Consensus statements on endoscopic ultrasound-guided tissue acquisition. Guidelines from the Asian Endoscopic Ultrasound Group.

OBJECTIVES: This consensus was developed by the Asian EUS Group (AEG), who aimed to formulate a set of practice guidelines addressing various aspects of endoscopic ultrasound-guided tissue acquisition (EUS-TA). METHODS: The AEG initiated the development of consensus statements and formed an expert panel comprising surgeons, gastroenterologists, and pathologists. Three online consensus meetings were conducted to consolidate the statements and votes. The statements were presented and discussed in the first two consensus meetings and revised according to comments. Final voting was conducted at a third consensus meeting. The Grading of Recommendations, Assessment, Development, and Evaluation system was adopted to define the strength of the recommendations and quality of evidence. RESULTS: A total of 20 clinical questions and statements regarding EUS-TA were formulated. The committee recommended that fine-needle biopsy (FNB) needles be preferred over conventional fine-needle aspiration (FNA) needles for EUS-TA of subepithelial lesions. For solid pancreatic masses, rapid on-site evaluation is not routinely recommended when FNB needles are used. For dedicated FNB needles, fork-tip and Franseen-tip needles have essentially equivalent performance. CONCLUSION: This consensus provides guidance for EUS-TA, thereby enhancing the quality of EUS-TA.

Isoformic PD-1-mediated immunosuppression underlies resistance to PD-1 blockade in hepatocellular carcinoma patients.

OBJECTIVE: Immune checkpoint blockade (ICB) has improved cancer treatment, yet why most hepatocellular carcinoma (HCC) patients are resistant to PD-1 ICB remains elusive. Here, we elucidated the role of a programmed cell death protein 1 (PD-1) isoform, Δ42PD-1, in HCC progression and resistance to nivolumab ICB. DESIGN: We investigated 74 HCC patients in three cohorts, including 41 untreated, 28 treated with nivolumab and 5 treated with pembrolizumab. Peripheral blood mononuclear cells from blood samples and tumour infiltrating lymphocytes from tumour tissues were isolated for immunophenotyping. The functional significance of Δ42PD-1 was explored by single-cell RNA sequencing analysis and validated by functional and mechanistic studies. The immunotherapeutic efficacy of Δ42PD-1 monoclonal antibody was determined in HCC humanised mouse models. RESULTS: We found distinct T cell subsets, which did not express PD-1 but expressed its isoform Δ42PD-1, accounting for up to 71% of cytotoxic T lymphocytes in untreated HCC patients. Δ42PD-1+ T cells were tumour-infiltrating and correlated positively with HCC severity. Moreover, they were more exhausted than PD-1+ T cells by single T cell and functional analysis. HCC patients treated with anti-PD-1 ICB showed effective PD-1 blockade but increased frequencies of Δ42PD-1+ T cells over time especially in patients with progressive disease. Tumour-infiltrated Δ42PD-1+ T cells likely sustained HCC through toll-like receptors-4-signalling for tumourigenesis. Anti-Δ42PD-1 antibody, but not nivolumab, inhibited tumour growth in three murine HCC models. CONCLUSION: Our findings not only revealed a mechanism underlying resistance to PD-1 ICB but also identified anti-Δ42PD-1 antibody for HCC immunotherapy.

Non-alcoholic fatty liver disease: the pathologist's perspective.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of diseases characterized by fatty accumulation in hepatocytes, ranging from steatosis, non-alcoholic steatohepatitis, to cirrhosis. While histopathological evaluation of liver biopsies plays a central role in the diagnosis of NAFLD, limitations such as the problem of interobserver variability still exist and active research is underway to improve the diagnostic utility of liver biopsies. In this article, we provide a comprehensive overview of the histopathological features of NAFLD, the current grading and staging systems, and discuss the present and future roles of liver biopsies in the diagnosis and prognostication of NAFLD.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Reliability of the nonalcoholic steatohepatitis clinical research network and steatosis activity fibrosis histological scoring systems.

