Study of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples.

Simultaneous quantitative measurement of mRNA of the WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples reflects the specific pathological proliferative activity in acute leukemia and their ratio is promising as a diagnostic marker. The transcriptome profile of acute leukemia cells is usually assessed using NGS or microarray techniques after a preliminary procedure for isolation of mononuclear cells. However, the results of using the multiplex PCR reaction for the simultaneous determination of all above mRNAs in whole blood samples have not been published so far. Determination of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in venous blood level samples by multiplex RT-PCR. The study included 127 blood samples from patients who diagnosis of acute leukemia was subsequently confirmed. In the comparison group, 87 samples of patients without oncohematological diagnosis were selected, including 31 samples (K1) with a normal blood formula and 56 samples (K2) with a violation of the cellular composition - anemia, leukocytosis and thrombocytopenia. RNA isolation and reverse transcription were performed using the Ribozol-D and Reverta-L kits (TsNIIE, Russia). Determination of the mRNA expression level of the WT1, BAALC, EVI1, PRAME and HMGA2 genes by multiplex real-time PCR using a homemade multiplex PCR kit. The mRNA level was characterized by high interindividual variation and did not correlate with the rate of circulating leukocytes or blood blasts. Expression of WT1 mRNA was observed in whole blood only in one patient from the control group and in 112 (88%) patients with leukemia and was combined with a decrease in the level of HMGA2 mRNA expression and BAALC mRNA values. In contrast to the control groups, patients with leukemia had higher levels of BAALC mRNA in AML and ALL, increased PRAME mRNA in AML and APL, but lower levels of HMGA2 in APL.

Use of the «BCR/ABL - multitest¼ kit in the algorithm of laboratory diagnostics of oncohematological diseases: economic aspects.

Abnormal mRNAs of the hybrid BCR-ABL gene in the majority of cases initiate the synthesis of proteins with a mass of 210 kDa (p210), 190 kDa (p190), and 230 kDa (p230). Expression of the p210 variant is most common in CML (95% of cases), while the p190 and p230 variants are less common (1-4%). On the contrary, p190 predominates in ALL. Measurement of BCR/ABL gene expression is included in clinical guidelines for the diagnosis of CML and ALL as sequential tests in accordance with their occurrence. At the same time, in the context of primary patients testing with suspected hematological malignancies with a low prevalence of BCR-ABL positive patients in the cohort of examined individuals, sequential testing is associated with low cost-effectiveness. Purpose: approbation of a parallel algorithm for detecting all three (p210, p190 and p230) using the multiplex RT-PCR format implemented in the «BCR/ABL-MULTITEST¼ reagent kit. We used anonymized blood samples from patients with suspected CML, as well as samples from ALL patients before starting therapy. Testing of blood samples was carried out using two variants of the algorithm: sequential determination of individual BCR-ABL transcripts and parallel determination using the developed set of reagents «BCR/ABL-MULTITEST¼. To detect the p210 transcript, a commercial kit «AmpliSens® Leukemia Quantum M-bcr-FRT¼ (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) was used. Simultaneously, a test was used to detect all three variants of BCR-ABL transcripts using the «BCR/ABL - MULTITEST¼ reagent kit based on a monochrome multiplex reaction «in one test tube¼. Reverse transcription were carried out using the REVERTA-L reagent kit (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) in accordance with the manufacturer's instructions. Using the reagent kits «BCR/ABL-MULTITEST¼ and «AmpliSens® Leukemia Quantum M-bcr-FRT¼ there is a high level of correlation of quantitative results of determining the chimeric transcript BCR-ABL р210 (r = 0.99). When using the proposed parallel algorithm with the primary use of the «BCR/ABL-MULTITEST¼ reagent kit, out of 95 patients with suspected CML, 9 samples with p210 transcript were identified, one with p190 BCR / ABL, and in one case a transcript variant characteristic of chronic neutrophilic leukemia - p230 BCR / ABL. The estimated cost for detecting one positive case of BCR-ABL when using the parallel diagnostic algorithm «BCR/ABL-MULTITEST¼ with a focused flow of studies is reduced by about 2 times due to a decrease in the amount of laboratory plastic used and the volume of the reaction mixture, as well as the absence of the need for repeated separate tests to detect p190 and p230. The use of the multiplex PCR-RT test system «BCR/ABL-MULTITEST¼ allows detecting in one test tube all three main variants of BCR-ABL transcripts - p210, p190, p230 and achieving significant resource savings when examining a cohort of patients with suspected CML and ALL and low frequency of positive samples.

