B-cell frequencies and immunoregulatory phenotypes in myeloproliferative neoplasms: Influence of ruxolitinib, interferon-α2, or combination treatment.

OBJECTIVE: Given a proposed role for PD-L1+ and IL-10-producing B-cell subsets in promoting certain cancers, we sought to characterize the frequency and phenotype of B cells in patients with chronic myeloproliferative neoplasms (MPNs) and the influence of ruxolitinib and interferon-α2 therapy. METHODS: We analyzed B-cell frequencies and phenotype in patients with MPNs (n = 107), before and during treatment with ruxolitinib (n = 29), interferon-α2 (n = 21), or the two drugs in combination (COMBI; n = 42) and healthy donors (HDs; n = 52) using flow cytometry. RESULTS: Myelofibrosis patients had lower lymphocyte counts and proportions of B cells than patients with essential thrombocythemia or polycythemia vera and HDs. The B-cell count correlated inversely with JAK2-V617F allele burden and spleen size and increased after ruxolitinib or COMBI treatment. The proportions of PD-L1+ B cells and PD-1+ B cells were significantly higher in patients with myelofibrosis or polycythemia vera than in HDs and decreased during ruxolitinib and COMBI treatment. The proportions of TNF-α+ and IL-6+ B cells were elevated in myelofibrosis patients. The proportion of IL-6+ B cells decreased, and the proportion of IL-10+ B cells increased during ruxolitinib treatment. CONCLUSION: B-cell frequency and phenotype were altered in MPN patients. Ruxolitinib therapy had marked effects on both frequency and phenotype.

Interferon-α induces marked alterations in circulating regulatory T cells, NK cell subsets, and dendritic cells in patients with JAK2V617F-positive essential thrombocythemia and polycythemia vera.

Long-term therapy with IFN-α2 is associated with sustained major molecular remissions in JAK2-positive ET and PV. The efficacy of IFN-α2 may be partly mediated by modulation of immune cells, which was investigated in twenty patients with ET (n = 6) and PV (n = 14). The frequency of CD4(+) CD25(+) Foxp3(+) T cells was significantly increased during IFN-α2 treatment in all patients (P < 0.0001). A significant expansion of the CD56(bright) NK cells (P = 0.0002) and a concomitant decrease in the frequency of CD56(dim) NK cells (P < 0.0001) were also detected. Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) were studied in nine patients, and decreased frequencies of both cell types were observed during the course of treatment. On both mDCs and pDCs, HLA-ABC expression was upregulated (P = 0.003), but decreasing expression levels of HLA-DR was detected on mDCs. The expression of CD40 (P = 0.002), CD83 (P = 0.03), and CD86 (P = 0.01) increased, but was confined to pDCs. Furthermore, PD-L1 expression was reduced on mDC (P = 0.003) and increased on pDCs (P = 0.02). No significant correlations were found between the changes in immune cells and hematological or molecular responses achieved in our cohort of patients. So forth, it remains to be revealed whether the profound changes in circulating immune cells contribute to the beneficial effects of long-term IFN-α2 treatment in some patients.

Expansion of circulating CD56bright natural killer cells in patients with JAK2-positive chronic myeloproliferative neoplasms during treatment with interferon-α.

