The Cytokinome Profile in Patients with Hepatocellular Carcinoma and Type 2 Diabetes

<div><p>Understanding the dynamics of the complex interaction network of cytokines, defined as ‘‘cytokinome’’, can be useful to follow progression and evolution of hepatocellular carcinoma (HCC) from its early stages as well as to define therapeutic strategies. Recently we have evaluated the cytokinome profile in patients with type 2 diabetes (T2D) and/or chronic hepatitis C (CHC) infection and/or cirrhosis suggesting specific markers for the different stages of the diseases. Since T2D has been identified as one of the contributory cause of HCC, in this paper we examined the serum levels of cytokines, growth factors, chemokines, as well as of other cancer and diabetes biomarkers in a discovery cohort of patients with T2D, chronic hepatitis C (CHC) and/or CHC-related HCC comparing them with a healthy control group to define a profile of proteins able to characterize these patients, and to recognize the association between diabetes and HCC. The results have evidenced that the serum levels of some proteins are significantly and differently up-regulated in all the patients but they increased still more when HCC develops on the background of T2D. Our results were verified also using a separate validation cohort. Furthermore, significant correlations between clinical and laboratory data characterizing the various stages of this complex disease, have been found. In overall, our results highlighted that a large and simple omics approach, such as that of the cytokinome analysis, supplemented by common biochemical and clinical data, can give a complete picture able to improve the prognosis of the various stages of the disease progression. We have also demonstrated by means of interactomic analysis that our experimental results correlate positively with the general metabolic picture that is emerging in the literature for this complex multifactorial disease.</p></div

Significant cytokines in some patient groups belonging to validation set.

<p>We report the significant molecule levels from controls, patients with type 2 diabetes (T2D), chronic hepatitis C (CHC), hepatocellular carcinoma (HCC) and hepatocellular carcinoma and type 2 diabetes (T2D-HCC) shown by means of box-and-whisker graphs. The boxes extend from the 25th to the 75th percentile, and the line in the middle is the median. The error bars extend down to the lowest value and up to the highest.</p

Metabolic pathways showing the constitutive proteins considered as significantly involved.

<p>Metabolic pathways showing the constitutive proteins considered as significantly involved.</p

Significant cytokines in some patient groups belonging to discovery set.

<p>We report the significant molecule levels from controls, patients with type 2 diabetes (T2D), chronic hepatitis C (CHC), hepatocellular carcinoma (HCC) and hepatocellular carcinoma and type 2 diabetes (T2D-HCC) shown by means of box-and-whisker graphs. The boxes extend from the 25th to the 75th percentile, and the line in the middle is the median. The error bars extend down to the lowest value and up to the highest.</p

Interactomic analysis of the significant molecules performed by means of the Ingenuity Pathway Analysis (IPA).

<p>The interactome shows the close functional association between significant cytokines (evidenced by yellow symbols) as well as the paths in which other functionally relevant molecules are also involved (evidenced by white symbols). Moreover, the six HUB nodes are evidenced by cyan symbols. On the left side the cellular localization of the molecules in the graph is shown.</p

Clinical and laboratory data of patients with type 2 diabetes (T2D), chronic HCV (CHC), HCC with HCV-related cirrhosis (HCC), and HCC with HCV-related cirrhosis and type 2 diabetes (T2D+HCC) belonging to discovery set.

<p>The corresponding patients belonging to validation set are indicated as T2D^V, CHC^V, HCC^V and T2D+HCC^V. We report the number of patients to whom the parameters refer. The related control ranges of the clinical data evaluated for the healthy donors, are also shown.</p

Comparison of cytokine serum levels between discovery and validation sets in patients and healthy controls.

<p>We report the results of all the performed statistical analysis obtained by the nonparametric Mann-Whitney U test in terms of U test and P values, by the Unparied t test in terms of P value, t, the number of degrees of freedom (df), the difference between the means, 95% confidence interval, and R squared, and by F test in terms of F, degrees of freedom for the numerator (DFn) and for the denominator (Dfd) and P value.</p

Statistical evaluation on the serum levels (expressed in pg/mL) of significant cytokines in the healthy controls and in four patient groups belonging to discovery set.

<p>We report for each cytokine the minimum and maximum values, the 25% and 75% Percentiles, the median, the mean, standard deviation, standard error, and the lower and upper 95% confidence intervals (CI).</p

Comparison of cytokine serum levels between patients and healthy controls in the discovery set.

<p>We report the results of all the performed statistical analysis obtained by the nonparametric Mann-Whitney U test in terms of U test and P values, by the Unparied t test in terms of P value, t, the number of degrees of freedom (df), the difference between the means, 95% confidence interval, and R squared, and by F test in terms of F, degrees of freedom for the numerator (DFn) and for the denominator (Dfd) and P value. In particular, we reported in bold the values of p<0.05 indicated with *, of p<0.01 with **, and of p<0.0001 with ***.</p

Study flow chart.

<p>Study flow chart.</p