Identifying the pathways that are significantly impacted in a given condition is 
a crucial step in understanding the underlying biological phenomena. All 
approaches currently available for this purpose calculate a P-value that aims to 
quantify the significance of the involvement of each pathway in the given 
phenotype. These P-values were previously thought to be independent. Here we 
show that this is not the case, and that many pathways can considerably affect 
each other's P-values through a "crosstalk" phenomenon. Although it is intuitive 
that various pathways could influence each other, the presence and extent of 
this phenomenon have not been rigorously studied and, most importantly, there is 
no currently available technique able to quantify the amount of such crosstalk. 
Here, we show that all three major categories of pathway analysis methods 
(enrichment analysis, functional class scoring, and topology-based methods) are 
severely influenced by crosstalk phenomena. Using real pathways and data, we 
show that in some cases pathways with significant P-values are not biologically 
meaningful, and that some biologically meaningful pathways with nonsignificant 
P-values become statistically significant when the crosstalk effects of other 
pathways are removed. We describe a technique able to detect, quantify, and 
correct crosstalk effects, as well as identify independent functional modules. 
We assessed this novel approach on data from four experiments involving three 
phenotypes and two species. This method is expected to allow a better 
understanding of individual experiment results, as well as a more refined 
definition of the existing signaling pathways for specific phenotypes.