Małgorzata Kuliszkiewicz-Janus

Lipid changes occuring in the course of hematological cancers

The relationship between plasma lipid levels and mortality from cardiovascular diseases has been shown in many studies, but there has been far less investigation into their relationship to non-cardiovascular diseases. The aim of this study was to investigate the lipid profile of individuals with hematological malignancies and its relationship to disease activity. 238 patients were included in the study: 84 with acute leukemia, 62 with non-Hodgkin lymphoma, 35 with Hodgkin’s lymphoma, 32 with multiple myeloma, and 25 with myeloproliferative syndrome. The HDL cholesterol level of the patients differed to that of the individuals in the control group in the active disease period for all the analyzed disorders, but only remained statistically significant in the acute leukemia and non-Hodgkin lymphoma groups during the remission period. Smaller differences were observed for the remaining lipid fractions, except for the triglyceride level, which increased in the active disease period in all the analyzed disorders except non-Hodgkin lymphoma. The most pronounced changes in the lipid fractions occurred in the HDL cholesterol level, and were the most remarkable for acute leukemia.

31P MRS analysis of the phospholipid composition of the peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) of patients with acute leukemia (AL).

The aim of this study was to evaluate the phospholipid concentration in acute leukemia (AL) blast cells from peripheral blood (PBMC) and bone marrow (BMMC). In vitro31P Nuclear Magnetic Resonance Spectroscopy (31P MRS) was used. The integral intensities of the resonant peaks and the phospholipid concentrations in PBMC and BMMC were analyzed. Differences in the phospholipid concentrations in cells from myeloblastic or lymphoblastic lines were also evaluated. This investigation was carried out on phospholipid extracts from PBMC and BMMC from 15 healthy volunteers and 77 patients with AL (samples taken at the moment of diagnosis). A significant decrease in sphingomyelin (SM) and phosphtidylserine (PS) was observed in the PBMC of patients with AL relative to the results for the healthy volunteers. For ALL, we found a significant decrease in the concentration of phosphatidylcholine plasmalogen (CPLAS), SM, PI+PE (phosphatidylinositol + phosphatidylethanolamine) and PS in comparison with the results for healthy volunteers and patients with AML. Experiments with BMMC cells revealed a significant decrease in the concentration of CPLAS, SM, PI+PE, and PS in ALL relative to AML. Additionally, a significant decrease in phosphatidylcholine (PC) concentration was observed in ALL compared to AML. If the phospholipid extracts were taken simultaneously from the same patient, there were no significant differences in the integral intensities and phospholipid concentrations between PBMC and BMMC.

A risk of essential thrombocythemia in carriers of constitutional CHEK2 gene mutations.

Germline mutations of the CHEK2 gene have been reported in some myeloid and lymphoid malignancies, but their impact on development of essential thrombocythemia has not been studied. In 16 out of 106 (15.1%) consecutive patients, newly diagnosed with essential thrombocythemia, we found one of four analyzed CHEK2 mutations: I157T, 1100delC, IVS2+1G>A or del5395. They were associated with the increased risk of disease (OR=3.8; P=0.002). The median age at ET diagnosis among CHEK2+/JAK2V617F+ patients was seven years lower than that among CHEK2−/JAK2V617F+ (52 vs. 59 years; P=0.04), whereas there was no difference in the medians of hematologic parameters between these groups. The results obtained suggest that CHEK2 mutations could potentially contribute to the susceptibility to essential thrombocythemia. The germline inactivation of CHEK2, as it seems, has no direct impact on the development of disease, but it could cause disruption of cell cycle checkpoints and initiate or support the cancerogenic process of essential thrombocythemia at a younger age.

The germline mutations of the CHEK2 gene are associated with an increased risk of polycythaemia vera

