patients with newly diagnosed metastatic pancreatic cancer. This study does not support the further evaluation of Hh inhibitors in this patient population. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01088815.

Axon Guidance Molecules Promote Perineural Invasion and Metastasis of Orthotopic Pancreatic Tumors in Mice.

BACKGROUND & AIMS: Little is known about mechanisms of perineural invasion (PNI) by pancreatic ductal adenocarcinomas (PDAs) or other tumors. Annexin A2 (ANXA2) regulates secretion of SEMA3D, an axon guidance molecule, which binds and activates the receptor PLXND1 to promote PDA invasion and metastasis. We investigated whether axon guidance molecules promote PNI and metastasis by PDA cells in mice. METHODS: We performed studies in a dorsal root ganglion (DRG) invasion system, wild-type C57BL/6 mice (controls), mice with peripheral sensory neuron-specific disruption of PlxnD1 (PLAC mice), LSL-KRASG12D/+;LSL-TP53R172H/+;PDX-1-CRE+/+ (KPC) mice, and KPC mice crossed with ANXA2-knockout mice (KPCA mice). PDA cells were isolated from KPC mice and DRG cells were isolated from control mice. Levels of SEMA3D or ANXA2 were knocked down in PDA cells with small hairpin and interfering RNAs and cells were analyzed by immunoblots in migration assays, with DRGs and with or without antibodies against PLXND1. PDA cells were injected into the pancreas of control and PLAC mice, growth of tumors was assessed, and tumor samples were analyzed by histology. DRG cells were incubated with SEMA3D and analyzed by live imaging. We measured levels of SEMA3D and PLXND1 in PDA specimens from patients with PNI and calculated distances between tumor cells and nerves. RESULTS: DRG cells increase the migration of PDC cells in invasion assays; knockdown of SEMA3D in PDA cells or antibody blockade of PLXND1 on DRG cells reduced this invasive activity. In mice, orthotopic tumors grown from PDA cells with knockdown of SEMA3D, and in PLAC mice, orthotopic tumors grown from PDA cells, had reduced innervation and formed fewer metastases than orthotopic tumors grown from PDA cells in control mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. CONCLUSIONS: DRG cells increase the migratory and invasive activities of pancreatic cancer cells, via secretion of SEMA3D by pancreatic cells and activation of PLXND1 on DRGs. Knockdown of SEMA3D and loss of neural PLXND1 reduces innervation of orthotopic PDAs and metastasis in mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. Strategies to disrupt the axon guidance pathway mediated by SEMA3D and PLXND1 might be developed to slow progression of PDA.

Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317.

The number of microspheres in Y90 radioembolization directly affects normal tissue radiation exposure.

PURPOSE: In Y90 radioembolization, the number of microspheres infused varies by more than a factor of 20 over the shelf-life of the glass radioembolization device. We investigated the effect of the number of Y90 microspheres on normal liver tissue. METHOD: Healthy pigs received lobar radioembolization with glass Y90 microspheres at 4, 8, 12, and 16 days post-calibration, representing a > 20× range in the number of microspheres deposited per milliliter in tissue. Animals were survived for 1-month post-treatment and the livers were explanted and scanned on a micro CT system to fully characterize the microscopic distribution of individual microspheres. A complete 3D microdosimetric evaluation of each liver was performed with a spatially correlated analysis of histopathologic effect. RESULTS: Through whole-lobe microscopic identification of each microsphere, a consistent number of microspheres per sphere cluster was found at 4, 8, and 12 days postcalibration, despite an 8-fold increase in total microspheres infused from days 4 to 12. The additional microspheres instead resulted in more clusters formed and, therefore, a more homogeneous microscopic absorbed dose. The increased absorbed-dose homogeneity resulted in a greater volume fraction of the liver receiving a potentially toxic absorbed dose based on radiobiologic models. Histopathologic findings in the animals support a possible increase in normal liver toxicity in later treatments with more spheres (i.e., ≥ day 12) compared to early treatments with less spheres (i.e., ≤ day 8). CONCLUSION: The microdosimetric evidence presented supports a recommendation of caution when treating large volumes (e.g., right lobe) using glass 90Y microspheres at more than 8 days post-calibration, i.e., after "2nd week" Monday. The favorable normal tissue microscopic distribution and associated low toxicity of first week therapies may encourage opportunities for dose escalation with glass microspheres and could also be considered for patients with decreased hepatic reserve.

