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Cytokines play a major role in driving immune responses in cancer and other diseases. There is a critical need to understand how they interact and regulate one another. In this study, we examine new approaches to understand meaningful networks between cytokines and their relevance to human disease. Effect size (ES) is a way of assessing the difference between two groups with respect to a continuous measure such as cytokines. Its use, relative to p-values, gives easier understanding to whether a difference is actually clinically important or not. Results of a study can be statistically significant but still be too small to be of any practical value, or statistically insignificant but still be too large to not be of any practical value; especially when multiple comparisons adjustments are applied. As opposed to p-values, ES is independent of sample size, and thus has recently attracted the attention of researchers. When modeling cytokines, we recommend the use of the A measure of stochastic superiority A = [#Y1 > Y2) + 0.5#(Y1=Y2)]/n1n2, due to Delaney and Vargha (2002), which is appropriate for the nonparametric Wilcoxon Test. The ES of interest from this measure is A/1 – A which gives the odds that an individual in one group will score higher than an individual in the other group. Our simulation indicated that odds= 1.253, 1.763, and 2.500 are equivalent to Cohen’s d = 0.2 (small), 0.5 (medium), and 0.8 (large). We applied this measure on a panel of 26 cytokines identified in 29 subjects (15 with Metastasis and 14 without Metastasis) with gastric cancer, and revealed 11 cytokines with large ES, five of which with non-significant Sidak adjusted p-values: (from largest to smallest) G-CSF, IL-6, IL-8, MCP-1, IL-10, TNF-a, GM-CSF, MIP-1a, IL-1b, MIP-1b, and VEGF.

Polarization in the CD4+ mediated T-helper cell immune response is a very well characterized phenomenon in immunology; towards either Th1, Th2, or Th17 pathways. However, to-date, this concept has neither been developed nor demonstrated in a robust statistical framework. In this study, we provide a novel statistical methodology to facilitate the idea of polarization within a multivariate setting, and apply it to luminal fluid cytokine data of 90 women from the Peruvian Amazon. The panel of cytokines and chemokines for this study were assayed using separate BioRad Multiplex Array kits. In our application, we show that soil transmitted helminth (STH) infection induces statistically significant Th2 polarization in the female genital tract. To provide statistical evidence of Th2 polarization in STH-infected women at the cervix, we compare the clustering of cytokines among STH-infected versus STH-uninfected women while examining the shift in the strength of correlation for Th2 cytokines with IL-4, which is seen as a central Th2 cytokine. The proposed statistical test is one that tests if, significantly, k>1 for f(L1)=f(L2) where L1 and L2 are the loadings in the principal component analysis (PCA) framework for the STH-infected and STH-infected groups respectively. The proposed method is a confirmatory one and could be supported by visual representation of the Th polarization through heat maps. It has the potential to become the reference or gold standard when studying the polarization phenomenon in immunology as it tailors the clinical characteristics and mechanisms associated with the entire panel of cytokines and chemokines in the context of a system.

Several studies have investigated the role of cytokines/chemokines in infection and chronic and autoimmune diseases using different statistical models. Generally, only two mediators are evaluated at a time, by comparing group-specific mean concentration differences. Restricted pairwise analysis from a large array of markers which inherently operate at a systems level to regulate immune response ignores important inter-correlation structures, compromising clinical and epidemiological inference. We propose the use of a rooted correlation network as a novel statistical method which allows group-specific comparisons of a panel of soluble immune markers while adjusting for the interaction complexity. We examined soluble immune markers in luminal fluid samples from 90 HPV infected female participants with and without soil transmitted helminth (STH) co-infection in Peru. We constructed a lower triangular correlation matrix using Spearman’s correlation with Sidak adjustment for both the STH-infected and -uninfected women. Rooted correlation network was constructed for the cytokines and chemokines that were insignificant in the non-helminth group but became significant in the helminth one. The generated cytokine network demonstrated statistically significant association of Th2 pathway cytokines with the canonical Th2 cytokine IL-4. Using this new statistical model, we demonstrate that the female genital tract of STH-infected women reveals polarization towards Th2 immune response (IL-25, IL-21, IL-5, IL-10, IL-8, and IL-31) in comparison to STH-uninfected women, which was not easily recognizable when using standard bivariate methods with multiple comparisons.