Droplet digital PCR for quantification of ITGA6 in a stool mRNA assay for the detection of colorectal cancers

AIM To investigate the use of droplet digital polymerase chain reaction(dd PCR) for detecting host m RNA markers in stools as a non-invasive test for colorectal cancer screening.METHODS dd PCR and quantitative PCR were compared side by side for their performance in the detection of ITGA6 and ITGA6 A transcripts in stool samples obtained from patients with various types of colorectal lesions(advanced adenomas and stage Ⅱ-Ⅳ colorectal cancers) and control(patients displaying no pathological findings) using duplex Taq Man reactions for both methods. ITGA6 and ITGA6 A were chosen for this proof-of-concept study based on their relative medium and low abundance in stool samples, respectively, as established in a previous study.RESULTS We found that the dd PCR and q PCR methods per-formed equally well in this Taq Man duplex assay for the detection of ITGA6 and ITGA6 A transcripts in stools of patients with colorectal lesions. For ITGA6, receiver operating characteristic(ROC) curve analysis showed comparable areas under the curve of 0.91(P < 0.0001) and 0.89-0.90(P < 0.0001) for the prediction of advanced adenomas and colorectal cancers, respectively. ITGA6 A, which was detected at very low levels in control patients, was found to be significantly elevated(over 40 times) in stage Ⅱ and Ⅲ colorectal cancers(P < 0.0002). Comparison of the two sets of data revealed a strong correlation of the copy numbers obtained by dd PCR and q PCR for both ITGA6 and ITGA6 A.CONCLUSION We found that ITGA6 and ITGA6 A detection in stools of patients with colorectal cancers with dd PCR is comparable to that of q PCR using Taq Man assays.