This trial was designed to define accuracy of ATP-CRA test in gastric cancer patients receiving paclitaxel and cisplatin chemotherapy, by comparing clinical response and ATP-CRA results in prospective, multi-center clinical trial. Primary end point was to assess accuracy of ATP-CRA results, and secondary endpoint was to find best method of defining in vitro chemosensitivity. Sample size was calculated to show sensitivity or specificity of ATP-CRA was more than 80%, to be 62 (α=0.05, δ=0.10). Considering 25% technical failure rate and 15% clinical drop out rate, total of 90 patients were to be enrolled.

Patients with chemotherapy-naïve, histologically or cytologically proven, metastatic or locally advanced gastric cancer not amenable to curative resection were enrolled for the study. Patient had to have at least 50 mg (endoscopic biopsy) or 250 mg (surgical biopsy) of tumor obtained, or at least 500 mL of ascites or pleural effusion, with more than 30% tumor cells to be included in the trial. Other inclusion criteria included age between 18-70, Karnofsky performance status: 60-100, bidimensionally measurable disease with more than 20 mm in computed tomography (CT) scan or 10 mm in radiography or physical examination and at least 12 weeks of life expectancy. Adjuvant chemotherapy given 6 months prior to enrollment was allowed and radiotherapy was allowed if the portal was outside of the measurable disease, with more than 4 weeks interval before enrollment. Patients who had active central nervous system metastasis, active infection, or other serious illness or medical conditions were excluded. Patients with less than 30% of tumor cells in malignant effusion were excluded from the study. This study was approved by local institutional review board (IRB) and all participants signed informed consent before enrollment. IRB approval numbers of participating centers are as follows; Seoul National University Bundang Hospital H-0306/104-009, Korea University Anam Hospital AN0328-001, Yeungnam University Medical Center 04-31-10, Seoul National University Boramae Hospital 06-2003-07, Seoul Paik Hospital 05-04-42, Korea Cancer Center Hospital 04-22(4), Seoul National University Hospital H-0306/104-009.

After obtaining informed consent for the study, tumor tissues were obtained by endoscopic biopsy, paracentesis of ascites, or excisional biopsy. Tumors were kept in transport media, containing Hank's balanced salt solution (HBSS, Gibco, Rockville, MD, USA), containing 100 IU/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL gentamicin and 2.5 µg/mL amphotericin B and 5% fetal bovine serum, and transported to central laboratory within 24 hr of procedure. Tumor cells were separated by previously described method (11). Briefly, tissues were washed, quantified, minced and then white blood cells and red blood cells were removed using Ficoll gradient centrifugation at 400 g for 15 min and using magnetic bead containing anti-CD45 antibody (Miltenyi Biotech, Auburn, CA, USA). Tumor cells were diluted and seeded in triplicate to a 96-well ultra-low attachment plate, at 2,000-20,000 cells/well. 100 µL of paclitaxel and cisplatin were added to the seeded cell cultures at ×5, 1, 0.5, 0.2, 0.1 times the individual test drug concentrations (2.5 µg/mL for cisplatin, 3 µg/mL for paclitaxel) determined by previous method (15, 16), and incubated for 48 hr in a CO₂ incubator. ATP in the cell lysates of treated and untreated control was measured using flash type luminescence measurements (Roche, Mannheim, Germany), and the inhibition rate was determined as the rate of ATP luminescence reduction in the treated group compared with the untreated control.

After obtaining tumor tissue, patients received combination chemotherapy consisting of paclitaxel 175 mg/m² and cisplatin 75 mg/m², on day 1 every 3 weeks for a maximum of 6 cycles. Tumor response was assessed by WHO criteria (17), using CT scan taken every 2 cycles. Both laboratory technicians and physicians were blinded to ATP-CRA results or clinical results. The chemosensitivity was assessed using 1) chemosensitivity index (CI) method, CI calculated by adding the percentage of tumor growth inhibition at each drug concentration tested (CI=[100×# concentration tested]-SUM [% cell suppression at given concentrations among 0.1×-5×]) (6); 2) comparison of in vitro area under the curve (AUC) at IC₅₀, drug concentration that achieve 50% growth inhibition in vitro, vs. clinical AUC (18, 19); 3) single concentration arbitrary criteria, which uses cut-off value of in vitro inhibition rate determined by Fisher's exact test, which discriminates clinical responders and non-responders (20); and 4) using mean value of growth inhibition rate using patient database (21). Patients were considered to be chemo-sensitive if they were sensitive to either one of the two drugs, defined by cut off values determined as above.