Development of a method for the determination of the JAK2 gene mRNA in venous blood and assessment of its diagnostic value in oncohematology.

Overactive JAK pathway signaling is a hallmark of immune diseases and critically affects on inflammation and coagulation. A number of mutations in the JAK2 gene act as driving forces of myeloproliferative neoplasms (MPN), the pathogenesis of certain variants of acute leukemia, a number of solid malignancies and cardiovascular diseases. Assays for quantifying JAK2 mRNA in circulating blood cells can be used as a marker associated with the activity of this enzyme. Development of an original method for detecting JAK2 mRNA in venous blood and assessment of the possible diagnostic value in chronic oncohematological diseases. The development of an RT-PCR method for determining the expression of the JAK2 gene mRNA in venous blood samples was carried out in accordance with the MIQE requirements. Primers and TaqMan probes were designed using the Primer3 program, taking into account the possibility of excluding subsequent DNase treatment. The stability of the investigated mRNA was assessed in vacutainers with different anticoagulants and depending on the storage time of the samples. The study of the expression of JAK2 mRNA in blood leukocytes of 41 patients with B-CLL, 16 patients with CML, 12 patients with multiple myeloma and 39 donors using the developed "real-time" PCR method. The study revealed a decrease in the level of JAK2 mRNA in venous blood samples in patients with primary CLL, but not with CML or with multiple myeloma. The level of the marker in the majority of patients with CLL after the start of therapy returned to the range typical for healthy people. It has been shown that the values of the relative expression of JAK2 mRNA are most stable in the range of 2 - 7 hours after taking blood in a vacutainer with EDTA. An original RT-PCR method was developed for the quantitative determination of JAK2 mRNA in venous blood samples, which meets the requirements of the MIQE system. Determination of JAK2 mRNA can be useful for clarifying the pathogenesis features of certain diseases involving impaired Janus kinase activity and can become a promising marker for prognosis and assessment of the effectiveness of therapy.

[Investigation of miR-155 level in the blood of patients with chronic lymphocytic leukemia and Ph-negative myeloproliferative neoplasms.]

MiR-155 is involved in various physiological processes in the cell, including hematopoiesis, immunity, inflammation and differentiation. Increased expression of miR-155 is observed in many malignant diseases, including lymphomas, acute myeloid leukemia and CLL. However, a comparative study of the miR-155 expression in the blood leukocytes in patients with chronic myeloid and lymphoproliferative diseases has not yet been carried out. To investigate the expression of miR-155 in the blood cells of patients with lympho- and ph-negative myeloproliferative neoplasms. MiR-155 expression were studied in the blood leukocytes of 28 patients with B-CLL, 52 patients with MPN and 51 donors by "real time" PCR method. The study revealed an increase in miR-155 in blood leukocytes in both patients with CLL and patients with MPN compared with the control group. In accordance with the results of the ROC analysis, the sensitivity and specificity of blood leukocytes testing on miR-155 expression level was 81.8% and 78.4%, respectively, for CLL and 55.1% and 82.4%, respectively, for MPN. At the same time, in patients with CLL who received therapy, the level of miR-155 was significantly lower compared with those who did not receive therapy. Thus, the involvement of miR-155 in the pathogenesis of chronic myeloid and lymphoproliferative diseases was demonstrated.