Role of baseline volumetric functional MRI in predicting histopathologic grade and patients' survival in hepatocellular carcinoma.

OBJECTIVES: We aimed to evaluate the role of volumetric ADC (vADC) and volumetric venous enhancement (vVE) in predicting the grade of tumor differentiation in hepatocellular carcinoma (HCC). METHODS: The study population included 136 HCC patients (188 lesions) who had baseline MR imaging and histopathological report. Measurements of vVE and vADC were performed on baseline MRI. Tumors were histologically classified into low-grade and high-grade groups. The parameters between the two groups were compared using Mann-Whitney U and chi-square tests for continuous and categorical parameters, respectively. Area under receiver operating characteristic (AUROC) was calculated to investigate the accuracy of vADC and vVE. Logistic regression and multivariable Cox regression were used to unveil the potential parameters associated with high-grade HCC and patient's survival, respectively. RESULTS: Lesions with higher vADC values and a higher absolute vADC skewness were more likely to be high grade on histopathology assessment (p = 0.001 and p = 0.0291, respectively). Also, vVE showed a trend to be higher in low-grade lesions (p = 0.079). Adjusted multivariable model including vADC, vVE, and vADC skewness could strongly predict HCC degree of differentiation (AUROC = 83%). Additionally, a higher Child-Pugh score (HR = 2.39 [p = 0.02] for score 2 and HR = 3.47 [p = 0.001] for score 3), vADC skewness (HR = 1.52, p = 0.02; per increments in skewness), and tumor volume (HR = 1.1, p = 0.001; per 100 cm3 increments) showed the highest association with patients' survival. CONCLUSIONS: vADC and vVE have the potential to accurately predict HCC differentiation. Additionally, some imaging features in combination with patients' clinical characteristics can predict patient survival. KEY POINTS: • Volumetric functional MRI metrics can be considered as non-invasive measures for determining tumor histopathology in HCC. • Estimating patient survival based on clinical and imaging parameters can be used for modifying management approach and preventing unnecessary adverse events.

Bariatric Arterial Embolization with Calibrated Radiopaque Microspheres and an Antireflux Catheter Suppresses Weight Gain and Appetite-Stimulating Hormones in Swine.

PURPOSE: To examine safety and efficacy of bariatric arterial embolization (BAE) with x-ray-visible embolic microspheres (XEMs) and an antireflux catheter in swine. MATERIAL AND METHODS: BAE with selective infusion of XEMs (n = 6) or saline (n = 4, control) into gastric fundal arteries was performed under x-ray guidance. Weight and plasma hormone levels were measured at baseline and weekly for 4 weeks after embolization. Cone-beam CT images were acquired immediately after embolization and weekly for 4 weeks. Hormone-expressing cells in the stomach were assessed by immunohistochemical staining. RESULTS: BAE pigs lost weight 1 week after embolization followed by significantly impaired weight gain relative to control animals (14.3% vs 20.9% at 4 weeks, P = .03). Plasma ghrelin levels were significantly lower in BAE pigs than in control animals (1,221.6 pg/mL vs 1,706.2 pg/mL at 4 weeks, P < .01). XEMs were visible on x-ray and cone-beam CT during embolization, and radiopacity persisted over 4 weeks (165.5 HU at week 1 vs 158.5 HU at week 4, P = .9). Superficial mucosal ulcerations were noted in 1 of 6 BAE animals. Ghrelin-expressing cell counts were significantly lower in the gastric fundus (17.7 vs 36.8, P < .00001) and antrum (24.2 vs 46.3, P < .0001) of BAE pigs compared with control animals. Gastrin-expressing cell counts were markedly reduced in BAE pigs relative to control animals (98.5 vs 127.0, P < .02). Trichrome staining demonstrated significantly more fibrosis in BAE animals compared with control animals (13.8% vs 8.7%, P < .0001). CONCLUSIONS: XEMs enabled direct visualization of embolic material during and after embolization. BAE with XEMs and antireflux microcatheters was safe and effective.