Targeting PPAR-gamma counteracts tumour adaptation to immune-checkpoint blockade in hepatocellular carcinoma.

OBJECTIVE: Therapy-induced tumour microenvironment (TME) remodelling poses a major hurdle for cancer cure. As the majority of patients with hepatocellular carcinoma (HCC) exhibits primary or acquired resistance to antiprogrammed cell death (ligand)-1 (anti-PD-[L]1) therapies, we aimed to investigate the mechanisms underlying tumour adaptation to immune-checkpoint targeting. DESIGN: Two immunotherapy-resistant HCC models were generated by serial orthotopic implantation of HCC cells through anti-PD-L1-treated syngeneic, immunocompetent mice and interrogated by single-cell RNA sequencing (scRNA-seq), genomic and immune profiling. Key signalling pathway was investigated by lentiviral-mediated knockdown and pharmacological inhibition, and further verified by scRNA-seq analysis of HCC tumour biopsies from a phase II trial of pembrolizumab (NCT03419481). RESULTS: Anti-PD-L1-resistant tumours grew >10-fold larger than parental tumours in immunocompetent but not immunocompromised mice without overt genetic changes, which were accompanied by intratumoral accumulation of myeloid-derived suppressor cells (MDSC), cytotoxic to exhausted CD8+ T cell conversion and exclusion. Mechanistically, tumour cell-intrinsic upregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) transcriptionally activated vascular endothelial growth factor-A (VEGF-A) production to drive MDSC expansion and CD8+ T cell dysfunction. A selective PPARγ antagonist triggered an immune suppressive-to-stimulatory TME conversion and resensitised tumours to anti-PD-L1 therapy in orthotopic and spontaneous HCC models. Importantly, 40% (6/15) of patients with HCC resistant to pembrolizumab exhibited tumorous PPARγ induction. Moreover, higher baseline PPARγ expression was associated with poorer survival of anti-PD-(L)1-treated patients in multiple cancer types. CONCLUSION: We uncover an adaptive transcriptional programme by which tumour cells evade immune-checkpoint targeting via PPARγ/VEGF-A-mediated TME immunosuppression, thus providing a strategy for counteracting immunotherapeutic resistance in HCC.

Detecting Early-Stage Liver Fibrosis Using Macromolecular Proton Fraction Mapping Based on Spin-Lock MRI: Preliminary Observations.

BACKGROUND: Liver fibrosis is characterized by macromolecule depositions. Recently, a novel technology termed macromolecular proton fraction quantification based on spin-lock magnetic resonance imaging (MPF-SL) is reported to measure macromolecule levels. HYPOTHESIS: MPF-SL can detect early-stage liver fibrosis by measuring macromolecule levels in the liver. STUDY TYPE: Retrospective. SUBJECTS: Fifty-five participants, including 22 with no fibrosis (F0) and 33 with early-stage fibrosis (F1-2), were recruited. FIELD STRENGTH/SEQUENCE: 3 T; two-dimensional (2D) MPF-SL turbo spin-echo sequence, 2D spin-lock T1rho turbo spin-echo sequence, and multi-slice 2D gradient echo sequence. ASSESSMENT: Macromolecular proton fraction (MPF), T1rho, liver iron concentration (LIC), and fat fraction (FF) biomarkers were quantified within regions of interest. STATISTICAL TESTS: Group comparison of the biomarkers using Mann-Whitney U tests; correlation between the biomarkers assessed using Spearman's rank correlation coefficient and linear regression with goodness-of-fit; fibrosis stage differentiation using receiver operating characteristic curve (ROC) analysis. P-value < 0.05 was considered statistically significant. RESULTS: Average T1rho was 41.76 ± 2.94 msec for F0 and 41.15 ± 3.73 msec for F1-2 (P = 0.60). T1rho showed nonsignificant correlation with either liver fibrosis (ρ = -0.07; P = 0.61) or FF (ρ = -0.14; P = 0.35) but indicated a negative correlation with LIC (ρ = -0.66). MPF was 4.73 ± 0.45% and 5.65 ± 0.81% for F0 and F1-2 participants, respectively. MPF showed a positive correlation with liver fibrosis (ρ = 0.59), and no significant correlations with LIC (ρ = 0.02; P = 0.89) or FF (ρ = 0.05; P = 0.72). The area under the ROC curve was 0.85 (95% confidence interval [CI] 0.75-0.95) and 0.55 (95% CI 0.39-0.71; P = 0.55) for MPF and T1rho to discriminate between F0 and F1-2 fibrosis, respectively. DATA CONCLUSION: MPF-SL has the potential to diagnose early-stage liver fibrosis and does not appear to be confounded by either LIC or FF. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 3.

