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Aim: To study efficacy of rituximab in patients with resistant B-cell lymphoma on high-dose chemotherapy., Material and Methods: From September 2000 to April 2002 we studied efficacy and tolerance of rituximab at different stages of high-dose chemotherapy. The treatment was given to 10 patients with histologically verified CD20+ non-Hodgkin's lymphoma: diffuse large-cell (n = 4), Berkitt's (n = 2), follicular (n = 3), mantle-cell (n = 1). Five patients with diffuse large-cell lymphoma and Berkitt's lymphoma had a primary resistant course of the disease, one patient with diffuse large-cell lymphoma had a refractory recurrence. Follicular and mantle-cell lymphomas were characterized by a resistant course and large tumor masses. The patients received 1-2 courses of induction chemotherapy with dexa-BEAM with collection of peripheral stem cells followed by high-dose chemotherapy (BEAM-9, CBV + mitoxantron-1) with transplantation of autologous stem blood cells. Rituximab infusion (375 mg/m2) was conducted before the collection of the stem cells, prior to high-dose chemotherapy and in posttransplantation period after recovery of hemopoiesis., Results: 4 patients achieved complete remission, 3-partial remission, 2 had progression and 1-stabilization. In mean follow-up 11 (2-20) months 7 of 10 patients were alive, overall survival being 15 +/- 2.4 months (95% confidence interval 10-19.7), median was not reached. 5 patients are in complete remission: 2 of them without further treatment, 3-after progression and repeat therapy including rituximab and interferon-alpha or rotuximab and CHOP chemotherapy., Conclusion: The addition of rituximab can improve the results of high-dose chemotherapy of patients with non-Hodgkin's lymphoma resistant to standard doses of cytostatics. Repeat use of this drug can be effective in some patients with progression after high-dose chemotherapy with rituximab.

The study of different epithelial antigen expression has been performed in 183 breast cancers. In 73 patients regional lymph nodes were studied as well. Panepithelial antigen Egp-34 (Mab HEA-125) was expressed in 100% of primary tumors and metastases. Antigen MUC-1 (Mab ICO-25) was identified in 93% of breast cancers. Monomorphic type of expression in tumor cells was typical for Egp-34, MUC-1 being in certain cases expressed as a proportion of cells. Additional immunohistochemical study of regional lymph nodes with Mab to Egp34 and MUC-1 provides a 10% increase in the rate of breast cancer metastases detection compared to histological examination alone. The carcinoembryonic antigen (CEA) was useful in identification of metastases only in CEA-positive breast cancers.

We have studied the reactivity patterns of a previously described pan-macrophage monoclonal antibody (MAb) D11 in 324 cases of acute leukemia and malignant lymphoma (ML). Reaction of D11 in tissue sections was restricted to histiocytes and macrophages. In non-Hodgkin's ML, D11 helped to confirm or to establish the histiocytic nature in 8 of 96 cases, i.e., in 4 of 6 histiocytic MLs; 2 of 13 anaplastic large-cell lymphomas; 1 of 4 large-cell immunoblastic clear-cell MLs; and 1 of 2 histiocytosis X cases. Positive reaction of D11 in acute lymphoblastic leukaemia (ALL) was found in 9 of 86 cases (all belonging to early B-lineage leukemia), of which 4 were CD34-positive and 5 co-expressed 1 or more myeloid/monocytic antigens. MAb D11 did not react in 42 cases of acute-myeloblastic-leukemia (AML) FAB variants M0-M5, except 1 acute mixed-lineage leukemia M1/pre-pre-B. Comparative study of the MAb D11 and a standard CD68 MAb KP- 1 showed that the antigens belong to different epitopes of different molecules.

Expression of new Mab D11 on blood and bone marrow cells was investigated in 85 hemoblastosis patients. In normals, antigen D11 is expressed on some monocytes and all tissue macrophages. D11 was noted on lymphoblasts of 5 out of 10 cases with B-cell ALL and of 1 case in B-lymphoid blast crisis of chronic myeloid leukemia. In T-ALL, ANLL, non-Hodgkin's lymphomas in leukemization stage, hairy cell leukemia and chronic lymphoid leukemia the cells were nonresponsive to Mab D11. Unlike D11 which have round nuclei, lymphoblasts D11+ have folded nuclei and more pronounced cytoplasmic basophilia. There were both B and myeloid antigens on D11+ blasts. ALL D11+ patients had extramedullary foci, more suppressed granulocytic and thrombocytic components of hemopoiesis, shorter remissions than those with ALL D11-.

