Intratumoraler Transfer der felinen Zytokin-Gene IL-2, IFN-gamma und GM-CSF unter Verwendung der Magnetofektion als neoadjuvante Behandlung des Fibrosarkoms der Katze

The prognosis for cats with fibrosarcoma is still poor as the
treatment options existing to date do not lead to satisfying
results. After sole surgical removal of the tumor, up to 70 % of
the cats develop local recurrences. Even with adjuvant radiation
and/or chemotherapy, the recurrence rate can just be reduced to at
most 40 %. In the present work an alternative treatment method in
terms of neoadjuvant immunostimulatory gene therapy should be
established. Via plasmids, three feline cytokine genes were
transferred into the tumor. The plasmids coded for feIL-2, feIFN-γ
and feGM-CSF. Using magnetofection, gene transfer should be
optimized. The aim of this clinical dose escalation study was to
define a well tolerated dose, which can be tested for its efficacy
in a subsequent phase-II trial. Therefore the cats were examined at
defined time points for treatment-related toxicity up to 180 days
after surgery. Two more check-ups on day 270 and 360 after surgery
were performed to elongate the observation period for local
recurrences. Prerequisite for a cat’s admission to the study was
the localization of the fibrosarcoma (primary tumor or recurrence)
on the trunk. The tumor had to be excised in one setting without
limb amputation. Affected cats were neither allowed to be
pretreated with chemo-, radiation- or gene therapy nor with
corticosteroids within the past six weeks. Further exclusion
criteria were hints for metastases or other severe illnesses which
reduce life expectancy to less than one year. Due to the potential
teratogenic effect of the expressed cytokines, pregnancy had to be
ruled out. Only cats with histopathologically confirmed
fibrosarcoma continued the study after surgery. As this was a
scientific study with a drug not registered yet, written informed
consent from the owners was a prerequisite for the participation of
each cat. Four treatment groups with defined dose escalation were
prospectively fixed. The dose of the feline cytokines was 15, 50,
150 and 450 µg per plasmid in group I, II, III and IV. The initial
dose (3 x 15 µg) was oriented to the total dose for small oral
melanomas in dogs (400 µg) established by DOW et al. and is 1/10 of
it. Plasmids as non-viral vectors were chosen for several reasons.
Their handling is not liable to such strict regulations as the
potentially more dangerous viral vectors. Their production is
simple and affordable. They induce fewer side effects than viral
vectors. Therefore plasmids are more suitable for application in
veterinary clinical practice. Equal amounts of the plasmids were
brought into 0.9 % saline. The positively charged magnetic
nanoparticles were brought into solution with aqua for injections
and were mixed with the plasmid formulation in a 1:1 ratio. This
formulation had a total volume of 500 µl and was injected twice
intratumorally in weekly intervals. Transfection was enhanced and
targeted to the tumor area by the application of a
neodymium-iron-boron magnet for the duration of 60 minutes. One
week after the second application, wide en-bloc resection of the
tumor was performed. Four cats were assigned to each treatment
group. As questionable toxicity occured in group IV, four more cats
were added to this group. A control group also consisting of four
cats received surgery without neoadjuvant therapy. For ethical
reasons, the application of empty plasmids was avoided so that in
this group surgery could be performed without delay. Medical care
of all the cats was carried out by the same team of internists,
anesthetists and surgeons. Clinical and laboratory parameters were
evaluated according to the VCOG-CTCAE system. All adverse events
were registered, classified with severity grades and correlated to
treatment. Plasma samples of all cats up to 14 days after surgery
were examined for the existence of feGM-CSF and feIFN-γ with
commercially available ELISA kits. Statistical analyses were
performed comparing the treatment groups themselves as well as
treatment groups and the control group regarding body weight, white
blood cells and differential blood counts. Only one cat out of
group IV showed adverse events during the neoadjuvant treatment
period, which were classified as grade 3 and which were probably
correlated to treatment (correlation grade 4). For this reason four
more cats were treated with the highest dose. None of these cats
showed side effects that could be correlate to treatment. The
occurrence of early recurrences in four cats of group IV was
outstanding, but of course, the expressiveness of this statement is
low regarding the small group sizes. However it is known that
biological drugs, especially IL-2, often do not have a linear
dose-response profile. Therefore the optimal effective dose lies
within a strictly defined area and is probably lower than the
maximal tolerated dose. The highest applied dose which is 450 µg
per plasmid was defined as a well tolerated dose. It can be safely
tested for its efficacy in a subsequent phase-II trial. Because of
the reflections mentioned above it would undoubtedly be convenient
to test the third dose (150 µg per plasmid) in parallel for its
effectiveness.