[Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots].

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.

[Expression of early hematopoietic markers in cord blood and mobilized blood].

G-CSF mobilized peripheral blood and cord blood are major sources of hematopoietic progenitor cells. These cells are characterized by the expression of "early" antigens. We have evaluated the coexpression of hematopoietic cell markers CD34, CD133, CD90, CDCP1, CD117 and activation antigen CD38 using multicolor flow cytometry. We show that (1) cells being positive for every single antigen form a separate population. (2) Percentage of cells expressing each "early" antigen are twice more in the cord blood than in the mobilized blood. The content of cells with complex progenitor phenotype (CD34+/CD38-/CD117, CD133+/CD34+/CD38-, CDCP1+/CD34+/CD38- etc.) is equal in mobilized and cord blood. (3) There are strong positive correlations between the expression of CD34, CD133, CD117 and CDCP1 in both groups. Positive correlation exists for CD90 with CD34, CD133, CDCP1 and CD117 only in cord blood and is not significant in mobilized blood. The analyses of early antigens coexpression with activation marker CD38 revealed that hypothesis on sequential activation and loss of expression of the aforementioned antigens is not confirmed. We assume that there is global regulation of the expression of CD34, CD133, CDCP1 and CD117. Yet expression of CD38 could be reversibly abolished during maturation of the hemapoetic cells and CD117 could be expressed not only on myeloid cells.

[Nine-year experience in the treatment of patients with diffuse large B-cell lymphosarcoma].

AIM: To ascertain indications to standard (CHOP-21/R-CHOP-21) and intensive (mNHL-BFM-90) treatment in patients with diffuse large B-cell lymphosarcoma (DLBCL) with involvement of lymphoid organs. MATERIAL AND METHODS: The trial, performed from January 2002 to December 2010, enrolled 139 DLBCL patients with affected lymph nodes (LN), tonsils, spleen, bone marrow (BM). The diagnosis was made according to WHO criteria. The patients were examined according to the protocol of lymphoproliferative diseases. Biopsy material from all 139 patients was studied immunohistochemically on paraffin blocks (LN, tonsils, spleen, BM) using a wide panel of antibodies. The same examinations of BM were made in all 18 cases of BM involvement. Cytogenetic examination was performed in 106 patients: 48 standard cytogenetic tests, 139 - FISH for t (14;18) as well as rearrangement of locus 3q27. Patients with a poor prognosis (n = 86, 61.8%) received intensive therapy according to mNHL-BFM-90 program. The signs of a poor prognosis were the following: massive tumor (tumor size more than 7.5 cm), invasion into the adjacent organs or tissues, stage III-IV disease by Enn-Erbor, high concentration of LDG. Patients without a poor prognosis (n = 53, 38.2%) received standard treatment CHOP-21 (n = 28) or R-CHOP-21 (n = 25). RESULTS: A complete remission without recurrences was achieved in all 53 patients without signs of unfavourable prognosis (100%). Overall 5-year survival was 96%, 2 patients died in remission of other causes. Of 86 patients with a poor prognosis a complete remission was achieved in 64 (74.4%) patients. Overall and recurrence-free 5-year survival was 65 and 86%, respectively. CONCLUSION: Standard treatment provided long-term complete remission in all the patients without poor prognosis. Intensive (mNHL-BFM-90) treatment produced the best results in generalized lesion without BM involvement. Overall 5-year survival was 84% in these patients and 12% in patients with BM involvement.

[Diffuse large B-cell lymphoma with primary involvement of mediastinal lymph nodes: diagnosis and treatment].

