Justyna Rybka

Low expression of microRNA-204 (miR-204) is associated with poor clinical outcome of acute myeloid leukemia (AML) patients

BackgroundAcute myeloid leukemia (AML) is a heterogeneous neoplasm of the bone marrow with poor prognosis. In clinical practice new prognostic factors are still needed. MicroRNAs (miRs), small endogenous noncoding RNAs, play an essential role in the development and progression of acute leukemia. The aim of the study was to evaluate miR-204 expression in patients with AML at diagnosis and after induction chemotherapy, in comparison to healthy controls. We also investigated, if miR-204 expression correlates with clinical features of AML patients.MethodsmiR-204 expression has been analyzed using RT-PCR in 95 bone marrow specimens from newly diagnosed AML patients in comparison to 20 healthy subject.ResultsWe showed down-regulated miR-204 expression in AML patients, which was associated with shorter patients’ survival. Higher expression of miR-204 in patients after induction therapy was correlated with complete remission achieving.ConclusionsWe showed low miR-204 expression in AML and found it to be an independent prognostic factor in this patient population.

Expression of microRNA-331 can be used as a predictor for response to therapy and survival in acute myeloid leukemia patients

AIM Aberrant expression of microRNAs (miRs) has been proved to have a role in acute myeloid leukemias (AML), but there is no information on miR-331 in AML. MATERIALS & METHODS miR-331 expression has been analyzed using reverse-transcription polymerase chain reaction (RT-PCR) in 95 bone marrow specimens from newly diagnosed AML patients in comparison with 20 healthy subjects. RESULTS miR-331 was upregulated in AML patients and its expression seemed to influence remission achieving and death risk. The time of remission duration in patients with complete remission was longer in subjects with miR-331 downregulation after induction chemotherapy. CONCLUSION we showed for the first time that miR-331 higher expression appears to be correlated with worse response to therapy and shorter survival of AML patients.

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.

Clinical response to azacitidine therapy depends on microRNA-29c (miR-29c) expression in older acute myeloid leukemia (AML) patients

Acute myeloid leukemia (AML) is a heterogeneous disease with different clinical course and prognosis. microRNA-29 (miR-29) family of non-coding small RNAs can play an important role in pathogenesis of AML, but also can influence response to therapy. The purpose of the study was to evaluate miR-29c expression in AML patients in relationship to clinical parameters and response to chemotherapy, including azacitidine. Materials and Methods miR-29c expression has been analyzed using RT-PCR in 95 bone marrow specimens from newly diagnosed AML patients in comparison to 20 healthy subject. Results We showed up-regulated miR-29c expression in AML patients which was linked also to higher risk of disease relapse after achieving complete remission. In subset of elderly AML patients treated with azacitidine, low miR-29c expression at diagnosis correlated with good response to therapy. Conclusions miR-29c is of prognostic value and influences response to azacitidine treatment in older AML patients.

The role of soluble HLA-G and HLA-G receptors in patients with hematological malignancies after allogeneic stem cell transplantation

Abstract HLA-G is a non-classical MHC class I molecule whose suppressive activity on immune effector cells is exerted due to interactions with receptors ILT2, ILT4 and KIR2DL4. These receptors are expressed mainly on NK cells and monocytes, and their intensity of expression changes depending on HLA-G level. HLA-G plays an important role in the development of tolerance following organ transplantations and bone marrow stem cell transplantations. HLA-G also participates in the modulation of the immune response during cancerogenesis. The aim of this study was to assess HLA-G level in blood serum, the percentage of NK cells and monocytes with expression of receptors for HLA-G (ILT2, ILT4, KIR2DL4 and NKG2D) in patients who received allogeneic stem cell transplantations, and their influence on the occurrence of graft-versus-host reaction. The study included 32 patients with bone marrow diseases (acute leukemias, myelodysplastic syndrome, chronic myeloid leukemia, paroxysmal nocturnal hemoglobinuria) who received allogeneic stem cell transplantations. We assessed the expression of receptors ILT2, ILT4, KIR2DL4 and NKG2D on monocytes and NK cells, as well as the level of HLA-G in blood serum in patients before conditioning, in the transplant hematopoietic reconstitution period following allogeneic bone marrow stem cell transplantation. The percentage of NK cells with expression of KIR2DL4, ILT2 and ILT4 receptors was higher in patients with 0–I grade GVHD than in patients with II–IV grade GVHD. The percentage of monocytes with expression of ILT4 and ILT2 receptors was higher in patients with 0–I grade GVHD than in patients with II–IV grade GVHD. The level of HLA-G in patients’ blood serum was higher after the stem cell transplantation compared with the period before transplantation. HLA-G level and HLA-G receptors are related to intensity of GVHD and may play the role of a prognostic factor for the development of GVHD and the clinical course of this reaction.