BACKGROUND AND AIM: We aimed to determine whether lobular inflammation and ballooning grades in the Non-alcoholic Steatohepatitis Clinical Research Network (NASH CRN) scoring system can be directly translated into the same for the Steatosis Activity Fibrosis scoring system (SAF) and to look at intra-observer and inter-observer agreement for each individual histological component and for diagnosis of non-alcoholic steatohepatitis (NASH) using the two scoring systems. METHODS: Four pathologists from two Asian centers scored 20 digitalized slides, twice using the NASH CRN, twice using the SAF. Intra-observer and inter-observer agreement was analyzed using Fleiss' kappa, weighted kappa, or Cohen kappa, where appropriate. RESULTS: The intra-observer discrepancy rate when using the NASH CRN compared with the SAF was higher than when using the individual scoring system for lobular inflammation (15% comparing both scoring systems vs 10% and 1.8% for the NASH CRN and the SAF, respectively) and hepatocyte ballooning (33.8% vs 12.5% and 5%, respectively), but not for diagnosis of NASH (6.3% vs 6.3% and 0%, respectively). Intra-observer and inter-observer agreement was substantial to almost perfect, except for inter-observer agreement for lobular inflammation and diagnosis of NASH, which was only fair to moderate in most instances. CONCLUSION: These findings do not support the direct inter-translation between the NASH CRN and the SAF. However, the diagnosis of NASH during examinations using the NASH CRN may be comparable with diagnosis of NASH using the SAF, vice versa. The inter-observer agreement for lobular inflammation and NASH diagnosis needs to be improved.

Unique molecular characteristics of NAFLD-associated liver cancer accentuate ß-catenin/TNFRSF19-mediated immune evasion.

BACKGROUND & AIMS: The hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), can lead to the development of hepatocellular carcinoma (HCC). Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations. METHODS: Herein, tumor-normal pairs from 100 patients diagnosed with NAFLD-HCC were subject to next-generation sequencing. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of the underlying CTNNB1 S45P driver mutation. A syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mutand TNFRSF19 in reshaping the tumor microenvironment. RESULTS: Mutational processes operative in the livers of patients with NAFLD inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. Chromatin immunoprecipitation-sequencing integrated with transcriptome and immune profiling revealed a unique transcriptional axis, wherein CTNNB1mut leads to an upregulation of TNFRSF19 which subsequently represses senescence-associated secretory phenotype-like cytokines (including IL6 and CXCL8). This phenomenon could be reverted by the Wnt-modulator ICG001. CONCLUSIONS: The unique mutational processes in the livers of patients with NAFLD and NAFLD-HCC allude to a "field effect" involving a gain-of-function role of CTNNB1 mutations in immune exclusion. LAY SUMMARY: The increasing prevalence of metabolic syndrome in adult populations means that NAFLD is poised to be the major cause of liver cancer in the 21st century. We showed a strong "field effect" in the livers of patients with NAFLD, wherein activated ß-catenin was involved in reshaping the tumor-immune microenvironment.

A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma.