Aberrant cholesterol metabolic signaling impairs antitumor immunosurveillance through natural killer T cell dysfunction in obese liver.

Obesity is a major risk factor for cancers including hepatocellular carcinoma (HCC) that develops from a background of non-alcoholic fatty liver disease (NAFLD). Hypercholesterolemia is a common comorbidity of obesity. Although cholesterol biosynthesis mainly occurs in the liver, its role in HCC development of obese people remains obscure. Using high-fat high-carbohydrate diet-associated orthotopic and spontaneous NAFLD-HCC mouse models, we found that hepatic cholesterol accumulation in obesity selectively suppressed natural killer T (NKT) cell-mediated antitumor immunosurveillance. Transcriptome analysis of human liver revealed aberrant cholesterol metabolism and NKT cell dysfunction in NAFLD patients. Notably, cholesterol-lowering rosuvastatin restored NKT expansion and cytotoxicity to prevent obesogenic diet-promoted HCC development. Moreover, suppression of hepatic cholesterol biosynthesis by a mammalian target of rapamycin (mTOR) inhibitor vistusertib preceded tumor regression, which was abolished by NKT inactivation but not CD8+ T cell depletion. Mechanistically, sterol regulatory element-binding protein 2 (SREBP2)-driven excessive cholesterol production from hepatocytes induced lipid peroxide accumulation and deficient cytotoxicity in NKT cells, which were supported by findings in people with obesity, NAFLD and NAFLD-HCC. This study highlights mTORC1/SREBP2/cholesterol-mediated NKT dysfunction in the tumor-promoting NAFLD liver microenvironment, providing intervention strategies that invigorating NKT cells to control HCC in the obesity epidemic.

Progranulin mediates immune evasion of pancreatic ductal adenocarcinoma through regulation of MHCI expression.

Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8+ T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC.

Association of genetic variations with NAFLD in lean individuals.

BACKGROUND & AIMS: How adiposity influences the effect of genetic variants on non-alcoholic fatty liver disease (NAFLD) in the Asian population remains unclear. We aimed to study the association between genetic risk variants and susceptibility/severity of NAFLD in the lean, overweight and obese individuals. METHODS: Nine hundred and four community subjects underwent proton-magnetic resonance spectroscopy and transient elastography examination. Lean (<23 kg/m2 ), overweight (23-24.9 kg/m2 ) and obesity (≥25 kg/m2 ) were defined according to the body mass index cut-offs for Asians. NAFLD was defined as intrahepatic triglycerides ≥5%. PNPLA3, TM6SF2, MBOAT7 and 9 other gene polymorphisms were analysed by rhAMPTM SNP assays. RESULTS: Five hundred and twenty-nine (58.5%), 162 (17.9%) and 213 (23.6%) subjects were lean, overweight and obese, respectively. The prevalence of NAFLD was 12.4%, 41.4% and 59.1% in the three groups (P < .001). Amongst those with NAFLD, lean subjects (30.3%) were more likely to carry the PNPLA3 rs738409 GG genotype than overweight (17.9%) and obese subjects (17.4%) (P = .003). Compared with the CC genotype, the GG genotype was associated with the greatest increase in the risk of NAFLD in lean subjects (odds ratio [OR] 6.04), compared with overweight (OR 3.43, 95% CI [1.06, 11.14]) and obese subjects (OR 2.51, 95% CI [0.93, 6.78]). Additionally, the TM6SF2 rs58542926 TT genotype was associated with reduced serum triglycerides only in lean subjects. A gene-BMI effect was not observed for the other gene polymorphisms. CONCLUSIONS: The PNPLA3 rs738409 gene polymorphism has a greater effect on liver fat in Asian lean individuals than in overweight or obese ones.