Alterations of biomechanics in cancer and normal cells induced by doxorubicin

Mechanical properties of biological structures play an important role in regulating cellular activities and are critical for understanding metabolic processes in cancerous cells and the effects of drugs. For some cancers, such as acute myeloid leukaemia, chemotherapy is one of preferential methods. However, due to the lack of selectivity to cancer cells, cytostatic agents cause toxicity to normal tissues. Here, we study the effect of doxorubicin (DOX) on the mechanical properties of DNA molecules, leukemic blast cells and erythrocytes, using optical tweezers. In addition, we controlled the subcellular distribution of the drug by confocal microscopy. Our results indicated that doxorubicin affects mechanical properties of cellular structures. In all cases the drug reduced mechanical strength of examined objects. For the leukemic cells the drug subcellular distribution was predominantly nuclear with some particulate cytoplasmic fluorescence. In erythrocytes, doxorubicin showed fluorescence mainly in cytoplasm and plasma membrane. The lowering of blast cells stiffness may be due to the interaction of doxorubicin with nuclear structures, especially with nucleic acids, as our studies with DNA confirmed. In addition, it is known that DOX inhibits the polymerization of actin and thus cytoskeletal modification may also be important in reducing of cell mechanical strength. In the case of erythrocytes - the non-nucleated cells, a significant effect on the decrease of cell stiffness, besides the cytoskeleton, may have the interaction of the drug with the cell membrane. Experiments with model phospholipid membranes confirmed that observed increase in cell elasticity originates, among other things, from the drug incorporation in the lipid membrane itself. The lowering of mechanical strength of leukemic cells may have an significant impact on the effectiveness of chemotherapy. However, the fact that doxorubicin interacts not only with proliferating cancer cells, but also with the health ones may explains the high toxicity of the drug at the therapeutic concentrations. Our observations also suggest that chemotherapy with doxorubicin may decrease the risk of vascular complications in acute leukemia, due to increasing the cell elasticity.

The predictive role of interim PET after the first chemotherapy cycle and sequential evaluation of response to ABVD in Hodgkin’s lymphoma patients—the Polish Lymphoma Research Group (PLRG) Observational Study

Background Interim PET after two ABVD cycles (iPET2) predicts treatment outcome in classical Hodgkins lymphoma. To test whether an earlier assessment of chemosensitivity would improve the prediction accuracy, we launched a prospective, multicenter observational study aimed at assessing the predictive value of iPET after one ABVD (iPET1) and the kinetics of response assessed by sequential PET scanning. Patients and methods Consecutive patients with newly diagnosed classical Hodgkins lymphoma underwent interim PET scan after one ABVD course (iPET1). PETs were interpreted according to the Deauville score (DS) as negative (-) (DS 1-3) and positive (+) (DS 4, 5). Patients with iPET1 DS 3-5 underwent iPET2. Results About 106 early (I-IIA) and 204 advanced (IIB-IV) patients were enrolled between January 2008 and October 2014. iPET1 was (-) in 87/106 (82%) or (+) in 19/106 (18%) of early, and (-) in 133/204 (65%) or (+) in 71/204 (35%) of advanced stage patients, respectively. Twenty-four patients were excluded from response analysis due to treatment escalation. After a median follow-up of 38.2 (3.2-90.2) months, 9/102 (9%) early and 43/184 (23%) advanced patients experienced a progression-free survival event. At 36 months, negative and positive predictive value for iPET1 were 94% and 41% (early) and 84% and 43% (advanced), respectively. The kinetics of PET response was assessed in 198 patients with both iPETs. All 116 patients with iPET1(-) remained iPET2(-) (fast responders), 41/82 with IPET1(+) became iPET2(-) (slow responders), and the remaining 41 stayed iPET2(+) (non-responders); progression-free survival at 36 months for fast, slow and non-responders was 0.88, 0.79 and 0.34, respectively. Conclusion The optimal tool to predict ABVD outcome in HL remains iPET2 because it distinguishes responders, whatever their time to response, from non-responders. However, iPET1 identified fast responders with the best outcome and might guide early treatment de-escalation in both early and advanced-stage HL.

