CDC25c expression in patients with myelofibrosis is associated with stronger myeloproliferation and shorter overall survival.

BACKGROUND: Cell division cycle 25c (CDC25c) is a gene coding a phosphatase controlling entry into mitosis upon activation through Polo-like kinase 1 (PLK1) and serves as a key regulator of cell division. The CDC25c was reported to be dysregulated in some malignant diseases, but its role in myelofibrosis has not yet been elucidated. METHODS: We have retrospectively investigated CDC25c mRNA expression in bone marrow aspirates of 43 patients with myelofibrosis (28 primary [PMF] and 15 secondary myelofibrosis [SMF]) and 12 controls. RESULTS: CDC25c mRNA expression did not significantly differ between PMF, SMF and controls (median ∆CT 3.08 vs 2.86 vs 2.29 for PMF, SMF and controls, respectively; P = 0.162). Patients presenting with higher CDC25c mRNA expression were of older age (P = 0.037), had statistically significantly higher white-blood-cells (P = 0.017), larger liver size (P = 0.022), higher absolute neutrophil (P = 0.010), monocyte (P = 0.050), basophil (P = 0.012), and eosinophil counts (P = 0.013). Patients presenting with high CDC25c mRNA expression had statistically significantly inferior overall survival compared to low CDC25c expression group (HR = 2.99; P = 0.049). Median overall survival was not reached in patients with low and was 44 months in patients with high CDC25c expression. CONCLUSION: Our data suggest that higher CDC25c expression is associated with more proliferative phenotype of myelofibrosis and is prognostic of worse survival. Future studies investigating these interesting associations are warranted.

Inflammatory Gene Expression in Neck Perivascular and Subcutaneous Adipose Tissue in Men With Carotid Stenosis.

The inflammatory phenotype of neck adipose tissue (NAT) might reflect its involvement in the pathogenesis of carotid atherosclerosis. We investigated inflammatory gene expression in the subcutaneous and the perivascular (pericarotid) adipose tissue from patients with carotid stenosis (CS) undergoing endarterectomy and a control group of patients without significant carotid atherosclerosis undergoing thyroid surgery. Only male patients were included (n = 13 in each study group). Clinical and biochemical data along with serum leptin, adiponectin, and monocyte chemoattractant protein 1 (MCP-1) were collected. Adipose tissue samples were obtained from both the subcutaneous and pericarotid compartments. Real-time polymerase chain reaction was used to measure gene expression of macrophage markers and adipokines. The CS group had higher subcutaneous and pericarotid visfatin gene expression and higher pericarotid expression of MCP-1 and CD68 genes. The ratio between pericarotid CD206 and CD68 gene expression was similar between study groups. Adiponectin gene expression in both NAT compartments did not differ between groups, but it was negatively associated with body weight. These observations suggest that NAT, and especially the pericarotid compartment, express enhanced inflammatory properties in patients with CS, but the proportion of anti-inflammatory macrophages in advanced atherosclerosis seems to be maintained.

Diagnostic validation of two SARS-CoV-2 immunochromatographic tests in Slovenian and Croatian hospitals.

AIM: To diagnostically validate two point-of-care (POC) rapid antigen tests for SARS-CoV-2 by comparing their results with those of laboratory-based real-time polymerase chain reaction tests (RT-PCR). METHODS: The study enrolled 455 patients from two Slovenian and two Croatian hospitals. The NADAL COVID-19 Ag Test (Nal von Minden, Moers, Germany) and ALLTEST COVID-19 Antigen Test (Hangzhou ALLTEST Biotech Co., Ltd, Hangzhou, China) were diagnostically validated in emergency care departments of two Slovenian hospitals, while only ALLTEST COVID-19 Antigen Test was validated in two Croatian hospitals. RESULTS: The antigen test results were in very good agreement with the RT-PCR results (Cohen's Kappa between 0.747 and 0.891 for the NADAL COVID-19 and between 0.820 and 0.954 for the ALLTEST COVID-19). The NADAL COVID-19 Ag Test had the sensitivity between 66.67% and 92.31%, with a negative predictive value between 85.51% and 99.2%. The ALLTEST COVID-19 Antigen Test had the sensitivity between 81.39% and 91.11%, with a negative predictive value between 85.45% and 98.78%. CONCLUSION: The antigen tests are practical and reliable screening assays for SARS CoV-2 in emergency care departments. Both antigen tests can be used as screening tests to reduce the number of patients waiting for RT-PCR results. Even more, they can be used to quickly isolate COVID-19 patients and reduce hospital transmissions.

