Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

Colonic epithelial cell diversity in health and inflammatory bowel disease.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

A prospective study on the prevalence of MASLD in people with type-2 diabetes in the community. Cost effectiveness of screening strategies.

BACKGROUND AND AIMS: As screening for the liver disease and risk-stratification pathways are not established in patients with type-2 diabetes mellitus (T2DM), we evaluated the diagnostic performance and the cost-utility of different screening strategies for MASLD in the community. METHODS: Consecutive patients with T2DM from primary care underwent screening for liver diseases, ultrasound, ELF score and transient elastography (TE). Five strategies were compared to the standard of care: ultrasound plus abnormal liver function tests (LFTs), Fibrosis score-4 (FIB-4), NAFLD fibrosis score, Enhanced liver fibrosis test (ELF) and TE. Standard of care was defined as abnormal LFTs prompting referral to hospital. A Markov model was built based on the fibrosis stage, defined by TE. We generated the cost per quality-adjusted life year (QALY) gained and calculated the incremental cost-effectiveness ratio (ICER) over a lifetime horizon. RESULTS: Of 300 patients, 287 were included: 64% (186) had MASLD and 10% (28) had other causes of liver disease. Patients with significant fibrosis, advanced fibrosis, and cirrhosis due to MASLD were 17% (50/287), 11% (31/287) and 3% (8/287), respectively. Among those with significant fibrosis classified by LSM≥8.1 kPa, false negatives were 54% from ELF and 38% from FIB-4. On multivariate analysis, waist circumference, BMI, AST levels and education rank were independent predictors of significant and advanced fibrosis. All the screening strategies were associated with QALY gains, with TE (148.73 years) having the most substantial gains, followed by FIB-4 (134.07 years), ELF (131.68 years) and NAFLD fibrosis score (121.25 years). In the cost-utility analysis, ICER was £2480/QALY for TE, £2541.24/QALY for ELF and £2059.98/QALY for FIB-4. CONCLUSION: Screening for MASLD in the diabetic population in primary care is cost-effective and should become part of a holistic assessment. However, traditional screening strategies, including FIB-4 and ELF, underestimate the presence of significant liver disease in this setting.

Metabolic Profile Reflects Stages of Fibrosis in Patients with Non-Alcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease worldwide, with fibrosis stage being the main predictor for clinical outcomes. Here, we present the metabolic profile of NAFLD patients with regards to fibrosis progression. We included all consecutive new referrals for NAFLD services between 2011 and 2019. Demographic, anthropometric and clinical features and noninvasive markers of fibrosis were recorded at baseline and at follow-up. Significant and advanced fibrosis were defined using liver stiffness measurement (LSM) as LSM ≥ 8.1 kPa and LSM ≥ 12.1 kPa, respectively. Cirrhosis was diagnosed either histologically or clinically. Fast progressors of fibrosis were defined as those with delta stiffness ≥ 1.03 kPa/year (25% upper quartile of delta stiffness distribution). Targeted and untargeted metabolic profiles were analysed on fasting serum samples using Proton nuclear magnetic resonance (1H NMR). A total of 189 patients were included in the study; 111 (58.7%) underwent liver biopsy. Overall, 11.1% patients were diagnosed with cirrhosis, while 23.8% were classified as fast progressors. A combination of metabolites and lipoproteins could identify the fast fibrosis progressors (AUROC 0.788, 95% CI: 0.703-0.874, p < 0.001) and performed better than noninvasive markers. Specific metabolic profiles predict fibrosis progression in patients with nonalcoholic fatty liver disease. Algorithms combining metabolites and lipids could be integrated in the risk-stratification of these patients.

TET2 Drives 5hmc Marking of GATA6 and Epigenetically Defines Pancreatic Ductal Adenocarcinoma Transcriptional Subtypes.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples. METHODS: We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalin-embedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo. RESULTS: We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. CONCLUSIONS: We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival.

Serum keratin 19 (CYFRA21-1) is a prognostic biomarker in severe alcoholic hepatitis.

