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HLA- C*12:138 differs from HLA- C*12:03:01:01 by a single change, resulting in an amino acid substitution. [ABSTRACT FROM AUTHOR]

Introduction: Human cytomegalovirus (Orthoherpesviridae: Herpesvirales: Cytomegalovirus: Cytomegalovirus humanbeta 5 ) (HCMV) is one of the most commonly detected viruses in recipients of allogeneic hematopoietic stem cell (allo-HSCT) transplants. However, the emergence of resistance to antiviral drugs such as ganciclovir (GCV) poses a challenge in managing these patients. This study aims to investigate the prevalence and impact of mutations in the HCMV UL97 gene associated with resistance to GCV on the course of infection among allo-HSCT patients., Materials and Methods: The study examined the association between UL97 mutations and the clinical course of HCMV infection in allo-HSCT patients. Genetic sequencing was performed to identify mutations, and their impact on viral replication and resistance to GCV was assessed., Results and Discussion: Six mutations were identified (D490A, T502A, C592G, C592F, E596G, C603W). C592G, C592F, E596G, and C603W are associated with resistance to antiviral drugs, while D490A and T502A described for the first time. When comparing patients with wild-type and those carrying the mutant variant, several parameters of peripheral blood were significantly lower in the former group. The median time to peak viral load following allo-HSCT, duration of viremia, and rate of virological response to high-dose therapy also differed significantly between the two groups., Conclusion: It was shown that approximately one third (4 out of 14) of allogeneic stem cell transplant recipients had mutations associated with resistance to GCV. Patients carrying the mutant variant of HCMV had longer viremia and took longer to achieve a negative virological test result after starting high-dose therapy. Performing genotyping may help make more evidence-based therapeutic decisions.

Introduction: Infection caused by cytomegalovirus (CMV) is a serious problem for patients with weakened immunity, including patients with hematopoietic depression. The cases of complications associated with cytomegalovirus require antiviral therapy. However, during the natural mutation process, especially with prolonged use of drugs in suboptimal doses, CMV strains resistant to the action of antiviral drugs (such as ganciclovir, valganciclovir) may occur. Hypothetically, the emergence of resistance in the virus may cause a more aggressive course of infection, the ineffectiveness of antiviral therapy and, as a result, an increase in the number of deaths. In this regard, timely detection of mutations that can potentially lead to the resistance of the virus to antiviral drugs during hematopoietic stem cell transplantation (HSCT), as well as during organ and tissue transplantation, may be important when making a therapeutic decision. We describe three clinical cases for which the dynamics of the appearance of a mutant strain of CMV by the UL97 gene, which correlates with the viral load and clinical picture, is analyzed.The aim of the study was to determine the timing of the occurrence of mutations in CMV phosphotransferase UL97 gene associated with resistance to antiviral drugs in patients with hemoblastoses after allogeneic hematopoietic stem cell (allo-HSCs) transplantation., Material and Methods: The study included 48 samples of CMV DNA isolated from the peripheral blood of three allo-HSCs recipients with CMV infection who were treated in the clinics of the FSBI «National Medical Research Center for Hematology» of the Ministry of Health of Russia with oncohematological diseases during 2015-2017. Patients received conditional codes (PR, PD, and FS). Mutations associated with antiviral therapy (AVT) resistance were identified in all patients. Sanger sequencing was used for mutation detection. The obtained DNA sequences were analyzed using Nucleotide BLAST and Genome compiler software. Mutations were searched in MRA mutation resistance analyzer software. The nucleotide sequences were compared with the UL97 reference sequence of the Merlin CMV strain using this software environment., Results and Discussion: For all patients in whom the virus strains containing C592G (PR), C607F (PD) and C603W (FS) mutations were detected, the timing of the mutation occurrence was determined at days 187, 124 and 1184, respectively. The emergence of mutations with a high resistance factor was shown to be accompanied by an increase in viral load (VL), the appearance of a clinical picture characteristic of CMV infection and a lack of an adequate response to therapy with ganciclovir and its derivatives., Conclusion: Using these results, it is proposed to develop the test system based on random polymerase chain reaction (rPCR) to detect mutations in the most frequently encountered codons: M460I/V, C592G, A591V, A594T/V, L595F/S, C603W. Given that the data on the prevalence of these mutations were obtained from foreign sources, it is advisable to conduct similar studies on the frequency of mutations in the UL97 gene among the population of the Russian Federation in order to improve the quality and accuracy of test systems.

The b-cell chronic lymphatic leukemia is the most common among all lymphatic proliferative diseases and is characterized by significant variability of its clinical course. The mutation status of genes of variable region of heavy chains of immunoglobulins (IgVH) is the most reliable prognostic factor forecasting time until beginning of treatment in case of b-cell chronic lymphatic leukemia. However its detection nowadays is inaccessible for routine diagnostics. Among surrogate markers of mutation status the indicator of expression of ZAP-70 by tumor cells estimated using flow cytofluorometry. However, in publications there are different guidelines concerning the technique of mentioned marker. To establish the optimal approach to evaluation of expression of ZAP-70 the peripheral blood samples of 5I patients with b-cell chronic lymphatic leukemia and 10 healthy persons were analyzed. The comparison with the results of detection of mutation status of IgVH-genes revealed the advantage of applying the technique of calculation of MFI ratio during interpretation of data of expression of ZAP-70 obtained with flow cytofluorometry. In this framework, the indicator of expression of ZAP-70 can be applied in assessing the course of disease and time until the beginning of treatment of b-cell chronic lymphatic leukemia.

