The present prospective study was performed using 15 patients with colorectal cancer treated with 5-FU combined chemotherapy at The Tobu Chiiki Hospital. All patients were subjected to standardized clinical practice, according to the Japanese Society for Cancer of the Colon and Rectum Guidelines 2016 for the Treatment of Colorectal Cancer (22). 5-FU combined chemotherapy included modified FOLFOX6 (mFOLFOX6) (n=6): Oxaliplatin 85 mg/m² for 2 h, leucovorin (folinic acid) 200 mg/m² for 2 h, 5-FU 400 mg/m² bolus and 5-FU 2,400 mg/m² continuous intravenous infusion for 46 h; or FOLFIRI (n=9): Irinotecan 150 mg/m² for 2 h, leucovorin 150 mg/m² for 2 h, 5-FU 400 mg/m² bolus and 5-FU 2,400 mg/m² continuous intravenous infusion for 46 h. All patients received the dose of 5-FU based on their BSA (5-FU 400 mg/m² bolus and 5-FU 2,400 mg/m² continuous intravenous infusion for both mFOLFOX6 and FOLFIRI regimens). The Eastern Cooperative Oncology Group Performance Status (ECOG-PS) classification (23) was used to evaluate the performance status (PS) of each patient. Patients were required to have a PS ≤2. The criteria for the classification of PS were as follows: i) PS 0, fully active, able to carry on pre-disease performances without restriction; ii) PS 1, restricted in physically strenuous activity but ambulatory and is able to carry out work of a light or sedentary nature; and iii) PS 2, ambulatory and capable of all selfcare but unable to carry out any work activities; up and about more than 50% of waking hours. All the patients included in the present study were treated under the guidance of professional medical staff members. Patients were required for 4 days in admission for treatment and were readmitted every 2 weeks for 3 months (a total of six admissions). The present study was approved by the Ethics Committee of Tobu Chiiki Hospital (approved December 21, 2015; IRB nos. 15 and 4), and performed between January 1, 2017 and March 31, 2017. The present study was conducted in accordance with the principles of the amended ‘Declaration of Helsinki and Ethical Guidelines for Epidemiological Research’ (established by the Japanese Government in 2008) (24). Written informed consent was obtained from all patients before they were enrolled in the present study. Patients with PS >2 or requiring radiation or dialysis, or those unable to provide informed consent were excluded.

Venous blood samples (10 ml) were collected three times during admission (prior to the start of infusion, and 22 and 40 h after the start of continuous 5-FU infusion). 5-FU plasma levels were measured using the My-5FU^® assay, a competitive homogeneous nanoparticle agglutination immunoassay (FALCO Biosystems Ltd.). This assay is used for patients receiving 5-FU by continuous infusion to facilitate PK dose adjustment at the next cycle and drug monitoring to achieve an optimal plasma level of the drug. The assay uses two reagents and when they are mixed, the nanoparticles aggregate together. Therefore, the amount of aggregation of nanoparticles determines the 5-FU concentration in plasma samples. 5-FU plasma levels at 0 (prior to the start of infusion), 22 and 40 h after the start of continuous 5-FU infusion in each patient were calculated as median values of six admissions and statistically analyzed.

Tumor response to treatment was classified according to Response Evaluation Criteria in Solid Tumors Group (RECIST 1.1) criteria (25) and was evaluated after the 3 month monitoring period. A complete response required the disappearance of every lesion. A partial response required a ≥30% reduction in the cross-sectional area of all lesions. Stable disease required a lesion size decrease <30%. Progressive disease categorization encompassed any situation in which any one lesion increased in cross-sectional size by >25% or a new lesion appeared.

Adverse events during chemotherapy were evaluated just after the infusion of 5-FU (for 46 h) on the 4th day of admission, according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (21), based on physical examination and laboratory tests. The severity of adverse events was graded between 1 and 5. Grade 1 consists of the following criteria: i) Mild; ii) asymptomatic or mild symptoms; iii) clinical or diagnostic observations only; and iv) intervention not indicated. Grade 5 is associated with death. Adverse events were assessed on the last day before discharge for each patient.

The plasma levels of 5-FU are presented as box-and-whisker plots (containing the minimum, first quartile, median, third quartile and maximum values), unless otherwise stated. The 5-FU plasma levels were measured three times (0 h prior to the start of infusion, 22 and 40 h after the start of continuous 5-FU infusion) in each patient, calculated as median values of six admissions, and statistically analyzed. Statistical significance was determined by one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism 7 (GraphPad Software, Inc.). Statistical significance for the different tumor responses (complete response, partial response, stable disease and progressive disease) as well as the response rate and disease control rate between mFOLFOX6 and FOLFIRI was determined by a bivariate analysis using JMP 13 software (SAS Institute, Inc.). P<0.05 was considered to indicate a statistically significant difference.

