Colorectal cancer screening: Assessment of CEACAM6, LGALS4, TSPAN8 and COL1A2 as blood markers in faecal immunochemical test negative subjects

Abstract

Prevention is essential to reduce Colorectal Cancer (CRC) mortality. We previously reported a panel of four genes: CEACAM6, LGALS4, TSPAN8, COL1A2 (CELTiC) able to discriminate patients with CRC. Here, we assessed the CELTiC panel by quantitative polymerase chain reaction, in the blood of 174 healthy subjects, who resulted negative to the faecal immunochemical test (FITN). Using non-parametric statistic and multinomial logistic models, the FITN were compared to previously analysed subjects: 36 false positive FIT (NFIT), who were negative at colonoscopy, 36 patients with low risk lesions (LR) and 92 patients with high risk lesions or CRC (HR/CRC). FITN showed a significantly lower expression of the four genes when compared to HR/CRC. Moreover, FITN showed a significantly lower expression of TSPAN8 and COL1A2 compared to NFIT and LR patients. The multinomial logistic model confirmed that TSPAN8 alone specifically discriminated FITN from NFIT, LR and HR/CRC, while LGALS4 was able to differentiate FITN from false positive FIT. Finally, ROC curves analysis of the comparisons between FITN and HR/CRC, LR or NFIT reported AUC greater than 0.87, with a sensitivity and specificity of 83% and 76%, respectively. The CELTiC panel was confirmed a useful tool to identify CRC patients and to discriminate false FIT positive subjects.

Keywords: Blood mRNA; CEACAM6; COL1A2; Faecal immunochemical test; LGALS4; TSPAN8.

Graphical abstract

Fig. 1

Graphical representation of the technical variability assay. Three samples were selected. Each sample was divided into 10 aliquots (replicates). For each sample, four aliquots (replicates) were extracted in one day to calculate within-assay variability. The other six aliquots were extracted on two different days (three replicates per day). After extraction of all the replicates of each sample, total RNA was reverse-transcribed and cDNA amplified on different days.

Fig. 2

Enrollment and outcomes. Study plan describing admission of 174 subjects with negative fecal immunochemical test (FITN). The distribution of cases of 164 samples collected in our previous studies , is also reported.

Fig. 3

Box-plot of the quantification cycles (ΔCq), normalised on the housekeeping gene, of the CELTiC markers for the four groups analysed. CEACAM6, carcinoembryonic antigen-related cell-adhesion molecule 6; LGALS4, lectin galactoside-binding soluble 4; TSPAN8, tetraspanin 8; COL1A2, collagen type I alpha 2 chain. FITN, healthy FIT negative; NFIT, negative-colonoscopy FIT-positive; LR, low risk; HR/CRC, high risk/colorectal cancer. * indicate significant difference between groups (p < 0.05).

Fig. 4

ROC curves of the CELTiC panel for the comparisons of FITN vs the other groups. AUC, area under curve; Se, sensitivity; Sp, specificity; FITN, healthy FIT negative; NFIT, negative-colonoscopy FIT-positive; LR, low risk; HR/CRC, high risk/colorectal cancer.