Progression-free Survival Is Accurately Predicted in Patients Treated with Chemotherapy for Epithelial Ovarian Cancer by the Histoculture Drug Response Assay in a Prospective Correlative Clinical Trial at a Single Institution

Patients and Methods

Patients. One-hundred and forty-eight patients with epithelial ovarian cancer with International Federation of Gynecology and Obstetrics (FIGO) stage III to IV, underwent primary cytoreductive surgery followed by combination chemotherapy of taxol and carboplatin between January 2007 and December 2012 at the Asan Medical Center (Seoul, Korea). All patients gave informed consent for HDRA testing. The study was approved by the Institutional Review Board of the Asan Medical Center (S2012-1977-0001). Of the 148 patients, a total of 104 had finished cycles of adjuvant chemotherapy and were not treated with neoadjuvant chemotherapy and did not have a coexisting malignancy. The median age of those patients was 58 years (36-80 years). The median follow-up duration was 26.0 months (1.5-66.0 months). The FIGO stage distribution was as follows: three in stage IIIa, 1 in stage IIIb, 76 in stage IIIc, and 24 in stage IV. The histological type showed 84 patients with papillary serous type; 41 with serous type; four with clear cell carcinoma; and five with mixed mullerian malignant tumor, while one had undifferentiated type (Table I).

Cytoreductive surgery. Patients diagnosed with ovarian cancer underwent a complete staging operation and the tumors were classified according to FIGO. The surgical staging operation included total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, appendectomy, and para-aortic and pelvic lymphadenectomy. Further, more aggressive resection of tumor-invaded organs, such as intestinal resection, colectomy, liver segmentectomy or splenectomy, was performed in selected cases with obvious tumor invasion. All 104 patients had undergone optimal debulking operations such that the size of residual tumor after surgery was less than 1 cm³.

Chemotherapy administration. Three to four weeks after surgery, all patients started adjuvant chemotherapy with taxol (175 mg/m²) and carboplatin (5 areas under the curve [AUC]), which was administered once every three weeks. Chemotherapy was halted if fever, thrombocytopenia, or neutropenia occurred.

The histoculture drug response assay. Tumor samples were obtained during cytoreductive surgery and aseptically washed in Hanks' balanced salt solution (HBSS; GIBCO, Gaithersburg, MD, USA) and minced into fragments approximately 0.5 mm in diameter. Necrotic and non-viable portions were excluded using 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2-tetrazolium (MTS; Sigma, St. Louis, MO, USA) staining. The viable cancer tissues were weighed on a chemical balance and placed onto collagen gels (Gelfoam®; Pharmacia & Upjohn, Kalamazoo, MI, USA) immersed in 1 ml RPMI-1640 medium (GIBCO) supplemented with 20% fetal calf serum (Biowest, Nuaillé, France) and drugs in 24-well plates. Six and four replicates were concurrently run for the control and treatment groups, respectively. After incubation for 72 h at 37°C with 5% CO₂, 100 μl 0.06% collagenase type I (Sigma) in HBSS and 0.2% MTT (Sigma) in PBS containing 50 mM sodium succinate (Wako Ind., Tokyo, Japan) were added to each well. Plates were incubated for another 4 h, the medium removed, and 0.5 ml dimethyl sulfoxide (DMSO) (Sigma) 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. In our study, the IR cut-off value for positive response was previously determined as ≥30% (17, 18).

Assessment of chemosensitivity with the HDRA and clinical response. To compare chemosensitivity of taxol and carboplatin in the HDRA with clinical response to these drugs, the 104 patients were divided into three groups by the HDRA results for each drug: drug resistant [IR greater than −0.5 standard deviation (SD) from the mean IR]; intermediate (IR between −0.5 SD and +0.5 SD from the mean IR); or sensitive (IR more than 0.5 SD from the mean IR). With this classification, all patients were further categorized into two groups as either sensitive to both taxol and carboplatin (SS), or resistant to one or both drug (R).

Between these two groups, inhibition rates for taxol and carboplatin, the decrease in the CA-125 level after the second and sixth cycle of chemotherapy, progression free survival (PFS) and overall survival (OS) were analyzed and compared. CA-125 levels were measured before surgery and after every cycle of chemotherapy. Imaging [(CT) or (PET/CT)] was done before surgery and after every three cycles of chemotherapy to examine the degree of disease control. PFS was defined as the interval between the date of debulking surgery and the date of imaging. In the patients without progression, PFS was defined as the duration from the date of surgery to the last follow-up. OS was defined as the interval between the date of debulking surgery and the date of death if the patient died of disease or the last follow-up date if the patient was still alive with or without disease.

Statistical analysis. Age, stage, surgical and chemotherapeutic treatment, CA-125 level, PFS and OS of each patient were all collected based on medical records. The χ² test and Fisher's exact test were performed to compare the characteristics of patients and response rate. The Kaplan–Meier method was used for PFS and OS. Data analysis was performed using SPSS ver.19.0 (SPSS Inc., Chicago, IL, USA). A p-value less than 0.05 was considered to be statistically significant.

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Figure 1.

Average inhibition rate (IRs) of tumors for drugs in the HDRA for 104 ovarian cancer. BLM: Bleomycin; VP16: etoposide; ADR: adriamycin; TOP: topotecan; GEM: gemcitabine; lFS: ifosphamide; BEL: belotecan; CDDP: cisplatin; DTAX: detaxel; CBP: carboplatin.