An additional challenge that has not been met by the current literature will be identifying use patterns for specific substance in the presence of poly-drug use. Although some of the studies mentioned above were able to identify the presence of poly-drug use through objective measures such as cotinine and hydroxyl-THC ELISA [101], it remains unclear how best to control for the presence of poly-drug use in epigenetic analyses targeted toward biomarker development. Specifically, with respect to smoking, the high rate of comorbid smoking among users of other substances and the relatively strong epigenetic signature of smoking may obscure detection of signals from other compounds. To overcome these challenges, careful study design using objective markers of use are essential. If methylation signatures of substances such as cocaine and opioids are relatively subtle, misclassification of use patterns in case versus control designs has the potential to seriously damage the power of such studies. From a bioinformatics perspective, it is also likely that more sophisticated algorithms will need to be developed to carefully distinguish the epigenetic signatures of different poly-drug use patterns, for example cannabis smoking in individuals who also smoke tobacco.
