[Molecular diagnosis angioimmunoblastic T-cell lymphoma].

AIM: to determine molecular diagnostics routine for different tissue samples in angioimmunoblastic T-cell lymphoma. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Subcutaneous panniculitis-like T-cell lymphoma: The authors' experience of diagnosis and treatment]. / Podkozhnaya pannikulitopodobnaya T-kletochnaya limfoma. Sobstvennyi opyt v diagnostike i lechenii.

The paper gives the data of clinical, histological, immunohistochemical, and molecular studies and the results of positron emission tomography in 3 cases of subcutaneous panniculitis-like T-cell lymphoma (SPTCL). It shows the high efficiency of a GEM-P regimen in the treatment of patients with SPTCL.

Evolution of Tumor Clones in Adult Acute Lymphoblastic Leukemia.

Clonal instability of a tumor cell population in acute lymphoblastic leukemia (ALL) may complicate the monitoring of a minimal residual disease (MRD) by means of patient-specific targets identified at the disease onset. Most of the data concerning the possible instability of rearranged clonal TCR and IG genes during disease recurrence were obtained for ALL in children. The appropriate features of adult ALL, which are known to differ from those of childhood ALL in certain biological characteristics and prognosis, remain insufficiently studied. The aim of this study was to assess the stability of IG and TCR gene rearrangements in adult ALL. Rearrangements were identified according to the BIOMED-2 protocol (PCR followed by fragment analysis). Mismatch in clonal rearrangements at onset and relapse was identified in 83% of patients, indicating clonal instability during treatment. Clonal evolution and diversity of IG and TCR gene rearrangements may be one of the tumor progression mechanisms. New rearrangements may emerge due to residual VDJ-recombinase activity in tumor cells. Also, many clonal IG and TCR gene rearrangements may be present at different levels at a diagnosis, but less abundant clones may be "invisible" due to limited detection sensitivity. Later, major clones may disappear in the course of chemotherapy, while others may proliferate. Investigation of clonal evolution and heterogeneity in ALL and their impact on the treatment efficacy will contribute to the identification of new prognostic factors and the development of therapeutic approaches.

[Hepatosplenic T-cell lymphoma: The problems of diagnosis and treatment]. / Gepatolienal'naia T-kletochnaia limfoma: problemy diagnostiki i lecheniia.

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 αß 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).

Clonal rearrangements and Malignant Clones in Peripheral T-cell Lymphoma.

AIM: To assess the feasibility and informative value of T-cell clonality testing in peripheral T-cell lymphoma (PTCL). PATIENTS AND METHODS: Biopsies of involved sites, blood, and bone marrow samples from 30 PTCL patients are included in the study. Rearranged TCRG and TCRB gene fragments were PCR-amplified according to the BIOMED-2 protocol and analyzed by capillary electrophoresis on ABI PRISM 3130 (Applied Biosystems). RESULTS: TCRG and TCRB gene clonality assay was valuable in confirming diagnosis in 97% of PTCL patients. T-cell clonality assay performed on blood or bone marrow samples reaffirmed lymphoma in 93% of cases, whereas morphological methods were informative in 73% of cases only. We observed multiple TCRG and TCRB gene rearrangements, loss of certain clones in the course of the disease, as well as acquisition of new clones in 63% of PTCL cases, which can be attributed to the genetic instability of the tumor. CONCLUSION: TCRG and TCRB gene clonality assay is beneficial for the diagnosis of PTCL. However, the presence of multiple clonal rearrangements should be considered. Clonal evolution in PTCL, particularly acquisition of new clones, should not be treated as a second tumor. Multiple TCRG and TCRB gene rearrangements may interfere with minimal residual disease monitoring in PTCL.

[PCR definition of B-cell clonality: which electrophoretic method is better].

AIM: To compare diagnostic efficacy of B-cell clonality determination in application of different electrophoretic methods. MATERIAL AND METHODS: B-cell clonality was determined with different techniques in 89 patients having B-cell lymphoma (n = 48), B-cell lymphoma in remission (n = 11), reactive processes (n = 24), lymphogranulomatosis and T-cell lymphoma (n = 6). Healthy donors served control. Clonality was defined by rearrangements of Ig heavy chain genes with usage of primers to FR2 region of Ig gene. Electrophoretic methods were the following: agarose electrophoresis and SSCP. RESULTS: Clonality was detected in the presence of more than 5.9% clonal cells from total count of mononuclear cells or more than 20% of clonal cells from total number of B-lymphocytes in the sample. We achieved detectability of B-clonality in B-cell tumors 87.5% (42 of 48 cases). False negative tests were also investigated with kits of primers to FR1 and FR3 regions, but clonality was not determined. Among reactive processes, non-B-cell tumors and healthy donors clonality was found in 1 of 41 cases. This patient had acute respiratory viral infection. Agarose electrophoresis and SSCP test results coincided. CONCLUSION: Determination of B-cell clonality with agarose electrophoresis proved to be a simple, reliable, cost-effective and reproducible method of differential diagnosis of tumor and reactive lymphoproliferation. This method is not suitable for verification of tumor remission or assessment of minimal residual disease because of its low sensitivity.

[Determination of T-cell clonicity by a T-cell receptor gamma-chain: final data].

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.