The molecular mechanisms underlying the development of myeloproliferative neoplasms (MPNs) are still not sufficiently well understood, although the association of MPNs pathogenesis with some gene alterations, especially somatic, have been reported (Tefferi, 2010; Bench et al, 2013). Recently, we demonstrated a strong association of germline mutations in the CHEK2 tumour suppressor gene with the increased risk of essential thrombocythaemia (ET) (Janiszewska et al, 2012). CHEK2 plays a key role in cell cycle regulation, coordination of DNA repair and apoptosis (Bartek & Lukas, 2003). In the general Polish population, four founder CHEK2 mutations (p.I157T, c.444+1G>A, c.1100delC and del5395) occur with 5 8% frequency, therefore it is possible to provide a reliable assessment of cancer risk in patient groups (Cybulski et al, 2007). The present study investigated the impact of these mutations on the risk of polycythaemia vera (PV). Such research has not been carried out so far. We also analysed the relationship between CHEK2 mutations and the JAK2 p.V617F mutation, and their relation to patient age, haematological features at PV diagnosis and a family history of cancer. The analysis included 106 consecutive patients, newly diagnosed with PV according to World Health Organization 2008 criteria (Tefferi & Vardiman, 2008) at two Polish haematology centres (Wrocław and Toru n). The median age of the cohort was 62 years. All molecular and statistical analyses were performed as previously described (Janiszewska et al, 2012). The gene mutations were investigated in DNA from peripheral blood (PB) and buccal swabs of patients, and in DNA from PB of 312 healthy persons, which formed the control group. The JAK2 p.V617F was present in 92 5% of patients, being homozygous in 27 6%. It was not found in buccal swabs of patients and in PB of controls. A CHEK2 mutation was found in 15 (14 1%) patients (Table I), 14 of whom were heterozygous and one was homozygous (p.I157T). The frequency of CHEK2 mutations was similar to that previously reported in ET patients (15 1%) (Janiszewska et al, 2012). All mutations were present both in DNA from PB and buccal swabs of patients, confirming their constitutional nature. The risk of PV was three times higher in CHEK2-positive patients, compared to both the control group [odds ratio (OR) = 3 0, P = 0 004] and general Polish population (OR = 2 7, P = 0 001) (Table I), and was similar to the previously reported risk of ET (Janiszewska et al, 2012). Thus, the estimated risk of PV and ET was found to be two times higher than the risk of breast cancer in women with CHEK2 mutations (Cybulski et al, 2007). The c.444+1G>A protein-truncating mutation was found at a frequency that was two times lower than the p.I157T missense mutation, but it was more strongly associated with the risk of PV. No patient had c.1100delC or del5395 (Table I). Both detected mutations are related to the highly conserved forkhead homology-associated (FHA) domain of the CHEK2 protein, functionally important for interactions with other proteins in response to DNA damage. The I157T protein variant has a disrupted ability to bind TP53 (p53) and BRCA1. The c.444+1G>A eliminates part of the FHA domain and the entire kinase activation domain, resulting in premature protein-truncation. These alterations, even in heterozygotes, may lead to reduction or loss of the gene function (Bartek & Lukas, 2003). Eight (53%) CHEK2-positive patients originated from families with at least one case of colon, stomach, breast, prostate or larynx cancer, and five (33%) from families with no known solid cancer. One of two remaining CHEK2-positive patients had a daughter with thrombocytopenia, and the mother of the second had acute leukaemia. No CHEK2 mutation was detected in one family with three consecutive generations of PV. JAK2 p.V617F was found in 13 CHEK2-positive patients. It this group, the median age at PV diagnosis was 7 years higher than in CHEK2-negative/JAK2 p.V617F-positive patients, but this difference was not statistically significant (Table II). Conversely, the median age at diagnosis in CHEK2-positive/JAK2 p.V617F-positive ET patients was 7 years lower than in CHEK2-negative/JAK2 p.V617F-positive cases, and this difference was statistically significant (P = 0 04) (Janiszewska et al, 2012). These results need to be corroborated in larger groups of patients, however they suggest that congenital CHEK2 mutations may have an impact on the later PV onset, and are associated with the neoplastic process in PV. The subgroups of patients with or without JAK2 p.V617F were too small for reliable statistical analysis. Significantly lower median levels of haemoglobin (P = 0 022) and haematocrit (P = 0 0009), i.e. basic parameters for a correct diagnosis of PV, were found in CHEK2positive/JAK2 p.V617F-positive patients compared to correspondence

Altered plasma fibrin clot properties in essential thrombocythemia

Abstract Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (Ks), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991 × 103/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced Ks (−38%, p < 0.0001) and prolonged CLT (+34%, p < 0.0001) were observed in ET. The differences remained significant after adjustment for fibrinogen and platelet count. ET was associated with a slightly shorter lag phase (−5%, p = 0.01) and higher maximum absorbency of the turbidimetric curve (+6%, p < 0.001). The ET patients had higher plasma P-selectin by 193% (p < 0.00001) and platelet factor 4 (PF4) by 173% (p < 0.00001), with higher P-selectin observed in 19 (44%) patients with JAK-2 gene V617F mutation. Higher t-PA (+20%, p < 0.001), 23% higher plasminogen activator inhibitor-1, PAI-1 (+23%, p < 0.01) and unaltered thrombin-activatable fibrinolysis inhibitor, plasminogen and α2-antiplasmin activity were found in the ET group. Ks inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.

Platelet-activating factor changes in phospholipid extracts from the plasma, peripheral blood mononuclear cells and bone marrow mononuclear cells of patients with acute leukemia — A 31P MRS in vitro study

The aim of this investigation was to evaluate the changes in PAF concentrations in the plasma, PBMC and BMMC of patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The plasma was from 23 healthy volunteers (HV) and 44 patients with AL (16 ALL, 28 AML). The PBMC were from 15 HV and 55 patients with AL (18 ALL, 37 AML), and the BMMC from 40 patients with AL (11 ALL, 29 AML). Methanol-chloroform phospholipid extraction from 60 × 106 cells (PBMC or BMMC) was performed according to a modified version of Folch’s method. 31P MRS data was obtained on an AMX 300 Bruker spectrometer (7.05 T). The PAF concentration in the plasma of the patients with ALL or AML was lower than that for the healthy volunteers. The PAF concentration in the plasma of the patients with ALL did not differ significantly from that of the patients with AML. In the case of both the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers.

