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Background and Aims: Efficacy of chimeric antigen receptor (CAR) T cells for treating solid tumors, including HCC, remains a challenge. Nanobodies are emerging building blocks of CAR T cells due to their small size and high expression. Membrane proximal sites have been shown as attractive epitopes of CAR T cells. However, current CAR formats are not tailored toward nanobodies or targeting membrane distal epitopes. Approach and Results: Using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced ß catenin levels in HCC cells. Conclusion: Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane proximal epitope, lead to robust T-cell signaling and induce swift and durable eradication of HCC tumors. [ABSTRACT FROM AUTHOR]

Suboptimal adherence to guidelines for hepatocellular carcinoma (HCC) surveillance among high‐risk patients is a persistent problem with substantial detriment to patient outcomes. While patients cite cost as a barrier to surveillance receipt, the financial burden they experience due to surveillance has not been examined. We conducted a retrospective administrative claims study to assess HCC surveillance use and associated costs in a US cohort of insured patients without cirrhosis but with hepatitis B virus (HBV) infection, monitored in routine clinical practice. Of 6831 patients (1122 on antiviral treatment, 5709 untreated), only 39.3% and 51.3% had received any abdominal imaging after 6 and 12 months, respectively, and patients were up to date with HCC surveillance guidelines for only 28% of the follow‐up time. Completion of surveillance was substantially higher at 6 and 12 months among treated patients (51.7% and 69.6%, respectively) compared with untreated patients (36.9% and 47.6%, respectively) (p < 0.001). In adjusted models, treated patients were more likely than untreated patients to receive surveillance (hazard ratio [HR] 1.75, 95% confidence interval [CI] 1.53–2.01, p < 0.001), and the proportion of those up to date with surveillance was 9.7% higher (95% CI 6.26–13.07, p < 0.001). Mean total and patient‐paid daily surveillance‐related costs ranged from $99 (ultrasound) to $334 (magnetic resonance imaging), and mean annual patient costs due to lost productivity for surveillance‐related outpatient visits ranged from $93 (using the federal minimum wage) to $321 (using the Bureau of Labor Statistics wage). Conclusion: Use of current HCC surveillance strategies was low across patients with HBV infection, and surveillance was associated with substantial patient financial burden. These data highlight an urgent need for accessible and easy‐to‐implement surveillance strategies with sufficient sensitivity and specificity for early HCC detection. [ABSTRACT FROM AUTHOR]

Prostate‐specific membrane antigen (PSMA) is a validated target for molecular diagnostics and targeted radionuclide therapy. Our purpose was to evaluate PSMA expression in hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatic adenoma (HCA); investigate the genetic pathways in HCC associated with PSMA expression; and evaluate HCC detection rate with 68Ga‐PSMA‐11 positron emission tomography (PET). In phase 1, PSMA immunohistochemistry (IHC) on HCC (n = 148), CCA (n = 111), and HCA (n = 78) was scored. In a subset (n = 30), messenger RNA (mRNA) data from the Cancer Genome Atlas HCC RNA sequencing were correlated with PSMA expression. In phase 2, 68Ga‐PSMA‐11 PET was prospectively performed in patients with treatment‐naïve HCC on a digital PET scanner using cyclotron‐produced 68Ga. Uptake was graded qualitatively and semi‐quantitatively using standard metrics. On IHC, PSMA expression was significantly higher in HCC compared with CCA and HCA (P < 0.0001); 91% of HCCs (n = 134) expressed PSMA, which principally localized to tumor‐associated neovasculature. Higher tumor grade was associated with PSMA expression (P = 0.012) but there was no association with tumor size (P = 0.14), fibrosis (P = 0.35), cirrhosis (P = 0.74), hepatitis B virus (P = 0.31), or hepatitis C virus (P = 0.15). Overall survival tended to be longer in patients without versus with PSMA expression (median overall survival: 4.2 vs. 1.9 years; P = 0.273). FGF14 (fibroblast growth factor 14) mRNA expression correlated positively (rho = 0.70; P = 1.70 × 10‐5) and MAD1L1 (Mitotic spindle assembly checkpoint protein MAD1) correlated negatively with PSMA expression (rho = −0.753; P = 1.58 × 10‐6). Of the 190 patients who met the eligibility criteria, 31 patients with 39 HCC lesions completed PET; 64% (n = 25) lesions had pronounced 68Ga‐PSMA‐11 standardized uptake value: SUVmax (median [range] 9.2 [4.9‐28.4]), SUVmean 4.7 (2.4‐12.7), and tumor‐to‐liver background ratio 2 (1.1‐11). Conclusion: Ex vivo expression of PSMA in neovasculature of HCC translates to marked tumor avidity on 68Ga‐PSMA‐11 PET, which suggests that PSMA has the potential as a theranostic target in patients with HCC. [ABSTRACT FROM AUTHOR]