BACKGROUND AND AIMS: Up to 30% of patients with severe alcoholic hepatitis (sAH) die within 3 months of presentation. The degree of ductular reaction, characterized by accumulation of biliary and liver progenitor cells, confers a poor prognosis. Keratin fragments are established serological surrogates of liver injury and keratin 19 (K19) is a histological marker of the ductular reaction. We assessed the relationship between serum K19 levels (viz. CYFRA21-1), histology and outcome in patients with sAH. METHODS: Serum CYFRA21-1 was quantified in pre-treatment serum samples from 824 patients enrolled in the STOPAH trial. The cohort was randomly divided into two groups to test mortality associations; histological analyses were performed using the 87 cases with suitable samples. RESULTS: CYFRA21-1 levels were elevated in sAH and strongly predicted alcoholic steatohepatitis (ASH) on biopsy (area under the receiver operated characteristic [AUROC] 0.785 [95% Confidence Interval 0.602-0.967]) and 90-day survival (AUROC 0.684/0.693). The predictive ability of CYFRA21-1 was comparable with the model of end-stage liver disease (MELD) score and was independently associated with survival in multivariable analysis. CYFRA21-1 moderately correlated with hepatocellular injury markers M30/M65 but displayed a more robust predictive ability. A combination of MELD and CYFRA21-1 conferred a modest improvement in the AUROC value (0.731/0.743). CONCLUSIONS: In sAH serum, CYFRA21-1 levels associate with the presence of ASH on biopsy and independently predict 90-day survival. As a single marker performance is comparable to established scoring systems. Therefore, CYFRA21-1, which is available in many clinical laboratories, may become a useful component of prognostic models.

Increased Body Mass Index and Type 2 Diabetes Are the Main Predictors of Nonalcoholic Fatty Liver Disease and Advanced Fibrosis in Liver Biopsies of Patients With Human Immunodeficiency Virus Monoinfection.

BACKGROUND: Liver disease is an important cause of morbidity and mortality in people living with human immunodeficiency virus (PLWH), of which nonalcoholic fatty liver disease (NAFLD) is an increasingly recognized cause. There are limited data investigating NAFLD in HIV monoinfection and histologically defined disease. We aimed to identify who is at risk of fibrosis, NAFLD, and nonalcoholic steatohepatitis (NASH) among PLWH and explore the diagnostic accuracy of noninvasive markers of fibrosis. METHODS: This was a retrospective, cross-sectional, international, multicenter study including patients with HIV monoinfection, without chronic viral hepatitis or other known causes of chronic liver disease, who underwent liver biopsy for abnormal liver biochemistry and/or clinical suspicion of liver fibrosis. RESULTS: A total of 116 patients from 5 centers were included. Sixty-three (54%) had NAFLD, of whom 57 (92%) had NASH. Overall, 36 (31%) had advanced fibrosis (≥F3) and 3 (3%) had cirrhosis. Of the 53 cases without NAFLD, 15 (28%) had advanced fibrosis. Collagen proportionate area was similar between cases with and without NAFLD (3% vs 2%). Body mass index was independently associated with NAFLD (aOR, 1.2; 95% CI, 1.08-1.34), and type 2 diabetes was independently associated with advanced fibrosis (aOR, 3.42; 95% CI, 1.00-11.71). The area under the curve for advanced fibrosis was 0.65 and 0.66 for both NAFLD Fibrosis Score (NFS) and FIB-4. Cutoff values of -1.455 (NFS) and 1.3 (FIB-4) have negative-predictive values of 0.80 and 0.82, respectively. CONCLUSIONS: Advanced fibrosis is strongly associated with type 2 diabetes in PLWH. Serological markers require further optimization.

Activation and transcriptional profile of monocytes and CD8+ T cells are altered in checkpoint inhibitor-related hepatitis.

