PKCα/ZFP64/CSF1 axis resets the tumor microenvironment and fuels anti-PD1 resistance in hepatocellular carcinoma.

BACKGROUND & AIMS: Despite remarkable advances in treatment, most patients with hepatocellular carcinoma (HCC) respond poorly to anti-programmed cell death 1 (anti-PD1) therapy. A deeper insight into the tolerance mechanism of HCC against this therapy is urgently needed. METHODS: We performed next-generation sequencing, multiplex immunofluorescence, and dual-color immunohistochemistry and constructed an orthotopic HCC xenograft tumor model to identify the key gene associated with anti-PD1 tolerance. A spontaneously tumorigenic transgenic mouse model, an in vitro coculture system, mass cytometry, and multiplex immunofluorescence were used to explore the biological function of zinc finger protein 64 (ZFP64) on tumor progression and immune escape. Molecular and biochemical strategies like RNA-sequencing, chromatin immunoprecipitation-sequencing and mass spectrometry were used to gain insight into the underlying mechanisms of ZFP64. RESULTS: We showed that ZFP64 is frequently upregulated in tumor tissues from patients with anti-PD1-resistant HCC. Elevated ZFP64 drives anti-PD1 resistance by shifting macrophage polarization toward an alternative activation phenotype (M2) and fostering an inhibitory tumor microenvironment. Mechanistically, we primarily demonstrated that protein kinase C alpha (PKCα) directly phosphorylates ZFP64 at S226, leading to its nuclear translocation and the transcriptional activation of macrophage colony-stimulating factor (CSF1). HCC-derived CSF1 transforms macrophages to the M2 phenotype to drive immune escape and anti-PD1 tolerance. Notably, Gö6976, a protein kinase inhibitor, and lenvatinib, a multi-kinase inhibitor, reset the tumor microenvironment and restore sensitivity to anti-PD1 by blocking the PKCα/ZFP64/CSF1 axis. CONCLUSIONS: We propose that the PKCα/ZFP64/CSF1 axis is critical for triggering immune evasion and anti-PD1 tolerance. Inhibiting this axis with Gö6976 or lenvatinib overcomes anti-PD1 resistance in HCC. LAY SUMMARY: Despite remarkable treatment progress, most patients with hepatocellular carcinoma respond poorly to anti-PD1 therapy (a type of immunotherapy). A deeper insight into the tolerance mechanisms to this therapy is urgently needed. Herein, we unravel a previously unexplored mechanism linking tumor progression, macrophage polarization, and anti-PD1 resistance, and offer an attractive novel target for anti-PD1 combination therapy, which may benefit patients with hepatocellular carcinoma.

Efficacy of Berberine in Patients with Non-Alcoholic Fatty Liver Disease.

OBJECTIVES: A randomized, parallel controlled, open-label clinical trial was conducted to evaluate the effect of a botanic compound berberine (BBR) on NAFLD. METHODS: A randomized, parallel controlled, open-label clinical trial was conducted in three medical centers (NIH Registration number: NCT00633282). A total of 184 eligible patients with NAFLD were enrolled and randomly received (i) lifestyle intervention (LSI), (ii) LSI plus pioglitazone (PGZ) 15mg qd, and (iii) LSI plus BBR 0.5g tid, respectively, for 16 weeks. Hepatic fat content (HFC), serum glucose and lipid profiles, liver enzymes and serum and urine BBR concentrations were assessed before and after treatment. We also analyzed hepatic BBR content and expression of genes related to glucose and lipid metabolism in an animal model of NAFLD treated with BBR. RESULTS: As compared with LSI, BBR treatment plus LSI resulted in a significant reduction of HFC (52.7% vs 36.4%, p = 0.008), paralleled with better improvement in body weight, HOMA-IR, and serum lipid profiles (all p<0.05). BBR was more effective than PGZ 15mg qd in reducing body weight and improving lipid profile. BBR-related adverse events were mild and mainly occurred in digestive system. Serum and urine BBR concentrations were 6.99ng/ml and 79.2ng/ml, respectively, in the BBR-treated subjects. Animal experiments showed that BBR located favorably in the liver and altered hepatic metabolism-related gene expression. CONCLUSION: BBR ameliorates NAFLD and related metabolic disorders. The therapeutic effect of BBR on NAFLD may involve a direct regulation of hepatic lipid metabolism. TRIAL REGISTRATION: ClinicalTrials.gov NCT00633282.

