Bożena Jaźwiec

Circulating sCD138 and some angiogenesis-involved cytokines help to anticipate the disease progression of early-stage B-cell chronic lymphocytic leukemia.

Syndecan-1 (CD138) is a transmembrane heparin sulfate proteoglycan expressed on distinct stages of differentiation of B-lymphoid cells. Its prognostic value in B-cell chronic lymphocytic leukemia (B-CLL) has not been evaluated so far. The serum concentration of sCD138 and some angiogenesis-involved cytokines: vascular endothelial growth factor (VEGF), basis fibroblast growth factor (bFGF), and endostatin were studied in 52 previously untreated patients with B-CLL. We found that bFGF and sCD138 levels were significantly higher in B-CLL patients than in controls. In patients with sCD138 level or endostatin level below the median value the lymphocyte count was higher than in patients with serum level of those cytokines above the median value. In patients with progressive disease bFGF level was significantly higher and sCD138 level significantly lower than in patients with stable one. Moreover, high sCD138 level was associated with longer lymphocyte doubling-free survival, and, on the limit of statistical significance, a high endostatin level was associated with shorter progression-free survival. We conclude that serum sCD138 level is increased in early stage B-CLL patients and may have a positive prognostic value as to the dynamics of the disease.

The expression of Toll-like receptors in patients with acute myeloid leukemia treated with induction chemotherapy

Toll-like receptors play an important role in the host defense against microorganisms. TLRs are mainly expressed in human immune-related cells, such as monocytes, neutrophils, macrophages, dendritic cells, T cells, B cells and NK cells. The expression or up-regulation of TLRs has been demonstrated in some tumors and tumor cell lines but the role of TLRs in pathogenesis and development of acute leukemias remains unclear. The aim of this study was to evaluate the expression of TLR2, TLR4 and TLR9 and their significance as prognostic factors in patients with acute leukemias treated with induction chemotherapy. 103 patients with newly diagnosed acute myeloid leukemia (AML) were evaluated (47 females and 56 males). The median age of patients was 51 years. Using quantitative reverse transcriptase PCR, the mRNA expression of genes TLR2, TLR4 and TLR9 was measured. The mRNA expression of TLR2 and TLR4 was significantly higher in patients with NR than in patients with CR and CRi. We especially observed that mRNA expression of TLR2 and TLR4 was significantly higher in patients with myelomonocytic and monoblastic acute leukemia than in patients with other types of AML. The mRNA expression of TLR2 and TLR4 was higher in AML patients than in healthy individuals, although there was no statistically significant difference. Patients with higher mRNA expression of TLR2 and TLR4 had significantly shorter OS than patients with lower mRNA expression of TLR2 and TLR4. Multivariate analysis showed that mRNA expression of TLR2 and the age of patients were independent factors associated with treatment response. Our results suggest that TLRs could be an independent prognostic factor for response rate after induction therapy in patients with acute myeloid leukemias.

Expression of PIM-2 and NF-κB genes is increased in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is associated with complete remission rate and overall survival

INTRODUCTION PIM-2 is a proto-oncogene that encodes for a serine/threonine kinase that interacts with various signaling molecules. PIM-2 is highly expressed in neoplastic tissues and in leukemic and lymphoma cell lines, which is consistent with its role during oncogenic transformation. The nuclear factor kappa B (NF-κB) pathway appears to be deregulated in a variety of tumors, with sustained activity of NF-κB leading to apoptotic resistance in tumor cells. The aim of this study was to investigate whether expression of PIM-2 and NF-κB is altered in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS One hundred forty-three patients were included: 91 with AML and 52 with ALL, aged 18-84 (median 46.7). Eighty-three patients (51 AML and 32 ALL) reached complete remission (CR). Bone marrow samples were collected at the time of diagnosis. Control samples were obtained from 24 healthy donors. We analyzed PIM-2 and NF-κB expression by RQ-PCR analysis. RESULTS Expression of both PIM-2 and NF-κB in all leukemia patients and subgroups was significantly higher than in controls. AML patients who reached CR expressed PIM-2 and NF-κB at significantly lower levels than did patients with primary resistance to chemotherapy and who did not reach CR (NCR). Survival analysis revealed that in AML patients with higher expression of PIM-2 the overall survival (OS) was significantly shorter than in patients with lower expression. CONCLUSION Our data indicate that PIM-2 and NF-κB gene expression is increased in patients with AML and ALL. Moreover, high PIM-2 expression is associated with CR rate and OS in AML patients.