In recent years, major molecular remissions have been observed in patients with JAK2-positive chronic myeloproliferative neoplasms (MPNs) after therapy with IFN-α. IFN-α is known to have altering effects on immune cells involved in immune surveillance and might consequently enhance anti-tumor immune response against the JAK2-mutated clone. The objective of this study was to investigate circulating levels and phenotype of natural killer cells in 29 JAK2-positive MPN patients during IFN-α treatment. Furthermore, functional studies of NK cells upon target-cell recognition and cytokine stimulation were performed. The CD56(bright) and CD56(dim) NK cell subtypes display different properties in terms of cytokine production and cytotoxicity, respectively. Our results show a significant increase in the proportion of CD56(bright) NK cells and a decreasing CD56(dim) population during treatment with IFN-α compared to patients that are untreated, treated with hydroxyurea and healthy controls, P < 0.0001. Furthermore, an overall increase in cytokine-dependent (IL-12 and IL-15) IFN-γ expression by CD56(dim) NK cells during IFN-α treatment was observed. In contrast, our data indicate a compromised NK cell response to target-cell recognition during treatment with IFN-α in four patients. We also report low levels of circulating NK cells in untreated patients compared to healthy donors, patients treated with hydroxyurea and IFN-α, P = 0.02. Based on our findings, one might speculate whether treatment with IFN-α skews the human NK population toward a helper type that may assist in CD8(+) T cell priming in lymphoid tissues at the expense of their immediate cytotoxic functions in peripheral blood and tissues.

Cytokine release syndrome caused by antineoplastic treatment with CAR-T and T-cell engaging therapies.

T-cell-based immunotherapy has recently evolved as a treatment option for a number of haematological malignancies and is also being developed in solid tumours. A common side effect of chimeric antigen T-cell therapy (CAR-T) and treatment with T-cell engagers is cytokine release syndrome (CRS), which is a potentially life-threatening condition characterized by release of inflammatory mediators. The treatment of CRS is similar to that of other hyper-inflammatory conditions and involves supportive treatment as well as immunosuppressive therapy. The risk of CRS can be mitigated by step-up dosing and immunosuppressive pre-treatment, as argued in this review.

Increase in circulating CD4⁺CD25⁺Foxp3⁺ T cells in patients with Philadelphia-negative chronic myeloproliferative neoplasms during treatment with IFN-α.

Recent reports have described complete or major molecular remission in patients with polycythemia vera after long-term treatment with the immunomodulatory agent IFN-α2. Accordingly, there are reasons to believe that the immune system is a key player in eradicating the JAK2 mutated clone in these patients. Foxp3(+) regulatory T cells play a pivotal role in maintaining immune homeostasis and, importantly, preventing immune reactivity to self-antigens; however, their suppressive activity can compromise an effective antitumor immune response, and high frequencies of regulatory T cells in peripheral blood have been reported in both hematologic and solid cancers. We have analyzed the number, phenotype, and function of circulating CD4(+)CD25(+)Foxp3(+) T cells in patients with chronic myeloproliferative neoplasms. Surprisingly, we found a marked expansion of this subset of lymphocytes in patients treated with IFN-α2 (13.0%; 95% confidence interval [CI] 10.8% to 15.2%) compared with healthy donors (6.1%; 95% CI 4.9% to 7.2%), patients with untreated chronic myeloproliferative neoplasms (6.9%; 95% CI 5.8% to 7.4%), or patients treated with hydroxyurea (5.8%; 95% CI 4.3% to 7.4%; P < .0001).

Whole-blood transcriptional profiling of interferon-inducible genes identifies highly upregulated IFI27 in primary myelofibrosis.

Gene expression profiling studies have unraveled deregulation of several genes that might be of pathogenetic importance for the development and phenotype of the Philadelphia-negative chronic myeloproliferative neoplasms. In the context of interferon-alpha2 as a promising therapeutic agent, we focused upon the transcriptional profiling of interferon-associated genes in patients with essential thrombocythemia (ET) (n = 19), polycythemia vera (PV) (n = 41), and primary myelofibrosis (PMF) (n = 9). Using whole-blood transcriptional profiling and accordingly obtaining an integrated signature of genes expressed in several immune cells (granulocytes, monocytes, B cells, T cells, platelets), we have identified a number of interferon-associated genes to be significantly deregulated but with a highly significant deregulation of interferon-inducible gene 27 (IFI27) (ET, PV, and PMF, fold change 8, 16, and 30, respectively). The striking deregulation of IFI genes may reflect a hyperstimulated but insufficient immune system being most enhanced in patients with advanced myelofibrosis, in whom the IFI27 gene displayed an exceedingly high expression. The interferon signature may reflect primary myelofibrosis as the burn-out phase of chronic inflammation which ultimately elicits clonal evolution and expansion owing to an exaggerated but incompetent antitumor immune response. Finally, IFI27 may be a novel biomarker of disease activity and tumor burden in patients with CMPNs.

