Enlarged Memory T-Cell Pool and Enhanced Th1-Type Responses in Chronic Myeloid Leukemia Patients Who Have Successfully Discontinued IFN-alpha Monotherapy

Peer reviewe

Chronic Myeloid Leukemia Patients in Prolonged Remission following Interferon-alpha Monotherapy Have Distinct Cytokine and Oligoclonal Lymphocyte Profile

Peer reviewe

Treatment of Chronic Myelomonocytic Leukemia with 5-Azacytidine: Case Reports

Epigenetic therapy with hypomethylating agent (5-azacytidine; AZA) is common in the management of specific subtypes of myelodysplastic syndrome (MDS), but there are only few studies in chronic myelomonocytic leukemia (CMML) patients. In this paper our experience with 3 CMML patients treated with AZA is described. In one patient transfusion independency was observed after 4 treatment cycles; in one case a partial response was recorded, but a progression to acute myeloid leukemia (AML) after 13 AZA cycles has appeared. In one patient, AZA in reduced dosage was administered as a bridging treatment before allogeneic stem cell transplantation (ASCT), but in the control bone marrow aspirate (before ASCT) a progression to AML was recorded. Future studies are mandatory for evaluation of new molecular and clinical features which could predict the efficiency of hypomethylating agents in CMML therapy with respect to overall survival, event-free survival, quality-adjusted life year, and pharmacoeconomy

Interferon-α Revisited: Individualized Treatment Management Eased the Selective Pressure of Tyrosine Kinase Inhibitors on BCR-ABL1 Mutations Resulting in a Molecular Response in High-Risk CML Patients.

Bone marrow transplantation or ponatinib treatment are currently recommended strategies for management of patients with chronic myeloid leukemia (CML) harboring the T315I mutation and compound or polyclonal mutations. However, in some individual cases, these treatment scenarios cannot be applied. We used an alternative treatment strategy with interferon-α (IFN-α) given solo, sequentially or together with TKI in a group of 6 cases of high risk CML patients, assuming that the TKI-independent mechanism of action may lead to mutant clone repression. IFN-α based individualized therapy decreases of T315I or compound mutations to undetectable levels as assessed by next-generation deep sequencing, which was associated with a molecular response in 4/6 patients. Based on the observed results from immune profiling, we assumed that the principal mechanism leading to the success of the treatment was the immune activation induced with dasatinib pre-treatment followed by restoration of immunological surveillance after application of IFN-α therapy. Moreover, we showed that sensitive measurement of mutated BCR-ABL1 transcript levels augments the safety of this individualized treatment strategy

Detection of Unknown and Rare Pathogenic Variants in Antithrombin, Protein C and Protein S Deficiency Using High-Throughput Targeted Sequencing

The deficiency of natural anticoagulants—antithrombin (AT), protein C (PC), and protein S (PS)—is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected—SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels

The cytotoxicity of NK-cells from IFN-OFF and IFN-ON patients is impaired.

<p>MNCs were used as effector cells and K562 cells as target cells. The NK-cell percentage was determined by flow cytometry and the number of effector MNCs was counted accordingly. Cells were co-incubated for 6 h at +37°C at effector:target ratios 4∶1 and 8∶1. The graphs present alive K562 cells after the co-incubation with effector cells.</p

Function of T-cell subsets in IFN-OFF patients.

<p><b>A)</b> T-cells were stimulated with anti-CD3, –CD28 and -CD49d and after 6 h incubation, cell surface markers and intracellular cytokines were stained and analyzed with flow cytometry. The cytokine secretion of CD4+ T-cell subsets (effector and central memory, naïve and temra cells) was analyzed separately. The percentage values present the proportion of cytokine secreting cells from all CD4+ cells showing that the effector and central memory T-cells are the main cytokine producers. <b>B)</b> The proportion of cytokine secreting effector and central memory CD4⁺ T-cells were also analyzed separately and the % values show the proportion of positive cells from a subset in question. <b>C)</b> Cytokine secretion of CD8⁺ T-cell subsets and <b>D)</b> the proportion of cytokine secreting effector and central memory CD8⁺ T-cells separately. <b>E)</b> Degranulation of CD8⁺ T-cells was analyzed by CD107a/b expression with flow cytometry after 6 h incubation with anti-CD3, -CD28 and -CD49d. Unstimulated MNCs were used as a control.</p

Patient characteristics.

*<p> = Pregnant. Abbreviations: CP, chronic phase; m, male; f, female; dg, diagnosis; cont, continued; MU, million unit; MR4.0, molecular response 4.0, CMR, Complete Molecular Response; MMR, Major Molecular Response; Auto-PBSCT, autologous peripheral blood stem cell transplantation.</p

NK-cells from IFN-OFF patients have a mature phenotype.

<p>NK-cell proportions and surface markers CD62L, CD57 and CD27 were analyzed with flow cytometry and absolute NK-cell counts were counted from total lymphocyte numbers. <b>A)</b> The proportion of NK-cells in IFN-OFF and IFN-ON patients <b>B)</b> Absolute amount of NK-cells in IFN-ON and IFN-OFF patients <b>C)</b> The proportion of CD56^DIM NK-cells from CD56 NK-cells in IFN-ON and IFN-OFF patients <b>D)</b> Absolute amounts of CD56^DIM NK-cells in IFN-ON and IFN-OFF patients. <b>E)</b> CD57 expression in CD56^DIM NK-cells. <b>F)</b> CD62L expression in CD56^DIM NK-cells <b>G)</b> CD27 expression in CD56^DIM NK-cells. White dots represent pregnant patients in IFN-ON group (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087794#pone-0087794-t001" target="_blank">Table 1</a>).</p

CD4⁺ T-cells from IFN-OFF patients are more prone to secrete TNF-α/IFN-γ than CD4⁺ T-cells from the healthy.

<p>T-cells were stimulated with anti-CD3, –CD28 and -CD49d. After 6 h incubation, cell surface markers and intracellular cytokines were stained and analyzed with flow cytometry. <b>A)</b> Non-stimulated and stimulated TNF-α/IFN-γ secretion of CD3+ T-cells. Shown are 3 representative patients from each group (IFN-OFF, IFN-ON and healthy). Percentages are calculated from CD4⁺ T-cell population. <b>B)</b> TNF-α/IFN-γ secretion of CD4⁺ T-cells in all patients <b>C)</b> TNF-α/IFN-γ secretion of CD8⁺ T-cells in all patients. White dots represent pregnant patients in IFN-ON group (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087794#pone-0087794-t001" target="_blank">Table 1</a>).</p