Insufficient T cell infiltration into noninflamed tumors, such as hepatocellular carcinoma (HCC), restricts the effectiveness of immune-checkpoint blockade (ICB) for a subset of patients. Epigenetic therapy provides further opportunities to rewire cancer-associated transcriptional programs, but whether and how selective epigenetic inhibition counteracts the immune-excluded phenotype remain incompletely defined. Here, we showed that pharmacological inhibition of histone deacetylase 8 (HDAC8), a histone H3 lysine 27 (H3K27)-specific isozyme overexpressed in a variety of human cancers, thwarts HCC tumorigenicity in a T cell-dependent manner. The tumor-suppressive effect of selective HDAC8 inhibition was abrogated by CD8+ T cell depletion or regulatory T cell adoptive transfer. Chromatin profiling of human HDAC8-expressing HCCs revealed genome-wide H3K27 deacetylation in 1251 silenced enhancer-target gene pairs that are enriched in metabolic and immune regulators. Mechanistically, down-regulation of HDAC8 increased global and enhancer acetylation of H3K27 to reactivate production of T cell-trafficking chemokines by HCC cells, thus relieving T cell exclusion in both immunodeficient and humanized mouse models. In an HCC preclinical model, selective HDAC8 inhibition increased tumor-infiltrating CD8+ T cells and potentiated eradication of established hepatomas by anti-PD-L1 therapy without evidence of toxicity. Mice treated with HDAC8 and PD-L1 coblockade were protected against subsequent tumor rechallenge as a result of the induction of memory T cells and remained tumor-free for greater than 15 months. Collectively, our study demonstrates that selective HDAC8 inhibition elicits effective and durable responses to ICB by co-opting adaptive immunity through enhancer reprogramming.

Artificial intelligence in prediction of non-alcoholic fatty liver disease and fibrosis.

Artificial intelligence (AI) has become increasingly widespread in our daily lives, including healthcare applications. AI has brought many new insights into better ways we care for our patients with chronic liver disease, including non-alcoholic fatty liver disease and liver fibrosis. There are multiple ways to apply the AI technology on top of the conventional invasive (liver biopsy) and noninvasive (transient elastography, serum biomarkers, or clinical prediction models) approaches. In this review article, we discuss the principles of applying AI on electronic health records, liver biopsy, and liver images. A few common AI approaches include logistic regression, decision tree, random forest, and XGBoost for data at a single time stamp, recurrent neural networks for sequential data, and deep neural networks for histology and images.

Chemotherapy-induced recruitment of myeloid-derived suppressor cells abrogates efficacy of immune checkpoint blockade.

BACKGROUND & AIMS: Immune checkpoint blockade (ICB) has been approved for treatment of hepatocellular carcinoma (HCC). However, many patients with advanced HCC are non-responders to ICB monotherapy. Cytotoxic chemotherapy has been proposed to modulate the tumor microenvironment (TME) and sensitize tumors to ICB. Thus, we aimed to study the combination of cytotoxic chemotherapy and ICB in an orthotopic HCC model. METHODS: Preclinical orthotopic HCC mouse models were used to elucidate the efficacy of 5-fluorouracil (5-FU) and ICB. The mice were intrahepatically injected with RIL-175 or Hepa1-6 cells, followed by treatment with 5-FU and anti-programmed cell death ligand 1 (PD-L1) antibody. Myeloid-derived suppressor cells (MDSCs) were depleted to validate their role in attenuating sensitivity to immunotherapy. Flow cytometry-based immune profiling and immunofluorescence staining were performed in mice and patient samples, respectively. RESULTS: 5-FU could induce intratumoral MDSC accumulation to counteract the infiltration of T lymphocytes and natural killer cells, thus abrogating the anti-tumor efficacy of PD-L1 blockade. In clinical samples, MDSCs accumulated and CD8+ T cell numbers decreased following transarterial chemoembolization. CONCLUSION: 5-FU can trigger the accumulation of immunosuppressive MDSCs, impairing the response to PD-L1 blockade in HCC. Our data suggest that the combination of specific chemotherapy and ICB may impair anti-tumor immune responses, warranting further study in preclinical models and consideration in clinical settings. LAY SUMMARY: Our findings suggest that some chemotherapies may impair the anti-tumor efficacy of immunotherapy. Further studies are required to uncover the specific effects of different chemotherapies on the immunological profile of tumors. This data will be critical for the rational design of combination immunotherapy strategies for patients with hepatocellular carcinoma.

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis.

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

A Sequential Algorithm Combining ADAPT and Liver Stiffness Can Stage Metabolic-Associated Fatty Liver Disease in Hospital-Based and Primary Care Patients.

