Intraoperative hyperthermic chemotherapy perfusion for malignant pleural mesothelioma: an in vitro evaluation

Objectives: Hyperthermic chemotherapy perfusion has been used in the treatment of both pleural and peritoneal mesothelioma without an extensive basic science foundation. Clinical data are limited with no prospective randomized trials to support the use of this potentially toxic therapy. We sought to generate basic scientific support for this clinical practice and to define the optimal conditions for use in future clinical trials.

Methods: Growth of a variety of in vitro established cell lines, including a hyperthermia-sensitive Chinese hamster ovary (CHO)-K1 cell line, a normal lung fibroblast line (MRC-5), a lung cancer line (A549), and 3 human mesothelioma cell lines (NCI-H28, NCI-H2052, and MSTO-211H), was assessed using a novel tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt [MTS]) and an electron coupling reagent (phenazine methosulfate), which measures the absorbance at 490 nm of a formazan product reduced from MTS by living cells (MTS metabolic assay), or a standard dilution clonogenic assay, which enumerates colony-forming units of more than 50 cells. Each cell line was plated into flasks and then exposed to varying combinations of chemotherapy agents and hyperthermia (37°C-45°C). The cells then were harvested and assessed in either assay. The role of chemotherapeutic agents currently most commonly used in clinical practice, including cisplatin, gemcitabine, and pemetrexed, was assessed with and without simultaneous heat exposure.

Results: Conditions initially were explored using hyperthermia alone in CHO-K1, A549, and NCI-H28 cell lines using temperatures of 37°C, 42°C, and 45°C for 20, 40, and 60 minutes, respectively. This showed a reproducible dose-response curve in CHO-K1 cells with increasing temperature producing lower survival to only 1.5% of the control at 45°C for 60 minutes (P < .01). The A549 cells also showed a response but only at the highest temperature, and the NCI-H28 cells showed a more modest reduction to 65% at 45°C for 60 minutes (P < .01). When the 2 assays were directly compared, the MTS assay failed to detect differences between groups and therefore was discontinued from the remainder of these experiments. Next, hyperthermia was limited to the physiologic limit of 42°C, and the addition of chemotherapy was assessed. Doses were chosen on the basis of prior pharmacokinetic data from studies showing a maximum tissue/blood level of 200 ng/mL for cisplatin pleural instillation and were thought to more accurately reflect actual tumor levels. Cisplatin alone modestly reduced the clonogenic potential to 26%, 16.4%, and 13.6% at 42°C, respectively, for 60 minutes (P < .01); however, this was only a further reduction of 29.6%, 33.8%, and 34.2%, respectively, from the cisplatin alone control. Therefore, most of the reduction was attributable to chemotherapy and not hyperthermia. With combinations of cisplatin/gemcitabine and cisplatin/pemetrexed, the effect was larger, with reduction to 9.6%, 0%, and 0%, respectively (P < .01) (incremental reduction of 16.5%, 0%, and 0%, respectively, due to hyperthermia). Cisplatin/pemetrexed produced essentially identical results.

Conclusions: Intrapleural chemotherapy seems to be most effective when using 2 drug combinations. All mesothelioma cell lines showed no particular sensitivity to heat. The use of hyperthermia alone or with chemotherapy produces at best only a modest effect and does not necessarily support its current clinical use.