Higher AURKA and PLK1 expression are associated with inferior overall survival in patients with myelofibrosis.

Aurora-kinase-A (AURKA), BORA and Polo-like-kinase-1 (PLK1) are regulating cell-cycle control and promotion of mitosis entry. AURKA contributes to Janus-kinase-2 (JAK2) activation and increased AURKA protein levels were reported in CD34+ and CD41+ cells of myeloproliferative neoplasm patients, leading to aneuploidy and aberrant megakaryopoiesis. We aimed to investigate AURKA, BORA and PLK1 mRNA expression in unfractionated bone-marrow aspirates of 43 patients with myelofibrosis (28 primary-/PMF, 15 secondary-myelofibrosis/SMF) and 12 controls and to assess their clinical correlations. AURKA expression did not significantly differ between myelofibrosis and controls (P = 0.466). Higher AURKA expression was significantly associated with higher absolute monocyte-count (P = 0.024) and shorter overall survival (HR = 3.77; P = 0.012). Patients with both PMF and SMF had lower BORA expression than controls (P = 0.009). Higher BORA expression was significantly associated with absence of constitutional symptoms (P = 0.049), absence of circulatory blasts (P = 0.047), higher monocyte- (P = 0.040) and higher eosinophil-counts (P = 0.016) and had neutral effect on survival (P > 0.05). PLK1 expression did not significantly differ between myelofibrosis and controls (P = 0.103). Higher PLK1 expression was significantly associated with higher white-blood-cell-count (P = 0.042) and inferior overall survival (HR = 5.87; P = 0.003). In conclusion, AURKA, BORA and PLK1 are involved in pathogenesis of myelofibrosis and may affect survival. Future studies investigating these interesting associations are warranted.

Neutral effect of Glioma-associated oncogene-1 expression on survival in myelofibrosis.

This study retrospectively analyzed glioma-associated oncogene 1 (GLI­1) mRNA expression in unfractionated bone marrow aspirates of 32 patients with myelofibrosis and 16 controls. It was found that GLI­1 expression did not significantly differ between primary, secondary myelofibrosis and controls (median difference in threshold cycles ∆CT 7.2, 7.3 and 6.9, respectively; P = 0.864), as well as that survival curves of myelofibrosis patients with higher/lower GLI­1 expression showed multiple overlaps and overall comparable course (P = 0.651). The results suggest that general upregulation of GLI­1 does not seem to be a feature of the disease and are in line with modest biological and clinical effects observed with inhibitors of Hedgehog signaling pathway in patients with myelofibrosis.

Neck adipose tissue - tying ties in metabolic disorders.

Upper body adipose tissue accumulation has been associated with clustering of metabolic disorders and increased cardiovascular risk. Neck circumference (NC) indicated that subcutaneous adipose tissue (SAT) in that region is an independent pathogenic depot that might account for the additional risk missed by visceral adipose tissue (VAT). Neck adipose tissue (NAT) is not only one more ectopic depot but has several particular features that might modulate its metabolic role. Besides a controversial impact on obstructive apnea syndrome, neck fat encompasses carotid arteries as an important perivascular adipose tissue (PVAT) depot. With dysfunctional changes in obesity, physiologic vascular regulation is lost and inflammatory signals accelerate atherogenesis. Unexpected was the discovery of brown and beige adipocytes in the neck of human adults. When stimulated, brown adipose tissue (BAT) dissipates energy through thermogenesis and it is associated with other favorable metabolic effects. Moreover, the neck is the region where the browning mechanism was disclosed. With this unique plastic nature, NAT revealed multiple ties, challenging dynamics and potential new therapeutic targets that might have significant implications on metabolic outcomes and vascular risk.

Assessing serum albumin concentration, lymphocyte count and prognostic nutritional index might improve prognostication in patients with myelofibrosis.

