Preservation of KIT genotype in a novel pair of patient-derived orthotopic xenograft mouse models of metastatic pediatric CNS germinoma

Abstract

Metastatic intracranial germinoma is difficult to treat. Although the proto-oncogene KIT is recognized as one of the most frequent genetic abnormalities in CNS germinoma, the development of new target therapeutic agents for CNS germinoma is hampered by the lack of clinically-relevant animal models that replicate the mutated or over-expressed KIT. CNS germinoma tumor cells from five pediatric patients were directly implanted into the brains of Rag2/severe combined immune deficiency mice. Once established, the xenograft tumors were sub-transplanted in vivo in mouse brains. Characterization of xenograft tumors were performed through histologic and immunohistochemical staining, and KIT mutation analysed with quantitative pyro-sequencing. Expression of putative cancer stem cell markers (CD133, CD15, CD24, CD44, CD49f) was analyzed through flow cytometry. Two patient-derived orthotopic xenograft (PDOX) models (IC-6999GCT and IC-9302GCT) were established from metastatic germinoma and serially sub-transplanted five times in mouse brains. Similar to the original patient tumors, they both exhibited faint expression (+) of PLAP, no expression (-) of β-HCG and strong (+++) expression of KIT. KIT mutation (D816H), however, was only found in IC-9320GCT. This mutation was maintained during the five in vivo tumor passages with an increased mutant allele frequency compared to the patient tumor. Expression of putative cancer stem cell markers CD49f and CD15 was also detected in a small population of tumor cells in both models. This new pair of PDOX models replicated the key biological features of pediatric intracranial germinoma and should facilitate the biological and pre-clinical studies for metastatic intracranial germinomas.

Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma

“…PCR reaction amplified a 236-bp genomic fragment spanning BRAF codon 600 on exon 15. Pyrosequencing was performed on a PSQ 96 (Qiagen) as previously described [ 56 ]. The pyrogram sequence was analyzed using sequencing primer PySeq 5′-CCACTCCATC GAGATT-3′ and dispensation order –CTAGCATGCTGT–.…”

Maximizing the potential of aggressive mouse tumor models in preclinical drug testing

“…Our clinical staging system was able to monitor progressive weakness and differentiate it from acute neurological deficits. In creating PDOX brain tumor mouse models 1 , 2 , we found these parameters most useful in determining the general health and neurological deficits of mice. Mild neurological signs related to the effects of brain tumor, impairing agility, focal weakness and gait disturbance (not affecting animal overall health fitness and function) were classified under a separate category of neurological deficits (Figs.…”

“…Aggressive tumors in PDOX mouse models progress rapidly in the limited murine life-span 1 , 2 . Though these PDOX models are accurate representation and replicas of the original patients’ disease, they are not used because the mice succumb quickly in the disease course and this translates to a narrow therapeutic window to administer drugs in preclinical testing.…”

Molecular exploration of paediatric intracranial germinomas from multi-ethnic Singapore

“…Next, there is a paucity of established pre-clinical models commercially available for IG research. At the time of this writing, there is only 1 published study involving the successful establishment of patient-derived IG cell lines in the laboratory [ 30 ]. As part of the effort to overcome such knowledge gaps, ongoing work in our laboratory is underway to develop such translationally relevant models, especially to elucidate the relationship demonstrated by our in silico findings.…”