AIM: To diagnose diffuse large B-cell lymphosarcoma (DLBCLS) with primary involvement of the mediastinal lymph nodes (LN) and to evaluate the efficiency of aggressive polychemotherapy (PCT). SUBJECTS AND METHODS: The study included 15 patients (6 men and 9 women aged 18 to 70 years; median 38 years) followed up at the Hematology Research Center, Russian Academy of Medical Sciences, in 2004 to 2009. Three and 12 patients had Stages II and IE DLBCLS, respectively. B symptoms were found in 14 (93.4%) patients. Increased lactate dehydrogenase (LDH) concentrations were detectable in 14 (93.4%) patients; tumors of 10 cm or more (bulky disease) were seen in 11 (73.3%). Enlarged cervical, supraclavicular, and axillary lymph nodes were found in 9 (60%) patients; lung involvement via extension in 9 (60%), and invasion into the pericardium in 5 (33.3%) and soft tissues of the anterior thoracic wall in (13.3%). There were no signs of involvement of extranodal organs at the moment of diagnosis. All the 15 patients received PCT according to the modified NHL-BFM-90 program: 4 to 6 courses depending on the response to the therapy; 10 (66.6%) and 5 (33.3%) patients had 4 and 6 courses, respectively; for consolidating purpose, 11 (78.5%) patients were prescribed radiotherapy applied to the mediastinum in a cumulative dose of 36 Gy due to the fact that they had a residual mass. RESULTS: Thirteen (86.6%) patients achieved a complete remission (CR). Primary PCT resistance was confirmed in one case. Another patient was stated to have near-complete remission. No recurrences were notified during the follow-up. The mean CR duration was 24.5 (range 2-49) months. CONCLUSION: DLBCLS with primary LN involvement is an individual nosological entity to be differentiated from primary mediastinal large B-cell lymphosarcoma. In most cases, DLBCLS shows signs of a poor prognosis, which makes it necessary to perform aggressive PCT.

[A morphometric analysis of the nuclei and nucleoli in tumor cells in lymphogranulomatosis, diffuse large B-cell lymphoma and anaplastic large cell lymphoma].

AIM: To make a comparative morphometric analysis of the nuclei and nucleoli of tumor cells in lymphogranulomatosis (LGM), diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL) for differential diagnosis of these lymphomas. MATERIAL AND METHODS: Biopsy material (lymph node biopsies) was frozen in hexane, fixed and stained, then microscopic pictures were made. RESULTS: Mean area of tumor cell nuclei in LGM was 97.25 +/- 68.77 mcm2, in DLBCL and ALCL--55.89 +/- 20.13 mcm2 and 70.31 +/- 34.64 mcm2, respectively. The area differences were significant (p < 0.001). Hodgkin's and Berezovsky-Rid-Sternberg cell bucleoli area was the largest (11.44 +/- 7.83 mcm2). The nucleoli of the former are larger than those of the latter. Mean area of the nucleoli in DLBCL was 3.05 +/- 1.58, in ALCL--5.53 +/- 4.94 mcm2. The differences are significant (p < 0.001). CONCLUSION: Nucleoli in Hodgkin 's cells are significantly larger than those in the tumor cells in ALCL and DLBCL and the nucleoli with the area more than 12 mcm2 can be used in differential diagnosis between LGM and DLBCL but not between LGM and ALCL.

[Treatment of life-threatening airway obstruction in patients with malignant lymphomas].

Case histories of patients with hemoblastosis admitted to a hospital for acute respiratory failure due to tumor-induced obstruction were retrospectively analyzed. The causes of obstruction, antitumor therapy, methods for provision of airway patency, and major critical syndromes were analyzed. Ten patients with life-threatening tumor-induced airway obstruction were hospitalized from 1995 to 2007. The patients suffered from malignant lymphomas in all cases. The causes of impaired airway patency were the compression of the trachea and large bronchi with a tumor, lymph nodes, affected thyroid, as well as the superior vena cava syndrome, and tumor-induced lesion of soft tissues of the neck and chest. Airway patency was effected with tracheal intubation in 9 cases, it was maintained by noninvasive mask ventilation in 1 patient. Translaryngeal tracheal intubation and tracheostomy were used for artificial ventilation (AV) in 6 and 3 patients, respectively. Multidrug therapy (MDT) was performed in all the patients. Airway patency restored in 9 patients after MDT initiation. The duration of AV was 5.8 +/- l.7 days. The length of stay in an intensive care unit was 90%; intrahospital survival was 70%; 28-day and 3-year survivals were 90 and 24%, respectively. Two of the 3 patients who had undergone were observed to have serious complications (cicatrical stenosis, tracheoesophageal fistula). Hemoblastosis patients with tumor-induced obstruction need for provision of airway patency and for immediate initiation of MDT and/or radiotherapy. Orothracheal or nasotracheal intubation of the trachea is the methods of choice in providing airway patency. Tracheostomy is not indicated in most cases. Short-term prognosis is good in this cohort of patients. Long-term prognosis is determined by a lot of factors of which tumor sensitivity to cytostatics and radiation is most important.

