In this prospective study, we measured plasma levels of sIL2-Rα, TIM-3, ST-2, intercellular adhesion molecule (sICAM-1), IFN-γ, and IL-6 at a fixed time point after allogeneic hematopoietic transplantation; that is, day +18 after allogeneic hematopoietic transplantation.

The present study included 95 patients, who underwent allogeneic hematopoietic transplantation at our institution between January 2013 and September 2017. It was a biological study aimed to assess at day +18 an array of cytokines in the plasma, as well as the frequency of clonogenic precursors in marrow aspirates. The study was approved by the Ethical Committee of our institution (35/2013VE), as an observational study. All patients received relevant information and gave consent.

Diagnoses included acute leukemia (n = 60), multiple myeloma or lymphoma (n = 16), and others (n = 19). Diagnoses were grouped into two categories: acute leukemia, lymphoma, and multiple myeloma (AL/LYM/MM) and aplastic anemia, myelodysplastic syndromes, and chronic myeloproliferative neoplasms (AA/MDS/MPN). Conditioning schedules were classified as myeloablative (MA) or reduced intensity conditioning (RIC), according to recently proposed criteria (13). MA conditioning was used in 82.5% of cases, and RIC was used in 17.5%. Intravenous busulfan (12.8 mg/kg), plus either fludarabine 160 mg/m² or cyclophosphamide 120 mg/kg, comprised the most commonly used MA conditioning regimen (42% of all MA conditioning). A further 8% of MA conditioning comprised total body irradiation (12 Gy) plus cyclophosphamide.

In 84% of cases, GVHD prophylaxis was cyclosporine + short course methotrexate (MTX). The MTX was routinely administered in four doses after matched unrelated donor (MUD) transplantation, or transplantation from an identical family donor, from whom the source was hematopoietic progenitor cells obtained from peripheral blood stem cells. Three doses of MTX were administered to patients with transplants from identical family donors, from whom the source was bone marrow. Anti-thymocyte globulin was routinely used only after MUD transplantation. The GVHD prophylaxis was grouped into two categories, CSA + MTX + ATG vs. others forms of prophylaxis. Criteria for acute GVHD scoring and treatment have been previously reported (12). Clonogenic precursors (colony forming unit–granulocyte, monocyte [CFU-GM] and burst-forming unit–erythroid [BFU-e]) in the marrow were studied on day +30 (n = 39). Demographic and disease-related features of patients are reported in Table 1. At the time of analysis, median follow-up for patients still alive was 198 weeks (range, 99–344 weeks).

Blood was drawn on day +18/+19 after transplantation, and plasma was obtained by centrifugation within 2 h. Samples were stored at −70°C until further analysis. Cytokines were assayed by automated ELISA, and each sample was tested in duplicate. A titration curve was constructed for known concentrations of various cytokines in the plasma, obtained from the kit manufacturer. Both ST-2 and TIM-3 were assayed using Bio-Rad ELISA kits, while high sensitivity IFN-γ, IL-6, and sIL2-Rα assays were conducted using Diaclone ELISA kits. The ELISAs were conducted by one of the authors (VF) in a central laboratory at our hospital, which specializes in this type of assay, and data analysis was supervised by AEM. Owing to missing data, TIM-3 and sIL2-Rα results were available for only 75/95 patients.

Comparisons of cytokine concentrations between groups, or other data with a non-normal distribution, were performed using the non-parametric Mann–Whitney U-test. Median and interquartile ranges (IQR) were used to describe the data. The values of cytokines as continuous variables were tested using a Cox proportional hazard model for OS, and a Fine and Gray proportional hazard model for competing events test for TRM and relapse risk (RR).

Receiver operating characteristic (ROC) curves were used to identify cutoff values for cytokine levels and determine the best combination of sensitivity and specificity with respect to OS. These cutoff values were used to divide patients into two groups. Gray's test was used for comparison of the cumulative incidence of competing risks (TRM and RR). A value of p ≤ 0.05 was considered to indicate statistically significant differences. Statistical analyses were performed using the StatView 5.0 (Cary, NC) or R software (EZR, version 3.1.1; 2014, R Foundation for Statistical Computing, Vienna, Austria).