INTRODUCTION: Metabolic-associated fatty liver disease is common, with fibrosis the major determinant of adverse outcomes. Population-based screening tools with high diagnostic accuracy for the staging of fibrosis are lacking. METHODS: Three independent cohorts, 2 with both liver biopsy and liver stiffness measurements (LSMs, n = 254 and 65) and a population sample (n = 713), were studied. The performance of a recently developed noninvasive algorithm (ADAPT [age, diabetes, PRO-C3 and platelets panel]) as well as aspartate aminotransferase-to-platelet ratio index, fibrosis-4, nonalcoholic fatty liver disease fibrosis score, and LSM was used to stage patients for significant (≥F2) and advanced (≥F3) fibrosis. RESULTS: In the hospital-based cohorts, the N-terminal propeptide of type 3 collagen (Pro-C3) increased with fibrosis stage (P < 0.0001) and independently associated with advanced fibrosis (odds ratio = 1.091, 95% confidence interval [CI]: 1.053-1.113, P = 0.0001). ADAPT showed areas under the receiver operating characteristics curve of 0.831 (95% CI: 0.779-0.875) in the derivation and 0.879 (95% CI: 0.774-0.946) in the validation cohort for advanced fibrosis. This was superior to the existing fibrosis scores, aspartate aminotransferase-to-platelet ratio index, fibrosis-4, BARD (BMI, aspartate aminotransferase to alanine aminotransferase ratio [AAR], diabetes), and nonalcoholic fatty liver disease fibrosis score in most comparisons and comparable with LSM. Serial use of ADAPT and LSM had diagnostic accuracy of 92.5%, with 98% and 100% negative predictive value in the derivation and validation cohorts, respectively. In the population cohort, PRO-C3 associated with advanced fibrosis (P = 0.04), while ADAPT had a negative predictive value of 98% for excluding advanced fibrosis. DISCUSSION: PRO-C3 and ADAPT reliably exclude advanced fibrosis in low-risk populations. The serial combination of ADAPT with LSM has high diagnostic accuracy with a low requirement for liver biopsy. The proposed algorithm would help stratify those who need biopsies and narrow down those patients who would need to be referred to specialty clinics.

EBV-encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy. The high expression of BART-miRNAs (miR-BARTs) during latent EBV infection in NPC strongly supports their pathological importance in cancer progression. Recently, we found that several BART-miRNAs work co-operatively to modulate the DNA damage response (DDR) by reducing Ataxia-telangiectasia-mutated (ATM) activity. In this study, we further investigated the role of miR-BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down-regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2-3p, BART12, BART17-5p and BART19-3p in BRCA1 expression. The ectopic expression of these four miR-BARTs suppressed endogenous BRCA1 expression in EBV-negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR-BARTs activities in C666-1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR-BART17-5p and miR-BART19-3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR-BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.

NADPH oxidase 5α promotes the formation of CD271 tumor-initiating cells in oral cancer.

Oral tongue squamous cell carcinoma (OTSCC) has a distinctive cell sub-population known as tumor-initiating cells (TICs). CD271 is a functional TIC receptor in head and neck cancers. The molecular mechanisms governing CD271 up-regulation remains unclear. Oxidative stress is a contributing factor in TIC development. Here, we explored the potential role of NADPH oxidase 5 (NOX5) and its regulatory mechanism on the development of CD271-expressing OTSCC. Our results showed that the splice variant NOX5α is the most prevalent form expressed in head and neck cancers. NOX5α enhanced OTSCC proliferation, migration, and invasion. Overexpression of NOX5α increased the size of OTSCC xenograft significantly in vivo. The tumor-promoting functions of NOX5α were mediated through the reactive oxygen species (ROS)-generating property. NOX5α activated ERK singling and increased CD271 expression at the transcription level. Also, NOX5α reduces the sensitivity of OTSCC to cisplatin and natural killer cells. The findings indicate that NOX5α plays an important part in the development of TIC in OTSCC.

