Carbonic Anhydrase IX Expression Is Associated with Improved Outcome of High-dose Interleukin-2 Therapy for Metastatic Renal Cell Carcinoma

Patients and Methods

Study design, patient eligibility and treatment. The medical record database was retrospectively searched for mRCC patients treated with HD IL-2 between January 1996 through December 2006 at the University of Minnesota Medical Center, Fairview in Minneapolis, Minnesota, USA. The purpose of this analysis was to determine whether the University of Minnesota's CR and PR were comparable to previously published data. This study included adult patients (18 years or older) with histologically documented RCC. Patients eligible for HD IL-2 therapy were required to have an Eastern Cooperative Oncology Group performance score between 0-2 due to the cardiopulmonary toxicity of IL-2. This retrospective chart review was approved by the University of Minnesota's Institutional Review Board.

HD IL-2 was given intravenously in a non-intensive care unit hospital ward. Every eight hours, a bolus of HD IL-2 at a dose of 600,000 IU/kg was administered over 15 minutes. Patients were monitored for side-effects related to capillary leak syndrome, including hypotension and pulmonary edema. These bolus infusions were repeated at eight-hour intervals as tolerated by the patient, with a maximum of 14 doses per cycle. Patients were discharged when hemodynamically stable, and brought back to the hospital usually 5-10 days later to repeat the course of treatment. Treatment was continued until either CR, or the disease no longer responded, or the patient could no longer tolerate the side-effects.

Evaluating response. Computed tomography imaging of the chest, abdomen, and pelvis was performed to assess treatment response (using Response Evaluation Criteria in Solid Tumors (RECIST)) (13) after every two cycles. In brief, CR was defined as disease that had disappeared and stayed in remission for at least one month. PR was defined as disease that had regressed by greater than 30% in tumor size. Stable disease (SD) was classified as disease that had regressed in size by less than 30% or grown by no more than 20%. Disease progression was indicated when the tumor had increased by more than 20% in size. Mixed response was defined as having at least one site of tumor progression and one site of tumor response.

Data collection. The data collected from the patients' medical records included: gender, pathological diagnosis, history of nephrectomy, sites of metastasis, Memorial Sloan-Kettering Cancer Center (MSKCC) prognostic scores (14), history of prior systemic treatment, response to therapy, number of doses per cycle, number of cycles of treatment received and time to disease progression.

Tumor tissue staining for carbonic anhydrase IX. Four-micrometer sections of formalin-fixed, paraffin-embedded tissue from primary kidney tumor were cut and placed on charged glass slides. Tissue sections were deparaffinized and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide. CAIX was identified using rabbit polyclonal antihuman antibody (ab15086; Abcam Inc. Cambridge, MA, USA) at a concentration of 1:1000. Heat antigen retrieval was carried out in 10 mM citrate buffer (pH 6.0). The immunogen for the polyclonal antibody was a synthetic peptide made to a sequence within residues 359-459 of human CAIX. Staining was performed in an automated immunostainer (Ventana Benchmark XT; Ventana, Tucson, AZ, USA). An indirect biotin-streptavidin system was used to detect the primary antibody (New DAB detection kit; Ventana). One representative block per tumor was selected and stained.

CAIX expression and staining intensity were evaluated by a pathologist (J.C.M.) without knowledge of patient outcome. CAIX expression was recorded as the percentage of tumor cells that stained positively for CAIX. Intensity was scored as absent (0), weak (+1), moderate (+2) or strong (+3) for the area of the section demonstrating maximal staining (Figure 1). CAIX tumor expression was analyzed using the cut-off point for low (85%) and high (>85%) expression previously identified by Bui et al. (10)

Statistical methods. For the purposes of these analyses, patients were grouped based on clinical benefit of therapy. Patients were considered to have benefited from therapy if they had a PR, mixed or CR. Patients with no response or SD were considered not to have benefited from therapy. Patients with MSKCC prognostic scores of ‘2’ or ‘3’ were grouped together for the analyses due to the small number of patients.

Fisher's exact test was used to determine if histology, metastatic site, prior treatment and prior nephrectomy were related to clinical benefit of therapy. The Spearman correlation coefficient was used to estimate correlation between the response type and MSKCC prognostic score. Both were assumed ordinal, with the total score ranging from 0-3 and the response ranging from no response to CR.

Time to disease progression (TTDP) was calculated from study entry date to the date of progression or was censored at the date of last contact for patients who remained disease free. TTDP was summarized using Kaplan-Meier methods (15). Cox proportional hazards models and log-rank statistics were used to evaluate the relationship between clinical benefit of therapy measured by TTDP and MSKCC prognostic score. The log-rank test was used to compare the survival curves by CAIX stain. All analyses were performed using SAS version 9.1. (SAS Institute Inc., Cary, NC, USA). P-values less than 0.05 were considered statistically significant.