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to determine molecular diagnostics routine for different tissue samples in angioimmunoblastic T-cell lymphoma.Molecular studies were performed for 84 primary AITL patients. The median age was 61 year (29-81); the male to female ratio was 48/36. T-cell and B-cell clonality was assessed by GeneScan analysis of rearranged T-cell receptor (TCRG, TCRB) and immunoglobulin heavy chain genes. For the quantitative determination of cells with RHOA G17V mutation real - time polymerase chain reaction (PCR) with allele - specific LNA modified primers was used.In lymph nodes rearrangements of T-cell receptor genes were determined in 76 (90.5%) of 84 patients and were absent in 8 (9.5%) cases. Identification of the same clonal products of the TCRG and TCRB genes in the lymph node and in peripheral blood and/or bone marrow indicated the prevalence of the tumor process and was observed in 64.7% of patients. Clonal products in peripheral blood and/or bone marrow different from those in the lymph node indicated reactive cytotoxic lymphocyte population and were noted in 58.8% of AITL cases. Simultaneous detection of T- and B-cell clonality in the lymph node was observed in 20 (24.7%) of 81 patients. Cells with RHOA G17V mutation were detected in lymph node in 45 (54.9%) of 82 patients. The use of allele - specific PCR with LNA modified primers revealed presence of the tumor cells in peripheral blood in 100% and in bone marrow in 93.9% of patients with G17V RHOA mutation in the lymph nodes.The validity of different molecular assays performed on certain tissue samples for the diagnosis of angioimmunoblastic T-cell lymphoma has been evaluated. Quantitative allele - specific PCR assay for RHOA G17V mutation based on LNA modified primers possesses sufficient sensitivity for tumor process prevalence evaluation and minimal residual disease monitoring.Цель: определить показания и последовательность проведения молекулярных исследований при ангиоиммунобластной Т-клеточной лимфоме (АИТЛ) в различном биологическом материале. Материалы и методы. Молекулярные исследования проведены у 84 первичных больных АИТЛ. Медиана возраста составила 61 (29-81) год, соотношение мужчины/женщины - 48/36. Оценку T-клеточной и В-клеточной клональности проводили по реаранжировкам генов цепей T-клеточного рецептора и тяжелых цепей иммуноглобулинов. Для количественного определения клеток с мутацией G17V гена RHOA применяли полимеразную цепную реакцию (ПЦР) в реальном времени с аллель - специфичными LNA модифицированными праймерами. Результаты. В биоптатах лимфатических узлов реаранжировки генов цепей Т-клеточных рецепторов определены у 76 (90,5%) из 84 больных и отсутствовали в 8 (9,5%) случаях. Выявление одинаковых клональных продуктов амплификации генов TCRG и TCRB в биоптате лимфатического узла и в образцах периферической крови и/или костного мозга свидетельствовало о лейкемизации опухолевого процесса и наблюдалось у 64,7% больных. Определение в образцах периферической крови и/или костного мозга клональных продуктов, отсутствующих в лимфатическом узле, обусловлено реактивной цитотоксической популяцией лимфоцитов и наблюдалось в 58,8% случаев АИТЛ. Одновременное выявление Т- и В-клеточной клональности в биоптате лимфатического узла отмечалось у 20 (24,7%) из 81 больного. Клетки с мутацией G17V гена RHOA выявлены в биоптатах лимфоузлов у 45 (54,9%) из 82 пациентов. Применение метода аллель - специфичной ПЦР с LNA модифицированными праймерами позволило выявить лейкемизацию опухолевого процесса у 100% и поражение костного мозга - у 93,9% пациентов с выявленной мутацией в лимфоузлах. Заключение. В результате выполненной работы определены показания и последовательность проведения молекулярных исследований при АИТЛ в различном биологическом материале. Количественная аллель - специфичная ПЦР с LNA модифицированными праймерами для определения мутации G17V гена RHOA обладает высокой чувствительностью, позволяет верифицировать лейкемизацию опухолевого процесса и проводить мониторинг минимальной резидуальной болезни во время терапии.