Macromolecular proton fraction mapping based on spin-lock magnetic resonance imaging.

PURPOSE: In MRI, the macromolecular proton fraction (MPF) is a key parameter of magnetization transfer (MT). It represents the relative amount of immobile protons associated with semi-solid macromolecules involved in MT with free water protons. We aim to quantify MPF based on spin-lock MRI and explore its advantages over the existing MPF-mapping methods. METHODS: In the proposed method, termed MPF quantification based on spin-lock (MPF-SL), off-resonance spin-lock is used to sensitively measure the MT effect. MPF-SL is designed to measure a relaxation rate (Rmpfsl ) that is specific to the MT effect by removing the R1ρ relaxation due to the mobile water and chemical exchange pools. A theory is derived to quantify MPF from the measured Rmpfsl . No prior knowledge of tissue relaxation parameters, including T1 or T2 , is needed to quantify MPF using MPF-SL. The proposed approach is validated with Bloch-McConnell simulations, phantom, and in vivo liver studies at 3.0T. RESULTS: Both Bloch-McConnell simulations and phantom experiments show that MPF-SL is insensitive to variations of the mobile water pool and the chemical exchange pool. MPF-SL is specific to the MT effect and can measure MPF reliably. In vivo liver studies show that MPF-SL can be used to detect collagen deposition in patients with liver fibrosis. CONCLUSION: A novel MPF imaging method based on spin-lock MRI is proposed. The confounding factors are removed, and the measurement is specific to the MT effect. It holds promise for MPF-sensitive diagnostic imaging in clinical settings.

Development of a Novel Inflammation-Based Index for Hepatocellular Carcinoma.

BACKGROUND: The aim of current study was to (1) construct and validate a novel hepatocellular carcinoma (HCC)-specific inflammatory index; (2) compare the performances of the Integrated Liver Inflammatory Score (ILIS) to existing 4 inflammatory indices in HCC; (3) explore the association between the inflammatory indices and systemic/intratumoral inflammatory markers. METHODS: Two cohorts from Hong Kong (HK; n = 1,315) and Newcastle (n = 574) were studied. A novel index was constructed from the HK training set (n = 627). The index was constructed from the training set by combing independent prognostic circulating parameters, followed by validating in the validation set of HK cohort (n = 688) and the Newcastle cohort. Its prognostic performance was compared to 4 inflammatory indices, namely, the neutrophil to lymphocyte ratio, platelet-to-lymphocyte ratio, prognostic nutrition index, and systemic immune-inflammation index, were compared in the HK cohort. Circulating cytokines and intratumoral gene expression were analyzed in a subset of patients with available samples and correlated with the inflammatory indices. RESULTS: In the training set of the HK cohort, the ILIS, was generated: -0.057 × albumin (g/L) + 0.978 × log (Bilirubin, µmol/L) + 1.341 × log (alkaline phosphatase, IU/L) + 0.086 × Neutrophil (109/L) + 0.301 × log (alpha-fetoprotein, µg/L). With cutoff of 2.60 and 3.87, the ILIS could categorize patients into 3 risk groups in the both validation cohorts. ILIS outperforms other inflammatory indices and remains an independent prognosticator for overall survival after adjustment with Barcelona Clinic Liver Cancer (hazard ratio 31.90, p < 0.001). The ILIS had the best prognostic performances as compared to other inflammatory indices. In exploratory analyses, the ILIS correlated with circulating inflammatory cytokines (e.g., IL-8) but not with any intratumoral inflammatory gene expression. CONCLUSIONS: ILIS is an HCC-specific prognostic index built on 5 readily available blood parameters. Its versatility is validated both Eastern and Western population of HCC. The score is correlated with levels of circulating cytokines.

Positive Hepatitis B Core Antibody Is Associated With Cirrhosis and Hepatocellular Carcinoma in Nonalcoholic Fatty Liver Disease.

