Importance Four assays have been registered with the FDA to detect PD-L1 to enrich for patient response to anti-PD-1/PD-L1 therapies. The tests use four separate PD-L1 antibodies on two separate staining platforms and have their own scoring systems which raises questions about their similarity and potential cross-utilization. Objective We compared the performance of four PD-L1 platforms, including two FDA-cleared assays and two laboratory developed tests (LDTs). Design Four serial histology sections from 90 archival NSCLCs were distributed to three sites that performed the following IHCs: 1) 28-8 antibody on Dako Link 48; 2) 22c3 antibody on Dako Link 48; 3) SP142 antibody on Ventana Benchmark; and 4) E1L3N antibody on Leica Bond. Slides were scanned and scored by thirteen pathologists by estimating the percentage of malignant and immune cells expressing PD-L1. Intraclass correlation coefficients (ICC) and paired and mixed effects statistical analyses were performed to compare antibodies and pathologists scoring of tumor and immune cells. Results The SP142 Ventana assay was an outlier with a significantly lower mean score of PD-L1 expression in both tumor and immune cells. Pairwise comparisons showed the 28-8 and E1L3N were not significantly different, but that 22c3 showed a slight but statistically significant reduction in tumor cell labeling. Evaluation of ICC between antibodies to quantify inter-assay variability using the average of thirteen pathologists scores for tumor shows very high concordance between antibodies for tumor cell scoring (0.813) and lower levels of concordance for immune cell scoring (0.277). When examining inter-pathologists variability for any single antibody, the concordance between pathologists’ reads for tumor ranged from ICC of 0.83 to 0.88 for each antibody while the ICC from immune cells for each antibody ranged from 0.17 to 0.23. Conclusions The assay using the SP142 antibody is a clear outlier detecting significantly less tumor cell and immune cell PD-L1 expression. Antibody 22c3 shows slight yet statistically significantly lower staining than either 28-8 or E1L3N, but this significance is only detected when using the average of thirteen pathologist scores. Pathologists show excellent concordance when scoring tumor cells stained with any antibody, but poor concordance for scoring immune cell staining.

Fructose intake from added sugars correlates with the epidemic rise in obesity, metabolic syndrome, and nonalcoholic fatty liver disease. Fructose intake also causes features of metabolic syndrome in laboratory animals and humans. The first enzyme in fructose metabolism is fructokinase, which exists as two isoforms, A and C. Here we show that fructose-induced metabolic syndrome is prevented in mice lacking both isoforms but is exacerbated in mice lacking fructokinase A. Fructokinase C is expressed primarily in liver, intestine, and kidney and has high affinity for fructose, resulting in rapid metabolism and marked ATP depletion. In contrast, fructokinase A is widely distributed, has low affinity for fructose, and has less dramatic effects on ATP levels. By reducing the amount of fructose for metabolism in the liver, fructokinase A protects against fructokinase C-mediated metabolic syndrome. These studies provide insights into the mechanisms by which fructose causes obesity and metabolic syndrome.ketohexokinase | hepatic steatosis | insulin | leptin

Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with high fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild type or fructokinase knockout mice were fed a low fat (11%), high fat (36%) or high fat (36%) and high sucrose (30%) diet for 15 weeks. Both wild type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence for hepatic inflammation on a high fat diet compared to a low fat diet. In contrast, wild type mice fed a high fat and high sucrose diet developed more severe hepatic steatosis with low grade inflammation and fibrosis, as noted by increased CD68, TNF-alpha, MCP-1, alpha-smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice. Conclusion An additive effect of high fat and high sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis (NASH).

Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver.

Carbohydrates with high glycemic index are proposed to promote the development of obesity, insulin resistance and fatty liver, but the mechanism by which this occurs remains unknown. High serum glucose concentrations glucose are known to induce the polyol pathway and increase fructose generation in the liver. Here we show that this hepatic, endogenously-produced fructose causes systemic metabolic changes. We demonstrate that mice unable to metabolize fructose are protected from an increase in energy intake and body weight, visceral obesity, fatty liver, elevated insulin levels and hyperleptinemia after exposure to 10% glucose for 14 weeks. In normal mice, glucose consumption is accompanied by aldose reductase and polyol pathway activation in steatotic areas. In this regard, we show that aldose reductase deficient mice were protected against glucose-induced fatty liver. We conclude that endogenous fructose generation and metabolism in the liver represents an important mechanism whereby glucose promotes the development of metabolic syndrome.

LAG-3 is expressed on TILs in tumor tissues of some patients with NSCLC. Its expression was higher in nonadenocarcinoma and correlated with PD-1/PD-L1 expression. LAG-3 positivity or both LAG-3 and PD-L1 positivity was correlated with early postoperative recurrence. LAG-3 was related to poor prognosis.

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Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation –either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.