The specificity of monoclonal antibodies (MoAbs) ICO in normal T and B cell differentiation has been described. The use of standard MoAbs CD19, CD5, CD7 was taken into account in modification of immunologic classification of lymphoblastic tumors. Analysis in 336 patients with ALL has been done to compare the use of E-rosettes, xenogeneic antisera and MoAbs in T-cell leukemia and lymphoma diagnosis. There are evident differences in the frequencies of some immunological subvariants of lymphoblastic leukemia as compared to lymphoma.

The antigen detectable with monoclonal antibodies ICO-11 was studied in cells of 33 patients with acute lymphoblastic leukemia (ALL) and 47 patients with acute non-lymphoblastic leukemia (ANLL). The incidence rate of antigen-positive cases in ALL comprised 39.4%, in ANLL 35.4%. The presence of antigen in blasts in ALL did not influence the disease prognosis. The presence of ICO-11+ blasts in ANLL was characteristic of the group of patients with more favourable prognosis: a higher survival rate, due to a higher frequency of remissions, a longer life-time in the absence of remission. Basing on the clinico-hematological data ICO-11+ group could not be identified as more favourable prognostically.

ICO - 10 (Thy-1 antigen) and K 20 (gp 120/200) monoclonal antibodies proved suitable for immunophenotyping solid tumors. The following typical antigen combinations were identified with regard to reaction with those antibodies: ICO - 10+ K 20- (neuroblastoma, malignant schwannoma, neurosarcoma and soft tissue sarcomas); K 20+ ICO - 10- (cancer of the tongue and esophagus, adenoma of the adrenal cortex, adenocarcinoma of the stomach, hepatoblastoma and nephroblastoma); ICO - 10+ K 20+ (immature teratoma and cervical and esophageal leiomyoma) and ICO - 10+ K 20- (malignant fibrous histiocytoma).

Fitness of immunofluorescence and enzyme immunoassay for tests with centrifuged acetone-fixed cell preparations was assessed. All the 11 monoclonal antibodies (MCA) used reacted with cellular antigens in centrifuged cell preparations. HEA-125 and IKO-25 MCA proved to be the most informative; they permitted detection of individual tumor cells in serous exudation and specify the cytologic diagnosis.

Monoclonal antibodies (Mab) ICO-13 of IgM isotype reacted in indirect surface immunofluorescence test with 88.5 +/- 3.4% of thymocytes and 7.1 +/- 2.4% of T cells, but failed to react with other peripheral blood cells from healthy donors. The antigen disappeared in cells stored at 4 degrees C overnight or at -196 degrees C in liquid nitrogen. The antigen detected by Mab ICO-13 was expressed on blast cells of acute lymphoblast leukemias and lymphomas. The antigen was absent in chronic lymphoblast leukemia and blast crisis of chronic myeloid leukemia.

The panel of monoclonal antibodies (MCA) ICO-16, ICO-17, ICO-18, ICO-19, ICO-20, ICO-27, ICO-28 IgG2a isotypes to CD38 antigen was obtained. MCA discovered the antigen with 45 kD molecular mass, expressed on the surface of 100% thymocytes, 43-53% lymphocytes, 32-46% monocytes. All obtained MCA blocked the binding each other with thymocyte of man. MCA reacts in complement-dependent cytotoxic test. The antigen CD38 is expressed on blast cells of patients with T-cells subset of ALL.

The goal of the investigation was to further characterize ICO-II monoclonal antibodies. ICO-II have been shown to block NK activity of mononuclear cells from the blood of healthy donors against K-562 and Molt-4 target cells, cytotoxic T lymphocytes induced in a 7-day mixed lymphocyte culture, the reaction of lymphocyte blast cell transformation to phytohemagglutinin and the formation of En-rosettes. The molecular weight of the antigen detected by ICO-II is 180 KD. ICO-II are shown to detect alpha-subunit of human lymphocyte function-associated antigen-I (LFA-I).