The new effective protocols of treatment of chronic B-cell lymphatic leukemia, including purine analogs and monoclonal antibodies, provide robust remissions under this disease. Accordingly, the requirements to remission quality assessment are changed too. In particular the assessment of minimal residual disease is obligatory. To assess minimal residual disease in terms of quantity in case of chronic B-cell lymphatic leukemia the technique of polymerase chain reaction was applied in real time with patient-specific primers from the area of V-D-J combinations of genes of heavy chain of immunoglobulin. The study included samples from 60 patients suffering of chronic B-cell lymphatic leukemia. In 15 of them (25%), it was impossible to apply neither the sequence analysis of genes of heavy chain of immunoglobulin nor the fitting of patient-specific primer. The results of quantitative determination of minimal residual disease were obtained in 45 patients (55 tests). The minimal residual disease was detected in 30 of 55 samples (54.5%) and was not detected in 25 of 55 samples (45.5%). At the same time, the quantitative determination of minimal residual disease was implemented in regard to the initial level of neoplastic cells. The method sensitivity qualified by serial dilutions, consisted 10(-5) or 1 neoplastic cell to 100 000 normal cells. The comparative analysis was applied to the results of determination of minimal residual disease using two methods -polymerase chain reaction in real time using patient-specified primers and four-color flow cytofluometry. The determination of minimal residual disease with both methods was implemented in 37 patients (45 tests). The results of both methods matched in 93.3% (42 tests out of 45) with maximal disparity of one degree. Then Spearman factor consisted 0.87 (p < 0.0001). In 3 out of 45 tests (6.7%) neoplastic cells were detected with only one method. In the first case, it was the method of four-color flow cytofluometry and in other two cases it was polymerase chain reaction in real time. Therefore, the detection of minimal residual disease under chronic B-cell lymphatic leukemia using the method of polymerase chain reaction in real time is rather sensitive and specific and correlates with the results received with the method of four-color flow cytofluometry. The results are the same in the case of using anti-CD20 monoclonal antibodies under treatment.

Aim: To study prognostic factors in previously untreated patients receiving FC regimen (fludarabine plus cyclophosphamide)., Material and Methods: We conducted a retrospective analysis of B-CLL patients observed in Hematology Research Center of Russia (Moscow) and Faculty Therapy Clinic of St. Petersburg State Medical University (St. Petersburg). All patients received FC regimen as a first line treatment (fludarabine 50 mg plus cyclophosphamide 250 mg/m2 for 3 days intravenously, repeated every 28 days)., Results: 54 patients were included into the study. The median age was 57.5 yrs (range 40-78 yrs). There were 38 males and 16 females. Before the treatment 22% patients had Binet stage A, 41%--stage B and 37%--stage C. 62% patients had unmutated subtype of B-CLL and 38% mutated subtype. 12 patients (22%) received less than 4 cycles of chemotherapy. In 8 patients (15%) there were significant delays between cycles (more than 2 months). In the whole cohort the median overall survival calculated from the time of treatment initiation was 57.4 months, the median progression free survival--24 months, and the median relapse free survival--27 moths. Mutational status of immunoglobulin variable region genes significantly influenced survival. In patients with unmutated subtype the median progression free survival was 23.6 months, while in patients with mutated subset it was not reached: 75% survival at 22.7 months (p = 0.027). Difference in progression free survival by stages (A versus B+C, A+B versus C) was not significant., Conclusion: Our data show that mutational status of immunoglobulin variable region genes remains a significant prognostic factor in patients receiving combined therapy with cyclophosphamide and fludarabine.

Aim: To test diagnostic efficacy of T-cell clonicity determination by a gamma-chain of T-cell receptor (TCR)., Material and Methods: The examination covered 426 patients (458 tests). T-cell tumors were detected in 132 patients. The samples from 294 patients in whom T-cell tumors were not found were referred to the laboratory for a differential diagnosis. Clonicity was determined by gamma-chain of TCR in the test for conformation polymorphism of one-chain DNA fragments. All the tests were made in one laboratory., Results: Sensitivity of the method, found by analysis of different delusions of the cell line Jurkat in selected polyclonal CD3+ cells is 10%. The results were of 3 kinds: clonal, doubtful and polyclonal. In patients free of T-cell tumors there were 15 (5%), 34 (11%) and 258 (84%) false positive, doubtful and true negative results. False positive results were most frequent in an acute phase of infectious mononucleosis--in 8 (33%) of 24 patients. 127 (84%) true positive, 5 (3%) doubtful and 19 (13% 0 false negative results were documented in patients with T-cell lymphoma. The occurrence of false negative results was the highest in anaplastic CD30+ T-cell lymphomas--in 6 (46%) of 13 cases., Conclusion: Diagnostic efficacy of the method is 92%, but in 10% the result is doubtful. Main reason of false negative results is a small number of tumor cells in tissue samples. The main reason of false positive results is prevalence of one or some T-cell clones in the presence of immune response caused by viruses, autoimmune diseases and, possibly, depletion of bone marrow in aplastic syndromes.