Development and Applicability of Integrative Tumor Response Assays for Metastatic Colorectal Cancer

Patients and Methods

Patient selection. A total of 129 patients with metastatic CRC were prospectively enrolled. All patients underwent palliative surgery between December 2011 and December 2013. Eligibility criteria included histologically-proven colorectal adenocarcinoma, Eastern Cooperative Oncology Group performance status ≤2, age ≤80 years, and expectation of palliative chemotherapy. Patients were excluded if they had received preoperative chemotherapy or radiotherapy. We excluded nine cases in which ITRA results were incomplete because of insufficient tumor samples. Baseline demographic and clinicopathological characteristics are described in Table I. The median follow-up period was 28 months (range=0-64 months). By the end of the study period, only 42 patients had undergone second-line chemotherapy using regimens that were evaluated with ITRA. The outcomes of those who had both first-line and second-line chemotherapy using tested drug regimens were evaluated for correlation with ITRA results. ITRA results were correlated with clinical response (Figure 1). The study protocol was approved by the Institutional Review Board of Asan Medical Center (Registration No: 2012-0222), in accordance with the Declaration of Helsinki.

Investigated drug combinations and concentrations. The regimens tested included 5-fluorouracil (5-FU) with leucovorin and oxaliplatin (FX), 5-fluorouracil with leucovorin and irinotecan (FR), and these combinations with biologically targeted drugs. First-line chemotherapy regimens were FX or FR and second-line regimens were FX, FR, or one of these combined with a targeted agent (Table II). The targeted drugs used were bevacizumab (Avastin®; Roche, Basel, Switzerland) and cetuximab (Erbitux®; Merck, Munchen, Germany). The combined regimens were FX with bevacizumab or cetuximab, and FR with bevacizumab or cetuximab. The doses initially used were based on the usual clinical dose as determined from pharmacokinetic and pharmacodynamic parameters obtained through phase I studies and empirical assays (8, 9). Median inhibitory concentrations (IC₅₀) of tested drugs were determined as those causing a 50% dose-dependent reduction in cell viability in complete growth medium after 48 h of continuous exposure to the drug. The cutoff concentrations of the six anticancer drugs used to determine in vitro sensitivity and resistance were 50 μg/ml for 5-FU, 10 μg/ml for leucovorin, 20 μg/ml for irinotecan, 40 μg/ml for oxaliplatin, 20 μg/ml for bevacizumab, and 20 μg/ml for cetuximab, the same as in our previous study of HDRA (5).

Integrative tumor-response assay. ITRA was theoretically based on the HDRA procedure, but in vitro evaluation of resistance to the first-line anticancer drugs was sequentially repeated. In the following description of the procedure, the first three steps for ITRA were similar to those for HDRA. The details for HDRA were the same as in our previous work, except for the tumor volume (5, 10). The final two steps were specific for ITRA.

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Figure 1.

Selection of patients with metastatic colorectal carcinoma. ITRA, Integrative tumor-response assay; HDRA, histoculture drug-response assay.

1. Preparation of tissue samples. At least two sections of tumor tissue (1 cm in diameter) were obtained from fresh surgically resected specimens. This was a larger volume than we had previously collected for HDRA only. Tumor samples were aseptically washed in Hank's balanced salt solution (HBSS; Gibco, Gaithersburg, MD, USA). Collagen sponge gels manufactured from pig skin (Gelfoam®) were purchased from Pharmacia & Upjohn (Kalamazoo, MI, USA). The cancerous portions of the specimen were minced with scissors into pieces approximately 1 mm in diameter. Necrotic and non-viable portions were excluded using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-tetrazolium (MTS; Sigma, St. Louis, MO, USA) staining. Viable cancer tissues were weighed on a chemical balance and placed on the prepared collagen gel surfaces (Gelfoam®; Pharmacia & Upjohn) in 96-well plates (Figure 2).

2. Tissue culture with first-line anticancer drugs. In this study, first-line chemotherapy regimens were confined to FX and FR. Three replicates were concurrently run for the control and treatment groups. The first-line anticancer drugs were dissolved in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (FBS, Biowest, Nuaille, France) in a humidified atmosphere containing 95% air and 5% CO₂. The drug solutions were added to each well (Figure 2A), and the plates were incubated for 3 days at 37°C.

3. Determination of sensitivity to first-line drugs. After histoculture, 900 μl of HBSS containing 0.1-mg/ml collagenase (type I; Sigma) and 100 μl of 0.2% MTT (Sigma) solution dissolved in phosphate-buffered saline was added to each culture well. Plates were incubated for another 4 h, the medium removed, and 0.5 ml of dimethyl sulfoxide (Sigma) was added to each well to extract MTT formazan. Extracts from each well (100 μl) were transferred to a 96-well plate and the absorbance was measured at 540 nm using a microplate reader (VersaMax, Sunnyvale, CA, USA). The rate of inhibition of cancer-cell viability (IR) was calculated using the following equation: IR (%)=(1 − mean absorbance of treated wells per gram of tumor/mean absorbance of control wells per gram of tumor) ×100. The IR cutoff value for a positive response was previously determined to be ≥30% (11, 12) and this was used the present study.

4. Tissue culture with second-line anticancer drugs. After the measurement of IR for first-line drug regimens (HDRA), the culture media and first-line drugs were removed. Three replicates were concurrently run for the control and treatment groups. Second-line anticancer drugs were dissolved in RPMI-1640 medium (Gibco) containing 10% FBS (Biowest) in a humidified atmosphere containing 95% air and 5% CO₂. The drug solutions were added to each well (Figure 2B), and the plates were incubated for another 2 days at 37°C.

5. Determination of sensitivity to second-line drugs. After the second histoculture, the IR for each drug regimen was measured in the same manner with an MTT end-point assay. An exemplary final ITRA report is shown in Figure 3.

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Figure 2.

Arrangement of a 96-well plate for first- and second-line treatment regimens for metastatic colorectal cancer. C, Control; FX, 5-fluorouracil (5-FU) + leucovorin + oxaliplatin; FR, 5-FU + leucovorin + irinotecan; FXA, FX + bevacizumab; FXE, FX + cetuximab; FRA, FR + bevacizumab; FRE, FR + cetuximab.

Evaluation of tumor response. Each patient's response to treatment was assessed every three or four chemotherapy cycles with consistent imaging techniques such as computed tomography or magnetic resonance imaging. According to the Revised RECIST (Response Evaluation Criteria in Solid Tumors) guideline (version 1.1, 2009) (13), complete response (CR) was defined as the disappearance of all metastatic lesions and a partial response (PR) as a reduction of at least 30% of the sum of the longest diameter of metastatic lesions, with no evidence of new lesions. Progressive disease (PD) was defined as an increase by at least 20% of the sum of the longest diameters of either metastatic or recurrent lesions. Stable disease was reported when the criteria for either PR or PD were not met. The primary endpoint was a correlation between the ITRA results and the clinical response in response rate (RR); this was defined as positive for tumor responses of CR or PR.

Statistical analysis. A cross-table analysis employing Pearson's chi-square test or Fisher's exact test, as appropriate, was used to compare categorical variables. A p-value of 0.05 was considered statistically significant. All calculations were carried out using SPSS software (version 21.0; IBM Corp., Armonk, NY, USA).