Abstract
This research project employed the use of cost-effective genomic methods to improve translational lymphoid cancer research in Aotearoa New Zealand (New Zealand), with a strong emphasis on the development of synergistic relationships between research facilities and clinical laboratories. Three individual case studies were conducted, which involved applying genomics to different research scopes including clinical diagnostics, basic cancer research, and therapeutic research. Overall, this research project demonstrated the importance of collaborative efforts between researchers and diagnostic scientists to advance translational lymphoid cancer research.
In New Zealand, genomic technology in the clinical management of lymphoid malignancies is primarily restricted to cytogenetic studies; with next-generation sequencing (NGS) testing often outsourced to overseas laboratories. New Zealand patients are at an inequitable disadvantage, thus, the implementation of a local diagnostic NGS solution with clinical utility for use with lymphoid malignancies was needed. The Delphi method was employed to obtain consensus from clinicians surrounding the use of NGS for lymphoid cancers. Results indicated a strong need for a New Zealand-based solution. However, one barrier preventing the direct validation and implementation of a custom NGS panel in Wellington was the lack of a local diagnostic laboratory with NGS accreditation. To address this need, the AmpliSeq for Illumina Cancer Hotspot panel v2 was validated for routine diagnostic use. Following this, a pilot validation study for a customised lymphoid malignancy NGS panel, designed through the aid of clinician consensus, was conducted. The results demonstrated the feasibility of implementing a cost-effective NGS panel to help address the inequitable gap in sequencing technology for New Zealanders with lymphoid malignancies.
Jurkat clone E6-1 cells, a cell line commonly used in T cell research, are derived from a paediatric case of acute leukaemia. There are limited data that illustrate the genomic variability between cell lines in different laboratories at various levels of genomic resolution. The results presented in this research project conduct a genome-wide survey of the Jurkat clone E6-1 cell line between various laboratories around New Zealand, with a comparison of their genomic landscapes made back to reference Jurkat clone E6-1 cells from the supplier. Karyotype results showed striking differences between each instance of the Jurkat clone E6-1 cell line. These results were all confirmed through DNA microarray-based methods. Whole exome sequencing was then conducted to discover mutational events with a single nucleotide resolution. The differences between each instance of the in-use Jurkat clone E6-1 cell lines were marked, and the functional consequences of the detected aberrations were explored by whole transcriptomic sequencing. These results confirmed that such a high variability of mutational events occurring at the DNA level in each of the Jurkat clone E6-1 cell lines resulted in the differential expression of key genes associated with T cell functionality and maintaining genomic stability. Overall, these results indicated that translational research facilities need to monitor their cell lines with higher scrutiny than before, and can do so in a cost-effective manner through collaborative efforts with diagnostic facilities.
Genomic approaches are often employed to reinforce the safety profiles of genome-modified cell therapies such as chimeric antigen receptor (CAR) T cell therapy. Regulatory agencies recommend controlling transgene integration numbers in clinical CAR T cell products, with a limit of five insertion events per cell to minimize the risk of secondary oncogenesis. Transgene copy numbers in CAR T cell products are commonly evaluated by PCR-based methods, but one major limitation is that these approaches provide no indication of transgene copy numbers at a single cell resolution. This research project addressed this issue by taking a genomic approach to transgene copy number detection using fluorescence in situ hybridization (FISH); a method primarily used in diagnostic facilities. Results showed that a FISH-based approach is an effective method of transgene copy number visualisation and enumeration at a single cell resolution in CAR T cells. Whilst further optimisation is required, this assay holds potential for use as another routine safety release assay in the clinical management of patients with lymphoid malignancies eligible for CAR T cell therapy.
In summary, this research project presents evidence that cost-effective genomic technology is required to advance multiple scopes of translational lymphoid cancer research. Additionally, the results indicate a strong need to continue building synergistic relationships between clinical and translational research facilities within New Zealand. By utilizing the genomic expertise in both the health sector and academic facilities, translational research in New Zealand may progress faster than previously possible.