Plasma IL-35 in Mature T-Cell Neoplasms: An Exploratory Single-Centre Study

2.1. Study Cohort, Plasma Collection, and Cytokine Measurement

Diagnostic plasma samples included in the study were collected between 2020 and August 2025. This exploratory, non consecutive cohort comprised 20 adult patients with histologically confirmed mature T-cell neoplasms selected according to the availability of stored diagnostic EDTA plasma. Inclusion required: (i) a histologically confirmed mature T-cell neoplasm; (ii) an EDTA plasma sample obtained at diagnosis and available in sufficient quantity for IL-35 analysis; and (iii) retrievable clinical and survival data. Patients lacking histological confirmation, an appropriate diagnostic plasma sample, or adequate outcome data were not eligible for the analysis. Diagnostic terminology was harmonized for manuscript reporting according to the fifth edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5). The cohort included 18 patients with systemic T-cell neoplasms and two with primary cutaneous T-cell neoplasms (P15, CTCL; P17, mycosis fungoides with large-cell transformation). Clinical data, including diagnosis, prognostic scores, first-line treatment, best response, hematopoietic stem-cell transplantation, survival status, and cause of death, were retrospectively retrieved from the medical records. The survival database was locked on October 1, 2026. Overall survival was calculated to death or the last documented clinical contact on or before this cutoff date; patients alive at their last documented contact were censored at that date. Following the updated data lock, all patient level censored survival times were rechecked against the source diagnosis and follow-up dates and the survival analyses were repeated. Because the overall cohort included two primary cutaneous T-cell neoplasms, a disease scope sensitivity analysis was performed after excluding P15 and P17, leaving 18 patients with systemic disease.

Peripheral blood was collected into EDTA containing vacutainer tubes and processed under standardized pre-analytical conditions for plasma cytokine analysis. Samples were centrifuged at 500 × g for 10 minutes at room temperature (20 °C), brake disabled, to facilitate peripheral blood mononuclear cell (PBMC) separation while preserving plasma integrity. Plasma fractions from individual tubes were carefully aspirated without disturbing the cellular interface, pooled into a single 15 mL polypropylene tube, and gently mixed by inversion. The pooled plasma was subsequently subjected to a second centrifugation at 2,000 × g for 10 minutes at room temperature, with brake disabled, to remove residual platelets and cellular debris and obtain platelet-depleted plasma. Following clarification, plasma was aliquoted into 2.0 mL polypropylene cryovials and stored at −80 °C until downstream analyses.

Plasma concentrations of human interleukins IL-2, IL-10, and IL-35 were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturers’ validated protocols. IL-2 and IL-10 levels were measured using ELISA kits from Thermo Fisher Scientific (Waltham, MA, USA; Cat. Nos. BMS221-2 and BMS215-2, respectively), while plasma IL-35 concentrations were measured using the Human IL-35 ELISA Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. EEL067). EEL067 is a solid-phase sandwich ELISA designed by the manufacturer to detect and quantify human IL-35 in serum and plasma. In this sandwich format, the target is captured by an immobilized antibody and subsequently recognized by a detection antibody, while intervening washing steps remove unbound sample components and detection reagents before signal development. The manufacturer defines IL-35 as the heterodimer composed of IL-12α/p35 and EBI3. According to the manufacturer, the analytical sensitivity of the assay is 9.38 pg/mL and the validated assay range is 15.63–1,000 pg/mL. Because a substantial proportion of samples yielded estimated concentrations below the validated quantitative range, values below the analytical sensitivity were not interpreted as precise quantitative measurements. For survival analyses, IL-35 was therefore primarily categorized according to the manufacturer's analytical sensitivity (≥9.38 vs. <9.38 pg/mL). A sensitivity analysis using the lower boundary of the validated assay range (≥15.63 vs. <15.63 pg/mL) was also performed. Samples previously reported as below the detection limit were included in the below-threshold category.

Plasma samples were obtained from EDTA anticoagulated peripheral blood processed under the standardized pre-analytical conditions described above. Briefly, plasma samples and standards were added to pre-coated 96-well microplates and incubated with biotinylated detection antibodies. After sequential washing steps to remove unbound material, horseradish peroxidase (HRP)–conjugated streptavidin was applied, followed by chromogenic substrate addition. The enzymatic reaction was terminated using stop solution, and optical density was measured at 450 nm using a Sunrise Basic microplate reader (Tecan, Männedorf, Switzerland) with Magellan v6.5 software. Cytokine concentrations were determined by interpolation from standard curves generated for each assay. All samples and standards were analyzed in duplicate. Assays were performed in accordance with the manufacturers’ quality control specifications, and samples yielding absorbance values outside the linear range of the standard curve were re-assayed after appropriate dilution. Plasma samples from eleven patients were analyzed for IL-2 and IL-10, while twenty patients were included in the IL-35 analysis.