Background. Angioimmunoblastic T-cell lymphoma (AITL) is the rare lymphoproliferative disorder traditionally thought to be associated with EBV. This original study gives data about serological and molecular markers of herpes viruses in primary AITL patients. Materials and methods. The review includes analysis of clinical and laboratory features of 40 primary AITL patients. Peripheral blood, lymph node, bone marrow and bronco-alveolar aspirate samples were tested by ELISA, PCR and in situ hybridization. Results. Laboratory markers of the acute herpetic infection were detected in 29 (72.0%) out of 40 patients. Primary infection was detected in 8 cases: 6 - primary EBV, 1 - HCMV and 1-EBV and HCMV simultaneously. Anti-HSV 1,2 IgM were observed in 15 (37.5%) patients. EBV small non-coding RNAs (EBER) was positive in 27 (71.1%) out of 38 cases. The comparison of the detection of EBER and markers of acute EBV infection showed good correlation (p

Nodal anaplastic ALK-negative large cell lymphoma (nALCL, ALK-) is a Т-cell lymphoma that is characterized by aggressive clinical course and low sensitivity to СНОР (cyclophosphamide, doxorubicin, vincristine, prednisolone) and other chemotherapy regimen. In the article we present a literature review and describe our clinical case of nALCL, ALK-. For the first time a combination of Brentuximab vedotin with modified program NHL-BFM-90 was used as a first-line therapy. As a result of immunochemotherapy a complete antineoplastic effect was obtained. For consolidation of this effect high-dose chemotherapy with following autologous blood stem cell transplantation was performed. The chosen treatment tactics allowed to achieve a complete remission in a medium risk group patient.

Contex. Angioimmunoblastic T-cell lymphoma (AITL) is a rare form of non-Hodgkins lymphoma, characterized by generalized lymphadenopathy, hepatosplenomegaly and dysproteinemia. Hypergammaglobulinaemia is revealed in 50-83% pts with AITL. However, the characteristics of immunoglobulinopathies observed in AITL are scarce. Objective: The aim of the study was to characterize quantitative and qualitative immunoglobulinopathies in patients with AITL at the onset of the disease. Patients and methods. 55 patients with newly diagnosed AITL were enrolled in the study, the male/female ratio was 30/25; median age was 61 (29-81) years. Diagnosis was based on standard WHO criteria. Immunochemical studies of blood serum included serum protein electrophoresis/immunofixation, nephelometric quantification of total immunoglobulins, serum free light chain assay. Results. Quantitative and qualitative immunoglobulinopathies were determined in 49 (89,1%) of 55 pts. Quantitative immunoglobulinopathies were revealed in 47 (85.5%) of 55 cases, qualitative - in 14 (25,5%). Combination quantitative and qualitative immunoglobulinopathies was observed in 12 (21,8%) of 55 pts. The detected immunoglobulinopathies were divided into 4 groups: polyclonal hypergammaglobulinaemia, hypogammaglobulinaemia, oligoclonal gammapathy, and monoclonal gammapathy. Polyclonal hypergammaglobulinaemia was marked in 41 (74.5%) of 55 pts, elevated level of IgG was determined in 27 (49,15%) of 55 cases, IgM - in 18 (32,7%) and IgA - in 21 (38.2%). Interestingly, polyclonal IgE hypergammaglobulinaemia was detected in 12 (48,0%) of 25 cases of performed studies. Hypogammaglobulinaemia was detected in 8 (14,5%) of 55 cases. Oligoclonal gammapathy was determined in 4 (7.3%) of 55 pts. Monoclonal gammapathy was revealed in 11 (20,0%) of 55 cases. The amount of monoclonal immunoglobulin varied from 2.6 to 14.1 g/l. Monoclonal immunoglobulin Gk was detected in 5 of 11 pts, Gλ - in 2, Mλ - in 2, Mk - in 2. Monoclonal gammapathy was accompanied by polyclonal hypergammaglobulinaemia in 9 of 11 cases, hypogammaglobulinaemia - in 2. Conclusions. Quantitative and qualitative immunoglobulinopathies are observed in most patients at the onset of AITL. Quantitative abnormalities were determined more often than qualitative. Monoclonal gammapathy can be a manifestation of lymphoproliferation and other concomitant disorders. The prognostic value of immunochemical parameters is still unclear and requires dynamic observation and study.

Background: Angioimmunoblast T-cell lymphoma (AITL) is a rare T-cell lymphoproliferative disease that is accompanied by generalized lymphadenopathy, hepatosplenomegaly, intoxication symptoms and extranodal lesions. The extranodal manifestations of the disease frequently involve various skin changes. One of the first such manifestations is maculopapular rashes observed in about half of AITL patients and usually preceding the appearance of lymphadenopathy. Other forms of skin lesions accompany the disease considerably less frequently.Aim: To characterize the range of skin changes in patients suffering from AITL, to establish a correspondence between the nature of skin changes and their histological picture.Materials and methods: 54 AITL patients were being treated at the National Research Centre for Hematology from 2000 to 2017, with the male/female ratio being 30/24. The median age was 61 (29–81) years.Results: Changes in the skin were observed in 24 (44.4 %) of 54 AITL patients, out of whom 18 (75 %) and 6 (25 %) were male and female patients, respectively. Maculopapular rash was observed in 22 (91.7 %) out of 24 patients. The morphological and molecular investigations of skin biopsy specimens exhibiting maculopapular rash demonstrated nonspecific reactive changes. Patients with maculopapular rash demonstrated an increase in the level of total (polyclonal) IgE. Specific skin lesions detected in 8 (14.8 %) cases were represented by a ‘livedo reticularis’, focal skin hyperpigmentation, erythroderma, left eyelid tumour and tumour in 3, 2, 1, 1 and 1 cases, respectively.Conclusion: Maculopapular rash frequently observed in AITL patients is a reactive process not associated with a specific skin lesion. Specific skin lesions in AITL are much less common and can be represented by various forms. In some AITL cases, skin changes of the reactive and tumour nature can be simultaneously observed.