High frequencies of circulating memory T cells specific for calreticulin exon 9 mutations in healthy individuals.

Mutations in exon 9 of the calreticulin gene (CALR) frequently occur in patients with chronic myeloproliferative neoplasms (MPN). Patients exhibit spontaneous cellular immune responses to epitopes derived from the mutant CALR C-terminus, and CALR-mutant-specific T cells recognize autologous CALR-mutant malignant cells. This study investigated whether CALR-mutant-specific T cells occur naturally in CALRwt MPN-patients and in healthy individuals. Specific immune responses against epitopes in the mutant CALR peptide sequence were detected in both CALRwt MPN-patients and in healthy individuals. Healthy donors displayed more frequent and stronger CALR-mutant specific T-cell responses compared to the responses identified in CALR-mutant MPN-patients. Several T-cell responses were identified in healthy donors directly ex vivo. Importantly, by running functional analyses on live-sorted immune cells from healthy donors, we showed that circulating CALR-mutant-specific immune cells are T-memory cells. These findings suggest, that healthy individuals acquire a CALR exon 9 mutation, but the immune system reacts and clears the mutant cells, and during this reaction generates CALR-mutant specific T-memory cells. We believe that these findings provide the evidence for tumor immune surveillance in MPN.

Spontaneous T-cell responses against Arginase-1 in the chronic myeloproliferative neoplasms relative to disease stage and type of driver mutation.

Compelling evidence supports the existence of a profound immune dysregulation in patients with chronic myeloproliferative neoplasms (MPN). Increased Arginase-1 expression has been described in MPN patients and in solid cancers. This increase contributes to an immunosuppressive tumor microenvironment in MPN patients because of L-arginine depletion by Arginase-1-expressing regulatory cells and cancer cells, which subsequently limits the activation of circulating effector cells. In the present study, we demonstrate that Arginase-1-derived peptides are recognized by T cells among peripheral mononuclear blood cells from MPN patients. We characterized the Arginase-1-specific T cells as being CD4+ and found that the magnitude of response to the Arginase-1 peptides depends on disease stage. Activation of Arginase-1-specific T cells by vaccination could be an attractive novel immunotherapeutic approach to targeting malignant and suppressive cells in MPN patients in combination with other immunotherapeutics.

Spontaneous T-cell responses against the immune check point programmed-death-ligand 1 (PD-L1) in patients with chronic myeloproliferative neoplasms correlate with disease stage and clinical response.

The Chronic Myeloproliferative Neoplasms (MPN) are cancers characterized by hyperinflammation and immune deregulation. Concurrently, the expression of the immune check point programmed death ligand 1 (PD-L1) is induced by inflammation. In this study we report on the occurrence of spontaneous T cell responses against a PD-L1 derived epitope in patients with MPN. We show that 71% of patients display a significant immune response against PD-L1, and patients with advanced MPN have significantly fewer and weaker PD-L1 specific immune responses compared to patients with non-advanced MPN. The PD-L1 specific T cell responses are CD4+ T cell responses, and by gene expression analysis we show that expression of PD-L1 is enhanced in patients with MPN. This could imply that the tumor specific immune response in MPN could be enhanced by vaccination with PD-L1 derived epitopes by boosting the anti-regulatory immune response hereby allowing tumor specific T cell to exert anti-tumor immunity.

A randomised controlled trial of cognitive-behaviour therapy for clinical perfectionism: a preliminary study.

