Discussion

PS of RMS is rare in children and still represents an oncological and therapeutic challenge . Due to recent promising clinical reports HIPEC (in combination with CRS) has been established as a novel treatment option for PS of RMS . Besides significant improvement of the technical performance of HIPEC in children there is still no consistent treatment plan regarding the best choice of chemotherapy and concentration, optimal drug combination and treatment duration. Additionally, it remains unclear whether CRS or HIPEC are the main factors for treatment success. Here, we established a feasible and reproducible murine model for the evaluation of HIPEC in pediatric RMS for the first time. At this juncture there is no animal model available allowing repeatable experimental studies of HIPEC within the context of disseminated intraperitoneal pediatric RMS.
We were able to demonstrate a constant and reliable intraperitoneal tumor growth of human alveolar RMS (cell line RH-30) in NOD/LtSz-scid IL2Rγnullmice. This model resembles pediatric RMS in terms of organ invasion and intraperitoneal tumor spread very well. To evaluate the tumor dissemination, we adapted the peritoneal carcinomatosis index (PCI) modelled on Jacquet and Sugarbaker et al. for this animal model. Furthermore, we developed a feasible and easy way for the application of liquid hyperthermic intraperitoneal chemotherapy in mice . MRI scans confirmed the tumor growth and showed the performance of HIPEC in vivo for the first time. Besides our animal model there are only few recent comparable HIPEC mouse models but using non pediatric tumor entities like adult ovarian cancer or performing HIPEC as pressurized aerosol . Especially pressurized intraperitoneal chemotherapy (PIPAC) has not been applied in children.
Using TUNEL-assay method we demonstrate different penetration depth (30 - 40 µm) of the HIPEC treatment depending on cisplatin and/or hyperthermia. Early concentration- or temperature-dependent anti-tumor effects of cisplatin-based HIPEC on the tumors could be revealed immediately after treatment. The observed penetration depth of cisplatin-based HIPEC is considerably more superficial as those published by Goodman et al . . However, cisplatin has a favorable area under the curve ratio (maximal the chemotherapeutic dose, minimal systemic toxicity) for the intraperitoneal application but there is no clear statement regarding the penetration depth of cisplatin-based HIPEC for pediatric RMS till now . In the past, cisplatin was used as single therapeutic agent in different concentrations for HIPEC in children suffering from intraperitoneal RMS . Whereas the synergy between heat and drug cytotoxicity could be confirmed, the optimal concentration of cisplatin for HIPEC remains unclear . Until now cisplatin concentrations for HIPEC derive from those for intravenous chemotherapy (15-30 mg/m²) knowing that higher cisplatin dosage with respectively higher effectiveness and tolerable toxicity are feasible . As final experiment ending immediately after HIPEC it was not possible to capture late anti-tumor effects after intraperitoneal chemotherapy lavage knowing that cellular signal cascades and apoptosis pathway need more time .
Several limitations should be pointed out in this study. Due to ethics committee approval CRS before HIPEC was not performed. Focusing the feasibility of a HIPEC animal model for pediatric RMS, we did not do any cytoreductive surgery before HIPEC compared to existing clinical studies. The recommendation of administration of HIPEC after CRS is generally based on clinical studies for adults whereas it remains unclear if either CRS or HIPEC or the combination of both determines the therapeutic success in children . Nevertheless, the observed intraperitoneal tumor growth in our animal model was a disseminated miliary tumor spread (maximal 3 to 4 cm in diameter) compared to an incomplete tumor resection in children. In combination with HIPEC residual tumor of 2,5 cm can be tolerated with the same outcome due to local tumor control through tumor penetration of HIPEC . All the few existing data regarding CRS and HIPEC in children were lifted from restricted clinical case reports . Ethical considerations constrain the systematic in vivo study of HIPEC in children regarding optimal drug concentration and combination. With the establishment of an appropriate animal model this limitation can now be overcome. Our model allows valid additional insights on the possible efficiency of HIPEC in pediatric RMS to improve existing treatment strategy and manage the therapeutic challenge.
In summary, we were able to establish the first animal model for evaluation of HIPEC in pediatric rhabdomyosarcoma in mice. Using TUNEL-method early cisplatin- and hyperthermia-dependent apoptotic effects at the tumor surface (penetration depth 30 – 40 µm) can be detected. Additionally, statistically significant reduction of early tumor proliferation depends on cisplatin or hyperthermia. This model allows valid additional insights on the possible efficiency of HIPEC in RMS so further studies using other drug combination, concentration and cytoreduction will follow.