Discussion

PS of RMS is rare in children and its therapy represents an oncological challenge . HIPEC (in combination with CRS) represents a therapeutic but experimental option for PS of RMS . The exact extent of PS and succeeded tumor control (whether by CRS or HIPEC or both) are crucial for the outcome. Due to the still sobering prognosis an optimization of HIPEC by photodynamic therapy might be beneficial. Here, we tested the combination of HIPEC with the promising photosensitizer HYP in pediatric RMS for the first time.
Based on previous studies with HYP and RMS, we used our HIPEC mouse model of intraperitoneal disseminated pediatric RMS to evaluate the feasibility and benefits combining HYP and HIPEC in vivo . Besides reliable intraperitoneal tumor growth of human alveolar RMS and good tolerance of HIPEC and i.p. HYP-PVP25 we could demonstrate a selective uptake and accumulation of HYP in RMS even after HIPEC. Under fluorescence guidance with blue light tumor bulks exhibited strong red fluorescent signals with clear contrast between tumor margins and surrounding healthy tissue. The performance of HYP-PVP25-based PDD resulted in an optimized tumor detection and consequently significant higher PDD-PCI compared to the evaluation of PCI without PDD. Compared to our study Urla et al . described an even better HYP-PVP25-based tumor detection ratio (1.6 - fold via laparotomy following laparoscopyvs . 1.2 - fold laparotomy) coming from complicated measuring of the PCI via laparoscopy in small dimension of a mouse compared to one via laparotomy . Using the KARL STORZ D-light C system surgical and photodynamic procedures could be carried out easily. Devised for the minimal invasive approach KARL STORZ D-light C system can be used for performing HYP-PVP25-based diagnostic laparoscopy. Staging laparoscopy enhanced by HYP might represent a new method for the pre-operative assessment of peritoneal surface malignancies to prevent unnecessary laparotomy and reduce postoperative morbidity in children .
Besides the macroscopic visible selective HYP uptake we could proof a HYP penetration across the tumor surface. The accumulated HYP persisted in the tumor even after HIPEC and showed a strong signal at the periphery followed by decreased intensity towards inner cell layers. Knowing that HYP fluorescence signal can be detected after 20 minutes of incubation with accumulation in tumor tissue after 60 minutes inin vitro studies the i.p. application of HYP-PVP25 was performed four hours before PDD/T in our mouse model . Whereas Urla et al.injected HYP 24 hours before PDD intravenously in a similar RMS mouse model we see the i.p. application with fast intraperitoneal distribution more suitable for PS of RMS .
Although the HYP uptake is thought to be tumor-selective a negligible HYP evidence was seen only microscopically in pancreas and fatty tissue ingrown by the harvested tumor. Similar findings were seen in other studies regarding liver and necrotic tissue . The lipophilic molecular structure of HYP with strong avidity to cholesterol (as one of the most essential part of natural cell membranes) is the major hypothesis of the selectivity for HYP in these tissues . The apparent accumulation of HYP in RMS might derive from intracellular co-transport of HYP with cholesterol during generally high metabolic activity in neoplastic tissue . Our study supports the anti-tumor effect of HYP-PVP25-based PDT, which already has been reported in various studies for different malignancies . In addition to HYP other photosensitive agents such as indocyanine green (ICG) and 5 - aminolevulinic acid (5-ALA) should be mentioned at this point: Whereas ICG seems to have primarily diagnostic properties for tumor visualization, Ritz et al. and Urla et al. demonstrated the superiority of HYP vs. 5-ALA regarding phototoxicity and visualization in medulloblastoma and RMS .
Using TUNEL-assay we demonstrated early apoptotic effects at the outer tumor surface by HIPEC without PDT, increased to deeper cell layers after PDT. This effect was seen especially combining PDT with 60 mg/m² cisplatin and hyperthermia of 42 °C. 9 - 11 cell layers with reduced proliferation appeared as sharply bounded stripe of the tumor surface in the corresponding PDT exposed sector only after HIPEC. The deepest penetration depth of PDT after HIPEC (50 µm) was seen after the application of 60 mg/m² cisplatin heated up to 42 °C. The observed penetration depth of PDT after cisplatin-based HIPEC was 10 µm deeper than after cisplatin-based HIPEC without PDT (40 µm, Wagner et al .) but considerably more superficial as those demonstrated by Goodmanet al (1 - 3 mm) . This study was published regarding peritoneal malignancies in adults without having included pediatric RMS . Additionally, as our experiment ended immediately after PDT it was not possible to capture late anti-tumor effects after PDT with or without HIPEC knowing that cellular signal cascades and apoptosis pathway need more time . Nevertheless, we could demonstrate the promising diagnostic and synergistic anti-tumor properties of HYP-PVP25 combined with HIPECin vivo . Moreover, the additional application of HYP-PVP25 is not to be expected reducing the tolerability of HIPEC. HYP has a low side effect profile not having showed severe side effects in other mouse models or in the experimental treatment of glioblastoma or bladder cancer in clinical studies . Photodermatitis, as major side effect caused by hypericism, can appear only at higher systemic HYP concentration typically during antidepressant therapy . The HYP doses (intraperitoneal or intravenous), which have been used for intraoperative PDD/T by Ritz et al. or in our study, were considerably lower than in the therapy of depression (0.1 mg/kg body wight vs. 1000 mg) . Therefore, the photosensitizer HYP also in combination with HIPEC should be tolerated without adverse side effects in children.
Several limitations should be pointed out. Due to ethics committee approval CRS before HIPEC or PDT was not performed. Focusing the feasibility of combining HYP-based PDD/T with HIPEC in vivo , we did not do any cytoreductive surgery compared to existing clinical HIPEC studies. Nevertheless, the observed intraperitoneal tumor spread in our study might be comparable to a situation of incomplete tumor resection in children, in which HYP-PVP25-based PDD/T could offer additional enhancement. One major limitation of the concept of PDT is the restriction to the outer surface of tissue as light permeates only into superficial cell layers. The full therapeutic potential of HYP can only be unfolded after activation by an appropriate illumination . As HYP shows a good penetration with distribution even to deeper cell layers of the tumor it can be utilized as carrier system, coupled with agents . To overcome the limitation of PDT, HYP was successfully radioiodinated revealing additional anti-tumor properties in RMS treated with targeted radiotherapy (131I-HYP) in vitro, so furtherin vivo studies are required .
As translational treatment approach, HYP-PVP25 could be combined with CRS and HIPEC for PS of RMS without major changes: After preoperative i.p. application of HYP-PVP25 the photosensitizer accumulates in tumor tissue allowing better detection of the exact tumor dissemination also facilitating tumor resection under fluorescence guided PDD. Combined with HIPEC, PDT of incomplete resected tumors or its margins might enhance local tumor control due cytotoxicity of photoactivated HYP. Based on this study the possible enhancement of HIPEC in RMS by a combination with HYP-PVP25 could improve the existing treatment strategy and consequently the outcome of the patient.
In summary, HYP as fluorescent photosensitizer offers an intraoperative diagnostic advantage detecting the exact intraperitoneal tumor dissemination. The combination of cisplatin-based HIPEC with HYP-PVP25 was feasible in the designed animal model for PS of RMS. TUNEL-method and Ki-67 staining could demonstrate promising synergistic anti-tumor properties of HYP-PVP25 combined with HIPEC. As a safe translation to clinical treatment of children is limited, further studies combining HYP, its coupled derivates and HIPEC are required to get additional insights on the possible efficiency of this approach.