Perfectionism can be a problem in its own right and it can impede the progress of treatment of Axis I disorders. This study reports on a preliminary randomised controlled trial of cognitive-behaviour therapy (CBT) for "clinical perfectionism". Twenty participants were randomly assigned to either immediate treatment (IT) (n=10) or a wait list (NL) (n=10). Treatment consisted of ten sessions of CBT over eight weeks. Two participants did not complete the follow-up assessments (10%). Fifteen of the original 20 participants (75%) were clinically significantly improved after treatment and the effect size was large (1.8). Treatment gains were maintained at 8-week and 16-week follow-up.

The impact of interferon-alpha2 on HLA genes in patients with polycythemia vera and related neoplasms.

Gene expression profiling in Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have unraveled significant deregulation of several immune and inflammation genes of potential importance for clonal evolution. Other mechanisms might be downregulation of major histocompatibility class I and II genes used by tumor cells to escape antitumor T-cell-mediated immune responses. Several genes encoding human leukocyte antigen (HLA) class I and II molecules have been shown to be significantly downregulated. Upregulation of HLA genes is considered one of the mechanisms of action of interferon (IFN)-alpha2, but regulation of these genes during IFN-alpha2 treatment in MPNs has never been studied. Our findings show a significant upregulation of several HLA genes of importance for tumor immune surveillance by IFN-alpha2 treatment in MPNs. This mechanism might enhance the cytotoxic potential of immune cells against MPNs and explain the induction of minimal residual disease by IFN-alpha2 treatment in these patients.

Statins in the treatment of polycythaemia vera and allied disorders: an antithrombotic and cytoreductive potential?

Thrombohaemorrhagic complications are major clinical problems in the classical chronic Ph-negative myeloproliferative disorders (CMPDs), polycytaemia vera (PV), essential thrombocythaemia (ET) and idiopathic myelofibrosis (IMF), contributing significantly to morbidity and mortality. Pathophysiologically these disorders are characterized by clonal myeloproliferation, myeloaccumulation and a propensity to develop myelofibrosis and neoangiogenesis in both the bone marrow and spleen. Based upon in vitro and in vivo studies of the effects of statins (antithrombotic, antiproliferative, proapoptotic and antiangiogenic), this review focuses on the translation of these effects into potential clinical benefits of statin therapy in patients with CMPDs.

Minimal residual disease after long-term interferon-alpha2 treatment: a report on hematological, molecular and histomorphological response patterns in 10 patients with essential thrombocythemia and polycythemia vera.

Essential thrombocythemia (ET) and polycythemia vera (PV) are Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) characterized by the JAK2 V617F mutation, which can be found in more than 98% of PV patients and in ∼ 50% of ET patients. Assessment of the JAK2 V617F allele burden by a highly sensitive quantitative PCR (qPCR) assay appears to be a useful tool for monitoring minimal residual disease (MRD) and evaluating treatment efficacy. This report expands and substantiates existing data, showing that IFN-alpha2 is a highly potent immunomodulating agent capable of inducing MRD with low-burden JAK2 V617F, major molecular response (MMR), complete hematological remission (CHR) and complete histomorphological normalization of the bone marrow in a sub-set of patients with ET and PV after long-term treatment (≥ 3.5 years). Furthermore, long-lasting hematological, molecular and histomorphological remissions are sustained after discontinuation of IFN-alpha2 for up to ∼ 5-6 years.

A 7-Gene Signature Depicts the Biochemical Profile of Early Prefibrotic Myelofibrosis.

