Chronic myeloid leukemia patients in prolonged remission following interferon-α monotherapy have distinct cytokine and oligoclonal lymphocyte profile

Abstract

Before the era of tyrosine kinase inhibitors (TKIs), interferon-alpha (IFN-α) was the treatment of choice in chronic myeloid leukemia (CML). Curiously, some IFN-α treated patients were able to discontinue therapy without disease progression. The aim of this project was to study the immunomodulatory effects of IFN-α in CML patients in prolonged remission and isolate biological markers predicting response. Due to rarity of patients on IFN-α monotherapy, a relatively small cohort of patients still on treatment (IFN-ON, n = 10, median therapy duration 11.8 years) or had discontinued IFN-α therapy but remained in remission for >2 years (IFN-OFF, n = 9) were studied. The lymphocyte immunophenotype was analyzed with a comprehensive flow cytometry panel and plasma cytokine levels were measured with multiplex bead-based assay. In addition, the clonality status of different lymphocyte subpopulations was analyzed by TCR γ/δ rearrangement assay. Median NK-cell absolute number and proportion from lymphocytes in blood was higher in IFN-OFF patients as compared to IFN-ON patients or controls (0.42, 0.19, 0.21×10(9)/L; 26%, 12%, 11%, respectively, p<0.001). The proportion of CD8+ T-cells was significantly increased in both patient groups and a larger proportion of T-cells expressed CD45RO. Most (95%) patients had significant numbers of oligoclonal lymphocytes characterized by T-cell receptor γ/δ rearrangements. Strikingly, in the majority of patients (79%) a distinct clonal Vγ9 gene rearrangement was observed residing in γδ(+) T-cell population. Similar unique clonality pattern was not observed in TKI treated CML patients. Plasma eotaxin and MCP-1 cytokines were significantly increased in IFN-OFF patients. Despite the limited number of patients, our data indicates that IFN-α treated CML patients in remission have increased numbers of NK-cells and clonal γδ(+) T-cells and a unique plasma cytokine profile. These factors may relate to anti-leukemic effects of IFN-α in this specific group of patients and account for prolonged therapy responses even after drug discontinuation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow cytometry analysis of basic lymphocyte subpopulations.

Immunophenotyping was done with 6-color flow cytometry from healthy controls (n = 16), IFN-α-treated CML patients (IFN-ON, n = 10), and CML patients who had discontinued IFN-α therapy (IFN-OFF, n = 9). Figures represent relative proportions of analyzed cells and corresponding absolute numbers can be found in Table S1. Statistical significance of differences in continuous variables was assessed by a non-parametric analysis of variance using the Kruskal-Wallis test (P-values reported in figures) and in case of a significant main effect, pairwise comparisons of patient groups were calculated using Dunn's multiple comparison test (statistically significant differences between groups are marked with asterisks and line). For the analysis of Vγ9+ cells (panel F), samples from 5 healthy controls, 4 IFN-ON, and 5 IFN-OFF patients were available. The figures also include results from four patients with myeloproliferative neoplasm (MPN), but they were not included in the statistical analysis.

Figure 2. Clonal Vγ9 rearrangement in γδ+ T-cells.

Example of Vδ2Jγ9 clonality detection in patient number 4 (Table 1). PCR with Vg2-Jg1.2 primers was carried out on FACS sorted cell fractions and the clonal products were identified by heteroduplex analysis. Lane 1, DNA ladder; lane 2 αβ+ T-cell fraction; lane 3 γδ+ T-cell fraction; lane 4 pool of healthy controls showing polyclonal smear; lane 5, water control. Clonal PCR product in the γδ+ T-cell fraction is shown with a white arrow.

Figure 3. Plasma IP-10, IL-6, IL-12, eotaxin, MCP-1, and IFN-γ cytokine levels.

Plasma levels of 25 cytokines were measured with a multiplex bead-based cytokine assay (Luminex®) from healthy controls (n = 10), IFN-α-treated CML patients (IFN-ON, n = 10), and CML patients who had discontinued IFN-α therapy (IFN-OFF, n = 9). Statistical significance of differences in continuous variables was assessed by a non-parametric analysis of variance using the Kruskal-Wallis test (P-values reported in figures) and in case of a significant main effect, pairwise comparisons of patient groups were calculated using Dunn's multiple comparison test (statistically significant differences between groups are marked with asterisks and line). The figures also include results from four patients with myeloproliferative neoplasm (MPN), but they were not included in the statistical analysis.

Figure 4. Characteristics of a predictor variable for IFN-α discontinuation.

A. Example of a hypothetical variable differentiating patients who can stop IFN-α therapy (empty circle) from those who cannot in the IFN-ON group. B. NK-cell count as a potential predictor variable. The figure presents absolute NK-cell numbers in different patient groups. Patients still using IFN-α therapy were divided in 2 groups based if their absolute NK-cell count was above (IFN-ON NK-high) or below (IFN-OFF NK-low) 0.2×109/l. C–H. Biomarker profile of NK-high and NK-low groups. Patients using IFN-α therapy (IFN-ON) were divided in 2 groups based on the absolute NK-cell count in the peripheral blood. In NK-high group, the absolute NK-cell count was above 0.2×109/l and in NK-low group below the given limit. Other variables are presented based on the group division by NK-cell number and IFN discontinued patients as a separate cohort (IFN-OFF).