Abstract
Purpose :
Intravitreal chemotherapy is part of the conservative treatment strategy for intraocular retinoblastoma management. The technique developed by Munier F. et al (Br J Ophthalmol., 2012) proposes several recommendations to minimize extraocular tumor dissemination after the injection of the drugs.
Despite well documented tumor regression, it is necessary to assess for potential risks of tumor cell dissemination from the needle track after the injection. Thus, our aim was to develop a specific, sensitive and simple technique of tumor cell assessment in the wound after intravitreal injections in retinoblastoma patients treated in two reference clinical centers in Argentina and Switzerland.
Methods :
After performing the intravitreal injection of chemotherapy followed by cryotherapy, a 3mm diameter circular paper filter (Whatman®42 Sigma-Aldrich) was placed on the scar and another piece of paper on the cryo-tip probe. Then, each paper was placed in a tube and PBS were added. After 1min, the supernatant was removed and the tube with the paper was subjected to freeze and thaw cycles. Then, cDNA was obtained by RT-PCR (SuperscriptIII, Life Technologies) to evaluate the expression of a lineage-specific gene marker CRX (cone-rod homeobox-containing gene). RTq-PCR was performed using SYBR®Green Master Mix (Life Technologies) and a Real-Time PCR system (ABI). A calibration curve was performed using WERI-Rb1 (ATCC). CRX was considered positive if Ct value of each replica was less than 40 and S.D between replicates was less than 0.5.
The study protocol and informed consent were approved by the by the Institutional Review Boards of both countries.
Results :
The developed technique allows detecting as low as one retinoblastoma cell. Primer specificities were confirmed by melting curve analysis with single peaks. The efficiencies of CRX and gapdh genes were between 80 and 120 % with associated correlation coefficients ≥0.95.
A total of 19 cycles of intravitreal chemotherapy were evaluated until the present report. In all cases, samples from the wound of injection or the cryo-tip probe resulted CRX negative.
Conclusions :
The developed technique is precise and sensitive allowing detecting as low as one retinoblastoma cell. This technique is tool to aid in assessing the performance and safety of intravitreal injection of chemotherapy in intraocular retinoblastoma management.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.