2.2. Statistical Analysis

Continuous variables were summarized using medians and interquartile ranges (IQRs), and categorical variables as counts and percentages. Overall survival (OS) was calculated from the date of diagnosis to death from any cause or the last documented clinical contact on or before the data-lock date of October 1, 2026; patients alive at their last documented contact were censored at that date. Because several IL-35 measurements fell below the validated quantitative range of the assay, survival analyses did not treat low estimated concentrations as precise continuous measurements. The primary IL-35 analysis therefore categorized patients according to the manufacturer's analytical sensitivity (≥9.38 vs. <9.38 pg/mL); samples reported as below the detection limit were retained in the <9.38 pg/mL group. Kaplan–Meier curves were compared using the log-rank test, and univariable Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Tied event times in the Cox proportional hazards regression were handled using the Breslow method. An assay-based sensitivity analysis used the lower boundary of the validated quantitative range (≥15.63 vs. <15.63 pg/mL). An additional disease scope sensitivity analysis excluded the two patients with primary cutaneous T-cell neoplasms (CTCL and mycosis fungoides with large-cell transformation), leaving 18 patients with systemic disease. Given the small number of deaths, no multivariable model was used for these categorical analyses. IL-2 and IL-10 were analyzed descriptively because of the limited number of quantifiable measurements. All tests were two-sided, with p < 0.05 considered statistically significant. Statistical analyses were performed using Python 3.13.5, with pandas 2.2.3, NumPy 2.3.5, SciPy 1.17.0, and statsmodels 0.14.6.

3.1. Patient Characteristics

The study included 20 patients with mature T-cell neoplasms. The median age at diagnosis was 54 years (range, 40–78; IQR, 45–60), and 14 patients (70%) were male. ALK-negative anaplastic large cell lymphoma (ALK-negative ALCL) and adult T-cell leukemia/lymphoma (ATLL) were the most frequent diagnoses, each accounting for 5 cases (25%). Nodal T-follicular helper cell lymphoma (nTFHL), including two cases of angioimmunoblastic type, was diagnosed in 3 patients (15%), while peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), was diagnosed in 2 (10%). The remaining cases included monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), enteropathy-associated T-cell lymphoma (EATL), extranodal NK/T-cell lymphoma (ENKTL), cutaneous T-cell lymphoma (CTCL), and mycosis fungoides with large-cell transformation, with one case each. Baseline demographic and diagnostic characteristics are summarized in Table 1.

3.2. Plasma IL-35 Concentrations

Estimated plasma IL-35 concentrations showed marked interpatient heterogeneity, ranging from 1.16 to 153.94 pg/mL among samples for which a numerical concentration was obtained. The cohort median was 12.67 pg/mL. However, 11 of 19 numerical estimates were below the lower boundary of the validated assay range (15.63 pg/mL), and 8 were below the manufacturer's analytical sensitivity (9.38 pg/mL). These low-end numerical estimates were therefore retained for descriptive presentation only and were not interpreted as precise quantitative concentrations in the survival analyses.

Using the manufacturer's analytical sensitivity, 11 patients had IL-35 values ≥9.38 pg/mL and 9 were classified below this threshold, including the patient whose result was reported as below the detection limit. Individual IL-35 results, prognostic scores, and survival outcomes are presented in Table 2.

3.3. Association Between Plasma IL-35 and Overall Survival

Using the manufacturer's analytical sensitivity of 9.38 pg/mL as an assay-based threshold, 11 patients had IL-35 values ≥9.38 pg/mL and 9 had values below this threshold. Three of 11 patients in the ≥9.38 pg/mL group died during follow-up, compared with 7 of 9 patients in the <9.38 pg/mL group. Kaplan–Meier analysis showed longer OS in the ≥9.38 pg/mL group (log-rank p = 0.029; Figure 2). In univariable Cox regression, IL-35 ≥9.38 pg/mL was associated with a lower hazard of death (HR 0.26, 95% CI 0.07–0.99; p = 0.049).

Figure 1.

Figure 2.

The result was consistent in a sensitivity analysis using 15.63 pg/mL, the lower boundary of the validated quantitative assay range. Eight patients had IL-35 values ≥15.63 pg/mL, of whom 1 died, compared with 9 deaths among 12 patients below 15.63 pg/mL. The ≥15.63 pg/mL group had longer OS (log-rank p = 0.012), with a univariable HR for death of 0.12 (95% CI 0.015–0.92; p = 0.041).

Because the overall cohort included two patients with primary cutaneous T-cell neoplasms (P15, CTCL; P17, mycosis fungoides with large-cell transformation), a disease scope sensitivity analysis was restricted to the 18 patients with systemic T-cell neoplasms. The association between higher IL-35 and longer OS was preserved. Using the primary analytical-sensitivity threshold of 9.38 pg/mL, IL-35 ≥9.38 pg/mL was associated with a lower hazard of death (HR 0.23, 95% CI 0.058–0.89; p = 0.033; log-rank p = 0.016). Using 15.63 pg/mL, the lower boundary of the validated quantitative range, the association remained evident (HR 0.076, 95% CI 0.009–0.61; p = 0.015; log-rank p = 0.0015). These disease-scope analyses were considered exploratory because of the small number of patients and events.

3.4. Plasma IL-2 and IL-10 Concentrations

IL-2 and IL-10 were evaluable in 11 patients and were quantifiable in only three patients each; because of the small number of measurable values, these cytokines were considered descriptive and were not subjected to formal survival analysis.