Chronic Lymphocytic Leukaemia: uncovering immunity during first-line treatment

B cell Chronic Lymphocytic Leukaemia (CLL) is the most common haematological cancer in Western countries. In health B cells rearrange immunoglobulin genes to make specific antibodies to fight an infection, and some change into memory B cells that can recall the antibody on re-exposure. This function gives B cells a central role in our learnt 'humoral' immunity. We are exposed to many different infections, so our bodies generate a 'repertoire' of different antibody-producing B cells. When CLL develops, the genes in one of these B cells become mutated and the cell proliferates often producing little or no functional antibody. This results in impaired humoral immunity, alongside cancer symptoms.
Recently new drugs called 'small molecule inhibitors' that fight CLL have lengthened responses compared to traditional 'cytotoxic' chemotherapy. They target molecules in CLL cells that make them proliferate for example BTK and BCL-2 (the targets of ibrutinib and venetoclax respectively). Inhibition of these molecules causes the CLL cells to die, and healthy B cells grow back ('reconstitute'). However, patients on prolonged small molecule inhibitor treatment have fewer B cells than normal and remain susceptible to infection and poor vaccination response. The importance of this has been highlighted by the recent pandemic: patients on ibrutinib and venetoclax have been shown to have lower rates of response to COVID-19 vaccination alongside lower antibody levels.
I aim to uncover how modern CLL treatment affect reconstituted B cells, and therefore how on or post-treatment humoral immunity is impacted by analysing differences in expressed DNA (RNA) in normal individuals and pre-treatment CLL cases, compared to treated cases. I will do this in the context of the FLAIR (Front-Line therapy in CLL: Assessment of Ibrutinib-containing Regimes) trial, the largest UK trial of modern CLL treatment (1576 patients). Ibrutinib regimens are compared to traditional chemotherapy: fludarabine, cyclophosphamide and rituximab; ibrutinib alone; ibrutinib and venetoclax. Although ibrutinib and venetoclax increase treatment efficacy, the pathways they target are active in healthy B cells, resulting in fewer B cells and immunity impairment. This is important because ibrutinib is efficacious and well tolerated as monotherapy or in combinations and venetoclax is now included in front-line and relapse treatment regimens. Therefore, to maintain a healthy remission while on treatment we need to understand how the normal reconstituting B cells are surviving, if the repertoire is affected by treatment, and whether we can optimise their function to enhance humoral immunity including vaccine responses.
I will use targeted single cell RNA sequencing (scRNAseq) to determine the effect of treatment on humoral immunity. I will identify the important RNAs using a bioinformatic method called parsimonious gene correlation network analysis (PGCNA) and apply it to both CLL and B cell RNA databases to build an evidence-based panel. I will perform scRNAseq on age-matched healthy controls and CLL pre-treatment samples and the FLAIR ibrutinib and ibrutinib-venetoclax treatment groups. I have already devised a cell separation strategy to separate and quantify small populations of normal B cells and will then use scRNAseq to perform targeted sequencing. I will then overlay single-cell B cell repertoire sequences and the panel results to demonstrate how repertoire is linked to RNA expression changes, and use bioinformatic analysis to uncover whether B cell reconstitution post treatment shows the same patterns of immune as normal age-matched or pre-treatment cases. I will overlay these results alongside FLAIR trial clinical data, to show whether reconstituting B cells are performing normal humoral immune functions and how these can be enhanced.

B cell Chronic Lymphocytic Leukaemia (CLL) is the most common leukaemia in Western countries. In the UK FLAIR is the largest trial of contemporary first-line therapy for CLL (n=1576); comparing venetoclax and ibrutinib, ibrutinib monotherapy and traditional chemoimmunotherapy (FCR). I aim to evaluate the effects of FLAIR trial treatments on reconstituted B cells to explore the impact of treatment on humoral immunocompetence. To achieve this, I will develop a targeted single-cell RNA sequencing panel (scRNAseq) using parsimonious gene correlation network analysis, a method that selects highest information content gene nodes across multiple networks. I will input bulk RNA and scRNAseq data to develop models of CLL alongside established B cell PGCNA networks, and identify up to 600 genes for the panel. Targeted scRNAseq has an advantage over agnostic scRNAseq because it reduces the risk of gene drop-out, so each relevant gene can be assessed at adequate depth. CD19+, ROR1 negative cells will be selected and run simultaneously with BCR and Abseq on the BD Rhapsody system. Novaseq6000 (20x10^9 reads, illumina) will be used for sequencing the libraries achieving a read-depth of at least 30,000 reads-per-cell, which is adequate to assess all expression features. Abseq will allow comparison of bioinformatically derived clusters with conventional flow-cytometry subsets to enable comparison of results with published data for validation, and each sample will have linked data from minimal residual disease analysis and mature clinical outcomes from the FLAIR trial. I will run normal age-matched and pre-treatment controls, as well as samples from ibrutinib and ibrutinib-venetoclax treatment groups. Samples will be taken 2 years or more into treatment to reflect stabilised B cell reconstitution. scRNAseq data will be analysed using t-SNE or UMAP clustering, compositional analysis including for graphical representation (scDC) and trajectory/pseudotime analysis (slingshot).