A total of 15 patients (5 female and 10 male) were enrolled in the present study. The patient characteristics are summarized in Table I. The mean age was 69.0±7.3 (mean ± SD, range of 52–79) years and the mean BSA was 1.57±0.18 m². Based on the ECOG-PS classification (23), 1 patient was categorized as PS 0, 11 patients were categorized as PS 1, and 3 patients were categorized as PS 2. Only 1 patient had a tumor located in the descending colon, 10 patients had a tumor located in the sigmoid colon and 4 patients had a tumor located in the rectum. In total, 4 patients had a pathological diagnosis of well differentiated adenocarcinoma, 10 patients were diagnosed with moderately differentiated adenocarcinoma and 1 patient was diagnosed with papillary adenocarcinoma, according to the Japanese Classification of Colorectal Carcinoma (8th Edition) (26). A total of 9 patients enrolled were treated for a primary tumor, while 6 patients were treated for a recurrent tumor. Among the 9 patients, 8 patients were diagnosed as Stage IV and 1 patient was diagnosed as Stage IIIb, based on the Japanese Classification of Colorectal Carcinoma (8th Edition) (26) (data not shown). The 9 patients were diagnosed with resectable tumors, and thus they were not treated for a neoadjuvant setting. A total of 14 patients had metastasis. The most frequent metastatic site (6 patients) was the liver; other metastasis sites included the lung (1 patient) and peritoneum (2 patients). A total of 5 patients had multiple metastatic sites. A total of 11 out of 15 patients underwent operations.

Before the start of infusion, the plasma level of 5-FU was 0 ng/ml in all patients (n=15). During infusion of 5-FU for 46 h, the level at 22 h was 460.5 ng/ml (median value), while the level at 40 h reached 682.0 ng/ml. The levels at 22 h and 40 h were significantly higher than that at 0 h (P<0.001; Fig. 1). Notably, the level at 40 h was significantly higher than that at 22 h (P<0.01).

Among the 15 patients, 6 patients underwent chemotherapy of the mFOLFOX6 regimen and 9 patients underwent chemotherapy of the FOLFIRI regimen. The tumor response, according to RECIST 1.1 criteria showed no significant differences between mFOLFOX6 and FOLFIRI in the categories of complete response, partial response, stable disease and progressive disease (Table II). The response rate (proportion of patients who achieved complete or partial response) and the disease control rate (proportion of patients who achieved complete, partial response or stable disease) also showed no significant differences between mFOLFOX6 and FOLFIRI.

The plasma levels of 5-FU in the patients with different tumor responses were examined. In the partial response/stable disease group, the 5-FU plasma level at 22 h after the start of infusion was 460.5 ng/ml (median value), while the level at 40 h reached 690.9 ng/ml (Fig. 2A). The levels at 22 and 40 h were significantly higher than at 0 h (P<0.001). Notably, the level at 40 h was significantly higher than at 22 h (P<0.01). In the progressive disease group, the 5-FU plasma level at 22 h after the start of infusion was 435.1 ng/ml, while the level at 40 h reached 525.9 ng/ml (Fig. 2B). The levels at 22 h and 40 h were significantly higher than at 0 h (P<0.05). However, the level at 40 h was not significantly higher than at 22 h.

Furthermore, the mean 5-FU plasma level at 40 h after the start of infusion between the partial response/stable disease group and the progressive disease group was compared (Fig. 2). The 5-FU plasma level of the partial response/stable disease group (776.2±286.9 ng/ml; mean ± SD) was higher than that of the progressive disease group (681.9±369.4 ng/ml; data not shown), although there was no significant difference between the two groups, as assessed by one-way ANOVA analysis with Bonferroni's post hoc test.

Table III shows the adverse events of grade ≥2 in the different tumor response groups; grade 2, according to CTCAE v5.0, which is defined according to the following criteria: i) Moderate; ii) minimal, local or noninvasive intervention indicated; and iii) limiting age-appropriate instrumental activities of daily living. In the partial response group, numbness was the most common adverse event (75%). In the stable disease group, numbness and diarrhea were observed (both 50%). In the progressive disease group, numbness (80%), appetite loss (60%) and hand foot syndrome (60%) were observed. Adverse events were evaluated after the infusion of 5-FU for 46 h on the 4th day of admission. Therefore, this suggests that the 5-FU plasma level at 40 h after the start of infusion may reflect the results of the adverse events.

Among the 15 patients, 13 patients were included in the adverse event level of grade ≥2. In the partial response/stable disease group, 8 out of 9 patients were included in the adverse event level of grade ≥2, whereas in the progressive disease group, 5 out of the 6 patients were included to have an adverse event level of grade ≥2. 5-FU plasma levels at 40 h after the start of infusion in the adverse event level of grade ≥2 (13 patients in the partial response/stable disease and progressive disease groups) reached 690.9 ng/ml (median value), while the 5-FU plasma level for the adverse event level of grade <2 (2 patients in the partial response/stable disease and progressive disease groups) was 591.6 ng/ml (Fig. 3). Although there was no statistical significance between the groups of grades ≥2 and <2, the 5-FU plasma level was ~100 ng/ml higher in the group of grades ≥2 than that in the group of grade <2.