Angioimmunoblastic T-cell lymphoma is a rare T-cell lymphoproliferative disease with generalized lymphadenopathy, hepatosplenomegaly, intoxication and polyclonal hypergammaglobulinemia. The persistence of plasma cells in peripheral blood can be a manifestation of both tumor and reactive processes. In our article, we described the case of angioimmunoblastic T-cell lymphoma with an increase in the number of peripheral blood plasma cells to 28 %, and in bone marrow to 9 %. The complex diagnostics, including plasma cells immunophenotyping, morphology of the lymph node biopsy and bone marrow samples, made it possible to verify the diagnosis of angioimmunoblastic T-cell lymphoma with polyclonal plasmacytosis.

Introduction. Аngioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma, characterized by abundant polymorphocellular infiltrate of lymph nodes with the small number of tumor CD4 + T-cells. AITL could often be misdiagnosed as reactive processes and other lymphomas, including Hodgkin’s lymphoma. Molecular methods of determining clonality are used to confirm the diagnosis. TCR gene rearrangements detected in the lymph nodes may not coincide with those detected in the bone marrow (BM), blood or skin, that makes their interpretation difficult. Recently discovered point somatic RHOA (Ras homolog gene family, member A) Gly17Val mutation is present in 53–71 % of angioimmunoblastic T-cell lymphomas. The aim of the study was to evaluate the number of RHOA Gly17Val mutated cells in different tissues of AITL patients and to correlate it with corresponding T-cell clonality results. Materials and methods . TCRG and TCRB gene rearrangements were PCR-amplified according to BIOMED-2 standardized protocol and analyzed by capillary electrophoresis on ABI Prism 3130. Gly17Val mutation was analyzed by quantitative allele-specific (qAS) TaqMan Real-Time PCR assay. Lymph nodes (LN) and skin biopsies, blood and BM samples were studied for 40 patients with AITL. Results. The clonal TCR gene rearrangements were found in 37 (92 %) of 40 patients in LN and in 26 (96 %) of 28 patients in BM, but they were not matched in length in LN and BM in 12 (46 %) of 26 patients. RHOA (Gly17Val) mutation in LN was revealed in 60 % (24 of 40) patients. Number of cells with RHOA mutation was highest in the LN (in average 26.7 % of the total cells), while in the BM RHOA mutation were undetectable (in 7 patients), or detected in 10 patients in a low quantity (in average 2 % of the total cells). Skin, blood and BM samples with the T-cell clonality peaks that differ from those found in the LN were also negative for the presence of cells having RHOA Gly17Val mutation. Also, in four patients we showed that clonal products in BM and blood that do not coincide with LN refer to CD8+ lymphocytes. Conclusions. The results of the study demonstrate that the quantitative determination of cells with the mutation of RHOA Gly17Val must be taken into account in staging the disease and interpreting the results of T-cell clonality assay. Clonal cells with rearrangements not matching those identified for the LN should be considered reactive.

Extranodal NK/T-cell lymphoma – a rare lymphoproliferative disease characterized predominantly by extranodal localization, aggressive course and low efficiency of conventional chemotherapy. Clinical presentation is diverse and depends on tumor lesion localization. In this article, a literature review and case report of patient with generalized extranodal NK/T-cell lymphoma with bone marrow involvement, unusual intracranial tumor localization, tumor leukemization and central nervous system-leukemia were presented. Adequate treatment strategy made it possible to achieve long-term complete remission in a patient with poor prognosis.

