Paper Details

ABSTRACT

Background:

HTLV-1 related adult T-cell leukemia-lymphoma (ATLL) is generally fatal and incurable with modern drugs. We recently completed a trial combining belinostat (BEL) with AZT and interferon (AI-BEL) for patients with aggressive ATLL and leukemic involvement (Clinicaltrials.gov NCT02737046). We hypothesized that reactivating HTLV-1 in ATLL and host virus infected cells with the HDAC inhibitor (BEL) in combination with interferon-based therapy would provoke anti-HTLV-1/ATLL immune responses leading to disease clearance. Safety and efficacy results were reported at ASH 2025. The primary endpoint was to determine the complete molecular response (CMR) rate by measuring clonality in the blood compartment using multiplex PCR for T-cell receptor (TCR) gene rearrangement; a transient or sustained disappearance of the ATLL clone was observed in 20% of subjects. ATLL cells harbor at least one clonally integrated HTLV-1 provirus, thus allowing the characterization of the original clone and it’s monitoring before and after therapy. To better depict the effects of AI-BEL on inducing deep molecular responses in ATLL, we examined clonal abundance in longitudinally collected samples by sequencing and quantifying the HTLV-1 integration sites (IS). Results were correlated with HTLV-1 proviral loads (PVLs) and multiplex PCR data.

Methods:

We measured the relative abundance of ATLL and HTLV-1 infected clones longitudinally in 15 patients with acute ATLL treated with AI-BEL by performing next generation sequencing (NGS) of HTLV-1 IS and PVLs by digital droplet PCR (ddPCR). DNA extracted from peripheral blood mononuclear cells (PBMCs) was sequenced using the GIGA Institute platform developed by Van den Broeke et al. [Lancet Microbe. 2025 Nov;6(11)] to precisely map and quantify HTLV-1 IS. PVLs were measured by ddPCR using HTLV1-Tax gene DNA primers and RNAse P as control reference housekeeping gene. Samples were multiplexed in the QIAcuity Digital PCR System (Qiagen) and data analyzed using QIAcuity Software Suite. PVLs were calculated according to formula described by Waters et al. (J Clin Virol 52:38-44). Results were correlated with multiplex PCR for TCR gene rearrangement studies performed according to BIOMED-2 report (Van Dogen et al. Leukemia 2003.17:2257-317).

Results:

On average HTLV-1 infected cells carry a single integrated provirus. Preliminary results of HTLV-1 IS NGS and correlation with HTLV-1 PVLs demonstrated a single clonal integration site in ATLL cells of 10/15 patients (67%), including 2 patients with CMR in blood compartment, 2 patients with complete response (CR), 2 with partial response (PR), 1 with stable disease (SD), and 3 with progressive disease (PD) after AI-BEL; 1 patient had evidence of an emerging second clone before progression. Multiple HTLV-1 IS (2 or 3) were found in single ATLL clones in 5/15 patients (33%), including 1 with CMR, 2 with PR, 1 with SD and 1 with PD.

Five patients (33%) carried ATLL clones with deletion in the 5’ long terminal repeat (5’LTR) of the provirus, including 1 patient with a CMR, 2 with PR, 1 with SD, and 1 with PD. Therefore, responses to AI-BEL occurred despite BEL not been able to potentially induce the HTLV-1 promoter for viral expression in ATLL cells carrying a defective provirus.

Two of 3 patients previously shown to have CMR by multiplex PCR for TCR after AI-BEL had evidence of residual ATLL by HTLV-1 IS NGS. Only 1 patient had an undetectable ATLL clone by HTLV-1 IS NGS, who is still disease-free after > 5 years. Therefore, HTLV-1 IS NGS could be more sensitive than standard PCR at detecting the presence of blood circulating clones after therapy.

In general, treatment response correlated with a decrease in HTLV-1 PVL. In some patients, massive decreases in PVLs were observed despite persistence of the dominant ATLL clone, suggesting a positive impact of AI-BEL in also clearing non-ATLL infected clones.

Conclusions:

HTLV-1 IS NGS is a powerful tool for characterizing deep molecular responses in ATLL patients undergoing therapy aimed at eradicating disease and appears to be more sensitive than standard multiplex PCR. HTLV-1 IS NGS is useful for determining the number of IS in single ATLL clones and provirus status (intact vs. defective) which could have a biological impact in the tumor. Measuring HTLV-1 PVLs is key for determining the number of HTLV-1 IS in ATLL clones and therapy impact on the quantity of blood-circulating HTLV-1 infected non-ATLL cells in patients.