[High-dose therapy of Burkitt's lymphoma in patients over 40 years of age].

AIM: To analyse efficacy and tolerance of high-dose polychemotherapy (PCT) of Berkitt's lymphoma (BL) in patients aged over 40 years. MATERIAL AND METHODS: High-dose PCT was given to 6 BL patients aged 41-56 years (median 48.1 years). RESULTS: Complete clinicohematological remissions were achieved in 4 patients. In two of them the treatment was discontinued after three blocks of PCT because of severe infectious complications. According to 4-12 month follow-up, remission continues. Remission was not achieved in two patients: one patient had primary resistance, the other died of sepsis after the second PCT course before remission. The time to remission did not correlate with age. Duration of myelotoxic agranulocytosis varied from 2 to 24 days. Duration of agranulocytosis did not correlate with age. Infections complicated 19 of 20 PCT blocks. Severity of complications caused withdrawal of three patients. CONCLUSION: BL is biologically heterogenous as it demonstrates different responses to BL-M-04 program. Causes of slow regression of tumor mass in some patients need further investigations. In spite of a great number of infectious complications high-dose therapy has no alternative.

[Efficacy of therapy of different variants of anaplastic large T-cell lymphomas].

AIM: To compare efficacy of NHL-BFM-90 and CHOP-like courses in the treatment of anaplastic large cell lymphoma (ALCL). MATERIAL AND METHODS: Twenty-two patients with ALCL participated in the study. The diagnosis was made basing on the findings of clinical, device, morphological, immunohistochemical and molecular-genetic examinations with application of a panel of monoclonal antibodies to CD30, ALK, CD3, CD4, CDS, CD7, CD34, CD15, CD68, CD20, CD45RO, CD45RA, Ki-67. 14 cases of 22 were negative by kinase of anaplastic lymphocytes (ALK-) and 8 were positive (ALK+). Mean age of ALK-ALCL patients was 39.6 +/- 4.1 years, of ALK+ALCL patients - 23.4 +/- 2.6 years. 14 patients were treated by the protocol NHL-BFM-90, 8 were initially treated with other schemes (CHOP, MACOP-B, BEACOPP and others). All 14 patients treated according to NHL-BFM-90 had ALCL stages III-IV with B-symptoms. 12 patients who completed treatment by the above protocol achieved complete remission after the forth course, 2 patients failed the treatment. Of 8 ALCL patients treated initially according to other schemes, a complete remission was achieved in 4 patients (2 had stage II). One of 4 patients with remission had recurrence. Four patients who had failed to achieve complete remission died of the disease progression. CONCLUSION: ALCL occurs more frequently in young and middle-aged patients. The disease has an aggressive course with rapid generalization. For such processes it is more preferable to use a modified protocol NHL-BFM-90.

[Primary diffuse large B-cell lymphosarcoma of the spleen].