High-Fat Diet Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites.

BACKGROUND AND AIMS: Dietary fat intake is associated with increased risk of colorectal cancer (CRC). We examined the role of high-fat diet (HFD) in driving CRC through modulating gut microbiota and metabolites. METHODS: HFD or control diet was fed to mice littermates in CRC mouse models of an azoxymethane (AOM) model and Apcmin/+ model, with or without antibiotics cocktail treatment. Germ-free mice for fecal microbiota transplantation were used for validation. Gut microbiota and metabolites were detected using metagenomic sequencing and high-performance liquid chromatography-mass spectrometry, respectively. Gut barrier function was determined using lipopolysaccharides level and transmission electron microscopy. RESULTS: HFD promoted colorectal tumorigenesis in both AOM-treated mice and Apcmin/+ mice compared with control diet-fed mice. Gut microbiota depletion using antibiotics attenuated colon tumor formation in HFD-fed mice. A significant shift of gut microbiota composition with increased pathogenic bacteria Alistipessp.Marseille-P5997 and Alistipessp.5CPEGH6, and depleted probiotic Parabacteroides distasonis, along with impaired gut barrier function was exhibited in HFD-fed mice. Moreover, HFD-modulated gut microbiota promotes colorectal tumorigenesis in AOM-treated germ-free mice, indicating gut microbiota was essential in HFD-associated colorectal tumorigenesis. Gut metabolites alteration, including elevated lysophosphatidic acid, which was confirmed to promote CRC cell proliferation and impair cell junction, was also observed in HFD-fed mice. Moreover, transfer of stools from HFD-fed mice to germ-free mice without interference increased colonic cell proliferation, impaired gut barrier function, and induced oncogenic genes expression. CONCLUSIONS: HFD drives colorectal tumorigenesis through inducing gut microbial dysbiosis, metabolomic dysregulation with elevated lysophosphatidic acid, and gut barrier dysfunction in mice.

A phase II clinical study on the efficacy and predictive biomarker of pegylated recombinant arginase on hepatocellular carcinoma.

BACKGROUND: Pegylated recombinant human arginase (PEG-BCT-100) is an arginine depleting drug. Preclinical studies showed that HCC is reliant on exogenous arginine for growth due to the under-expression of the arginine regenerating enzymes argininosuccinate synthetase (ASS) and ornithine transcarbamylase (OTC). METHODS: This is a single arm open-label Phase II trial to assess the potential clinical efficacy of PEG-BCT-100 in chemo naïve sorafenib-failure HCC patients. Pre-treatment tumour biopsy was mandated for ASS and OTC expression by immunohistochemistry (IHC). Weekly intravenous PEG-BCT-100 at 2.7 mg/kg was given. Primary endpoint was time to progression (TTP); secondary endpoints included radiological response as per RECIST1.1, progression free survival (PFS) and overall survival (OS). Treatment outcomes were correlated with tumour immunohistochemical expressions of ASS and OTC. RESULTS: In total 27 patients were recruited. The median TTP and PFS were both 6 weeks (95% CI, 5.9-6.0 weeks). The disease control rate (DCR) was 21.7% (5 stable disease). The drug was well tolerated. Post hoc analysis showed that duration of arginine depletion correlated with OS. For patients with available IHC results, 10 patients with ASS-negative tumour had OS of 35 weeks (95% CI: 8.3-78.0 weeks) vs. 15.14 weeks (95% CI: 13.4-15.1 weeks) in 3 with ASS-positive tumour; expression of OTC did not correlate with treatment outcomes. CONCLUSIONS: PEG-BCT-100 in chemo naïve post-sorafenib HCC is well tolerated with moderate DCR. ASS-negative confers OS advantage over ASS-positive HCC. ASS-negativity is a potential biomarker for OS in HCC and possibly for other ASS-negative arginine auxotrophic cancers. TRIAL REGISTRATION NUMBER: NCT01092091. Date of registration: March 23, 2010.

Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is an increasing healthcare burden worldwide. We examined the role of dietary cholesterol in driving NAFLD-HCC through modulating gut microbiota and its metabolites. DESIGN: High-fat/high-cholesterol (HFHC), high-fat/low-cholesterol or normal chow diet was fed to C57BL/6 male littermates for 14 months. Cholesterol-lowering drug atorvastatin was administered to HFHC-fed mice. Germ-free mice were transplanted with stools from mice fed different diets to determine the direct role of cholesterol modulated-microbiota in NAFLD-HCC. Gut microbiota was analysed by 16S rRNA sequencing and serum metabolites by liquid chromatography-mass spectrometry (LC-MS) metabolomic analysis. Faecal microbial compositions were examined in 59 hypercholesterolemia patients and 39 healthy controls. RESULTS: High dietary cholesterol led to the sequential progression of steatosis, steatohepatitis, fibrosis and eventually HCC in mice, concomitant with insulin resistance. Cholesterol-induced NAFLD-HCC formation was associated with gut microbiota dysbiosis. The microbiota composition clustered distinctly along stages of steatosis, steatohepatitis and HCC. Mucispirillum, Desulfovibrio, Anaerotruncus and Desulfovibrionaceae increased sequentially; while Bifidobacterium and Bacteroides were depleted in HFHC-fed mice, which was corroborated in human hypercholesteremia patients. Dietary cholesterol induced gut bacterial metabolites alteration including increased taurocholic acid and decreased 3-indolepropionic acid. Germ-free mice gavaged with stools from mice fed HFHC manifested hepatic lipid accumulation, inflammation and cell proliferation. Moreover, atorvastatin restored cholesterol-induced gut microbiota dysbiosis and completely prevented NAFLD-HCC development. CONCLUSIONS: Dietary cholesterol drives NAFLD-HCC formation by inducing alteration of gut microbiota and metabolites in mice. Cholesterol inhibitory therapy and gut microbiota manipulation may be effective strategies for NAFLD-HCC prevention.

Role of oxidative stress in clofazimine-induced cardiac dysfunction in a zebrafish model.

BACKGROUND: Clofazimine (CFZ), a riminophenazine, is now commonly used in the treatment of multidrug-resistant tuberculosis. However, its use may be potentially associated with cardiac dysfunction in some individuals. In this study, the zebrafish heart, by merit of its developmental and genetic characteristics being in homology with that of human, was chosen as an animal model for evaluation of such dysfunction. METHODS: Morphological and physiological parameters were used to assess cardiac dysfunction. Transcriptome analysis was performed, followed by validation with real-time quantitative PCR, for delineation of the relevant genomics. RESULTS: Exposure of 2 dpf zebrafish to 4 mg/L CFZ for 2 days, adversely affected cardiac functions including significant decreases in HR, SV, CO, and FS, with observable pathophysiological developments of pericardial effusion and blood accumulation in the heart, in comparison with the control group. In addition, genes which respond to xenobiotic stimulus, related to oxygen transport, glutathione metabolism and extracellular matrix -receptor interactions, were significantly enriched among the differentially up-regulated genes. Antioxidant response element motif was enriched in the 5000 base pair upstream regions of the differentially expressed genes. Co-administration of N-acetylcysteine was shown to protect zebrafish against the development of CFZ-induced cardiac dysfunction. CONCLUSIONS: This study suggests an important role of oxidative stress as a major pathogenetic mechanism of riminophenazine-induced cardiac dysfunction.

Genetic variation in ABCB5 associates with risk of hepatocellular carcinoma.

Expression of ATP-binding cassette B5 (ABCB5) has been demonstrated to confer chemoresistance, enhance cancer stem cell properties and associate with poor prognosis in hepatocellular carcinoma (HCC). The aim of this study was to evaluate the genetic variations of ABCB5 in HCC patients with reference to healthy individuals and the clinicopathological significance. A pilot study has examined 20 out of 300 pairs HCC and paralleled blood samples using conventional sequencing method to cover all exons and exon/intron regions to investigate whether there will be novel variant sequence and mutation event. A total of 300 HCC and 300 healthy blood DNA samples were then examined by Sequenom MassARRAY genotyping and pyrosequencing for 38 SNP and 1 INDEL in ABCB5. Five novel SNPs were identified in ABCB5. Comparison of DNA from blood samples of HCC and healthy demonstrated that ABCB5 SNPs rs75494098, rs4721940 and rs10254317 were associated with HCC risk. Specific ABCB5 variants were associated with aggressive HCC features. SNP rs17143212 was significantly associated with ABCB5 expression level. Nonetheless, the paralleled blood and tumour DNA sequences from HCC patients indicated that ABCB5 mutation in tumours was not common and corroborated the TCGA data sets. In conclusion, ABCB5 genetic variants had significant association with HCC risk and aggressive tumour properties.

