Contributions
Abstract: EP876
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
The genomic landscape of Burkitt lymphoma has largely been defined, but the clonal complexity and key molecular events driving disease development and/or relapse remain to be determined. In part this relates to the relative lack of patient samples, especially following relapse, and the limited number of representative experimental models.
Aims
To develop a novel approach to the modelling and study of sporadic Burkitt lymphoma biology we developed orthotopic patient-derived xenograft models. The aim is that such models can be used both to study the molecular drivers of disease development/relapse and for pre-clinical testing of new treatment approaches.
Methods
Samples from newly presenting or relapsed patients were collected and cryopreserved with informed consent for animal research. Thawed cells were injected into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratory) or Rag2-/-γc-/- (C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J, Jackson Laboratory). Tumour bearing animals were killed and serial transplantation performed. Parallel samples were taken for cytogenetic and molecular analysis.
Immunohistochemistry and cytogenetic analysis were performed within an NHS diagnostic laboratory setting. RNA and DNA were isolated and subjected to mRNA or exome sequencing using Illumina Novoseq or NextSeq platforms. Species specific transcripts were identified and murine reads removed using Disambiguate before further analysis. Exome data were analysed using GATK4.
Results
Viable cells from multiple sources (tumour, pleural effusion, bone marrow) were successfully cryopreserved and showed good viability. Patient material from all samples engrafted within 30-48 days. Serial transplantation across three (n=3) or four (n=1) generations was successful in all samples.
Immunohistochemical/cytogenetic analysis were consistent with the original pathological diagnoses. Transcriptomic analysis identified a metagene expression pattern consistent with published Burkitt lymphoma patient data, as distinct from diffuse large B cell lymphoma, confirming the representative nature of the models at this level.
Exome sequencing identified several mutations previously described to be recurrent in sporadic Burkitt lymphoma, including in the genes MYC, TP53, ARID1A, RHOA and ID3. Multiple pathogenic mutations in ID3 were identified in each of three seperate patient samples and analysis of variant allelic frequency across sequential xenograft generations demonstrated differential fitness within the subclonal populations.
Conclusion
Patient-derived xenograft models of Burkitt lymphoma are feasible and representative of the original disease, despite cryopreservation. Molecular analysis of these models provides an alternative approach to the study of drivers of disease progression and may be used to track clonal evolution in response to therapy.
Keyword(s): Burkitt's lymphoma, Xenotransplantation
Abstract: EP876
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
The genomic landscape of Burkitt lymphoma has largely been defined, but the clonal complexity and key molecular events driving disease development and/or relapse remain to be determined. In part this relates to the relative lack of patient samples, especially following relapse, and the limited number of representative experimental models.
Aims
To develop a novel approach to the modelling and study of sporadic Burkitt lymphoma biology we developed orthotopic patient-derived xenograft models. The aim is that such models can be used both to study the molecular drivers of disease development/relapse and for pre-clinical testing of new treatment approaches.
Methods
Samples from newly presenting or relapsed patients were collected and cryopreserved with informed consent for animal research. Thawed cells were injected into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratory) or Rag2-/-γc-/- (C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J, Jackson Laboratory). Tumour bearing animals were killed and serial transplantation performed. Parallel samples were taken for cytogenetic and molecular analysis.
Immunohistochemistry and cytogenetic analysis were performed within an NHS diagnostic laboratory setting. RNA and DNA were isolated and subjected to mRNA or exome sequencing using Illumina Novoseq or NextSeq platforms. Species specific transcripts were identified and murine reads removed using Disambiguate before further analysis. Exome data were analysed using GATK4.
Results
Viable cells from multiple sources (tumour, pleural effusion, bone marrow) were successfully cryopreserved and showed good viability. Patient material from all samples engrafted within 30-48 days. Serial transplantation across three (n=3) or four (n=1) generations was successful in all samples.
Immunohistochemical/cytogenetic analysis were consistent with the original pathological diagnoses. Transcriptomic analysis identified a metagene expression pattern consistent with published Burkitt lymphoma patient data, as distinct from diffuse large B cell lymphoma, confirming the representative nature of the models at this level.
Exome sequencing identified several mutations previously described to be recurrent in sporadic Burkitt lymphoma, including in the genes MYC, TP53, ARID1A, RHOA and ID3. Multiple pathogenic mutations in ID3 were identified in each of three seperate patient samples and analysis of variant allelic frequency across sequential xenograft generations demonstrated differential fitness within the subclonal populations.
Conclusion
Patient-derived xenograft models of Burkitt lymphoma are feasible and representative of the original disease, despite cryopreservation. Molecular analysis of these models provides an alternative approach to the study of drivers of disease progression and may be used to track clonal evolution in response to therapy.
Keyword(s): Burkitt's lymphoma, Xenotransplantation