Expression of microRNA‑181 determines response to treatment with azacitidine and predicts survival in elderly patients with acute myeloid leukaemia

MicroRNAs (miRs) are small non-coding RNAs that play important roles in cell differentiation and survival. Abnormal expression of miRs has been demonstrated in numerous types of cancer, including acute myeloid leukaemia (AML). The aim of the present study was to evaluate miR-181 expression at diagnosis and following the completion of chemotherapy in AML patients, with regard to clinical response and outcome, particularly in patients treated with azacitidine. miR-181 expression was analysed using reverse transcription-quantitative polymerase chain reaction in 95 bone marrow specimens from newly diagnosed AML patients and in 20 healthy subjects for comparison. The results revealed upregulated miR-181 expression in the total cohort of AML patients, which was correlated with longer survival. However, in a subset of older AML patients treated with azacitidine, low miR-181 expression at diagnosis was a predictor for complete remission and prolonged survival. The findings indicated that miR-181 has an important role in AML and determines response to azacitidine treatment in older AML patients.

Variations in genes involved in regulation of the nuclear factor – κB pathway and the risk of acute myeloid leukaemia

Genes involved in regulation of the nuclear factor – kappa B (NF‐κB) pathway are suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML). The present study aimed to assess the association between the NF‐κB1, TRAF3 and TLRs genes single nucleotide polymorphisms (SNPs) and disease susceptibility as well as progression in patients with AML. For this purpose 62 patients and 126 healthy individuals were genotyped for NF‐κB1 (rs28362491), TRAF3 (rs11160707; rs12147254), TLR2 (rs201786064), TLR4 (rs4986790; rs4986791) and TLR9 (rs5743836; rs187084) alleles. Three SNPs were found to be associated with the risk for the AML development. The TRAF3 (rs12147254) AA homozygosity (RR = 2.770, P = 0.0392), TLR9 (rs5743836) C wild‐type allele (RR = 2.542, P = 0.0096) as well as TLR9 (rs187084) T allele (RR = 13.396, P < 0.0001) and its homozygosity (RR = 11.805, P < 0.0001) were more frequent among patients with AML than healthy individuals. The associations of the rs187084 SNP were significant for both sexes. Moreover, patients who relapsed were more frequently characterized with the presence of the rs187084 TLR9 TT genotype (P = 0.045) or the rs12147254 TRAF3 A variant (P = 0.066). In conclusion, polymorphisms within the TLR9 and TRAF3 genes are associated with predisposition to AML and may affect the progression of the disease in the Polish population.

Polymorphisms within beta-catenin encoding gene affect multiple myeloma development and treatment

Recent studies have suggested that cereblon (CRBN) is essential for the anti-myeloma (MM) activity of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, and that dysregulation of Wnt/β-catenin pathway may be one of possible reasons of lenalidomide resistance. This prompted us to analyze the effect of polymorphisms within the genes coding for cereblon (CRBN (rs121918368 C>T)) and β-catenin (CTNNB1 (rs4135385 A>G; rs4533622 A>C)). MM patients (n=142) and healthy individuals (n=123) were genotyped using the Light SNiP assays. The presence of the CTNNB1 (rs4533622) A allele was more frequently detected in patients presented with stage II-III disease according to International Staging System (63/82 vs. 26/44, p=0.043) and Durie-Salmon criteria (75/99 vs. 14/26, p=0.049). The CTNNB1 (rs4135385) AA homozygosity was more frequent among patients with better response to CTD, i.e., cyclophosphamide-thalidomide-dexamethasone (18/23 vs. 32/60, p=0.047). Patients carrying the CTNNB1 (rs4533622) AA genotype were better responders to the first line therapy with thalidomide containing regimens (p<0.05). No significant association was observed between the effect of lenalidomide therapy and polymorphisms studied. However, the occurrence of neutropenia during lenalidomide therapy was more frequent among the CTNNB1 (rs4135385) AA carriers (p=0.019), while the CTNNB1 (rs4533622) AA homozygosity characterized patients with high grade (3-4) neutropenia (p=0.044). No association was found for the CRBN polymorphism. These results suggest that the CTNNB1 polymorphisms may affect the clinical course and response to chemotherapy in patients with multiple myeloma.