Recent studies have shown that a large proportion of patients classified as essential thrombocythemia (ET) actually have early primary prefibrotic myelofibrosis (prePMF), which implies an inferior prognosis as compared to patients being diagnosed with so-called genuine or true ET. According to the World Health Organization (WHO) 2008 classification, bone marrow histology is a major component in the distinction between these disease entities. However, the differential diagnosis between them may be challenging and several studies have not been able to distinguish between them. Most lately, it has been argued that simple blood tests, including the leukocyte count and plasma lactate dehydrogenase (LDH) may be useful tools to separate genuine ET from prePMF, the latter disease entity more often being featured by anemia, leukocytosis and elevated LDH. Whole blood gene expression profiling was performed in 17 and 9 patients diagnosed with ET and PMF, respectively. Using elevated LDH obtained at the time of diagnosis as a marker of prePMF, a 7-gene signature was identified which correctly predicted the prePMF group with a sensitivity of 100% and a specificity of 89%. The 7 genes included MPO, CEACAM8, CRISP3, MS4A3, CEACAM6, HEMGN, and MMP8, which are genes known to be involved in inflammation, cell adhesion, differentiation and proliferation. Evaluation of bone marrow biopsies and the 7-gene signature showed a concordance rate of 71%, 79%, 62%, and 38%. Our 7-gene signature may be a useful tool to differentiate between genuine ET and prePMF but needs to be validated in a larger cohort of "ET" patients.

Effect of a Brief Massage on Pain, Anxiety, and Satisfaction With Pain Management in Postoperative Orthopaedic Patients.

BACKGROUND: The majority of massage therapy studies have evaluated 20- to 45-minute interventions in nonsurgical patients. Studies are needed to evaluate the effects of a brief massage intervention that would be more clinically feasible for bedside clinicians to administer as an adjunct to pharmacologic pain management in acutely ill surgical patients. PURPOSE: To evaluate the impact of a brief massage intervention in conjunction with analgesic administration on pain, anxiety, and satisfaction with pain management in postoperative orthopaedic inpatients. METHODS: A convenience sample of postoperative orthopaedic patients was studied during two therapeutic pain treatments with an oral analgesic medication. A pretest, posttest, randomized, controlled trial study design, with crossover of subjects, was used to evaluate the effect of a 5-minute hand and arm massage at the time of analgesic administration. Each patient received both treatments (analgesic administration alone [control]; analgesic administration with massage) during two sequential episodes of postoperative pain. Prior to administration of the analgesic medication, participants rated their level of pain and anxiety with valid and reliable tools. Immediately after analgesic administration, a study investigator provided the first, randomly assigned treatment. Pain and anxiety were rated by the participant 5 and 45 minutes after medication administration. Satisfaction with pain management was also rated at the 45-minute time point. Study procedures were repeated for the participant's next requirement for analgesic medication, with the participant receiving the other randomly assigned treatment. Analysis of variance was used to determine whether pain, anxiety, and/or satisfaction with pain management differed between the two treatment groups and/or if treatment order was a significant factor. The level of significance for all tests was set at p < .05. RESULTS: Twenty-five postoperative patients were studied during two sequential episodes of pain, which required analgesic medication administration (N = 25 analgesic alone; N = 25 analgesic with massage). Patient ages ranged from 32 to 86 years (average ±SD = 61.2 ± 11.5 years). Pain and anxiety scores after medication administration decreased in both groups, with no significant differences found between the analgesic alone or analgesic with massage treatments (p > .05). Patient satisfaction with pain management was higher for pain treatment with massage than medication only (F = 6.8, df = 46, p = .012). CONCLUSION: The addition of a 5-minute massage treatment at the time of analgesic administration significantly increased patient satisfaction with pain management.

[Status and perspectives on chronic myeloproliferative neoplasm treatment]. / Status og perspektiver for behandling af de kroniske myeloproliferative neoplasier.

Polycythaemia vera, essential thrombocytosis and primary myelofibrosis are closely related, clonal myeloproliferative neoplasms. Our knowledge of the underlying molecular mechanisms driving these diseases has increased dramatically during the latest ten years. Traditionally, treatment of these malignancies has focused on lowering their inherent thromboembolic risk but with the discovery of the JAK2-V617F mutation and most recently the calreticulin mutations new therapeutic options such as interferon-alpha, JAK2-inhibitors and statins are being contemplated. This article reviews these new treatment options.