BACKGROUND & AIMS: Checkpoint inhibitor-related hepatitis (CPI-Hep) is an emerging clinical challenge. We aimed to gain insights into the immunopathology of CPI-Hep by comprehensively characterising myeloid and lymphoid subsets. METHODS: CPI-treated patients with or without related hepatitis (CPI-Hep; n = 22 and CPI-noHep; n = 7) were recruited. Phenotypic and transcriptional profiling of peripheral immune subsets was performed and compared with 19 healthy controls (HCs). In vitro monocyte-derived macrophages (MoMFs) were assessed for activation and cytokine production. CD163, CCR2, CD68, CD3, CD8 and granzyme B expression was assessed using immunohistochemistry/immunofluorescence (n = 4). RESULTS: A significant total monocyte depletion was observed in CPI-Hep compared with HCs (p = 0.04), along with a proportionate increase in the classical monocyte population (p = 0.0002) and significant upregulation of CCR2, CD163 and downregulation of CCR7. Soluble CD163 levels were significantly elevated in CPI-Hep compared with HCs (p <0.0001). In vitro MoMFs from CPI-Hep showed enhanced production of pro-inflammatory cytokines. CD8+ T cells demonstrated increased perforin, granzyme B, ICOS and HLA-DR expression in CPI-Hep. Transcriptional profiling indicated the presence of activated monocyte and enhanced effector CD8+ T cell populations in CPI-Hep. Immunohistochemistry demonstrated co-localisation of CD8+/granzyme B+ T cells with CD68+CCR2+/CD68+CD163+ macrophages in CPI-Hep liver tissue. CONCLUSIONS: CPI-Hep is associated with activation of peripheral monocytes and an enhanced cytotoxic, effector CD8+ T cell phenotype. These changes were reflected by liver inflammation composed of CD163+/CCR2+ macrophages and CD8+ T cells. LAY SUMMARY: Some patients who receive immunotherapy for cancer develop liver inflammation, which requires cessation of cancer treatment. Herein, we describe ways in which the white blood cells of patients who develop liver inflammation differ from those of patients who receive the same immunotherapy but do not experience liver-related side effects. Targeting some of the pathways we identify may help to prevent or manage this side effect and facilitate cancer treatment.

Histopathological diagnosis of tumour deposits in colorectal cancer: a Delphi consensus study.

AIMS: Tumour deposits (TDs) are an important prognostic marker in colorectal cancer. However, the classification, and inclusion in staging, of TDs has changed significantly in each tumour-node-metastasis (TNM) edition since their initial description in TNM-5, and terminology remains controversial. Expert consensus is needed to guide the future direction of precision staging. METHODS AND RESULTS: A modified Delphi consensus process was used. Statements were formulated and sent to participants as an online survey. Participants were asked to rate their agreement with each statement on a five-point Likert scale and also to suggest additional statements for discussion. These responses were circulated together with anonymised comments, and statements were modified prior to carrying out a second online round. Consensus was set at 70%. Overall, 32 statements reached consensus. There were concerns that TDs were currently incorrectly placed in the TNM system and that their prognostic importance was being underestimated. There were concerns regarding interobserver variation and it was felt that a clearer, more reproducible definition of TDs was needed. CONCLUSIONS: Our main recommendations are that the number of TDs should be recorded even if lymph node metastases (LNMs) are also present and that nodules with evidence of origin [extramural venous invasion (EMVI), perineural invasion (PNI), lymphatic invasion (LI)] should still be categorised as TDs and not excluded, as TNM-8 specifies. Whether TDs should continue to be included in the N category at all is controversial, and did not achieve consensus; however, participants agreed that TDs are prognostically worse than LNMs and the N1c category is suboptimal, as it does not reflect this.

Qualification of tumour mutational burden by targeted next-generation sequencing as a biomarker in hepatocellular carcinoma.