En bloc transdiaphragmatic lung resection for locally advanced hepatocellular carcinoma: a case report.

A 56-year-old man presented with an 11-cm hepatocellular carcinoma (HCC) at segment 7 of liver. To induce left liver hypertrophy, a sequential transarterial chemoembolization (TACE) and portal vein embolization before right hepatectomy were adopted. However, the tumor further increased in size despite TACE and invaded through the diaphragm to the right lung base. Anterior approach right hepatectomy with en bloc wedge resection of the involved right lower lobe of lung by endovascular staplers via transdiaphragmatic approach was performed. The diaphragmatic defect was closed with Goretex mesh. Patient made an uneventful recovery. Pathology confirmed a 12.5 cm poorly differentiated HCC invading through diaphragm to lung. During follow-up, patient developed a 6 cm recurrence at left lung base 17 months after surgery for which he received sorafenib therapy. However, the lung mass further increased in size with new liver recurrence at segment 3 despite treatment. He succumbed 2 years and 3 months after surgery.

Endoscopic ultrasound-guided tissue acquisition with or without macroscopic on-site evaluation: randomized controlled trial.

BACKGROUND: The use of macroscopic on-site evaluation (MOSE) to estimate the adequacy of a specimen for histological diagnosis during endoscopic ultrasound (EUS)-guided fine-needle tissue acquisition (FNTA) has recently been advocated. This study aimed to evaluate the diagnostic yield of MOSE compared with conventional EUS-FNTA without rapid on-site evaluation (ROSE). METHODS: This was an international, multicenter, prospective, randomized controlled study. After providing informed consent, consecutive adult patients referred for EUS-FNTA for solid lesions larger than 2 cm were randomized to a MOSE arm or to a conventional arm without ROSE. A designated cytopathologist from each center performed all cytopathological examinations for that center and was blinded to the randomization results. The primary outcome measure was the diagnostic yield, and the secondary outcomes included sensitivity, specificity, positive predictive value, negative predictive value, diagnostic accuracy, and the rate of procedure-related complications. RESULTS: 244 patients (122 conventional, 122 MOSE) were enrolled during the study period. No significant differences between the two arms were found in procedure time or rate of procedure-related adverse events. The diagnostic yield for the MOSE technique (92.6 %) was similar to that for the conventional technique (89.3 %; P  = 0.37), with significantly fewer passes made (median: conventional 3, MOSE 2; P  < 0.001). CONCLUSIONS: EUS-FNTA with the MOSE technique provided a similar diagnostic yield to conventional EUS-FNTA technique in the absence of ROSE but with fewer passes. This technique can be used when ROSE is not available.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas.

BACKGROUND & AIMS: Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. METHODS: Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. RESULTS: We determined the monoclonal origins of individual tumors using a single-sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phyloepigenetic analyses showed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis showed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features showed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin of recurrent HCCs allowed construction of a temporal map of genetic alterations that were associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that are associated with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution showed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the cancer genome atlas data set. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. CONCLUSIONS: We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that are associated with tumor progression and recurrence. We identified chromatin regulators that are up-regulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Pearls of experience for safe and efficient hospital practices in otorhinolaryngology-head and neck surgery in Hong Kong during the 2019 novel coronavirus disease (COVID-19) pandemic.