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.

Mitoxantrone changes spectrin-aminophospholipid interactions.

Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatitydcholine (PE/PC) monolayers. The change in Δπ induced by spectrins is several-fold larger in the presence of 72 nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5′-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16′-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.

Expression of bone morphogenetic proteins (BMPs) receptors in patients with B-cell chronic lymphocytic leukemia (B-CLL).

Bone morphogenetic proteins (BMPs) are multifunctional cytokines which belong to transforming growth factor β (TGF β) superfamily. They regulate proliferation, differentiation, and apoptosis in a variety of cells including hematopoietic cells. BMPs act because of binding to two types of serine/threonine kinase receptors: BMP type I receptors (IA and IB) and BMP type II receptor. Deregulation of BMPs signaling pathways has been reported in some of human cancers, but the role of BMPs in hematopoietic malignancies remains unknown. The aim of our study was to examine the percentage of expression of BMPs receptors on lymphocytes of patients with B‐cell chronic lymphocytic leukemia (B‐CLL). A total of 46 patients with B‐CLL (27 men and 19 women) and 10 healthy persons were evaluated. Freshly isolated mononuclear cells were incubated with antibodies against BMPs receptors: BMPRIA, BMPRIB, and BMPRII and examined in 2‐color flow cytometry. On cells of patients with B‐CLL, the percentage of expression of BMP RIA, BMP RIB, and BMP RII was significantly higher than in normal cells of the control group. The percentage of the expression of BMP RIA and BMP RIB was higher in patients with advanced stage of disease.

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

CD117 (c-kit) Expression on CD34+ Cells Participates in the Cytogenetic Response to Imatinib in Patients with Chronic Myeloid Leukemia in the First Chronic Phase

Background: Chronic myeloid leukemia (CML) biology seemed to be perfectly explored especially at the beginning of the tyrosine kinase inhibitors era. Later years with imatinib and second-generation tyrosine kinase inhibitors showed a variety of resistance mechanisms and it became obvious that the bcr-abl chimeric gene is not the only enemy to fight. Some studies assumed the decreased rate of programmed cell death (apoptotic) to be the primary mechanism by which BCR-ABL affects expansion of the leukemic clone in CML. Therefore, the aim of this study was to investigate the role of c-kit inhibition in treatment response. Methods: Cytogenetic analysis, real-time quantitative reverse-transcriptase polymerase chain reaction, flow-cytometric analysis and imatinib serum level quantification were applied. Results: The percentage of CD34+ cells expressing c-kit (CD117) isolated from bone marrow samples of 54 CML patients treated with standard-dose imatinib was significantly lower among imatinib responders. The fraction of apoptotic CD34+CD117+ cells in this patient group was significantly higher than in nonresponders. Conclusion: To achieve optimal treatment response in CML patients, the elimination of CD34+CD117+ may be necessary through an apoptotic pathway.

Expression of cyclin A and bone morphogenetic protein receptors and response to induction therapy in patients with acute leukemias

Abstract Bone morphogenetic proteins (BMPs) are multifunctional cytokines that belong to the transforming growth factor β (TGFβ) family. They participate in the regulation of growth, differentiation and apoptosis in a variety of cell types including hematopoietic lineages. To date, the role of BMPs in carcinogenesis has not been well known. Cyclin A is a cell cycle regulatory protein which plays the role of a parameter of cell proliferation in various types of carcinomas including hematological malignancies. The role of BMPRIA, BMPRIB, BMPRI and cyclin A in the pathogenesis of acute leukemias remains unclear. The aim of this study was to evaluate the expression of BMP receptors and cyclin A on blast cells and their possible relationship with clinical outcome. Seventy patients with acute leukemias (28 female and 42 male) and 10 aged-matched healthy controls were studied. All patients were examined before cytostatic treatment. The expression of BMP receptors and cyclin A was detected by flow cytometry. The results show that higher expression of BMPRIA, BMPRIB, BMPRII and cyclin A is related with a higher complete response (CR) rate, higher overall survival (OS) and lower relapse risk. The expressions of BMPRIA, BMPRIB, BMPRII and cyclin A could be useful as prognostic parameters of the proliferation status of acute leukemia cells, but further studies are needed to assess this phenomenon.

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.