The Number Of Peripheral Blood CD4+CD25+FOXP3+ Regulatory T-Cells Is a Marker Of Response In Patients With JAK2 V617F Positive Myeloproliferative Neoplasms Treated With Pegylated-Interferon-α

Treatment of patients with polycythemia vera (PV) or essential thrombocythemia (ET) with pegylated interferon-a (Peg-Infa) frequently induces hematologic remission and may also induce a decrease in the JAK2^V617Fallele burden and a reversion to polyclonal hematopoiesis. However, the mechanism(s) by which Peg-Infa generates these responses remain unclear. Possibilities include a direct suppression of clonal hematopoietic stem cells, a stimulation of quiescent residual normal stem cells or a beneficial alteration of the bone marrow immune microenvironment. We are currently exploring a variety of hypotheses and submit that Peg-Infa acts via a combination of these mechanisms. We have previously examined its role in the possible reversion to polyclonal hematopoiesis in patients treated in a Phase III trial for PV and ET with either Peg-Infa or hydroxyurea. Previous data has demonstrated that Peg-Infa treatment induces an increase in the number of circulating CD4+CD25+FOXP3+ regulatory T cells (Tregs). Tregs are an immunosuppressive T cell subset characterized by expression of FOXP3. They have an established influence on the state of the bone marrow immune microenvironment and appear to play a role in the suppression of anti-tumor immunity in a variety of malignant processes. Thus, they may have a previously unexplored influence on the pathophysiology of myeloproliferative neoplasms that is important in the context of Peg-Infa treatment. In this study, our aims were to serially monitor Tregs in a series of ET and PV patients treated with Peg-Infa to determine if treatment influenced Treg numbers and determine whether or not Tregs correlated with markers of response.

Peripheral blood samples we collected at various time intervals and assessed for the number of circulating Tregs, the JAK2^V617F allelic burden in granulocytes and, in females, the possible conversion of clonal to polyclonal hematopoiesis (detected by X-chromosome allelic usage ratio). The JAK2^V617F allelic burden was measured by quantitative allele-specific PCR on an Applied Biosystems instrument in peripheral blood granulocytes enriched using density gradient centrifugation. The number of Tregs was measured in peripheral blood by flow cytometry using a cocktail of antibodies including CD4, CD25 and FOXP3. The proportion of Tregs among lymphocytes was measured. Using this information, and the absolute lymphocyte count from the CBC data, the absolute number of circulating Tregs was calculated and expressed as #Tregs/microliter. The number of Tregs/microliter was also correlated with the QT-PCR-determined transcript levels of FOXP3.

In ongoing studies, 32 patients (15 females and 17 males) with PV and ET were treated with Peg-Infa. Following treatment, 15 of 23 patients with clinical follow-up demonstrated a 20% or greater decrease in the JAK2^V617F allelic burden, indicating that Peg-Infa had an effect on the JAK2^V617F-positive clone. Eight patients had no response or had an increase in JAK2^V617F levels. Thirteen out of 15 female patients were informative for clonality testing. Two of these 13 patients developed polyclonal hematopoiesis during therapy. In one of these patients, the JAK2^V617F allelic burden was unchanged, indicating that Peg-Infa had a selective effect on a pre-JAK2^V617F PV clone, while in the other patients Peg-Infa clearly decreased the JAK2^V617F-positive PV clone. There was a significant inverse correlation between the absolute number of peripheral blood Tregs and the JAK2^V617F allelic burden. We also found that the changes in Treg numbers correlated with FOXP3 mRNA in circulating monuclear cells. Similar ongoing studies are being performed in the marrow of these patients and in patients treated with hydroxyurea.

In conclusion, Peg-Infa induces changes in the number of circulating Tregs, in addition to the previously reported beneficial clinical effects including a decrease in the JAK2^V617F allelic burden and, in some instances, re-establishment of polyclonal hematopoiesis. Furthermore, we found that the number of peripheral blood Tregs is inversely correlated to the JAK2^V617Fallelic burden suggesting that it may serve as a marker of response and suggesting the Tregs may play a role in the pathophysiology of the disease.

First and second authors contributed equally

This work was supported by 1P01CA108671-O1A2 (NCI) MPD Consortium and the Leukemia & Lymphoma Society