HTLV‐1 cell‐free DNA in plasma as a potential biomarker in HTLV‐1 carriers and adult T‐cell leukemia‐lymphoma

Abstract Viral cell‐free DNA (cfDNA) in plasma has been widely evaluated for detecting cancer and monitoring disease in virus‐associated tumors. We investigated whether the amount of cfDNA of human T‐cell leukemia virus type 1 (HTLV‐1) correlates with disease state in adult T‐cell leukemia‐lymphoma (ATL). HTLV‐1 cfDNA in aggressive ATL was significantly higher than that in indolent ATL and asymptomatic carriers. Notably, patients with lymphoma type represented higher HTLV‐1 cfDNA amount than chronic and smoldering subtypes, though they had no abnormal lymphocytes in the peripheral blood. HTLV‐1 cfDNA can be a universal biomarker that reflects the expansion of ATL clones.

gave written informed consent in accordance with the Declaration of Helsinki. This study was approved by the existing institutional review board at Saga University. Plasma was spun down from whole-blood samples with an EDTA 2Na tube (3000 rpm for 20 min) within 4 h after blood collection, and cfDNA was extracted from 1 mL plasma using Maxwell RSC. Genomic DNA was extracted from PBMCs using the DNeasy kit (QIAGEN) for HTLV-1 PVL measurement. Droplet digital PCR (ddPCR) was performed using primers and a probe targeting a conserved region in HTLV-1 pX region as previously described [16].
PCR cycles were performed in a QX200 Droplet Digital PCR system (Bio-Rad) with the following settings: 95 • C for 10 min, followed by 39 cycles of 94 • C for 30 s, 58 • C for 60 s, and final 98 • C for 10 min and 4 • C for hold. Data were analyzed using QuantaSoft software (Bio-Rad).
Statistical significance was analyzed by Prism 9 software (version 9.4.1; GraphPad Software).
Patients with lymphoma type represented the highest HTLV-1 cfDNA amount in all clinical subtypes, though they had low values of HTLV-1 PVL in PBMCs. The cfDNA levels of lymphoma type were significantly higher than those of chronic, smoldering types, and asymptomatic carriers (lymphoma vs. chronic, p = 0.01; lymphoma vs. smoldering, p = 0.001; lymphoma vs. asymptomatic carriers, p < 0.0001). The cfDNA levels of lymphoma type also tended to be higher than that of acute type, though there was no significant difference (median, 625 and 1650 copies/mL in acute and lymphoma types, respectively, p = 0.111). The HTLV-1 cfDNA levels of acute type were significantly higher than those of smoldering type and asymptomatic carries (acute vs. smoldering, p = 0.032; acute vs. asymptomatic carriers, p < 0.0001), but were not significantly different from those of chronic type (p = 0.164). However, the two patients with chronic type having the highest HTLV-1 cfDNA amount (shown in circles in Figure 1A HTLV-1 cfDNA was highly correlated with sIL-2R, which reflects the overall tumor burden in ATL patients. The soluble form of IL-2R is generated by the proteolytic cleavage of membrane IL-2Rα from activated T cells [17]. The sIL-2R value is, therefore, influenced by not only the disease states of ATL but also infection and inflammatory disorders. In fact, we experienced two HTLV-1-infected carriers who developed diffuse large B-cell lymphoma presented with high values of sIL-2R (10,514 and 3545 U/mL), while HTLV-1 cfDNA was not detected in their serum (Table 1). Additionally, another patient with smoldering type ATL showed right axillary lymphadenopathy and an elevated sIL-2 level (1501 U/mL), and therefore, we suspected a transformation to aggressive ATL. However, histological analysis of the swollen lymph node revealed reactive lymphadenopathy, which is consistent with the low levels of HTLV-1 cfDNA in the patient (8.8 copies/mL).
These findings suggested that the amount of HTLV-1 cfDNA has a higher specificity for ATL diagnosis compared to that of sIL-2R.
HTLV-1 cfDNA is a potential biomarker in HTLV-1-infected individuals from asymptomatic carriers to aggressive ATL. We demonstrated

F I G U R E 1 Measurement of HTLV-1 cell-free DNA (cfDNA) in plasma and proviral loads in peripheral blood mononuclear cells (PBMCs). (A)
HTLV-1 cfDNA copies per milliliter of plasma were quantified using droplet digital PCR (ddPCR) in patients with four acute types, five lymphoma types, seven chronic types (two unfavorable and five favorable chronic types), nine smoldering types, and 42 asymptomatic carriers. HTLV-1 cfDNA were not detected in one patient with smoldering type and 19 with asymptomatic carriers. (B) HTLV-1 proviral loads in PBMCs were quantified using ddPCR in the same patients. (C) Correlation between HTLV-1 cfDNA and soluble interleukin-2 receptor (sIL-2R). A significant correlation between sIL-2R and HTLV-1 cfDNA levels was observed. (D) Correlation between HTLV-1 cfDNA and serum LDH. HTLV-1 cfDNA level was not correlated with LDH. The value of r indicates correlation coefficient. Each circle, triangle, and square represent an individual with aggressive ATL (acute, lymphoma, unfavorable chronic types), indolent ATL (favorable chronic and smoldering types), and asymptomatic carrier, respectively. The gray marks in (C) and (D) indicate the three individuals in Table 1. The bars indicate median values with 95% confidence interval. Statistical significance was obtained by Mann-Whitney U test. *p < 0.05, **p < 0.005, ***p < 0.0001. HTLV-1, human T-cell leukemia virus type 1; ns, not significant; nd, not detected.

TA B L E 1
Clinical information of two HTLV-1 carriers who developed DLBCL and a patient with smoldering ATL who was diagnosed with reactive lymphadenopathy.

Patient
Age Clinical subtype Histopathological diagnosis HTLV-1 cell-free DNA (copies/mL) sIL-2R (U/mL) LDH (U/L)  Figure 1B. The population of CADM1 + CD7 dim cells evaluated by flow cytometric analysis increases in patients with ATL development (from asymptomatic carriers to indolent ATL) [9], while the population of CADM1 + CD7cells expands in patients who progress from indolent to aggressive ATL [8]. However, this flow cytometric analysis is not useful for predicting the development of lymphoma-type ATL because of the lack of expansion of abnormal cells in PBMCs. Early detection of lymphoma-type ATL using PBMCs is also difficult even by quantification of T-cell receptor Vβ and the evaluation of clonality through next-generation sequencing. We demonstrated that the amount of HTLV-1 cfDNA was extremely high even in patients with lymphoma type ( Figure 1A), suggesting that HTLV-1 cfDNA may potentially be used as a surrogate biomarker for lymphomatous ATL, which may be a more superior method in this setting.
The histological features of lymph nodes in ATL patients are usually characterized by diffuse proliferation of atypical medium-sized to large pleomorphic cells [18]. However, several morphological variants have been described that mimic angioim-