BACKGROUND: Primary and secondary myelofibrosis (PMF and SMF) are malignant diseases of hematopoietic stem cell characterized by the neoplastic myeloproliferation and a strong inflammatory milieu. The prognostic nutritional index (PNI) integrates information on albumin and absolute lymphocyte count (ALC) and reflects the inflammatory, nutritional and immune status of a patient. The clinical and prognostic significance of albumin, ALC and PNI in patients with myelofibrosis has not been previously investigated. METHODS: We retrospectively analyzed a cohort of 83 myelofibrosis patients treated in our institution from 2006 to 2017. Albumin, ALC and PNI were assessed in addition to other disease specific markers. RESULTS: The PMF and SMF patients had significantly lower ALC and PNI but similar albumin compared to controls. Lower albumin was significantly associated with older age and parameters reflecting more aggressive disease biology (e.g. anemia, lower platelet levels, higher lactate dehydrogenase (LDH), circulatory blasts, transfusion dependency, blast phase disease), inflammation (higher C reactive protein (CRP), constitutional symptoms) and higher degree of bone marrow fibrosis. Lower ALC was significantly associated with lower white blood cells (WBC) and lower circulatory blasts. Low PNI was associated with lower albumin, lower ALC, anemia, lower WBCs, lower serum iron and lower transferrin saturation. There was no difference in albumin, ALC and PNI regarding the driver mutations. In multivariate analysis adjusted for age and gender, low albumin (hazard ratio [HR] = 4.61, P = 0.001), low ALC (HR = 3.54, P = 0.004) and Dynamic International Prognostic Scoring System (DIPSS) (HR = 2.45, P = 0.001) were able to predict inferior survival independently of each other. Accordingly, low PNI (HR = 4.32, P < 0.001) predicted poor survival independently of DIPSS (HR = 3.31, P < 0.001). CONCLUSION: Assessing albumin, ALC and PNI might improve prognostication in patients with myelofibrosis and could assist in recognition of patients under increased risk of death.

High absolute basophil count is a powerful independent predictor of inferior overall survival in patients with primary myelofibrosis.

OBJECTIVES: To investigate the clinical and prognostic significance of absolute basophil count (ABC) in patients with primary myelofibrosis (PMF). METHODS: We retrospectively investigated 58 patients with PMF treated in our institution in the period from 2006 to 2017. ABC was obtained in addition to other hematological and clinical parameters. Patients were separated into high and low ABC groups using the Receiver operating characteristic curve analysis. RESULTS: ABC was higher in PMF patients than in healthy controls (P < 0.001). Patients with high ABC had higher white blood cells (P < 0.001), higher red cell distribution width (P = 0.035), higher lactate dehydrogenase (P < 0.001), more frequently had circulatory blasts (P < 0.001), constitutional symptoms (P = 0.030) and massive splenomegaly (P = 0.014). ABC was also positively correlated with absolute monocyte count (AMC) (P < 0.001) and other components of differential blood count. There was no difference in ABC regarding driver mutations or degree of bone marrow fibrosis. Univariately, high ABC was significantly associated with inferior overall survival (hazard ratio (HR) 4.79, P < 0.001). This effect remained statistically significant (HR 4.27, P = 0.009) in a multivariate Cox regression model adjusted for age, gender, Dynamic International Prognostic Scoring System (HR 2.6, P = 0.001) and AMC (HR 8.45, P = 0.002). DISCUSSION: High ABC reflects higher disease activity and stronger proliferative potential of disease. ABC and AMC independently predict survival and therefore seem to reflect different underlying pathophysiologic processes. Hence, both have a potential for improvement of current prognostic scores. CONCLUSION: Basophils represent a part of malignant clone in PMF and are associated with unfavorable disease features and poor prognosis which is independent of currently established prognostic scoring system and monocytosis.

Increased mean platelet volume (MPV) is an independent predictor of inferior survival in patients with primary and secondary myelofibrosis.

Neoplastic megakaryopoiesis is a dominant feature of Philadelphia-chromosome-negative myeloproliferative neoplasms (Ph- MPNs), and elevated mean-platelet-volume (MPV) is a common finding in these diseases. The clinical and prognostic significances of MPV in patients with primary (PMF) and secondary myelofibrosis (SMF) have not been reported. We retrospectively analyzed 87 patients with myelofibrosis (66 with PMF, 21 with SMF) treated at our institution. MPV was recorded in addition to other hematological and clinical parameters. MPV was elevated in both PMF and SMF patients in comparison to controls, whereas there was no statistically significant difference between PMF and SMF. Elevated MPV was associated with lower platelets (P = 0.016), higher white blood cells (P = 0.015), higher percentage of circulatory blasts (P = 0.009), higher lactate dehydrogenase (P = 0.011), larger spleen size (P = 0.014) and higher Dynamic International Prognostic score category (P = 0.027), while there was no statistically significant association with driver mutations or degree of bone marrow fibrosis. Higher MPV was univariately associated with inferior overall survival in the whole cohort (HR = 3.82, P = 0.006), PMF (HR = 4.35, P = 0.007) and SMF patients (HR = 7.22, P = 0.034). These associations remained significant in multivariate analyses adjusted for DIPSS. Higher MPV is associated with more aggressive disease features and exhibits powerful independent prognostic properties in both PMF and SMF settings.