BACKGROUND & AIMS: Tumour mutational burden (TMB) predicts improved response and survival to immunotherapy. In this pilot study, we optimized targeted next-generation sequencing (tNGS) to estimate TMB in hepatocellular carcinoma (HCC). METHODS: We sequenced 48 non-paired samples (21 fresh-frozen [FF] and 27 paraffin-embedded [FFPE]), among which 11 FFPE samples were pretreated with uracil-DNA glycosylase (UDG). Thirty samples satisfied post-sequencing quality control. High/low TMB was defined by median number of mutations/Mb (Mut/Mb), across different minimum allele frequency (MAF) thresholds (≥0.05, ≥0.1 and ≥0.2). RESULTS: Eligible patients (n = 29) were cirrhotic (84%) with TNM stage I-II HCC (75%). FFPE samples had higher TMB (median 958.39 vs 2.51 Mut/Mb, P < .0001), estimated deamination counts (median 1335.50 vs 0, P < .0001) and C > T transitions at CpG sites (median 60.3% vs 9.1%, P = .002) compared to FF. UDG-treated samples had lower TMB (median 4019.92 vs 353 Mut/Mb, P = .041) and deamination counts (median 6393.5 vs 328.5, P = .041) vs untreated FFPE. At 0.2 MAF threshold with UDG treatment, median TMB was 5.48 (range 1.68-16.07) and did not correlate with salient pathologic features of HCC, including survival. CONCLUSION: While tNGS on fresh HCC samples appears to be the optimal source of tumour DNA, the low median TMB values observed may limit the role of TMB as a predictor of response to immunotherapy in HCC.

Stat2 loss disrupts damage signalling and is protective in acute pancreatitis.

The severity of sterile inflammation, as seen in acute pancreatitis, is determined by damage-sensing receptors, signalling cascades and cytokine production. Stat2 is a type I interferon signalling mediator that also has interferon-independent roles in murine lipopolysaccharide-induced NF-κB-mediated sepsis. However, its role in sterile inflammation is unknown. We hypothesised that Stat2 determines the severity of non-infective inflammation in the pancreas. Wild type (WT) and Stat2-/- mice were injected i.p. with caerulein or l-arginine. Specific cytokine-blocking antibodies were used in some experiments. Pancreata and blood were harvested 1 and 24 h after the final dose of caerulein and up to 96 h post l-arginine. Whole-tissue phosphoproteomic changes were assessed using label-free mass spectrometry. Tissue-specific Stat2 effects were studied in WT/Stat2-/- bone marrow chimera and using Cre-lox recombination to delete Stat2 in pancreatic and duodenal homeobox 1 (Pdx1)-expressing cells. Stat2-/- mice were protected from caerulein- and l-arginine-induced pancreatitis. Protection was independent of type I interferon signalling. Stat2-/- mice had lower cytokine levels, including TNF-α and IL-10, and reduced NF-κB nuclear localisation in pancreatic tissue compared with WT. Inhibition of TNF-α improved (inhibition of IL-10 worsened) caerulein-induced pancreatitis in WT but not Stat2-/- mice. Phosphoproteomics showed downregulation of MAPK mediators but accumulation of Ser412-phosphorylated Tak1. Stat2 deletion in Pdx1-expressing acinar cells (Stat2flox/Pdx1-cre ) reduced pancreatic TNF-α expression, but not histological injury or serum amylase. WT/Stat2-/- bone marrow chimera mice were protected from pancreatitis irrespective of host or recipient genotype. Stat2 loss results in disrupted signalling in pancreatitis, upstream of NF-κB in non-acinar and/or bone marrow-derived cells. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer.

Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues.