Orthotopic inflammation-related pancreatic carcinogenesis in a wild-type mouse induced by combined application of caerulein and dimethylbenzanthracene.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies, with a poor long-term prognosis, and effective therapeutic options are lacking. Observing the dynamics of the pathogenesis of pancreatic intraepithelial neoplasia (PanIN) and PDAC in tumor models can facilitate understanding of the molecular mechanisms involved in early PDAC. Furthermore, it can compensate for the research limitations associated with analyzing clinical specimens of late-stage PDAC. In this study, we orthotopically treated the pancreas with dimethylbenzanthracene (DMBA) combined with caerulein in wild-type C57BL/6 J mice to induce inflammation-related pancreatic carcinogenesis. We observed that DMBA and caerulein treatment induced a chronic consumptive disease, which caused a decrease in the relative body and pancreas weights, diminishing the health status of the mice and enhancing the inflammation-related histological changes. Moreover, mid-dose and high-frequency treatment with caerulein caused prolonged inflammatory damage to the pancreas and contributed to a permissive environment for the development of PDAC. CXCL12/CXCR4, CCL2/CCR2, and several cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α were upregulated in the tumor tissue of DMBA and caerulein-induced PDAC mice. This orthotopic mouse pancreatic carcinogenesis model mimic human disease because it reproduces a spectrum of pathological changes observed in human PDAC, ranging from inflammatory lesions to pancreatic intraepithelial neoplasia. Thus, this mouse model may improve the understanding of molecular mechanisms underlying the injury-inflammation-cancer pathway in the early stages of pancreatic carcinogenesis.

Comparison of diffusion-weighted MRI acquisition techniques for normal pancreas at 3.0 Tesla.

PURPOSE: We aimed to optimize diffusion-weighted imaging (DWI) acquisitions for normal pancreas at 3.0 Tesla. MATERIALS AND METHODS: Thirty healthy volunteers were examined using four DWI acquisition techniques with b values of 0 and 600 s/mm2 at 3.0 Tesla, including breath-hold DWI, respiratory-triggered DWI, respiratory-triggered DWI with inversion recovery (IR), and free-breathing DWI with IR. Artifacts, signal-to-noise ratio (SNR) and apparent diffusion coefficient (ADC) of normal pancreas were statistically evaluated among different DWI acquisitions. RESULTS: Statistical differences were noticed in artifacts, SNR, and ADC values of normal pancreas among different DWI acquisitions by ANOVA (P <0.001). Normal pancreas imaging had the lowest artifact in respiratory-triggered DWI with IR, the highest SNR in respiratory-triggered DWI, and the highest ADC value in free-breathing DWI with IR. The head, body, and tail of normal pancreas had statistically different ADC values on each DWI acquisition by ANOVA (P < 0.05). CONCLUSION: The highest image quality for normal pancreas was obtained using respiratory-triggered DWI with IR. Normal pancreas displayed inhomogeneous ADC values along the head, body, and tail structures.

Clinical strategies for differentiating autoimmune pancreatitis from pancreatic malignancy to avoid unnecessary surgical resection.

OBJECTIVE: The study aimed to determine a practical strategy for differentiating between autoimmune pancreatitis (AIP) and pancreatic malignancy in order to avoid unnecessary surgical resection. METHODS: Altogether, 19 patients with AIP or other pancreatic diseases underwent routine examinations including liver function test and carbohydrate antigen 19-9, computed tomography and/or magnetic resonance imaging. Serum immunoglobulin G (IgG) and/or IgG4 was determined in patients with clinically suspected or pathologically proven AIP. Patients with suspected AIP either received diagnostic steroid therapy or laparotomy (if malignant tumors could not be excluded). Surgery was not performed in patients with a definite diagnosis of AIP by fast intraoperative frozen biopsy. Those with confirmed AIP received steroid treatment. RESULTS: In total, 15 cases were finally confirmed as AIP with eight diagnosed preoperatively, five confirmed by surgical pathology (preoperatively misdiagnosed) and two by intraoperative biopsy. Of these 15 patients with AIP and one without AIP, 14 had elevated serum γ-globulin levels. It was proven by subsequent antibody tests that serum IgG or IgG4 were simultaneously increased. CONCLUSIONS: Elevated serum γ-globulin level can be used as a preoperative sentinel indicator for differentiating between IgG4-related AIP and pancreatic malignancy. Serum IgG or IgG4 tests should be further performed in those with elevated serum γ-globulin level, which helps to identify AIP in order to avoid unnecessary operation.