OBJECTIVES: Previous exposure to hepatitis B virus (HBV) may increase the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. We aim to study the impact of previous HBV infection on the severity and outcomes of patients with nonalcoholic fatty liver disease (NAFLD). METHODS: This was a multicenter study of 489 patients with biopsy-proven NAFLD and 69 patients with NAFLD-related or cryptogenic HCC. Antihepatitis B core antibody (anti-HBc) was used to detect the previous HBV infection. RESULTS: In the biopsy cohort, positive anti-HBc was associated with lower steatosis grade but higher fibrosis stage. 18.8% and 7.5% of patients with positive and negative anti-HBc had cirrhosis, respectively (P < 0.001). The association between anti-HBc and cirrhosis remained significant after adjusting for age and metabolic factors (adjusted odds ratio 2.232; 95% confidence interval, 1.202-4.147). At a mean follow-up of 6.2 years, patients with positive anti-HBc had a higher incidence of HCC or cirrhotic complications (6.5% vs 2.2%; P = 0.039). Among patients with NAFLD-related or cryptogenic HCC, 73.9% had positive anti-HBc. None of the patients had positive serum HBV DNA. By contrast, antihepatitis B surface antibody did not correlate with histological severity. DISCUSSION: Positive anti-HBc is associated with cirrhosis and possibly HCC and cirrhotic complications in patients with NAFLD. Because a significant proportion of NAFLD-related HCC may develop in noncirrhotic patients, future studies should define the role of anti-HBc in selecting noncirrhotic patients with NAFLD for HCC surveillance.

Performance of Simple Fibrosis Scores in Nonobese Patients With Nonalcoholic Fatty Liver Disease.

Because only a minority of patients with nonalcoholic fatty liver disease (NAFLD) have advanced fibrosis and would eventually develop liver-related complications, current guidelines recommend initial assessment with noninvasive tests of fibrosis.1-3 Most previous studies focused on overweight and obese patients. Despite a strong association between obesity and NAFLD, 3%-30% of people with relatively normal body mass index (BMI) may still have NAFLD.4,5 Hence, this study aims to evaluate the performance of the common noninvasive tests in non-obese (BMI <25 kg/m2) and obese (BMI ≥25 kg/m2) NAFLD patients.

Quantifying and monitoring fibrosis in non-alcoholic fatty liver disease using dual-photon microscopy.

OBJECTIVE: Fibrosis stage is strongly associated with liver-related outcomes and is a key surrogate endpoint in drug trials for non-alcoholic steatohepatitis. Dual-photon microscopy allows automated quantification of fibrosis-related parameters (q-FPs) and may facilitate large-scale histological studies. We aim to validate the performance of q-FPs in a large histological cohort. DESIGN: 344 patients with non-alcoholic fatty liver disease (NAFLD) underwent 428 liver biopsies (240 had paired transient elastography examination). Fibrosis stage was scored using the NASH Clinical Research Network system, and q-FPs were measured by dual-photon microscopy using unstained slides. Patients were randomly assigned to the training and validation cohorts to test the performance of individual q-FPs and derive optimal cut-offs. RESULTS: Over 25 q-FPs had area under the receiver-operating characteristics curves >0.90 for different fibrosis stages. Among them, the perimeter of collagen fibres and number of long collagen fibres had the highest accuracy. At the best cut-offs, the two q-FPs had 88.3%-96.2% sensitivity and 78.1%-91.1% specificity for different fibrosis stages in the validation cohort. q-FPs and histological scoring had nearly identical correlations with liver stiffness measurement, suggesting that the accuracy of q-FPs approached that of histological assessment. Among patients with paired liver biopsies, changes in the same q-FPs were associated with changes in fibrosis stage. At a median follow-up of 5.6 years, baseline q-FPs predicted liver-related events. CONCLUSION: q-FP is highly accurate in the assessment of fibrosis in NAFLD patients. This automated platform can be used in future studies as objective and reliable evaluation of histological fibrosis.