High-Throughput, Machine Learning-Based Quantification of Steatosis, Inflammation, Ballooning, and Fibrosis in Biopsies From Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Liver biopsy is the reference standard for staging and grading nonalcoholic fatty liver disease (NAFLD), but histologic scoring systems are semiquantitative with marked interobserver and intraobserver variation. We used machine learning to develop fully automated software for quantification of steatosis, inflammation, ballooning, and fibrosis in biopsy specimens from patients with NAFLD and validated the technology in a separate group of patients. METHODS: We collected data from 246 consecutive patients with biopsy-proven NAFLD and followed up in London from January 2010 through December 2016. Biopsy specimens from the first 100 patients were used to derive the algorithm and biopsy specimens from the following 146 were used to validate it. Biopsy specimens were scored independently by pathologists using the Nonalcoholic Steatohepatitis Clinical Research Network criteria and digitalized. Areas of steatosis, inflammation, ballooning, and fibrosis were annotated on biopsy specimens by 2 hepatobiliary histopathologists to facilitate machine learning. Images of biopsies from the derivation and validation sets then were analyzed by the algorithm to compute percentages of fat, inflammation, ballooning, and fibrosis, as well as the collagen proportionate area, and compared with findings from pathologists' manual annotations and conventional scoring systems. RESULTS: In the derivation group, results from manual annotation and the software had an interclass correlation coefficient (ICC) of 0.97 for steatosis (95% CI, 0.95-0.99; P < .001); ICC of 0.96 for inflammation (95% CI, 0.9-0.98; P < .001); ICC of 0.94 for ballooning (95% CI, 0.87-0.98; P < .001); and ICC of 0.92 for fibrosis (95% CI, 0.88-0.96; P = .001). Percentages of fat, inflammation, ballooning, and the collagen proportionate area from the derivation group were confirmed in the validation cohort. The software identified histologic features of NAFLD with levels of interobserver and intraobserver agreement ranging from 0.95 to 0.99; this value was higher than that of semiquantitative scoring systems, which ranged from 0.58 to 0.88. In a subgroup of paired liver biopsy specimens, quantitative analysis was more sensitive in detecting differences compared with the nonalcoholic steatohepatitis Clinical Research Network scoring system. CONCLUSIONS: We used machine learning to develop software to rapidly and objectively analyze liver biopsy specimens for histologic features of NAFLD. The results from the software correlate with those from histopathologists, with high levels of interobserver and intraobserver agreement. Findings were validated in a separate group of patients. This tool might be used for objective assessment of response to therapy for NAFLD in practice and clinical trials.

In Severe Alcoholic Hepatitis, Serum Keratin-18 Fragments Are Diagnostic, Prognostic, and Theragnostic Biomarkers.

INTRODUCTION: Up to 40% of patients with severe alcoholic hepatitis (AH) die within 6 months of presentation, making prompt diagnosis and appropriate treatment essential. We determined the associations between serum keratin-18 (K18) and histological features, prognosis, and differential response to prednisolone in patients with severe AH. METHODS: Total (K18-M65) and caspase-cleaved K18 (K18-M30) were quantified in pretreatment sera from 824 patients enrolled in the Steroids or Pentoxifylline for Alcoholic Hepatitis trial (87 with suitable histological samples) and disease controls. RESULTS: K18 fragments were markedly elevated in severe AH and strongly predicted steatohepatitis (alcoholic steatohepatitis) on biopsy (area under receiver operating characteristics: 0.787 and 0.807). Application of published thresholds to predict alcoholic steatohepatitis would have rendered biopsy unnecessary in 84% of all AH cases. K18-M30 and M65 were associated with 90-day mortality, independent of age and Model for End-stage Liver Disease score in untreated patients. The association for K18-M65 was independent of both age and Model for End-stage Liver Disease in prednisolone-treated patients. Modelling of the effect of prednisolone on 90-day mortality as a function of pretreatment serum K18 levels indicated benefit in those with high serum levels of K18-M30. At low pretreatment serum K18 levels, prednisolone was potentially harmful. A threshold of K18-M30 5 kIU/L predicted therapeutic benefit from prednisolone above this level (odds ratio: 0.433, 95% confidence interval: 0.19-0.95, P = 0.0398), but not below (odds ratio: 1.271, 95% confidence interval: 0.88-1.84, P = 0.199). Restricting prednisolone usage to the former group would have reduced exposure by 87%. DISCUSSION: In a large cohort of patients with severe AH, serum K18 strongly correlated with histological severity, independently associated with 90-day mortality, and predicted response to prednisolone therapy. Quantification of serum K18 levels could assist in clinical decision-making.