The 2019 novel coronavirus disease (COVID-19) epidemic originated in Wuhan, China and spread rapidly worldwide, leading the World Health Organization to declare an official global COVID-19 pandemic in March 2020. In Hong Kong, clinicians and other healthcare personnel collaborated closely to combat the outbreak of COVID-19 and minimize the cross-transmission of disease among hospital staff members. In the field of otorhinolaryngology-head and neck surgery (OHNS) and its various subspecialties, contingency plans were required for patient bookings in outpatient clinics, surgeries in operating rooms, protocols in wards and other services. Infected patients may shed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) particles into their environments via body secretions. Therefore, otolaryngologists and other healthcare personnel in this specialty face a high risk of contracting COVID-19 and must remain vigilant when performing examinations and procedures involving the nose and throat. In this article, we share our experiences of the planning and logistics undertaken to provide safe and efficient OHNS practices over the last 2 months, during the COVID-19 pandemic. We hope that our experiences will serve as pearls for otolaryngologists and other healthcare personnel working in institutes that serve large numbers of patients every day, particularly with regard to the sharing of clinical and administrative tasks during the COVID-19 pandemic.

Aspirin Reduces Colorectal Tumor Development in Mice and Gut Microbes Reduce its Bioavailability and Chemopreventive Effects.

BACKGROUND & AIMS: Alterations in the intestinal microbiota affect development of colorectal cancer and drug metabolism. We studied whether the intestinal microbiota affect the ability of aspirin to reduce colon tumor development in mice. METHODS: We performed studies with APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium to induce colorectal carcinogenesis. Some mice were given antibiotics to deplete intestinal microbes, with or without aspirin, throughout the entire experiment. Germ-free mice were studied in validation experiments. Colon tissues were collected and analyzed by histopathology, quantitative reverse-transcription polymerase chain reaction, and immunoblots. Blood samples and gut luminal contents were analyzed by liquid chromatography/mass spectrometry and an arylesterase activity assay. Fecal samples were analyzed by 16S ribosomal RNA gene and shotgun metagenome sequencing. RESULTS: Administration of aspirin to mice reduced colorectal tumor number and load in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium that had been given antibiotics (depleted gut microbiota), but not in mice with intact microbiota. Germ-free mice given aspirin developed fewer colorectal tumors than conventionalized germ-free mice given aspirin. Plasma levels of aspirin were higher in mice given antibiotics than in mice with intact gut microbiota. Analyses of luminal contents revealed that aerobic gut microbes, including Lysinibacillus sphaericus, degrade aspirin. Germ-free mice fed L sphaericus had lower plasma levels of aspirin than germ-free mice that were not fed this bacterium. There was an inverse correlation between aspirin dose and colorectal tumor development in conventional mice, but this correlation was lost with increased abundance of L sphaericus. Fecal samples from mice fed aspirin were enriched in Bifidobacterium and Lactobacillus genera, which are considered beneficial, and had reductions in Alistipes finegoldii and Bacteroides fragili, which are considered pathogenic. CONCLUSIONS: Aspirin reduces development of colorectal tumors in APCmin/+ mice and mice given azoxymethane and dextran sulfate sodium, depending on the presence of intestinal microbes. L sphaericus in the gut degrades aspirin and reduced its chemopreventive effects in mice. Fecal samples from mice fed aspirin were enriched in beneficial bacteria, with reductions in pathogenic bacteria.

Receptor tyrosine kinase fusions act as a significant alternative driver of the serrated pathway in colorectal cancer development.

Serrated polyps are a clinically and molecularly heterogeneous group of lesions that can contribute to the development of colorectal cancers (CRCs). However, the molecular mechanism underlying the development of serrated lesions is still not well understood. Here, we combined multiple approaches to analyze the genetic alterations in 86 colorectal adenomas (including 35 sessile serrated lesions, 15 traditional adenomas, and 36 conventional adenomatous polyps). We also investigated the in vitro and in vivo oncogenic properties of a novel variant of the NCOA4-RET fusion gene. Molecular profiling revealed that sessile serrated lesions and traditional serrated adenomas have distinct clinicopathological and molecular features. Moreover, we identified receptor tyrosine kinase translocations exclusively in sessile serrated lesions (17%), and the observation was validated in a separate cohort of 34 sessile serrated lesions (15%). The kinase fusions as well as the BRAF and KRAS mutations were mutually exclusive to each other. Ectopic expression of a novel variant of the NCOA4-RET fusion gene promoted cell proliferation in vitro and in vivo, and the proliferation was significantly suppressed by RET kinase inhibitors. All of these underscored the importance of mitogen-activated protein kinase (MAPK) pathway activation in the serrated pathway of colorectal tumorigenesis. In addition, we demonstrated that the kinase fusion may occur early in the precursor lesion and subsequent loss of TP53 may drives the transformation to carcinoma during serrated tumorigenesis. In conclusion, we identified kinase fusions as a significant alternative driver of the serrated pathway in colorectal cancer development, and detecting their presence may serve as a biomarker for the diagnosis of sessile serrated lesions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Virtual multiplex immunohistochemistry: Application on cell block of effusion and aspiration cytology.