Plasma levels of cytokines on day +18 are reported in Table 2. Patients with transplants from MUD or haploidentical donors had higher plasma levels of sIL2-Rα (p = 0.002), TIM-3 (p = 0.009), ICAM-1 (p = 0.03), and IL-6 (p = 0.05) than those who received transplants from HLA-identical siblings.

For all patients under investigation, the OS at 2 years was 58.6% (95% confidence interval [CI]), 48–67%), while the TRM at 2 years was 17.9% (95% CI, 10.9–26.3%). The overall RR at 2 years was 24.2% (95% CI, 16.1–33.3%). Factors important for OS were age (HR = 1.034, p = 0.01), marrow as the source of HSCs (HR = 2.053; 95% CI, 1.142–3.691; p = 0.01), use of a-GVHD prophylaxis other than CSA + MTX + ATG (HR 1.794; 95% CI, 1.011–3.184; p = 0.04) and AA/MDS/MPN diagnosis type (HR = 0.223; 95% CI, 0.054–0.920; p = 0.03). In contrast, no significant association was observed between OS and Haplo-MUD donor type, hematopoietic cell transplantation HCT-comorbidity score, disease stage, or conditioning type. When evaluated as continuous data, sIL2-Rα (HR = 1.005, p = 0.002) and TIM-3 (HR = 1.054, p = 0.0007) were also significantly associated with OS (Table 3). The levels of sIL2-Rα, IFN-gamma, and IL-6 were found to be important predictors of TRM (Table 3). No biomarkers were significantly associated with RR.

We wished to identify threshold levels of cytokines with clinical importance for predicting OS. Thus, the area under the curve (AUC) was calculated for the ROC curves (Supplementary Table 1) and the best cutoff values were identified. The ROC curve for TIM-3, with regard to the end point of OS, had an AUC of 0.616 (95% CI 0.488–0.744), with a cutoff of 950 pg/ml. The ROC curve for sIL2-Rα had an AUC of 0.605 (95% CI 485–0.726), with a cutoff of 8,100 pg/ml. The ROC curve for IL-6 had an AUC of 0.563 (9% CI 0.434–0.692), with a cutoff of 3,490 pg/ml. The ROC curve for IFN-γ had an AUC of 0.602 (95% CI 0.474–0.730), with a cutoff of 6,360 pg/ml (Supplementary Figures 1, 2).

The predictive power of cytokines for OS was then evaluated by grouping patients, based on these cutoff values. Data were then analyzed using a multivariable stepwise Cox proportional model, which included the variables age, source of HSCs, diagnoses categorized into two groups (AA/MDS/MPN vs. AL/LYM/MM), GVHD prophylaxis, and donor type. Factors significantly associated with OS were HSC source, diagnosis, GVHD prophylaxis, and levels of both TIM-3 and sIL2-Rα above their respective cutoff values (HSC source: p to remove, p = 0.003; diagnosis: p to remove, p = 0.01; GVHD prophylaxis other than CSA + MTX + ATG: p to remove, p = 0.002; TIM-3 over the threshold: p to remove, p = 0.001; sIL2-Rα over the threshold: p to remove, p = 0.008).

Patients with TIM-3 and sIL2-Rα levels over these thresholds had lower OS rates, according to univariable and multivariable analyses (Table 4).

When patients were grouped based on TIM-3 levels, according to the determined cutoff value, those with TIM-3 levels <950 pg/ml had a TRM of 5.3% (95% CI, 0.3–22%) vs. 23.7% (95% CI, 13.7–35.2%) in patients with higher plasma levels of this cytokine (p = 0.05). Further, TRM in patients with low levels of sIL2-Rα (<8,100 pg/ml) was 10.5% (95% CI, 4.2–20.1%) vs. 34.5% (95% CI, 17.7–51.9%) in the group with higher plasma levels of the same cytokine (p = 0.002).

Given the importance of sIL2-Rα and TIM-3 for OS and TRM, we constructed a composite scoring system, based on the frequency with which the two biomarkers showed levels over their respective threshold values. A score of 0 was attributed to patients with both TIM-3 and sIL2-Rα levels below the respective cutoff values; a score of 1 was attributed to patients with levels of only one of the two biomarkers over the threshold; and a score of 2 was attributed to patients with levels of both biomarkers above the respective cutoff values.