Mouse Models of Nonalcoholic Steatohepatitis: Toward Optimization of Their Relevance to Human Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) arises from a variable interplay between environmental factors and genetic determinants that cannot be completely replicated in animals. Notwithstanding, preclinical models are needed to understand NASH pathophysiology and test mechanism-based therapies. Among several mouse models of NASH, some exhibit the key pathophysiologic as well as histopathologic criteria for human NASH, whereas others may be useful to address specific questions. Models based on overnutrition with adipose restriction/inflammation and metabolic complications, particularly insulin resistance, may be most useful to investigate critical etiopathogenic factors. In-depth pathologic description is required for all models. Some models demonstrate hepatocyte ballooning, which can be confused with microvesicular steatosis, whereas demonstration of an inflammatory infiltrate and pattern of liver fibrosis compatible with human NASH is desirable in models used for pharmacologic testing. When mice with specific genetic strains or mutations that cause overeating consume a diet enriched with fat, modest amounts of cholesterol, and/or simple sugars ("Western diet"), they readily develop obesity with liver disease similar to human NASH, including significant fibrosis. Purely dietary models, such as high-fat/high-cholesterol, Western diet, and choline-deficient, amino acid-defined, are similarly promising. We share concern about using models without weight gain, adipose pathology, or insulin resistance/hyperinsulinemia and with inadequate documentation of liver pathology. NASH-related fibrosis is a key endpoint in trials of possible therapies. When studied for this purpose, NASH models should be reproducible and show steatohepatitis (ideally with ballooning) and at least focal bridging fibrosis, while metabolic factors/disordered lipid partitioning should contribute to etiopathogenesis. Because murine models are increasingly used to explore pharmacologic therapies for NASH, we propose a minimum set of requirements that investigators, drug companies, and journals should consider to optimize their translational value.

Development of hepatic pathology in GBV-B-infected red-bellied tamarins (Saguinus labiatus).

GB virus B (GBV-B) is a new world monkey-associated flavivirus used to model acute hepatitis C virus (HCV) infection. Critical for evaluation of antiviral or vaccine approaches is an understanding of the effect of HCV on the liver at different stages of infection. In the absence of longitudinal human tissue samples at defined time points, we have characterized changes in tamarins. As early as 2 weeks post-infection histological changes were noticeable, and these were established in all animals by 6 weeks. Despite high levels of liver-associated viral RNA, there was reversal of hepatic damage on clearance of peripheral virus though fibrosis was demonstrated in four tamarins. Notably, viral RNA burden in the liver dropped to near undetectable or background levels in all animals which underwent a second viral challenge, highlighting the efficacy of the immune response in removing foci of replication in the liver. These data add to the knowledge of GBV-B infection in New World primates which can offer attractive systems for the testing of prophylactic and therapeutic treatments and the evaluation of their utility in preventing or reversing liver pathology.

Mass spectrometry transanal minimally invasive surgery (MS-TAMIS) to promote organ preservation in rectal cancer.

BACKGROUND: Transanal minimally invasive surgery (TAMIS) is deployed for organ preservation in early rectal cancer and significant rectal polyps. Rapid evaporative ionisation mass spectrometry (REIMS) provides biochemical tissue analysis, which could be applied intraoperatively to give real-time tissue feedback to the surgeon and decrease the risk of an involved margin. However, the accuracy and feasibility of this approach have not been established. METHODS: In this prospective observational study, patients undergoing resection of rectal adenomas or carcinomas were recruited. An electrosurgical handpiece analysed tissues ex vivo using diathermy, with the aerosol aspirated into a Xevo G2-S ToF mass spectrometer. The relative abundance of lipids underwent predictive statistical modelling and leave-one-patient-out cross-validation. The outcomes of interest were the ability of REIMS to differentiate normal, adenomatous and cancerous tissue, or any disease subtype from normal. REIMS was coupled with TAMIS for in vivo sampling, assessing the accuracy of tissue recognition and distinguishing bowel wall layers. RESULTS: Forty-seven patients were included, yielding 266 spectra (121 normal, 109 tumour and 36 adenoma). REIMS differentiates normal, adenomatous and cancerous rectal tissues with 86.8% accuracy, and normal and adenomatous tissue with 92.4% accuracy and 91.4% accuracy when differentiating disease from normal. We have performed the first five in-man mass spectrometry augmented TAMIS (MS-TAMIS). In real time, MS-TAMIS can differentiate rectal mucosa and submucosa based on their relative abundance of triglycerides and glycerophospholipids. The ex vivo accuracy distinguishing diseased and normal tissues is maintained in vivo at 90%, with negative predictive value of 95%. The system identified a deep and lateral involved tumour margin during TAMIS. CONCLUSIONS: REIMS distinguishes rectal tissue types based on underlying lipid biology, and this can be translated in vivo by coupling it to TAMIS. There is a role for this technology in improving the efficacy of resection of early rectal cancers.

