HTLV-1 Carriers with Circulating Dominant Clones Harbor ATL-like Mutational and Transcriptomic Profiles Which Identify an Ultra-High Risk Group for Disease Transformation

Background: Identifying carriers of human T cell leukaemia virus type -1 (HTLV-1) at high risk of developing the aggressive T-cell malignancy adult T-cell leukaemia/lymphoma (ATL) is important as it will provide the opportunity for therapeutic intervention before disease transformation, which typically results in dismal outcomes. This further provides a rationale for broadening HTLV-1 screening. The pre-transformation processes take decades but remain elusive, with the main established risk factor being a high proviral load (proportion of infected mononuclear cells >4%) but this lacks specificity. We recently reported that ATL-like clonal T cell expansions can be identified by flow-cytometry based on T cell receptor (TCR)Vβ subunits in a subset of high-load HTLV-1 carriers ('oligoclonality-flow, OCI-flow') and that a high degree of oligoclonality (OCI flow-score >0.770), usually with a single dominant clone, predicts transformation risk. In this study, we characterize the mutational and transcriptomic profiles in these dominant clones in otherwise healthy carriers.

Methods: A cohort of 19 high proviral load HTLV-1 infected individuals was selected, of whom 9 had a high OCI-flow consistent with ATL-like clonal expansion, and 10 had low OCI-flow (<0.770). Flow-sorting was used to isolate hyper-expanded ATL-like clones based on the immunophenotype CD3+CD4+CCR4+CD26-TCRVβX+ (representing the major clone) as well sorting fractions CD3+CD4+CCR4+CD26-TCRVβOther (infected polyclonal cells), CD3+CD4+CCR4-/CCR4+CD26+ cells (uninfected populations- termed 'other CD4+') and CD33/19+ (germline) cells. RNA was extracted from all flow-sorted populations for sequencing (RNA-Seq). DNA was extracted from CD33+/19+ cells for whole exome sequencing (WES). TCRα and -β complementarity-determining region 3 (CDR3) sequences were extracted and analysed with MiXCR software. Variant calling was performed using Mutect2 using paired RNA ('tumor') and DNA from CD33/CD19+ cells ('normal') samples. Differential gene expression was performed using DeSeq2.

Results: Presence of a hyper-expanded major clone in high OCI samples was confirmed by analysis of CDR3 sequence diversity. Two of the nine subjects with a major clone also had an additional 'minor’ hyper-expanded T cell clone within the HTLV-1 infected polyclonal cell population. Eight of nine major clones (89%) harbored at least one mutation in a gene reported to be frequently mutated in ATL (VAF > 0.1, median 1 mutation, range 0-4). Both minor clones also harbored mutations in ATL driver genes. In total, sixteen mutations in genes that are frequently mutated in ATL were identified (Figure 1). These included mutations in CSNK2B (n =2), PRKCB (n =2), and CIC (n = 2) and CCR4 (n =1). Three subjects had mutations in a gene in the T-cell receptor/nuclear factor kappa B (TCR/NFkB) pathway, and all three subsequently transformed to ATL (median time since first detection of mutation(s) = 5.2 months). No other subject has yet transformed to ATL. There were no mutations detected in genes that are frequently mutated in ATL in any subject with low OCI-flow. Differential expression analysis demonstrated similarity in patterns of gene expression between major clones and ATL cells, and were distinct from patterns observed in infected polyclonal populations. Most differentially expressed genes in major clones were downregulated relative to uninfected CD4+ cells, and the majority (58%) of downregulated genes were downregulated in major clones compared to other HTLV-1 infected cells. Gene ontology analysis of differential gene expression in major clones showed enrichment of ATL-related pathways as well as suppression of pathways in lymphocyte differentiation and immune receptor activity.

Conclusions: High proviral load HTLV-1 carriers with high OCI-flow cytometry scores carry hyper-expanded T cell clones with mutational and transcriptomic profiles that resemble ATL prior to transformation. TCR/NFkB pathway mutations were detected in cases who subsequently developed ATL and may represent a later stage in the transformation process. This offers the potential to identify ultra high-risk HTLV-1 carriers for novel pre-emptive, therapeutic intervention.

Cook:Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.