Kaplan–Meier analysis evaluating OS in patients grouped according to the composite score, both unadjusted and adjusted, for the effects of diagnosis, GVHD prophylaxis, and HSC source, is presented in Figure 1. The OS rates were projected to be 95, 65, and 30% at 2 years in patients with scores of 0, 1, and 2, respectively (trend log-rank, p = 0.0001). Multivariate analysis indicated that patients with both sIL2-Rα and TIM-3 levels over the respective threshold values had an HR of 4.188 (95% CI, 1.948–9.004) for death, relative to all other patients (p = 0.0002) (Table 5).

Patients with scores of 0 (n = 14) had 2 years TRM rates of 7.1% (95% CI, 4–28%); those with scores of 1 (n = 37) had 2 years TRM rates of 10.8% (95% CI, 3.4–23.3%); and those with scores of 2 (n = 24) had 2 years TRM rates of 41.7% (95% CI, 21.5–60.7%) (Gray's test, p = 0.001) (Figure 2). Fulfillment of criteria for score 2 had a sensitivity of 0.688 (95% CI, 0.413–0.890) and a specificity of 0.780 (95% CI, 0.653–0.877) in predicting TRM (Table 6).

The combined score was also highly informative in the stratum of Haplo-MUD transplantation, both with respect to OS (Trend log-rank: p = 0.0001) and TRM (Gray's test p = 0.001) (Figure 3).

Results were not different if analysis was performed in the stratum of patients affected by acute leukemia. In fact, when we selected AML, ALL, and MDS patients (n 65), group 0 patients had, at 2 years, an OS, of 92.9% (CI 59.1–99.0%) and a TRM of 7.1% (CI 0.4–28.5%); group 1 patients had an OS of 58.3% (CI 40.6–72.4%) and a TRM of 10.8% (CI 3.4–23.3%); group 2 patients had an OS of 20.8% (CI 7.5–38.5%) and a TRM of 45.8% (CI 24.7–64.7%). Difference in OS was significant (log-rank: p = 0.00004) as difference in TRM was significant (Gray's test p = 0.001).

The ROC curve of this combined score had an AUC of 0.738 (95% CI, 0.637–0.840) for OS and an AUC of 0.744 (95% CI, 0.612–0.875) for TRM (Supplementary Figure 3).

The incidence of Grades II–IV a-GVHD during the first 100 days was higher in patients with scores of 2 vs all other patients (54 vs 36%, respectively); however, this difference was not significant. Further, the incidence of lower gastrointestinal tract involvement in GVHD was 33, 8.5, and 7.1% in patients with scores of 2, 1, and 0, respectively (p = 0.007) (Supplementary Table 2).

Patients presenting with scores of 2 at day +18 had a reduced number of total lymphocytes relative to all other patients (0.050 × vs. 0.220 ×10⁹/L, respectively; p = 0.0001), as well as a lower median absolute neutrophil count (0.345 × vs. 0.540 ×10⁹/L, p = 0.04). Evaluation of BFU-e growth, using marrow samples collected on day +30 in 39 patients, revealed significantly reduced levels of BFU-e in patients with scores of 2 (p = 0.005) (Supplementary Table 2). Score 2 patients had, in respect to all other patients, a higher need for blood red cell transfusion, median 5 units vs. 2 units (p = 0.009). A higher number of platelets transfusion were registered in score 2 patients, median 9 units vs. 4 units (p = 0.0002). Moreover, patients with scores of 2 had higher rates of fever between days +10 and +18 (58, vs. 45 and 14% in patients with scores of 1 and 0, respectively; p = 0.02). No differences were detected in the incidence of positive hemoculture (p = 0.70) or cytomegalovirus (CMV) reactivation rate during the first 25 days (p = 0.52) among the three groups.

Among the patients with scores of 0–1 (n = 51 patients), 20 died and five of those deaths were due to TRM. Of those five deaths due to TRM, four were ascribed to a-GVHD or infections. Among patients with scores of 2 (n = 24), 20 died, and 11 of those deaths were due to TRM; of those 11 deaths, eight were ascribed to a-GVHD or infections. Thus, deaths due to a-GVHD or infections were recorded for 4/51 (8%) patients in the group with scores of 0–1, and 8/24 (33%) in the group with a score of 2 (chi-squared, p = 0.001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.