[Liver disease spectrum in hospitalized type 2 diabetes and related risk factors analysis of non-alcoholic fatty liver disease].

OBJECTIVE: To explore the liver disease spectrum in patients with type 2 diabetes (T2DM) and the risk factors of non-alcoholic fatty liver disease (NAFLD). METHODS: From September 2009 to October 2011, 1069 hospitalized patients with T2DM in Department of Endocrinology and Metabolism were involved in the study. The history informations, results of laboratory examination, hepatic ultrasound and hepatic proton magnetic resonance spectrum ((1)H MRS) of all patients were collected to analysis. RESULTS: (1) The detectable rate of raised liver enzymes in T2DM patients was 28.7% (307/1069), composed mainly of NAFLD (39.4%, 121/307). After excluding the factors such as alcoholic abuse, viral hepatitis, the detect rate of raised liver enzymes in T2DM patients was 26.9% (185/688). (2) The detectable rate of fatty liver by ultrasound in T2DM patients was 56.7% (500/882), composed mainly of NAFLD (72.6%, 363/500), and the detectable rate of NAFLD was 58.0% (363/626). (3) The detectable rate of fatty liver by hepatic (1)H MRS was 72.8% (227/312), composed mainly of NAFLD (69.6%, 158/227). The detectable rate of NAFLD was 69.6% (158/227). (4) Of the three methods for diagnosing NAFLD, (1)H MRS had the highest detectable rate, followed by ultrasound, and the hepatic enzymes was the lowest. Set the hepatic (1)H MRS as gold diagnosing standard of NAFLD, the combination of hepatic enzymes and ultrasound increase the sensitivity. The optional cut-off points of ALT were 19.7 U/L (male, ROCAUC = 0.689, P < 0.01) and 17.0 U/L (female, ROCAUC = 0.727, P < 0.01). (5) Logistic stepwise regression analysis showed sex, BMI, hemoglobin, fasting C-peptide and uric acid (OR = 3.803, 1.195, 1.037, 2.896, 1.011, all P < 0.05) were positively correlated with NAFLD, and diabetes duration (OR = 0.948, P < 0.05) was positively correlated with NAFLD independently. CONCLUSIONS: The detectable rate of fatty liver was high in T2DM which was composed mainly of NAFLD. High abnormal liver enzymes detectable rate indicated that NAFLD with T2DM are prone to NASH.

Glomus tumor in the stomach: computed tomography and endoscopic ultrasound findings.

A 57-year-old man presented with intermittent dull abdominal pain after a period of 1 year. Abdominal computed tomography (CT) was performed. Except for the endoscopy, the work-up for possible medical causes remained inconclusive. An open-abdomen, partial surgical excision of the stomach was performed after the unsuccessful endoscopic resection. The pathology report revealed a glomus tumor of the stomach. Importantly, glomus tumors of the stomach are rare and are almost always benign. Therefore, the most important current role of imaging associated with the diagnostic approach and therapeutic plan for a glomus tumor is to differentiate it from other gastric submucosal tumors (SMTs). We report this case with representative radiologic findings, including CT and endoscopic ultrasound (EUS) reports, and also correlate them with clinical and pathologic presentations that can help in the early detection and differentiation of gastric SMTs from other SMTs. As such, the purpose of this report is to provide a better understanding of relevant CT and EUS features. Alternative treatments should be considered carefully according to the imaging results.

Evaluation of ADC measurements among solid pancreatic masses by respiratory-triggered diffusion-weighted MR imaging with inversion-recovery fat-suppression technique at 3.0T.

