Clinical significance of detecting circulating tumor cells in colorectal cancer using subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH)

Abstract

Circulating tumor cells (CTC) are useful in early detection of colorectal cancer. This study described a newly developed platform, integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), to assess CTCs in colorectal cancer. CTCs were detected by SE-iFISH in 40 of 44 preoperative colorectal cancer patients, and yielded a sensitivity of 90.9%, which was significantly higher than CellSearch system (90.9% vs. 43.2%, P=0.033). No significant association was found between tumor stage, survival and preoperative CTC number. CTCs were detected in 10 colorectal cancer patients one week after surgery; seven patients with decreased CTC numbers (compared with preoperative CTC number) were free of recurrence; whereas two of the three patients with increased CTC numbers had tumor recurrence. Moreover, CTCs were detected in 34 colorectal cancer patients three months after surgery; patients with CTC<2 at three months after surgery had significantly longer Progression Free Survival than those with CTC>=2 (P=0.019); patients with decreased CTC number (compared with preoperative CTC number) had significantly longer Progression Free Survival than those with increased CTC number (P=0.003). In conclusion, CTCs could be detected in various stages of colorectal cancer using SE-iFISH. Dynamic monitoring of CTC numbers could predict recurrence and prognosis.

Keywords: FISH; biomarker; circulating tumor cells; colorectal cancer; recurrence.

Figure 1. Identification of CTCs in colorectal cancer using the SE-iFISH platform

a. CK-/CD45+/DAPI+/CEP8=2 (WBC); b. CK-/CD45-/DAPI+/CEP8=3; c. CK-/CD45-/DAPI+/CEP8=4; d. CK-/CD45-/DAPI+/CEP8≥5; e. CK+/CD45-/DAPI+/CEP8≥3. DAPI, blue; CEP8, orange; CD45, red; CK, green; iFISH, immunostaining and fluorescence in situ hybridization; DAPI, (4′,6-diamidino-2-phenylindole); WBC, White blood cells; CEP8, Centromere Probe 8.

Figure 2. Detection of CTCs in colorectal cancer patients and healthy controls

a. CTCs enumeration in healthy controls and colorectal cancer. Number of CTCs in 44 colorectal cancer patients and 17 healthy controls were recorded. b. The ROC curves for CTCs enumeration to discriminate colorectal cancer patients from healthy controls. The cutoff value was defined as one CTCs in 7.5 mL of blood. c. The association of CTCs enumeration with pathological stage (TNM) in colorectal cancer patients. d. Comparison of CTC and CEA as blood-based markers. The red horizontal line indicated the CEA threshold of 5.0 ng/mL. e. Comparison of CTC and CA199 as blood-based markers. The red horizontal line indicated the CA199 threshold of 37 U/mL.

Figure 3. Comparison of preoperative and postoperative one week (P1W) CTCs to predict tumor recurrence in colorectal cancer patients

P1W CTC numbers decreased one week after surgery in the first 7 patients who had no tumor recurrence, whereas P1W CTC numbers increased after surgery in the last three patients, two of whom had tumor recurrence three months post-surgery, while another patient lost the follow up.

Figure 4. Kaplan-Meier curve analysis of PFS of colorectal cancer patients stratified by postoperative 3 months (P3M) CTC numbers

a. The average PFS in patients with P3M CTC< 2 was significantly better than those with P3M CTC≥2 (P= 0.019). b. The average PFS in patients with decreased P3M CTC number (compared with preoperative CTC number) was significantly better than those with increased P3M CTC number (P= 0.003).