Canonical Wnt/ß-Catenin Signaling Pathway Is Dysregulated in Patients With Primary and Secondary Myelofibrosis.

INTRODUCTION: ß-Catenin is a central effector molecule of the canonical wingless-related integration site (Wnt) signaling pathway. It is important for maintenance of stem cell homeostasis and its aberrant activation has been implicated in a wide array of malignant hematological disorders. There are few reports suggesting its dysregulation in Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs). PATIENTS AND METHODS: We analyzed ß-catenin mRNA expression in bone marrow (BM) aspirates of 29 patients with primary (PMF) and 4 patients with secondary, post Ph- MPN, myelofibrosis (SMF) using quantitative real-time polymerase chain reaction (qRT PCR). The control group consisted of 16 BM aspirates from patients with limited-stage aggressive non-Hodgkin lymphoma without BM involvement. We compared relative gene expression with clinical and hematological parameters. RESULTS: Relative expression of ß-catenin differed significantly among groups (P = .0002), it was significantly higher in patients with PMF and SMF than in the control group, but did not differ between patients with PMF and SMF. A negative correlation was found regarding hemoglobin level in PMF (P = .017). No association according to Janus kinase 2 (JAK2) V617F mutational status or JAK2 V617F allele burden was detected. CONCLUSION: Our results show for the first time that ß-catenin mRNA expression is increased in patients with PMF and SMF and its upregulation might potentiate anemia. A number of inflammatory cytokines associated with PMF are capable of mediating their effects through increased ß-catenin expression. Accordingly, ß-catenin can induce expression of a number of genes implicated in processes of cell cycle control, fibrosis, and angiogenesis, which are central to the PMF pathogenesis. Therefore, ß-catenin might represent an interesting new therapeutic target in these diseases.

Hemochromatosis gene mutations may affect the survival of patients with myelodysplastic syndrome.

OBJECTIVES: The recent availability of potent oral iron chelators is renewing an interest in the assessment of the possible impact of HFE genetics in MDS. METHODS: Thirty six newly diagnosed patients with MDS were studied for parameters of iron metabolism in addition to C282Y and H63D mutations of the HFE gene. RESULTS: Mutations were present in 11 out of 36 patients (31%), which were not different from our general population and were equally distributed among MDS subtypes. Mutated patients had higher ferritin levels (P = 0.039) and lower TIBC (P = 0.018). Ferritin was found to be higher for the untransfused mutated patients (P = 0.017), but not for transfusion-dependent patients in whom ferritin levels correlated significantly with the number of blood units received (P = 0.04). There was no difference in the number of blood units received between the mutated and wild type patients. A new observation made was that the mutated patients had a lower overall survival in addition to a poorer leukemia free survival (LFS) (P = 0.004 and P = 0.003, respectively). DISCUSSION: The HFE gene mutations are not more frequent in MDS patients. Iron overload in mutated patients was higher but there was no correlation found using supportive therapy for anemia. The effect of mutations on survival could be mediated by changes in iron metabolism. CONCLUSION: The HFE genotype may predict MDS prognosis and there is a need for further studies. It remains a challenging question if HFE mutated MDS patients should be considered for potent iron chelation therapy.

Genes Involved in Maintaining the Bone Marrow Stroma Are Dysregulated in Patients with Myelofibrosis: Lenalidomide Treatment Up-regulates SOCS3.

AIM: The purpose of the present study was to determine whether genes involved in the organization of the hematopoietic niche were dysregulated in patients with primary myelofibrosis (MF) treated with lenalidomide. MATERIALS AND METHODS: We used reverse-transcription quantitative polymerase chain reaction to study the expression of a set of genes involved in the organization of the hematopoietic niche in peripheral blood and bone marrow (BM) mononuclear cell (MNC) samples from 32 patients with primary MF who participated in a phase II trial of lenalidomide plus prednisone. RESULTS: At baseline (before treatment) cyclo-oxygenase 2 (COX2) was significantly up-regulated, while chemokine (C-X-C motif) receptor 4 (CXCR4), paired box 5 (PAX5) C-terminus, and hypoxia inducible factor 1A (HIF1A) were significantly down-regulated in BM MNCs from patients with primary MF compared to BM MNCs from healthy individuals. After 9 months of treatment, the expression of suppressor of cytokine signaling 3 (SOCS3) was significantly increased. CONCLUSION: Patients with primary MF showed aberrant expression of several genes involved in maintaining BM homeostasis and our findings suggest that treatment with lenalidomide plus prednisone up-regulates SOCS3.