PURPOSE: The objective of this paper was to investigate the value of apparent diffusion coefficients (ADCs) for differential diagnosis among solid pancreatic masses using respiratory triggered diffusion-weighted MR imaging with inversion-recovery fat-suppression technique (RT-IR-DWI) at 3.0 T. MATERIALS AND METHODS: 20 normal volunteers and 72 patients (Pancreatic ductal adenocarcinoma [PDCA, n=30], mass-forming pancreatitis [MFP, n=15], solid pseudopapillary neoplasm [SPN, n=12], and pancreatic neuroendocrine tumor[PNET, n=15]) underwent RT-IR-DWI (b values: 0 and 600 s/mm(2)) at 3.0 T. Results were correlated with histopathologic data and follow-up imaging. ADC values among different types of pancreatic tissue were statistically analyzed and compared. RESULTS: Statistical difference was noticed in ADC values among normal pancreas, MFP, PDCA, SPN and PNET by ANOVA (p<.001). Normal pancreas had the highest ADC value, then followed by PNET, PDCA, MFP and SPN. There was noticeable statistical difference in ADC values among PDCA, MFP and normal pancreas by Least Significant Difference (LSD) (p<.001). ADC of SPN was statistically lower than that of PNET (p=0.1800×10(-4)), PDCA (p=0.0300×10(-4)) and normal pancreas (p=0.0007×10(-4)). ADC of PNET was statistically lower than that of normal pancreas (p=0.0360) and higher than that of MFP (p=9.3000×10(-4)). CONCLUSIONS: ADC measurements using RT-IR-DWI at 3.0T may aid to disclose the histopathological pattern of normal pancreas and solid pancreatic masses, which may be helpful in characterizing solid pancreatic lesions.

Standardized ultrasound hepatic/renal ratio and hepatic attenuation rate to quantify liver fat content: an improvement method.

Accurate measures of liver fat content are essential for investigating the role of hepatic steatosis in the pathophysiology of multiple metabolic disorders. No traditional imaging methods can accurately quantify liver fat content. [(1)H]-magnetic resonance spectroscopy (MRS) is restricted in large-scale studies because of the practical and technological issues. Previous attempts on computer-aided ultrasound quantification of liver fat content varied in method, and the ultrasound quantitative parameters measured from different ultrasound machines were hardly comparable. We aimed to establish and validate a simple and propagable method for quantitative assessment of liver fat content based on the combination of standardized ultrasound quantitative parameters, using [(1)H]-MRS as gold standard. Totally 127 participants were examined with both ultrasonography (US) and [(1)H]-MRS. Ultrasound hepatic/renal echo-intensity ratio (H/R) and ultrasound hepatic echo-intensity attenuation rate (HA) were obtained from ordinary ultrasound images using computer program. Both parameters were standardized using a tissue-mimicking phantom before analysis. Standardized ultrasound H/R and HA were positively correlated with the liver fat content by [(1)H]-MRS (r = 0.884, P < 0.001 and r = 0.711, P < 0.001, respectively). Linear regression analysis showed ultrasound H/R could modestly predict the amount of liver fat (adjusted explained variance 78.0%, P < 0.001). The addition of ultrasound HA slightly improved the adjusted explained variance to 79.8%. Difference of estimated liver fat contents between different ultrasound machines and operators was reasonably well. Thus, computer-aided US is a valid method to estimate liver fat content and can be applied extensively after standardization of ultrasound quantitative parameters.

Small solid tumors (< or = 2 cm) of the pancreas: relative accuracy and differentiation of CT and MR imaging.

BACKGROUND/AIMS: To determine the relative accuracy of CT and MRI in characterization of solid pancreatic masses (< or = 2cm) and useful imaging appearance for differentiating small pancreatic duct adenocarcinoma (PDAC) from other small solid pancreatic neoplasms. METHODOLOGY: CT and MRI scans of 46 patients with evidence of small pancreatic solid tumor were retrospectively evaluated, who underwent CT (n=30), MRI (n=4), or both (n=12). Two gastrointestinal radiologists independently recorded specific morphological features of tumors and the most likely diagnosis. RESULTS: With respect to specific histopathological characterization, CT and MRI were equally accurate. The mean number of correct diagnoses made by the two reviewers was 37.5 (88.4%) of 42 cases for CT compared to 12 (75%) of 16 cases for MRI (p=0.388). Location in the pancreatic head (p=0.000), presence of dilatation of MPD (p=0.000), presence of dilatation of CBD (p=0.001) and enhancement pattern (p=0.000) were statistically significant for differentiating PDAC from the other small solid pancreatic tumors, while pancreatic atrophy (p=0.069) was statistically inadequate for differentiation, although it gave a 96.4% specificity for the diagnosis of PDAC. CONCLUSIONS: CT and MRI are similarly accurate in the characterization of small solid pancreatic tumors. Small PDAC has characteristic CT and MRI findings that differentiate it from other small solid tumors.

