Clinical Significance of 5-Fluorouracil Chemosensitivity Testing in Patients with Colorectal Cancer

Patients and Methods

Patients and data. Prospectively collected data were analyzed retrospectively for patients with T3 or T4 colorectal cancer without nodal involvement and node-positive rectal cancer without distant metastasis, who had undergone radical resection and treatment with 5-fluorouracil (5-FU)/leucovorin adjuvant chemotherapy at the Korea University Ansan Hospital between December 2005 and December 2012. D2 or D3 lymphadenectomies were routinely performed for all patients. Pathologic examinations were conducted by experienced pathologists and the pathologic stage was determined according to the 7th Edition of the American Joint Committee on Cancer guidelines. Patients who had familial colon cancer, a history of other malignancies, as well as patients who underwent surgical resection other than R0 resection, were excluded from this study. Patients who previously had colorectal cancer and those who had received prior chemotherapy, immunotherapy or radiotherapy were also excluded. All patients included in the study received chemotherapy within 6 weeks of surgery. The Institutional Review Board of Korea University Ansan Hospital approved this study (AS16142-002).

Chemotherapy. All patients received 5-FU/leucovorin chemotherapy as an adjuvant treatment. The regimen used in this study consisted of an intravenous continuous infusion of 5-FU (425 mg/m²) plus leucovorin (20 mg/m²), daily for 5 days, every 4 weeks, over 6 cycles, after surgical resection.

Follow-up. During chemotherapy, patients underwent routine follow-up examinations, including abdomen and pelvis computed tomography (CT), chest CT, carcinoembryonic antigen (CEA) measurement, routine blood analysis every 3 cycles and total colonoscopy after completion of chemotherapy. For patients who discontinued chemotherapy, routine follow-up examinations were performed after the last chemotherapy treatment and every 6 months after the operation for a total of a 2-year postoperative period, with the exception of the CEA measurement that was performed every 3 months at least for a postoperative period of 5 years.

HDRA with an MTT end-point. HDRA was performed according to the modified protocol of Hoffman et al. (4). Collagen gel sponges were cut into 10-mm cubes and placed in the wells of a 24-well plate. The sponges were then soaked with RPMI-1640 culture medium containing 20% fetal bovine serum and 25× Fungizone (Gibco, Grand Island, NY, USA). The fresh surgical specimens were washed 3 times with RPMI-1640 medium and then cut into 24, 1-mm cubes. Next, a tissue cube was placed on each of the gel sponges in each well. After 24-hour incubation, 5-FU was added to the plate and tested in the culture medium at final concentrations of 3, 12, 6.25, 12.5, 25, 50 and 100 μg/ml. Wells that did not contain the chemotherapeutic agent were used as a control. After culturing for another 6 days at 37°C in an atmosphere of 5% CO₂, 5 mg of 3-(4,5-dimethylthiazol-2-yl)02,5-diphenyl-2H tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 1 ml of phosphate-buffered saline containing 100 nM sodium succinate and 100 μl of the resulting solution was added to each well. After incubation for 3 hours at 37°C, the tissue cubes were transferred to new wells containing 1 ml of dimethyl sulfoxide (DMSO) for extraction of the MTT-formazan product. Then, 100 μl of the extract was transferred to each well of a 96-well plate and the absorbance of the wells was measured at 540 nm using a microplate reader. The wet weight of the tumor tissues was measured after extraction with DMSO. The tumor growth inhibition rate (IR) was calculated by the following equation: IR (%)=(1 − mean absorbance/g of the drug-treated wells/mean absorbance/g of the control wells) ×100. In this study, the tumor was regarded as sensitive to the chemotherapeutic agent when the IR was ≥30% at 5-FU concentrations >50 μg/ml (5, 6).

Statistical analysis. Clinicopathological analysis and comparison of toxicities were performed using the Student's t-test and chi-square test. Survival was analyzed using the Kaplan-Meier method with the log-rank test. The Cox proportional hazards model was applied to identify recurrence risk factors. All statistical analyses were performed using SPSS, version 20 (IBM Corp., Armonk, NY, USA).