AIM: To investigate characteristics of the course and efficacy of treatment of diffuse large B-cell lymphosarcoma (DLBL) with primary lesion of the spleen. MATERIAL AND METHODS: From 1998 to 2006, primary splenic lesion was registered in 15 of 120 patients with DLBL and affected lymph nodes (LN), spleen and Waldeyer's ring. The diagnosis was made according to WHO criteria. Of them 14 patients had splenectomy as the first stage of therapy. The operation was followed with 6 to 8 courses of CHOP-21 (8 patients), 4 courses of R-CHOP-21 and radiotherapy (one patient). One patient received 7 courses of CHOP-21 followed by splenectomy. Because of the presence of several signs of unfavourable prognosis 5 patients under 60 years were given intensive therapy: 4-6 courses of the modified program NHL-BFM-90, 2 of 5 patients received radiotherapy. RESULTS: All the patients with primary DLBL of the spleen had two and more signs of unfavourable prognosis: elevated concentration of serum LDG, size of the tumor more than 10 cm, high proliferative activity of tumor cells, B-symptoms, severe condition. Seven patients had centroblastic, 8--anaplastic variants of DLBL. Tumor cells in primary DLBL of the spleen had no specific immunophenotype. Complete remission of the disease was achieved in 9 (90%) of 10 patients treated on programs CHOP-21, R-CHOP-21, in 4 of 4 patients on the modified program NHL-BFM-90. Mean follow-up was 39.3 months (from 7 to 103 months). CONCLUSION: For primary DLBL of the spleen characteristic are long-term remissions on first line therapy according to CHOP-21 program irrespective of morphology and immunophenotype.

Expression level of lipoprotein lipase and dystrophin genes predict survival in B-cell chronic lymphocytic leukemia.

Mutational status of immunoglobulin variable region genes (VH-genes) is known as the strongest predictor of long term prognosis in B-CLL. However, applications in the routine clinical practice are time consuming, and therefore some other predictions are required. In this study, we have compared prognostic values of real time PCR quantification of the expression levels of four genes previously shown to be differentially expressed in V(H)-unmutated and mutated B-CLL subtypes: ZAP-70, ZBTB20, DMD and LPL. The study included 134 B-CLL patients. Expression levels of LPL and DMD genes were significantly correlated to mutational status, while expression levels of of ZAP-70 gene correlated only in CD19+ selected cases (N = 40). No correlation was observed for ZBTB20 gene. Expression levels of LPL and DMD predicted overall survival in the entire cohort of patients. Prognostic values of LPL gene expression levels were significant even for CLL patients with stage A. Quantitative RT-PCR assays for measuring LPL gene expression are robust enough to be introduced into routine clinical practice.

[The modified program NHL-BFM-90 in the treatment of patients with diffuse large B-cell lymphosarcoma].

AIM: To investigate efficacy of the modified protocol NHL-BFM-90 in patients with diffuse large B-cell lymphosarcoma (DLBCLS). MATERIAL AND METHODS: A total of 13 DLBCLS patients with stage II-IV of the disease with affection of lymph nodes at the disease onset (nodal lesion) and stage II with tumor size more than 10 cm (bulky disease) received first-line treatment according to the modified program NHL-BFM-90 from 2002 to 2005. The diagnosis was made by WHO criteria. RESULTS: A complete remission was achieved in 76.9% patients. Resistance to therapy was observed in the patients with bone marrow affection. The 2.5-year overall survival was 74%, 2-year event-free survival was 75% (the events were recurrence and resistance). Follow-up continued from 5 to 47 months. CONCLUSION: The efficacy of the modified protocol NHL-BFM-90 in DLBCLS patients with stage III-IV of the "nodal" disease and stage II of the "bulky" disease was high.

[Characteristics of T-cell immunity in patients with acute leukemia].

