4002 - Longitudinal CITE-Seq analyses of the peripheral blood identify transcriptomic signatures associated with progression and transformation under targeted therapy

Abstract Text: Background
BTK/PLCG2 mutations account for only 60% of ibrutinib resistance. To understand co-evolution of normal immune bystander-tumor cells in CLL patients under covalent BTKi therapy, and hopefully help predict clinical relapses/transformations through transcriptomic signatures in discrete subsets of circulating cells, we have applied Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq), a new powerful method to deeper analyze cellular protein and transcriptome in a single-cell sequencing approach, in 6 patients under therapy at various timepoints (before treatment, developing progressive disease (PD), and Richter’s transformation (RT)).

Patients and Methods
Cells samples from patients with PD or RT under targeted therapies were obtained from the Hematology Department (IUCT-O) with informed consent and referenced in INSERM cell bank. Blood cells were cryopreserved in liquid nitrogen. For the study, CITESeq analyses have been done on samples from patients naïve of treatment (n=2), or resistant to ibrutinib (RT, n=3; PD n=3).
Cells from the different samples were thawed with a method preserving cell surface epitopes. Flow cytometry analyses (using the same antibodies as in CITESeq experiment), were done to evaluate the expression of cell surface proteins. For each sample, cells were processed according to the CITESeq manufacturer instructions. Raw data sets were computed with Cell Ranger 3.0 and then loaded in a R session with the Seurat 3.0 toolkit package to produce UMAP maps. Data were then analyzed using Single-Cell Signature Explorer. To assess differentiation trajectories, cells from each sample were injected into a corresponding reference and then classified by their mapping on the reference trajectory.

Results:
Transcriptomic analyses of cells from micro-environment revealed differential expression of gene score related to activation, cytotoxicity and exhaustion in T lymphocytes in RT samples compared to PD (Fig1.A, B), as well as differentiation status in CD8 T lymphocytes (Fig1.C, D). Despite this CD8 differential gene expression profile, B leukemic cells from PD and RT samples were equally and highly sensitive to Glofitamab in vitro (Fig.1E). Our data also defined a specific gene signature in monocytes and NK cells from RT patients, related to an inflammatory microenvironment associated with TNF signaling.

Using Single cell signature scanner software, differential transcriptomic analyses between the patients allowed us to define a specific “RT” B cell signature (142 genes) (Fig.2A) and revealed hallmark signatures of TNFa signaling via NFKB (Fig.2B), hypoxia and CLL-BCR associated gene signature in RT samples. Among this signature, we observed an increase of CD83, CXCR4, BCL-2 (Fig.2C) associated with a decrease of MS4A1, CD79B, CD27 gene expression (Fig.2D). Flow cytometry analysis on a few numbers of patients showed a higher frequency of CD19+/5+/83+/79Bneg/20neg in RT samples.
Among patients presenting a PD, a significant percentage of B leukemic cells expressed “RT” signature (Fig.2E). Clinical data from these patients showed the development of a RT 8 months after stopping ibrutinib treatment (time-point of CITESeq analysis). These data showed that our specific “RT” signature could detect and predict B leukemic clones evolving to RT.

Conclusion
Altogether our results showed an increase of genes involved in TNF signaling pathway in all cellular populations in RT patients. In these patients, we identified new phenotypic markers and differences in T lymphocytes sub-populations. Inside “RT” gene signature, we defined some genes that could be related to new therapeutic targets and/or phenotypic characteristics. This will be analyzed in a new cohort of patients after targeted therapies (PD, RT) or before treatment to validate a predictive “RT” blood signature.

Keywords : progression/transformation, CITESeq, ibrutinib

Please indicate how this research was funded. : This work was funded by IUCT/CRCT translational program

Please indicate the name of the funding organization.: IUCT/CRCT Fondation Toulouse Cancer Sante