Clinical implications of heterogeneity in PD-L1 immunohistochemical detection in hepatocellular carcinoma: the Blueprint-HCC study.

Programmed cell death ligand-1 immunohistochemical detection (PD-L1 IHC) is a putative predictor of response to PD-1/PD-L1-targeted checkpoint inhibitors. However, there is no gold standard assay in hepatocellular carcinoma (HCC). We evaluated 5 PD-L1 IHC assay platforms (E1LN3, 28-8, 22c3, SP263 and SP142) in 100 HCCs reporting PD-L1 expression in malignant (M) and tumour-infiltrating immune cells (TICs) and non-tumorous cirrhotic tissues (NTICs). We found substantial inter-assay heterogeneity in detecting PD-L1 expression in M (R2 = 0.080-0.921), TICs (Cohen's κ = 0.175-0.396) and NTICs (κ = 0.004-0.505). Such diversity may impact on the reliability and reproducibility of PD-L1 IHC assays as a predictor of response to immune checkpoint inhibitors.

Focused Ultrasound Hyperthermia for Targeted Drug Release from Thermosensitive Liposomes: Results from a Phase I Trial.

Purpose To demonstrate the feasibility and safety of using focused ultrasound planning models to determine the treatment parameters needed to deliver volumetric mild hyperthermia for targeted drug delivery without real-time thermometry. Materials and Methods This study was part of the Targeted Doxorubicin, or TARDOX, phase I prospective trial of focused ultrasound-mediated, hyperthermia-triggered drug delivery to solid liver tumors ( ClinicalTrials.gov identifier NCT02181075). Ten participants (age range, 49-68 years; average age, 60 years; four women) were treated from March 2015 to March 2017 by using a clinically approved focused ultrasound system to release doxorubicin from lyso-thermosensitive liposomes. Ultrasonic heating of target tumors (treated volume: 11-73 cm3 [mean ± standard deviation, 50 cm3 ± 26]) was monitored in six participants by using a minimally invasive temperature sensor; four participants were treated without real-time thermometry. For all participants, CT images were used with a patient-specific hyperthermia model to define focused ultrasound treatment plans. Feasibility was assessed by comparing model-prescribed focused ultrasound powers to those implemented for treatment. Safety was assessed by evaluating MR images and biopsy specimens for evidence of thermal ablation and monitoring adverse events. Results The mean difference between predicted and implemented treatment powers was -0.1 W ± 17.7 (n = 10). No evidence of focused ultrasound-related adverse effects, including thermal ablation, was found. Conclusion In this 10-participant study, the authors confirmed the feasibility of using focused ultrasound-mediated hyperthermia planning models to define treatment parameters that safely enabled targeted, noninvasive drug delivery to liver tumors while monitored with B-mode guidance and without real-time thermometry. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Dickey and Levi-Polyachenko in this issue.

Fourier transform infrared spectroscopic imaging of colon tissues: evaluating the significance of amide I and C-H stretching bands in diagnostic applications with machine learning.

Fourier transform infrared (FTIR) spectroscopic imaging of colon biopsy tissues in transmission combined with machine learning for the classification of different stages of colon malignancy was carried out in this study. Two different approaches, an optical and a computational one, were applied for the elimination of the scattering background during the measurements and compared with the results of the machine learning model without correction for the scattering. Several different data processing pathways were implemented in order to obtain a high accuracy of the prediction model. This study demonstrates, for the first time, that C-H stretching and amide I bands are of little to no significance in the classification of the colon malignancy, based on the Gini importance values by random forest (RF). The best prediction outcome is found when supervised RF classification was carried out in the fingerprint region of the spectral data between 1500 and 1000 cm-1 (excluding the contribution of amide I and II bands). An overall prediction accuracy higher than 90% is achieved through the RF. The results also show that dysplastic and hyperplastic tissues are well distinguished. This leads to the insight that the important differences between hyperplastic and dysplastic colon tissues lie within the fingerprint region of FTIR spectra. In this study, computational correction performed better than optical correction, but the findings show that the disease states of colon biopsies can be distinguished effectively without elimination of Mie scattering effect. Graphical abstract.