Estrov and collaborators examine the role of fibrocytes in primary myelofibrosis and propose a novel therapeutic approach.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor clinical course and outcome. There is a constant need for new prognostic factors that could facilitate patient risk stratification. The aim of our research was to determine the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways in leukemic cells, their relation to P-glycoprotein (P-gp) expression/activity and their prognostic significance in adult de novo AML. A total of 118 patients with AML were enrolled in the study. In a multivariate Cox regression analysis we found that P-gp activity and Akt phosphorylation were independent poor prognostic factors of overall survival (OS). In contrast, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) represented a favorable prognostic factor of OS and relapse-free survival (RFS). A negative correlation between P-gp activity and p38 phosphorylation level was found, implying a possible role of this MAPK pathway in P-gp regulation. In addition, we found correlation between Akt and p38 phosphorylation levels, indicative of co-activation of two signaling cascades in AML.

AimTo evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients.MethodsConvenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay.ResultsHLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P < 0.001 and P = 0.002, respectively) and an abnormal serum FLC ratio (for both P < 0.001). In addition, abnormal HLC isotype level correlated with serum beta-2-microglobulin level (P = 0.038). In terms of prognosis, abnormal HLC isotype level and abnormal HLC ratio were significantly associated with shorter overall survival (P < 0.001 and P = 0.002, respectively). Interestingly, suppression of the uninvolved (polyclonal) isotype pair, but not other non-myeloma immunoglobulin isotypes, was also associated with a shorter overall survival (P = 0.021). In a multivariate analysis, an abnormal HLC ratio and β2-microglobulin level >3.5mg/L were independent risk factors for survival.ConclusionThe new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients' outcome.

Phenotypic characterization of T cells in myelofibrosis (MF) is intriguing owing to increased inflammation, markedly elevated pro-inflammatory cytokines, and altered distribution of T-cell subsets. Constitutive activation of Janus kinase-2 (JAK2) in the majority of MF patients contributes to the expression of the programmed cell death protein-1 (PD1) and T-cell exhaustion. We wondered whether T-cell activation affects treatment outcome of patients with MF and sought to determine whether the JAK1/2 inhibitor ruxolitinib affects the activation of T-cell subsets. T cells from 47 MF patients were analyzed and the percent of either helper (CD4+) or cytotoxic (CD8+) naive, central memory, effector memory, or effector T cells; and fractions of PD1-expressing cells in each subset were assessed. An increased number of T cells coexpressing CD4/PD1 and CD8/PD1 in MF compared to healthy controls (n=28) was found, and the T cells were significantly skewed toward an effector phenotype in both CD4+ and CD8+ subsets, consistent with a shift from a quiescent to an activated state. Over the course of ruxolitinib treatment, the distribution of aberrant T-cell subsets significantly reversed towards resting cell phenotypes. CD4+ and CD8+ subsets at baseline correlated with monocyte and platelet counts, and their PD1-positive fractions correlated with leukocyte counts and spleen size. Low numbers of PD1+/CD4+ and PD1+/CD8+ cells were associated with complete resolution of palpable splenomegaly and improved survival rate, suggesting that low levels of exhausted T cells confer a favorable response to ruxolitinib treatment.

Introduction: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm that develops de novo (primary myelofibrosis) or transforms from polycythemia vera or essential thrombocytosis. MF is characterized by stem cell-derived clonal myeloproliferation, abnormal cytokine expression, bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis, constitutional symptoms, cachexia, leukemic progression, and shortened survival. Several studies suggested that clonal monocytes play a role in the pathobiology of MF. However whether a specific monocyte subpopulation is predominantly present in MF is largely unknown. Traditionally, three subpopulations of CD14+ monocytes have been identified: classical (CD14++/CD16-), intermediate (CD14++/CD16+), and non-classical (CD14dim+/CD16++). Whether MF patients' monocyte subpopulations are different from those of normal individuals and how ruxolitinib treatment affects them has not been elucidated. Methods: Using flow cytometry we first assessed the distribution of the three monocyte subpopulations in the bone marrow (BM) of healthy individuals and MF and then assessed their distribution in BM samples from phase I/II clinical trial of ruxolitinib in patients with primary or secondary MF (Verstovsek S. etal. N Engl J Med 12:1117, 2010). Results: Using BM samples from 7 healthy individuals and 12 untreated MF patients we found a significant decrease in the percentage of monocytes (p =0.0061) in the mononuclear gate of untreated MF patients compared to normal individuals. However, the distribution of classical vs. non-classical monocyte subpopulations in MF was similar to that of normal BM (p =0.3, p =0.3, respectively). Remarkably, ruxolitinib treatment significantly altered the distribution of classical vs. non-classical monocyte subpopulations. During treatment (years 0-3, 3-6, 6-8) we identified a progressive increase in the percentage of monocytes in the mononuclear gate (p =0.1; p =0.04, and p =0.03, respectively) with a substantial increase in the non-classical monocyte subpopulation in years 0-3 and 3-6 of treatment (p= 0.04, p= 0.005, respectively) and a decrease in the classical monocytes (p =0.07, p =0.008, respectively). This trend reversed after 6-8 years of therapy (p =0.3, p =0.2, respectively). Importantly, in ruxolitinib-treated patients with a ≥50% spleen size reduction highest percentage of non-classical monocytes was observed during the first 3 years and years 3-6 of treatment (p =0.01, p =0.01, respectively). However during years 6-8 this difference was no longer detected and the percentage of non-classical monocytes was similar to the percentage detected in the pre-treatment BM samples (p =0.4). These changes correlated with response to ruxolitinib treatment. In patients with a <50% spleen size reduction the percentage of non-classical monocytes in years 0-3 of ruxolitinib treatment was significantly lower compared to patients with ≥50% spleen reduction (p =0.005), and patients with ≥50% spleen reduction show correlation between post-treatment spleen size and percentage of non-classical monocytes (p <0.0001, r=−0.4). Conclusions: Taken together, our results suggest that ruxolitinib induces a transition of classical to non-classical monocyte subpopulation during the first years of ruxolitinib treatment and that this effect correlates with the patients' clinical response. Further studies aimed at exploring the role of monocytes and their subpopulations in the pathobiology of MF are warranted. Disclosures No relevant conflicts of interest to declare.