Use of integrin alpha 6 transcripts in a stool mRNA assay for the detection of colorectal cancers at curable stages

Abstract

Objective: An important criterion for colorectal cancer (CRC) screening is the ability to detect lesions at a curable stage. In the present study, we have assessed the integrin α6 subunit transcript (ITGA6) as part of a stool assay for the detection of colorectal lesions.

Results: In comparison with control samples, ITGA6 levels were found to be significantly increased at all stages (P < 0.01). Receiver operating characteristic analysis revealed areas under the curve of 0.89 for the prediction of CRC with 81% sensitivity and 88% specificity and of 0.90 for the prediction of advanced adenomas (Ad) with 75% sensitivity and 88% specificity. The ITGA6A variant was also found to be increased relative to ITGA6 in stage II and III CRCs. Combining ITGA6 with other selected transcripts and/or immunochemical fecal occult blood test (iFOBT) results further increased sensitivity and specificity for the detection of colorectal lesions.

Patients and methods: ITGA6 detection used alone and under various combinations including detection of other mRNA markers and iFOBT was assessed on stool samples obtained from 175 patients (91 CRCs, 24 Ad and 60 healthy controls).

Conclusions: These data confirm the usefulness and reliability of an mRNA stool assay for the detection of colorectal lesions. The validation of additional candidate genes and their analysis in multiplex qPCR represents a powerful and robust approach that can be combined with iFOBT results to improve the detection of colorectal lesions.

Keywords: adenomas; biomarker; colorectal cancer; mRNA; non-invasive screening.

Figure 1. Detection of ITGA6 in stool samples of controls and patients diagnosed with Ad and stage I to IV CRC

A. For ITGA6, in comparison with controls (Ctrl), significant increases were observed in Ad and all CRC stages alone and in combination (I-IV and II+III). B. ROC curve analysis of ITGA6 detection in Ad, CRC II-III and CRC I-IV. Sensitivity and specificity are provided in % (95% CI). C–D. For the detection of ITGA6A, significant increases were observed for stage II-IV CRC individually and in combinations (I-IV and II-III) (C). ITGA6A/ITGA6 ratios were found to be significantly increased for stage II and III CRC (D). Results in A, C and D are expressed as median (interquartile range) of copy number relative to control patients. * P < 0.05 to *** P < 0.001 using the Kruskal-Wallis test.

Figure 2. Combining ITGA6 detection with iFOBT: the IF score

The IF score was calculated using an algorithm that combines ITGA6 and iFOBT. A. The results show a median score ≥ 3 for all types of lesions including Ad and stage I CRCs relative to controls; * P < 0.05 to *** P < 0.001 using the Kruskal-Wallis test. B. ROC curve analysis of the IF score for Ad, stage I-IV CRCs and stages II-III CRCs showing an overall increase in sensitivity and specificity for lesion detection as compared with ITGA6 alone (Figure 1B). Sensitivity and specificity are provided in % (95% CI).

Figure 3. Use of the IGM (ITGA6, GADD45B and MYC) score for the diagnosis of CRC

The IGM genes were evaluated individually and in combination by comparing specificities for 80% sensitivity in stage II-III and stage I-IV CRCs as well as the IGMF score, which combines IGM genes with iFOBT A. ROC curve analysis of the IGM and IGMF scores were determined for stage I-IV and II-III CRC B, C. Sensitivity and specificity provided in % (95% CI).