Development of in Vivo Models of Paediatric Burkitt Lymphoma Allowing Therapeutic Target Analysis and Drug Testing

Childhood sporadic Burkitt lymphoma (BL) is a highly aggressive mature B-cell lymphoma. Patients require intensive chemotherapy which results in prolonged periods of hospitalisation for distressing and dangerous side effects. Additionally those with refractory disease or those that relapse have an extremely poor prognosis. As a result, there is an urgent need to develop new targeted treatments that have undergone rigorous pre-clinical evaluation. The aim of this project is to develop improved models for target selection and pre-clinical validation to reduce the failure of translation of novel agents.

Two models are being developed. Firstly, xenotransplantation of BL cell lines is being used to optimise transplantation protocols and to provide an orthotopic assay for in vivo functional genomic (RNAi) screening. This is currently being used in genome-wide target identification screens. Secondly, cryopreservation of viable BL blasts is allowing development of a patient-derived xenograft model, suitable for validation of candidate targets by both RNAi and pharmacological inhibition and for testing novel therapeutic agents.

The sporadic BL cell lines CA46 and RAMOS have been transplanted into NOD/LtSz-scid IL-2Rg^-/- (NSG) and RAG2^-/-gc^-/- (RAG2) mice at reducing cell doses (10⁵, 10⁴ and 10³ cells/mouse). This approach will identify the optimal conditions for orthotopic engraftment. Prior to transplantation, cells were transduced with the lentiviral vector pSLIEW expressing green fluorescent protein (analysis and cell sorting) and firefly luciferase (in vivo bioluminescent imaging). Engraftment was then monitored in real-time using the Caliper in vivo imaging system (IVIS).

CA46 and RAMOS cell lines have been transduced with SLIEW, sorted to high purity (>90%) and engrafted in both NGS and RAG2 mice. IVIS imaging of mice transplanted with CA46 cells showed that rate of engraftment was relative to cell dose. All six mice showed a bioluminescent signal over the femurs by 3 weeks post transplantation, indicative of bone marrow (BM) engraftment. Analysis of BM by flow cytometry confirmed engraftment in the two mice analysed, as determined by GFP positivity and presence of the human B-cell markers CD10, CD19 and CD20. Renal enlargement was observed in all mice analysed, with dense infiltration by mature lymphoid blasts evident microscopically. Immunohistochemistry confirmed infiltration with human CD20 positive cells. Splenic enlargement was observed in five out of six mice and engraftment was again confirmed histologically. Onset of disease was rapid. Mice were killed when they showed signs of ill health (see table).

A similar pattern of engraftment was observed in NSG and RAG2 mice transplanted with RAMOS cells, although signal intensity was not relative to transplanted cell dose. Bioluminescent signal over the femur was detected in four out of six mice by 3 weeks post transplantation. NSG and RAG2 mice receiving 10³ cells, with no IVIS signal, were negative for BM engraftment by flow cytometry. Renal enlargement was not observed, however focal renal lesions were evident macroscopically in three out of six mice. These lesions were composed of human CD20 positive cells. Splenic enlargement was not observed, although a degree of splenic engraftment was identified histologically. Onset of disease was also rapid.

Following the initial cell line model development, we are now able to transplant the first patient-derived BL sample. If successful, primograft material will be labelled with luciferase (pSLIEW) and re-transplanted, allowing in vivo imaging. When combined with in vivo RNAi screening, this advanced pre-clinical model for target selection and validation may help reduce target drop-out during early phase clinical trials. This is a critical step, not only to avoid exposing children to ineffectual and potentially toxic drugs, but also to avoid delay in the development of new effective therapies.

CD20 positivity for spleen and kidney; none (-), < 10% (-/+), 10 - 50% (+), > 50% (++), 100% (+++). For BM engraftment (CD10, CD19 and CD20 positive); no engraftment (-), engrafted (+), not assessed (na).