AIM: To study a cytokine profile of cytotoxic T-cells and T-helpers at diagnosis of acute myeloid and lymphoblastic leukemia (AML and ALL) and at different stages of its treatment. MATERIAL AND METHODS: T-cell population of peripheral blood lymphocytes was studied in 19 AML patients at diagnosis, 13 AML patients in remission, 5 AML patients in developing recurrence and 5 ALL patients at diagnosis. The control group consisted of 10 healthy donors. A mononuclear fraction of peripheral blood cells was stained with monoclonal antibodies. Surface expression of CD3, CD4, CD8 antigens was estimated with the use of flow cytofluorimeter FACS Calibur. Part of the cells were cultured for production of activated lymphocytes. The cytokine profile of T-cells was investigated according to the modified method of Rostaing et al. in the population of CD3+CD8+ and CD3+CD8- T-cells using the flow cytofluorimeter FACS Calibur. The results were processed with the programs CellQuest and WinMDI, Statistica Module Switcher. RESULTS: The percentage of CD3+CD8+ cells producing interferon-gamma (IF-g) was the same in AML patients and donors. The percentage of CD3+CD8- cells (T-helpers) producing IF-g was similar in the patients at diagnosis and in complete remission being higher than in healthy donors. The number of CD3+CD8- cells producing IF-g was reduced in recurrence while the number of CD3+CD8+ and CD3+CD8- cells producing interleukin-4 (IL-4) increased. In the onset of the disease, ALL patients had significantly increased percentage of CD3+CD8- IL-4 cells. CONCLUSION: The results obtained evidence for different polarization of T-cell immunity in AML and ALL patients at the time of diagnosis, remission and recurrence: in the onset of AML there was an increase in the production of Th1--proinflammatory cytokines, in ALL--in percent of Th-2 cytokines. In AML recurrence the balance of cytokines shifted in the direction of Th-2 response.

[Flow fluorimetry in differential diagnosis of diffuse large B-cell lymphoma].

AIM: To analyse immunophenotype of diffuse large B-cell lymphoma (DLBCL) with flow cytometry. MATERIAL AND METHODS: Combinations of antibodies against the following antigens were used: CD3/ CD19/CD45, CD5/CD19/CD38, CD19/CD10/CD23, CD4/CD8/CD3, kappa/lambda/CD19, CD25/CD20/FMC7; CD43/CD22/CD20; CD79a/Ki-67/CD3; cytoplasmic kappa/lambda. The analysis was made on flow fluorimeter FacsCalibur using computer program CellQuest (Beckton Dickenson, USA). RESULTS: Specific coexpression of markers is not detectable in DLBCL, in the greatest degree the phenotype corresponds to lymphoma from the cells of the marginal zone. The study of cells with maximal direct light diffusion provides more precise assessment of clonality and proliferative potential of tumor cells than the analysis of the whole lymphocytic polygon. The proliferative index in 33 cases of DLBCL varied in the range 10-60%. CONCLUSION: Flow cytometry in most DLBCL cases allows identification of B-cell clonality, more precise assessment of a proliferative potential of the tumor.

[Acute lymphoblastic leukemias with aberrations of BCR-ABL genes].

AIM: To develop an original therapeutic strategy in Ph-positive acute lymphoblastic leukemia (ALL). MATERIAL AND METHODS: In November 2001 Hematological Research Center (HRC) initiated the study of chimeric BCR-ABL gene. During the first stage of the study (November 2001-July 2004), 18 primary ALL patients were recruited in HRC, from July 2004 to January 2005--16 patients in HRC, N.N. Burdenko Central Military Hospital, regional Samara hospital. The diagnosis of Ph-positive ALL was established in detection of translocation t(9;22) by standard cytogenetic test or fluorescent hibridization in situ with double signal (D-FISH), or by polymerase chain reaction with reverse transcription (RT-PCR). In detection of aberration of BCR-ABL gene the patients received stem hemopoietic cells, from June 2004 imatinib was added to chemotherapy in the period of induction and consolidation. RESULTS: Incidence rate of BCR-ABL-positive ALL by standard cytogenetic test and D-FISH makes up 20%, by RT-PCR--25%. Differences in chimeric transcripts detectability by different methods may be explained by different sensitivity of the methods. Complete hematological remissions were achieved in the majority of the patients (6 of 8) irrespective of imatinib administration. Achievement of molecular remission in BCR-ABL-positive ALL occurs also in standard chemotherapy but molecular remissions begin 2-4 months later than clinicohematological ones. CONCLUSION: In using imatinib combination with chemotherapy, molecular remission can be achieved simultaneously with hematological one. Long-term results will be analysed later.