ICO-63, new monoclonal antibodies (MCAB), used for immunodiagnosis, were produced by the standard hybridoma technique ICO-63 MCAB reacted with 50% granulocytes, 20% thrombocytes and endothelial cells, whereas they did not react with peripheral blood mononuclear cells, thymocytes and erythrocytes. The molecular weight of ICO-63 MCAB-identified antigen was about 100 kD. ICO-63 MCAB reacted with 12 out of 15 neuroblastomas, with some solid tissue sarcomas and melanomas, but did not react with tumours of the epithelial origin.

The hybridoma clone IKO-GM-1 was obtained as a result of fusion of P3X63Ag8.653 cells with splenocytes of BALB/c mice with the aid of 50% polyethyleneglycol. Antigen demonstrated by antibodies IKO-GM-1 expressed on 100% polymorphonuclear neutrophils, 54.4 +/- 3.1% monocytes, 25.9 +/- 2.4% mononuclears of healthy subjects' blood, on 11.1 +/- 1.0% T lymphocytes, on 14.6 +/- 1.6% T lymphocytes bearing Fc-receptor for IgG, on 49.3 +/- 8.2% enriched population of B lymphocytes and O cells. The treatment of healthy donors' mononuclears with antibodies IKO-GM-1 and complement blocked EK cellular activity against Molt-2 cells but not against K-562 cells. Antigen demonstrated by MAT IKO-GM-1 did not express on the colony-forming granulocyte or macrophagal cells. Antigen expressed on blast cells of patients with AMonoL, on those in part of patients with AML and AMML, on leukocytes of patients with chronic ML, on monocytes of a patient with chronic MonoL. Antigen was absent from blast cells of patients with ALL, LSA, on lymphocytes of patients with ChLL.

Expression of erythroid antigens identifying with the use of monoclonal antibodies (mab) HAE3 and HAE9 was studied in 333 patients with different types of hemoblastoses. Frequency of erythroid variants based on the reaction with mab was 5.7%. In 2.4% of cases erythroid markers were the only indication on the nature of leukemia. In 2.7% of cases the types of leukemia were accounted for as "mixed" ones, i.e. erythrolymphoid and erythromyeloid variants. Only in 0.6% of cases erythroid variant was detected by the morphological criteria.

Comprehensive analysis of skin infiltrate cells, carried out in malignant lymphomas of the skin, has included examinations with the use of monoclonal antibodies (MCA). Basing on the clinical, histologic, and immunologic findings, mycosis fungoides has been diagnosed in 9 patients and T-cellular lymphoblastic lymphosarcoma consisting of T-suppressors in 1. The findings evidence that (1) mycosis fungoides is a tumor consisting of OKT4 cells in the majority of cases; (2) mature T-cell markers are sometimes lost in the course of mycosis fungoides, this being parallelled by the emergence of antigens characteristic of earlier differentiation stages, e. g. corticothymocytic ones; (3) immunologic heterogeneity is a characteristic feature of malignant lymphomas of the skin; this may be explained by both: the tumor progress of the clone and the therapeutic pathomorphosis; (4) use of MCA to a wide spectrum of leukocytic differentiation antigens permits an accurate estimation of the nature of the tumor cells and of the cellular relationships; this helps understand the pathogenesis of malignant lymphomas of the skin and develop new treatment modalities.

To date, only anti-glycophorin-A monoclonal antibodies (MAbs) have been widely used as anti-erythroid probes in the diagnosis of leukemias. We have examined blood, bone-marrow and lymph-node samples from 474 patients, adults and children, with different hemopoietic malignancies, using a panel of MAbs including 2 anti-erythroid MAbs directed to glycophorin-A and an antigen of erythroblasts, Ag-Eb. MAb HAE9 directed against a human epitope of Ag-Eb has earlier been shown to be highly specific for immature erythroid cells. Of all the patients, 2.7% demonstrated glycophorin-A expression on blast cells, while anti-Ag-Eb MAb HAE9 reacted positively with cells from 6.0% of patients. Samples from 31 of 474 (6.5%) patients expressed one or both erythroid markers. Our results indicate that MAb HAE9 may be useful, in combination with anti-glycophorin-A MAbs, as an anti-erythroid probe for immunophenotyping human leukemias.