Elevation of liver enzymes within the normal limits and metabolic syndrome.

1. Metabolic syndrome is frequently associated with elevated liver enzymes. However, the current 'normal' limits for liver enzymes often fail to identify patients with metabolic syndrome and the associated non-alcoholic fatty liver disease (NAFLD). 2. In the present study, 1503 participants, aged between 18 and 95 years, were recruited from the physical examination centre of Shanghai Zhongshan Hospital and Shanghai Changfeng Community Health Centre. The association between liver enzymes within the 'normal' range and metabolic syndrome was investigated and optimal cut-off values for liver enzymes in metabolic syndrome were determined. We further compared the diagnostic performance of the new cut-off values for liver enzymes in metabolic syndrome and NAFLD with the traditional 'normal' range for liver enzymes. 3. Serum liver enzymes within the traditional 'normal' limits, especially alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (GGT), were correlated with most of components of the metabolic syndrome, as determined by Spearman's partial correlation analysis. Logistic regression analysis revealed that within the 'normal' range of liver enzymes, the frequency of metabolic syndrome was significantly increased in the higher quintile for ALT and GGT compared with the lowest quintile. Receiver operating characteristic curve analysis revealed that the optimal cut-off values for ALT, aspartate aminotransferase and GGT to identify metabolic syndrome were 26, 25 and 29 U/L, respectively, in men and 20, 23 and 21 U/L, respectively, in women. These values were much more effective in detecting patients with potential metabolic syndrome and NAFLD than the traditional cut-off values. 4. A slight elevation of liver enzymes within the 'normal' limits, especially ALT and GGT, indicates the presence of metabolic syndrome and NAFLD. Revision of the current normal limits for liver enzymes is advisable so that patients with potential metabolic disorders can be identified.

[Relationship between liver fat content and liver enzymes in individuals with various statuses of glucose metabolism].

OBJECTIVE: To study the relationship between liver fat content (LFC) and liver enzymes in individuals with various statuses of glucose metabolism. METHODS: A total of 109 subjects including with impaired glucose regulation (IGR) (n = 31), newly diagnosed type 2 diabetes (NT2DM) (n = 31) and normal glucose tolerance (NGT) (n = 47) were recruited. The level of LFC was measured by (1)H magnetic resonance spectroscopy ((1)H-MRS) to study the relationship between liver fat content (LFC) and alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP) and γ-glutamyltransferase (GGT). The receiver operating characteristic curve (ROC) was employed to obtain the optimal cut-off point of ALT to predict the occurrence of nonalcoholic fatty liver disease (NAFLD). RESULTS: (1) The levels of LFC were progressively raised in NGT, IGR and NT2DM groups respectively [3.83 (2.35 - 7.59)%, 12.82 (8.10 - 21.37)% and 21.99 (11.89 - 34.43)%, P < 0.01]; (2) the subjects were divided into four subgroups by the method of LFC quartile. And quartile subgroups Q1-4 were associated with the increase of LFC. Waist, BMI, systolic blood pressure, triglyceride, total cholesterol, fasting plasma glucose, OGTT 2 h postprandial glucose and HOMA-IR had a rising trend from Q2. But HDL-C showed a declining trend from Q2; (3) ALT and GGT significantly increased from Q3 (P < 0.01) while AST and AKP significantly increased in Q4 (P < 0.01); (4) adjusted by gender, age and body mass index (BMI), LFC was positively correlated with AST (r = 0.329, P < 0.05), ALT (r = 0.454) and GGT (r = 0.378) (All P < 0.01). But it was negatively correlated with AST/ALT (r = -0.364, P < 0.01); (5) the analysis of stepwise regression demonstrated that LFC was a predictor of ALT, AST, GGT and AST/ALT; (6) ALT had a ROC(AUC) of 0.813 (male) and 0.769 (female) (All P < 0.01). The optimal cut-off point of 23.5 U/L (male) and 17.5 U/L (female) might predict the occurrence of NAFLD. CONCLUSIONS: Liver enzymes are correlated with LFC even in normal range. The optimal cut-off point of 23.5 U/L (male) and 17.5 U/L (female) might predict the occurrence of NAFLD. The current used ALT upper limit could underestimate the NAFLD.