Bone marrow stroma-secreted cytokines protect JAK2(V617F)-mutated cells from the effects of a JAK2 inhibitor.

Signals emanating from the bone marrow microenvironment, such as stromal cells, are thought to support the survival and proliferation of the malignant cells in patients with myeloproliferative neoplasms (MPN). To examine this hypothesis, we established a coculture platform [cells cocultured directly (cell-on-cell) or indirectly (separated by micropore membrane)] designed to interrogate the interplay between Janus activated kinase 2-V617F (JAK2(V617F))-positive cells and the stromal cells. Treatment with atiprimod, a potent JAK2 inhibitor, caused marked growth inhibition and apoptosis of human (SET-2) and mouse (FDCP-EpoR) JAK2(V617F)-positive cells as well as primary blood or bone marrow mononuclear cells from patients with polycythemia vera; however, these effects were attenuated when any of these cell types were cocultured (cell-on-cell) with human marrow stromal cell lines (e.g., HS5, NK.tert, TM-R1). Coculture with stromal cells hampered the ability of atiprimod to inhibit phosphorylation of JAK2 and the downstream STAT3 and STAT5 pathways. This protective effect was maintained in noncontact coculture assays (JAK2(V617F)-positive cells separated by 0.4-µm-thick micropore membranes from stromal cells), indicating a paracrine effect. Cytokine profiling of supernatants from noncontact coculture assays detected distinctly high levels of interleukin 6 (IL-6), fibroblast growth factor (FGF), and chemokine C-X-C-motif ligand 10 (CXCL-10)/IFN-γ-inducible 10-kD protein (IP-10). Anti-IL-6, -FGF, or -CXCL-10/IP-10 neutralizing antibodies ablated the protective effect of stromal cells and restored atiprimod-induced apoptosis of JAK2(V617F)-positive cells. Therefore, our results indicate that humoral factors secreted by stromal cells protect MPN clones from JAK2 inhibitor therapy, thus underscoring the importance of targeting the marrow niche in MPN for therapeutic purposes.

Detection of t(14;18) by PCR of IgH/BCL2 fusion gene in follicular lymphoma from archived cytological smears.

According to WHO classification follicular lymphoma (FL) is a neoplasm composed of follicle centre (germinal centre) B-cells, which usually has at least a partially follicular pattern. Bone marrow (BM) infiltration by lymphoma occurs in 40-70% of cases at the time of diagnosis. The characteristic chromosomal translocation of follicular lymphoma is t(14;18)(q32;q21) with transposition of BCL2 oncogene to the regulatory region of immunoglobulin heavy chain gene IgH. Aim of this study was to determine the frequency of PCR detection of IgH/BCL2 in DNA samples isolated from archival cytological slides of lymph node aspirates, bone marrow and/or peripheral blood (PB) obtained from patients with histologically confirmed follicular lymphoma using primers and protocol proposed by BIOMED-2 consortium. We also compared molecular with cytomorphological findings in bone marrow/peripheral blood and tested this method of detection of IgH/BCL2 molecular marker in monitoring minimal residual disease (MRD) in routine clinical setting. DNA was successfully isolated from all archival cytological slides obtained by fine needle aspiration of lymph nodes as well as from 75% of smears of bone marrow aspirates from 19 patients. Fusion oncogene was detected in 10 of 19 patients (52%). For patients with PCR IgH/BCL2 positive lymph nodes, molecular test found BM infiltration in 5 cases (83%), while cytomorphology detected infiltration in three of eight cases (37%) available for comparison. May-Grünwald-Giemsa stained cytological smears can be used for PCR-based ancillary methods and the rate of detection of IgH/BCL2 rearrangement is similar to results reported for paraffin-embedded tissues. For patients with detectable baseline molecular marker, PCR is a highly suitable method for detection of bone marrow involvement and monitoring MRD.