Receiver operating characteristic (ROC) curve analysis and Fisher's exact test were performed to select analytic method with highest accuracy. By the method chosen, 2×2 table was constructed yielding sensitivity, specificity, positive and negative predictive value, and accuracy was calculated as proportion of true positive and true negative patients out of whole patients.

Survival curves were constructed using the Kaplan-Meier method. The log-rank test was used to compare survival probabilities between in vitro responders and non-responders. All statistical calculations were carried out by independent statisticians at CMIC Korea, Ltd., using SAS 8.1 (SAS Institute, Cary, NC, USA) and the SPSS Windows program version 15 (SPSS, Chicago, IL, USA). P values <0.05 were considered statistically significant.

From September 2003 to January 2006, a total of 71 patients signed informed consent and underwent biopsy from seven centers. Of the 71 patients, 23 were deemed ineligible due to not enough tumor cells (n=10), bacterial contamination (n=3), violation of enrollment criteria (n=6), and not receiving planned treatment (n=4), rendering 48 patients enrolled. Majority of specimens were endoscopic biopsies of primary tumor (46 out of 48) and remaining two were obtained from excisional biopsy and ascites. The study was originally designed to enroll 62 evaluable patients, but the trial was terminated and analyzed after enrolling 48 patients due to poor patient accrual.

The median age of the patients were 57 yr (range, 31-70) and other clinical characteristics are summarized in Table 1. No patients had received adjuvant chemotherapy or radiotherapy before enrollment. Of the 48 patients, 11 were inevaluable (4 due to inaccurate dose of chemotherapy, 6 were lost to follow up before response assessment, 1 culture failure and 1 secondary bacterial contamination during the assay) and 36 patients were evaluable for both in vitro and in vivo responses.

Thirty-six evaluable patients received total of 152 cycles of chemotherapy, median number of cycles received was four (1-6). Among 36 evaluable patients, response rate was 36.1% with 13 patients achieving partial response, 12 stable disease and 11 patients with progressive disease. Twenty-seven patients received second line chemotherapy upon progression. Median PFS was 4.2 months (95% CI: 3.4-5.0) and median OS was 11.8 months (95% CI: 9.7-13.8) for all enrolled patients.

Of the 48 patients enrolled, 46 specimens yielded chemosensitivity results, with two test failures; one secondary bacterial contamination during the assay, one culture failure. The success rate on the intention-to-assay basis was 75.4% (46 of 61, excluding ten patients with violation of enrollment criteria and who did not receive planned treatment). If the samples met all the inclusion criteria of more than 50 mg (endoscopic biopsy) or 250 mg (surgical biopsy), or at least 500 mL of ascites or pleural effusion, with more than 30% tumor cells, the success rate was as high as 95.8% (46 of 48 enrolled patients). Chemosensitivity was determined using four different methods described in the methods and ROC curve analysis was performed to define the method with highest accuracy (Table 2). Chemosensitivity index method, which used ×1.0 and ×5.0 concentrations and cut-off value of 49.9, were chosen as the method with highest accuracy of 77.8% (Table 2, Fig. 1). This method would define a patient as in vitro sensitive, if the sum of percentage of cell death at ×1.0 and ×5.0 times peak plasma concentrations of either paclitaxel or cisplatin were below cut-off value of 49.9.

Using chemosensitivity index method defined above, specificity of ATP-CRA was 95.7% (95% CI: 77.2-99.9%); sensitivity 46.2% (95% CI: 19.2-74.9%); positive predictive value 85.7% (95% CI: 42.1-99.6%); and negative predictive value was 75.9% (95% CI: 55.1-89.3%), respectively (Table 3). Other methods such as chemosensitivity index method using ×1 and ×5 concentrations, cut off point of 52.3, single concentration and arbitrary criteria at cisplatin ×1 or paclitaxel ×1 concentrations, cut off point of 52.7 yielded accuracy above 70% (Table 2).

Using the selected chemosensitivity index method and cutoff point, seven patients were defined as in vitro sensitive (S) and 29 patients as resistant (R). S group and R group showed similar baseline characteristics such as age, sex, stage, performance, and whether they received second line chemotherapy (Table 4). S group showed statistically significantly higher response rate compared to R group (85.7% vs. 24.1%, P=0.005). Progression free survival (median: 4.3 vs. 4.5 months, mean: 5.5 vs. 4.4 months, P=0.60) and overall survival (8.4 vs. 12.3 months, P=0.08) difference did not reach statistical significance (Fig. 2).