Whole blood transcriptional profiling reveals deregulation of oxidative and antioxidative defence genes in myelofibrosis and related neoplasms. Potential implications of downregulation of Nrf2 for genomic instability and disease progression.

The Philadelphia-negative chronic myeloproliferative neoplasms - essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) (MPNs) - have recently been shown to be associated with chronic inflammation, oxidative stress and accumulation of reactive oxygen species (ROS). Using whole blood transcriptional profiling, we report that several oxidative stress and anti-oxidative stress genes are significantly deregulated in MPNs. Among the twenty most up- and downregulated genes, ATOX1, DEFB122, GPX8, PRDX2, PRDX6, PTGS1, and SEPP1 were progressively upregulated from ET over PV to PMF, whereas AKR1B1, CYBA, SIRT2, TTN, and UCP2 were progressively downregulated in ET, PV and PMF (all FDR <0.05). The gene Nrf2, encoding the transcription factor nuclear factor erythroid 2-related factor 2 (NFE2L2 or Nrf2) was significantly downregulated in all MPNs. Nrf2 has a key role in the regulation of the oxidative stress response and modulates both migration and retention of hematopoietic stem cells (HSCs) in their niche. The patogenetic importance of Nrf2 depletion in the context of expansion of the hematopoietic progenitor pool in MPNs is discussed with particular focus upon the implications of concomitant downregulation of Nrf2 and CXCR4 for stem cell mobilization.

Transcriptional profiling of whole blood identifies a unique 5-gene signature for myelofibrosis and imminent myelofibrosis transformation.

Identifying a distinct gene signature for myelofibrosis may yield novel information of the genes, which are responsible for progression of essential thrombocythemia and polycythemia vera towards myelofibrosis. We aimed at identifying a simple gene signature - composed of a few genes - which were selectively and highly deregulated in myelofibrosis patients. Gene expression microarray studies have been performed on whole blood from 69 patients with myeloproliferative neoplasms. Amongst the top-20 of the most upregulated genes in PMF compared to controls, we identified 5 genes (DEFA4, ELA2, OLFM4, CTSG, and AZU1), which were highly significantly deregulated in PMF only. None of these genes were significantly regulated in ET and PV patients. However, hierarchical cluster analysis showed that these genes were also highly expressed in a subset of patients with ET (n = 1) and PV (n = 4) transforming towards myelofibrosis and/or being featured by an aggressive phenotype. We have identified a simple 5-gene signature, which is uniquely and highly significantly deregulated in patients in transitional stages of ET and PV towards myelofibrosis and in patients with PMF only. Some of these genes are considered to be responsible for the derangement of bone marrow stroma in myelofibrosis. Accordingly, this gene-signature may reflect key processes in the pathogenesis and pathophysiology of myelofibrosis development.

Whole blood transcriptional profiling reveals significant down-regulation of human leukocyte antigen class I and II genes in essential thrombocythemia, polycythemia vera and myelofibrosis.

Gene expression profiling studies in the Philadelphia-negative chronic myeloproliferative neoplasms have revealed significant deregulation of several immune and inflammation genes that might be of importance for clonal evolution due to defective tumor immune surveillance. Other mechanisms might be down-regulation of major histocompatibility (MHC) class I and II genes, which are used by tumor cells to escape antitumor T-cell-mediated immune responses. We have performed whole blood transcriptional profiling of genes encoding human leukocyte antigen (HLA) class I and II molecules, ß2-microglobulin and members of the antigen processing machinery of HLA class I molecules (LMP2, LMP7, TAP1, TAP2 and tapasin). The findings of significant down-regulation of several of these genes may possibly be of major importance for defective tumor immune surveillance. Since up-regulation of HLA genes is recorded during treatment with epigenome modulating agents (DNA-hypomethylators and DNA-hyperacetylators [histone deacetylase inhibitors]) and interferon-α2, our findings call for prospective transcriptional studies of HLA genes during treatment with these agents.