PRECLINICAL PLATFORM OF DISSEMINATED RETINOBLASTOMA FOR EVALUATION OF NEW DRUG TREATMENTS

ZUGBI, SANTIAGO; CASTAÑON, A; TORBIDONI, ANA; LUBIENIECKI FABIANA; SCHAIQUEVICH, PAULA; WINTER, URSULA; SAMPOR, CLAUDIA; SGROI, MARIANA; PODHAJCER OSVALDO L; ASCHERO, MARIA; CAFFERATA EDUARDO G; FANDIÑO ADRIANA; CHANTADA, GUILLERMO

Congreso; IV International Congress in Translational Medicine,; 2018

preclinical PLATFORM OF DISSEMINATED RETINOBLASTOMAFOR EVALUATION OF NEW DRUG TREATMENTS Retinoblastoma (RB) is the mostcommon intraocular cancer in children .In the absence of prompt diagnosis andappropriate treatment, retinoblastoma disseminates outside of the ocular globe.In lower-income countries, patients more often present with disseminateddisease due to late diagnosis. Patients with disseminated or metastatic diseaseare subject to high-dose chemotherapy but poor prognosis is expected in casesof tumor invasion into the central nervous system. Currently, there is nosecond line of treatment and thus, disseminated retinoblastoma is considered anunmet medical need.In order to study the tumor biologyand new or alternative pharmacological treatments there is a need of adequatepreclinical models that recapitulate the human disease. However, there are noavailable preclinical models of disseminated retinoblastoma. Thus, our aim wasto establish primary cells cultures from biopsies of different sites of tumordissemination and to develop and characterize an animal model after orthotopicinjection of those cells in order to resemble the patient´s condition.The study protocol and informedconsent were approved by the Institutional Review Board of Hospital dePediatría JP Garrahan (Protocol #838).A biopsy wasobtained from a metastatic patient heavily pretreated with unilateralretinoblastoma and disseminated disease in the cervical lymph node at the timeof tumor progression (LNP). Sampleswere mechanically processed to obtain a cellsuspension in neural stem cells medium. After the cell culture wasestablished, evaluations were performed to assess the origin of the cell usingdifferent types of lineage specific markers including diasylganglioside GD2,synaptophysin by immunohistochemistry and the expression of cone-rod homebox (CRX)through RTq-PCR. Doubling time (DT) was also calculated. Pharmacologicalcharacterization was performed by the determination of the concentration of melphanathat inhibits 50% of the cell viability (IC50). After complete characterization, LNPcells were injected into the posterior segment of the eye in 6-week old athymicmice. The mice were then enucleated and sacrificed once the tumor reached stage3 of tumor progression (complete ocular invasion, proptosis).  Eyes were conserved for histopathologicalstudies. Tissue samples (optic nerve, brain, bone marrow, lymph nodes and CSF) werecollected to assess for retinoblastoma dissemination quantifying CRX by meansof RT-qPCR.Primaryretinoblastoma cell line derived from dissemination into the lymph node (LNP) wassuccessfully established. The cells grow as neurospheres and marked positive toGD2 and synaptophysin and were positive for CRX mRNA.The median (range)IC50% of melphalan was 69.7µM (64.0-95.7) and the doubling time was 2.6 days.A total of 16 eyes were injectedwith LNP cells with a 90% of engraftment. Eye survival was 29 days. Positivedissemination of LNP cells was observed into 11 optic nerves of 16 treated eyesand into the lymph nodes of 6 of 12 treated animals. Bone marrow and brainsamples were negative for CRX.We were able to develop a uniquecell model derived from a metastatic patient with retinoblastoma. This cellline is much less sensitive to melphalan compared to commercial cell lines ofretinoblastoma and cells derived from intraocular tumors. as previous reportson the In addition, we established a novel orthotopic animal model ofdisseminated retinoblastoma. Eye survival was lower than that reported for eyestreated with commercial cell lines of retinoblastoma and cells derived fromintraocular tumors.These two models may beuseful to understand the progression and tumor dissemination and can be used to evaluate more advanced andefficient alternative treatment options on basis of translational studies inpediatric oncology.