Prognosis of hepatocellular carcinoma (HCC) could be affected by lack of or delayed therapy. We aimed to characterize the prevalence, correlates, and clinical impact of therapeutic underuse and delay in patients with HCC. Patients with HCC diagnosed between 2010 and 2017 were analyzed from the United States National Cancer Database. Logistic regression analysis identified factors associated with no and delayed (>90 days after diagnosis) HCC treatment. Cox proportional hazards regression with landmark analysis assessed the association between therapeutic delay and overall survival (OS), accounting for immortal time bias. Of 116,299 patients with HCC, 24.2% received no treatment and 18.4% of treated patients had delayed treatment. Older age, Black, Hispanic, lower socioeconomic status, earlier year of diagnosis, treatment at nonacademic centers, Northeast region, increased medical comorbidity, worse liver dysfunction, and higher tumor burden were associated with no treatment. Among treated patients, younger age, Hispanic, Black, treatment at academic centers, West region, earlier tumor stage, and receipt of noncurative treatment were associated with treatment delays. In multivariable Cox regression with a landmark of 150 days, patients with and without treatment delays had similar OS (adjusted hazard ratio [aHR], 1.01; 95% confidence interval [CI], 0.98‐1.04) with a median survival of 33.7 vs. 32.1 months, respectively. However, therapeutic delay was associated with worse OS in patients who had tumor, nodes, and metastases (TNM) stage 1 (aHR, 1.06; 95% CI, 1.01‐1.11) or received curative treatment (aHR, 1.12; 95% CI, 1.05‐1.18). Conclusion: One‐fourth of patients with HCC receive no therapy and one‐fifth of treated patients experience treatment delays. Both were associated with demographic, socioeconomic, and clinical characteristics of patients as well as facility type and region. The association between therapeutic delay and survival was stage and treatment dependent. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) has poor prognosis due to late‐stage, symptomatic presentation. Altered DNA methylation markers may improve diagnosis of CCA. Reduced‐representation bisulfite sequencing was performed on DNA extracted from frozen CCA tissues and matched to adjacent benign biliary epithelia or liver parenchyma. Methylated DNA markers (MDMs) identified from sequenced differentially methylated regions were selected for biological validation on DNA from independent formalin‐fixed, paraffin‐embedded CCA tumors and adjacent hepatobiliary control tissues using methylation‐specific polymerase chain reaction. Selected MDMs were then blindly assayed on DNA extracted from independent archival biliary brushing specimens, including 12 perihilar cholangiocarcinoma, 4 distal cholangiocarcinoma cases, and 18 controls. Next, MDMs were blindly assayed on plasma DNA from patients with extrahepatic CCA (eCCA), including 54 perihilar CCA and 5 distal CCA cases and 95 healthy and 22 primary sclerosing cholangitis controls, balanced for age and sex. From more than 3,600 MDMs discovered in frozen tissues, 39 were tested in independent samples. In the clinical pilot of 16 MDMs on cytology brushings, methylated EMX1 (empty spiracles homeobox 1) had an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.95‐1.0). In the clinical pilot on plasma, a cross‐validated recursive partitioning tree prediction model from nine MDMs was accurate for de novo eCCA (AUC, 0.88 [0.81‐0.95]) but not for primary sclerosing cholangitis–associated eCCA (AUC, 0.54 [0.35‐0.73]). Conclusion: Next‐generation DNA sequencing yielded highly discriminant methylation markers for CCA. Confirmation of these findings in independent tissues, cytology brushings, and plasma supports further development of DNA methylation to augment diagnosis of CCA. [ABSTRACT FROM AUTHOR]

Autophagy has been shown to be a key cellular event controlling tumor growth in different neoplasms including hepatocellular carcinoma (HCC). Although this biological role of autophagy has been clearly established, the mechanism underlying its regulation remains elusive. Here, we demonstrate a role of sulfatase 2 (SULF2), a 6‐O‐endosulfatase modulating various growth factors and cytokine‐related signaling pathways controlling tumor cell proliferation and survival, in the regulation of autophagy in HCC cells. SULF2 increased autophagosome formation, shown by increased LC3B‐II protein and green fluorescent protein–LC3 puncta. Increased fusion between autophagosomes and lysosomes/lysosomal enzymes, higher expression of lysosomal membrane protein, and an increase in autolysosomes were also shown by western blot, immunofluorescence, and electron microscopy of SULF2‐expressing cells, indicating enhanced autophagic flux. In contrast, RNA‐interference silencing of SULF2 in Huh7 cells induced lysosomal membrane permeabilization with diffuse cytosolic staining of cathepsin D and punctate staining of galectin‐3. Analysis of the mechanism showed that inhibition of lysosome‐associated protein transmembrane 4 beta (LAPTM4B), a gene induced by SULF2, resulted in decreased autophagosome formation, decreased fusion between autophagosomes and lysosomes, and increased lysosomal membrane permeabilization. Interestingly, down‐regulation of LAPTM4B also phenocopies the knockdown of SULF2, significantly reducing cell viability and colony formation. Conclusion: Our results demonstrate a role for SULF2 in the regulation of autophagic flux that is mediated through LAPTM4B induction in HCC cells, and provide a foundation for future translational efforts targeting autophagy in liver malignancies. [ABSTRACT FROM AUTHOR]