Objective: to estimate the rate, predictive and prognostic value of VHL gene alterations in the population of patients with sporadic metastatic renal cell carcinoma (mRCC).Subjects and methods. Paraffin embedded tumor tissue blocks were available from 88 patients with mRCC who had undergone antitumor therapy in 1994- 2010. Of them, 53 patients received only immunotherapy regimens with interferon (IFN)-α and 35 patients had targeted therapy with VEGFR inhibitors. VHL mutations were detected by polymerase chain reaction (PCR) for exons of 1-3, single-strand conformation polymorphism analysis of PCR products, and further sequencing. VHL gene methylation was determined by methyl-sensitive PCR.Results. Somatic mutations and/or promoter hypermethylation of the VHL gene were found in 23 (26%) patients; Of them, VHL gene mutations and promoter hypermethylation were found in 15 patients and 7 patients respectively. Mutation and promoter methylation VHL were simultaneously observed in one case. VHL gene mutations were detected only in patients with clear cell RCC while aberrant promoter methylation was seen in both clear cell and papillary RCC. With a median follow-up of 34 months (range, 2-127 months), the median time to progression (TTP) and median overall survival (OS) for the entire group of patients were 5.8 and 26.7 months, respectively. In patients with and without VHL gene alterations, the median TTP was 5.5 and 6.9 months, respectively (p = 0.15) and the median overall survival time was 22.0 and 34.5 months, respectively (p = 0.98). Moreover, the subgroup analysis revealed that VHL gene inactivation events had no impact on the objective response rate (ORR), TTP and OS in the subgroup of patients who received immunotherapy (n = 53) or antiangiogenic targeted therapy (n = 35) (p > 0.05).Conclusion. VHL gene mutations and/or promotor hypermethylation observed in 26% of patients with mRCC. These VHL gene alterations were neither prognostic nor predictive factors in mRCC patients during immunotherapy with IFN or antiangiogenic therapy with VEGFR inhibitors.

An immunofluorescence assay that enables the quantitative detection of the level and intensity of the expression of multidrug resistance marker Pgp in human solid tumors using flow cytometry and monoclonal antibody (clone 17F9, BD Pharmingen) was developed and adapted for clinical assessment. The analysis includes the following relevant task-specific stages. Single-cell suspension can be prepared from both surgical biopsy native tumor tissues and tumor specimens fixed in 4% formaldehyde. The assay allows one to verify results, even after the tumor material has been stored for 2 years. The cost of analysis can be decreased by increasing the period of cell incubation with antibodies and reducing the antibody concentration. The possibility of analyzing minimal tumor samples, e.g., endoscopic biopsy specimens, by decreasing the concentration of cells incubated with antibodies to 100000 cells/mL and the number of cells used in the flow cytometer to 1000 has also been shown. Despite its continuance, the developed assay does not require any changes in the investigator’s work schedule.

Molecular mechanisms underlying the formation of B-cell lymphomas in connection with processes associated with the maturation of B-lymphocytes are reviewed. The currently used diagnostic methods do not always distinguish lymphomas from reactive changes of the lymphoid tissue. The principle of the molecular genetic method of clonality detection in lymphocyte populations, technical problems, and the strategy of its application in clinical diagnostics of lymphomas are described in detail.

In the past decade, a notable advance has been made in the understanding of the pathogenesis of NK/T-cell lymphomas; however, their diagnosis remains difficult because of their rarity and clinical and morphological variabilities. The paper generalizes the ten-year experience of the Hematology Research Center, Ministry of Health of Russia, in diagnosing and treating hepatosplenic T-cell lymphoma (HSTL), considers the problems of differential diagnosis with other hematological diseases occurring with similar clinical and laboratory symptoms, and lays down current approaches to the diagnosis and treatment of this condition. A clinician's view of the problem of diagnosis and treatment of this disease is given. HSTL is shown to be a heterogeneous group of diseases differing in a T-cell receptor chain gene rearrangement, the clinical course of the disease, and overall survival (OS). According to our data, 3-year OS was 12%; the median survival was 26 months. Two-year OS for γδ and αβ HSTL was equal to 25 and 70%, respectively. The difference in OS for the variants of HSTL failed to reach statistical significance (because the sample might be insufficient).За последнее десятилетие достигнуты значительные успехи в понимании патогенеза NK/Т-клеточных лимфом, однако их диагностика остается затруднительной вследствие их редкости и клинико-морфологического разнообразия. В статье обобщен десятилетний опыт диагностики и лечения гепатолиенальной Т-клеточной лимфомы (ГЛТЛ) в ФГБУ 'Гематологический научный центр' Минздрава России, рассмотрены вопросы дифференциальной диагностики с другими гематологическими заболеваниями, протекающими со сходными клинико-лабораторными симптомами, сформулированы современные подходы к диагностике и лечению. Представлен взгляд клинициста на проблему диагностики и лечения данного заболевания. Показано, что ГЛТЛ - гетерогенная группа заболеваний, различающихся по реаранжировкам генов цепей Т-клеточного рецептора, клиническому течению заболевания, общей выживаемости (ОВ). По нашим данным, 3-летняя ОВ составила 12%, медиана продолжительности жизни - 26 мес. Для вариантов γδ и αβ ГЛТЛ 2-летняя ОВ равна 25 и 70% соответственно. Различие ОВ для вариантов ГЛТЛ не достигало статистической значимости (возможно, в силу недостаточного объема выборки).