Application of the apparent diffusion coefficient in magnetic resonance imaging in an assessment of the early response to treatment in Hodgkin's and non-Hodgkin's lymphoma : pilot study

Purpose Lymphoproliferative neoplasms are the largest and most frequently diagnosed entities in the group of haematological malignancies. The aim of the study was to assess whether apparent diffusion coefficient (ADC) measured on the first day of the second cycle of chemotherapy could be a predictor of prognosis and of the final treatment’s outcome. Material and methods The study included 27 patients with diagnosed Hodgkin’s and non-Hodgkin’s lymphoma, who had magnetic resonance (MR) performed with diffusion weighted imaging/apparent diffusion coefficient (DWI/ADC) before and on the first day of the second cycle of chemotherapy. Imaging was performed using a 1.5 T MR scanner. ADC was measured in lymphoma infiltration in the area of the lowest signal in the ADC map and the highest signal on β 800 images in post-treatment study. After that, the corresponding area was determined in a pre-treatment study and an ADC value was measured. Results The difference between ADC values in pre-treatment (ADC = 720 mm2/s) and post-treatment (ADC = 1059 mm2/s) studies was statistically significant (p < 0.001). Cutoff values for estimating response to treatment were established at the level of ADC 1080 mm2/s, and ADC to muscle ratio at 0.82 in post-treatment study. Patients with ADC > 752 mm2/s before treatment manifested lower probability of progression than patients with ADC < 752 mm2/s. Conclusions ADC measurement’s before treatment and on the first day of the second cycle of chemotherapy can be used as a prognostic marker in lymphoma therapy. ADC values lower than 1080 mm2/s and an increase of the ratio after the treatment can be considered as a marker of disease progression.

Peripheral circulatory disorders in essential thrombocythemia

A significant number of patients with essential thrombocythemia (ET) complain of symptoms including distal parts of the extremities (e.g., paresthesias or Raynauds phenomenon). The aim of the present study was to examine peripheral circulation in the upper extremities of individuals with ET. The study included 45 ET patients and 30 control subjects. All participants were subjected to thermography, photoplethysmography, impedance plethysmography, and applanation tonometry pulse wave analysis. The patients with ET differed significantly from the control subjects in terms of 3rd finger skin temperature (mean 31.04 vs. 32.45°C), skin temperature gradient (mean 1.82 vs. 0.11°C), photoplethysmographic amplitude (median 0.25 vs. 0.74%), and pulse waveform in the radial artery (more frequent occurrence of type B waveform). Pulse wave parameters correlated with the skin temperature gradient. The study findings imply the altered regulation of peripheral circulation in ET, including a decreased flow and an increased resistance.

Constitutional mutations of the CHEK2 gene are a risk factor for MDS, but not for de novo AML

CHEK2 plays a key role in cellular response to DNA damage, and also in regulation of mitosis and maintenance of chromosomal stability. In patients newly diagnosed with myelodysplastic syndrome (MDS, n = 107) or acute myeloid leukemia (AML, n = 117) congenital CHEK2 mutations (c.444 + 1G > A, c.1100delC, del5395, p.I157 T) were tested by PCR and sequencing analysis. The karyotype of bone marrow cells of each patient was assessed at disease diagnosis using classical cytogenetic methods and fluorescence in situ hybridization. The CHEK2 mutations were strongly associated with the risk of MDS (p < 0.0001) but not with the risk of de novo AML (p = 0.798). In CHEK2-positive MDS patients, two times higher frequency of aberrant karyotypes than in CHEK2-negative patients was found (71% vs. 37%, p = 0.015). In CHEK2-positive patients with cytogenetic abnormalities, subtypes of MDS: refractory anemia with excess blasts-1 or 2, associated with unfavorable disease prognosis, were diagnosed two times more often than in CHEK2-negative cases with aberrations (78% vs. 44%). In conclusion, the congenital CHEK2 inactivation is strongly associated with the risk of MDS and with a poorer prognosis of the disease. However, the chromosomal instability in AML is not correlated with the hereditary dysfunction of CHEK2.

The pathogenesis and available prevention optionsin patients with diabetic thrombophilia

Diabetes mellitus (DM), a growing health problem itself, is accompanied by an increased risk of cardiovascular and thrombotic complications. The imbalance between coagulation and fibrinolysis processes observed in patients with diabetes may be defined as diabetic thrombophilia. Several mechanisms are involved in the hypercoagulability state in diabetics, including endothelial cell damage, altered platelet structure and function, increased microparticle formation, different structure of fibrin clots, disturbances in the activity of coagulation factors, fluctuations in the concentrations of fibrinolysis activators and inhibitors, and qualitative changes of proteins due to glycation and oxidation processes. These all are the reasons why DM is the most common cause of acquired thrombophilia. Moreover, diabetes changes the efficacy of certain medications. Results of various trials seem to suggest that thrombolytic drugs are less effective in patients suffering from this disease. The impact of DM on the effectiveness of treatment with acetylsalicylic acid (ASA) remains unclear. Awareness of thrombotic complications in diabetic patients may enable earlier diagnosis and proper therapy.