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.