The expression of antigens on granulocyte-macrophagal colony-forming cells of patients with nonhematological diseases was studied. Treatment of bone marrow cells with murine monoclonal antibodies ICO-1 and ICO-11 led to statistically significant inhibition of the number of growing colonies. Monoclonal antibodies ICO-02, ICO-10, ICO-GM-1 and ICO-G-2 had no such effect.

The possibility to reveal the following types of neuroblastoma is described: with c-ALL-antigen; with Thy-1-antigen (ICO-10); with granulocytic antigen (ICO-G2); with B-cell antigen (IPO-10). Immunocompetent cells in the tumour may be studied with the use of monoclonal antibodies ICO-1, ICO-11, ICO-GM-1, OKT3, IPO-3.

Monoclonal antibodies (Mab) Ig G2a isotypes reacting in indirect immunofluorescence assay with 68.7 +/- 4.1% of thymocytes, 7% of T-cells and not determining the antigen on other blood cells were obtained. Mab ICO-20 reacted in complement-dependent cytotoxic test. The antigen was expressed on colony-forming cells of granulocyte-macrophage row. Mab ICO-20 reaction with 100% of thymocytes was defined by flow cytometry. Antigen molecular mass is 45000 Dalton. The antigen was expressed on blast cells of patients with ALL and AML. Mab ICO-20 reaction was more more often with T-cell ALL.

Mouse monoclonal antibodies (MAB) ICO-10 to Thy-1 antigen were obtained. MAB ICO-10 reacted in indirect immunofluorescence test with 5.7 +/- 0.8% human thymocytes. Antibodies did not react with granulocytes, monocytes, T- and non-T cells from peripheral blood, and with marrow cells of healthy donors. MAB ICO-10 reacted with blast cells from 25 of 53 patients with T-cell acute lymphoblastic leukemia (ALL), from 2 of 5 patients with B-cell ALL. This antigen was absent on blood and marrow cells from some patients with ALL, 80 patients with chronic lymphoid leukemia, 54 patients with chronic granulocytic leukemia at the stage of blastic crisis, 128 patients with acute nonlymphoblastic leukemia. Antibodies are specifically bound to thymocytes and spleen cells of Thy 1.1 and Thy 1.2 mice. MAB ICO-10 detect Thy-1 antigen expressed on human hematopoietic cells. MAB ICO-10 may be applied for human leukemia and lymphoma immune diagnosis.

Antigen expression determined by ICO-11 monoclonal antibodies was studied on leukemia cells from bone marrow, blood cells from patients with lymphoproliferative diseases and natural killer (NK) cells responsible for natural resistance. ICO-11 monoclonal antibodies were shown to recognize the antigen expressed on NK-cells, predecessors of T-cells (thymocytes), myelomonocytes (myeloblasts, monoblasts) and to block NK-cell activity.

ICO-1 Mab were obtained following BALB/c mouse immunization with 24-week human fetal thymocytes. Cloning for two times by the method of limited dilutions led to a hybridoma with a stable production of G3 isotype Mab. ICO-1 Mab have immunoprecipitated an antigen consisting of two polypeptide chains with molecular weight 29 and 34 kDal. The antigen expression was enhanced following PHA or alloantigen activation of blood mononuclear cells. ICO-1 Mab inhibited the alloantigenic response of blood mononuclear cells. Mab detected 29% of antigen-positive cells in the peripheral blood of healthy adults. When the reaction of ICO-1 Mab was compared with that of Mab against monomorphic Ia-like antigens OKLa, anti-Ia-BRL, BMA-021 and HLA-Dr on blood cells from healthy donors and patients with leukemia proved to be identical.

ICO-35 monoclonal antibodies (Mabs) were produced following BALB/c mouse immunization with peripheral blood cells from a patient with lymphoid type of chronic myeloid leukemia in blast crisis (SML BC) ICO-35. Mabs detect antigen on CD10-positive cells from patients with acute lymphoblastic leukemia, CML BC, CD10-positive cells of Reh line and are not bound to other cells. Comparative studies of reactivity of ICO-35 and K 503 Mabs to CD10 antigen revealed their similarity. However, in contrast to K503 ICO-35 Mabs do not react with granulocytes.