Clinical Characteristics of Cerebrovascular Pathology with Patients Suffering from Ph-Negative Myeloproliferative Disease

Background: Disturbances of microcirculation play a significant role in the development and progression of both acute and chronic cerebrovascular diseases (CVD) and may be associated with different hemogram abnormalities. One of the reasons of the prothrombogenic state of the endothelium is the increase in the number of blood corpuscles leading to (non-Ph) myeloproliferative disorders (MPD) including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PM). Materials and Methods: The study included 167 patients: 102 patients with Ph-MPD and the control group comprising 65 patients with CVD. According to MPD subtype, the patients were divided into three groups: patients with ET (37%, n = 38, male/female 7/31, age 52 ± 7 years), those with PV (40%, n = 41, male/female 20/21, age 50 ± 6 years) and those with PM (23%, n = 23, male/female 5/18, age 54 ± 4 years). Results: In 79% (n = 81) of cases in the study group (with Ph-MPD), patients had chronic CVD, with the most frequently identified symptoms being asthenia (92%) and headache (72%). Headache in Ph-MPD patients was more frequently (86%) associated with PM, while in patients with PV and ET it was equally distributed (70%). Neurological symptoms in 53% of cases were associated with focal changes of the brain on MRI localized in the subcortical area of the frontal and parietal lobes. Twenty-one (21%) patients suffered an acute cerebrovascular accident, 8 of them had thrombotic occlusion of one of the internal carotid arteries leading to hemispheric infarcts. Endothelial function (as measured by flow-dependent dilation of the brachial artery) was severely impaired in all study groups (median 5% with normal cut-off at 10%), the lowest degree of vasodilator activity being specific for patients with a history of stroke (p = 0.011). Conclusion: Patients suffering from MPD had asymptomatic focal changes in the brain in the absence of concomitant vascular disease (hypertension, atherosclerotic vascular disease, heart rhythm disorders) in 50% of cases. MPD, while remaining un- or underdiagnosed, presents a major concern in the cerebrovascular setting. A large number of thrombotic strokes occurring in patients with ET underline the necessity of early diagnostics and preventive therapy in these patients

Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells

Analysis of T-Cell Receptor-γ Gene Rearrangements Using Oligonucleotide Microchip: A Novel Approach for the Determination of T-Cell Clonality

T-cell clonality estimation is important for the differential diagnosis between malignant and nonmalignant T-cell proliferation. Routinely used methods include polymerase chain reaction (PCR) analysis of T-cell receptor-γ (TCR-γ) gene rearrangements followed by Genescan analysis, polyacrylamide gel electrophoresis, or heteroduplex analysis to visualize amplification products. Here, we present a new method for the analysis after PCR of TCR-γ rearrangements using hybridization on oligonucleotide microchip. A microchip was designed to contain specific probes for all functional variable (V) and joining (J) gene segments involved in rearrangements of the TCR-γ locus. Fluorescently labeled fragments of rearranged γ-chain from patients and donors were obtained in a multiplex nested PCR and hybridized with a microchip. The results were detected using a portable microchip analyzer. Samples from 49 patients with T-cell lymphomas or leukemias and 47 donors were analyzed for T-cell clonality by microchip and single-strand conformation polymorphism analysis, which served as a standard reference method. Comparison of two techniques showed full concordance of the results. The microchip-based approach also allowed the identification of V and J gene segments involved in the particular TCR-γ rearrangement. The sensitivity of the method is sufficient to determine 10% of clonal cells in the sample