BACKGROUND: With the continuous development of new antibodies, the use of immunohistochemistry (IHC) is becoming more often a requirement. IHC is frequently necessary for establishing cancer diagnosis and making therapeutic decisions. However, cytology specimens such as effusion fluid and fine-needle aspiration are highly variable in cellularity and contain background inflammatory and mesothelial cells. Compared to biopsy or excision specimens, tissue exhaustion and levels of sections not matching are more commonly encountered. We present a method of integrating whole-slide cell block image of multiple antibodies by digital image processing and reconstructing a virtual multiplex IHC image for enhanced interpretation. METHODS: Historic archived cell block preparations of carcinomas (n = 19) and melanoma (n = 1) and IHC performed with 3,3'-diaminobenzidine chromogen were reviewed. The slides were digitized by a whole-slide image scanner. Using ImageJ and self-developed code, the slides were aligned by image registration, layered, and recolored to reconstruct a virtual multiplex image, simulating a multiplex preparation with multiple chromogens. To quantity the performance of the image registration, the mean distance between the same cell clusters in aligned images were measured. RESULTS: All 20 cases were successfully registered. The mean distance between cell clusters after image registration was 8.40 +/- 5.52 µm. The reconstructed images were able to demonstrate coexpression of membranous, cytoplasmic, and nuclear antibodies. CONCLUSION: With virtual multiplex IHC, we were able to visualize coexpression of multiple antibodies without the added cost of multiplex IHC. Routine and historic slides, without additional tissue consumption, can be retrieved for image reconstruction. This technique is a low-cost adjunct to diagnosis in cytology for more efficient and accurate assessment of antibody coexpression and histochemical stains.

Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy. DESIGN: Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined. RESULTS: Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model. CONCLUSION: Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy.

"Interchangeability" of PD-L1 immunohistochemistry assays: a meta-analysis of diagnostic accuracy.

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.

Prediction of Survival Among Patients Receiving Transarterial Chemoembolization for Hepatocellular Carcinoma: A Response-Based Approach.

BACKGROUND AND AIMS: The heterogeneity of intermediate-stage hepatocellular carcinoma (HCC) and the widespread use of transarterial chemoembolization (TACE) outside recommended guidelines have encouraged the development of scoring systems that predict patient survival. The aim of this study was to build and validate statistical models that offer individualized patient survival prediction using response to TACE as a variable. APPROACH AND RESULTS: Clinically relevant baseline parameters were collected for 4,621 patients with HCC treated with TACE at 19 centers in 11 countries. In some of the centers, radiological responses (as assessed by modified Response Evaluation Criteria in Solid Tumors [mRECIST]) were also accrued. The data set was divided into a training set, an internal validation set, and two external validation sets. A pre-TACE model ("Pre-TACE-Predict") and a post-TACE model ("Post-TACE-Predict") that included response were built. The performance of the models in predicting overall survival (OS) was compared with existing ones. The median OS was 19.9 months. The factors influencing survival were tumor number and size, alpha-fetoprotein, albumin, bilirubin, vascular invasion, cause, and response as assessed by mRECIST. The proposed models showed superior predictive accuracy compared with existing models (the hepatoma arterial embolization prognostic score and its various modifications) and allowed for patient stratification into four distinct risk categories whose median OS ranged from 7 months to more than 4 years. CONCLUSIONS: A TACE-specific and extensively validated model based on routinely available clinical features and response after first TACE permitted patient-level prognostication.