[Apoptosis and neutrophils on the regulation of proliferation and differentiation ex vifo of myeloid cells with Ph-chromosome].

Human myeloid cells with Ph chromosome (Ph+ cells) from chronic myeloid leukemia (CML) in the course of proliferation and differentiation ex vivo are regulated under alternation of cell proliferation and neutrophil maturation stages by consecutive blocking and inducing apoptosis with of neutrophils participation as well bcr/abl, bax and bcl2 genes expression. Apoptosis regulation of three main Ph+ cells types from CML patients depends on alternation sequences of proliferation (1) and maturation (2) cell stages and realized by two ways. The first one is performed by consecutive blocking and inducing apoptosis under 2/1/2 stage alternation. The way is not described early. Neutrophils accumulation correlates with apoprosis blocking. Apoptosis level enhances under neutrophils exhausted. Apoptosis blockage allows cells to proliferate and, thus, to form new portion of neutrophils with consecutive regular their death as well a consequent alternation of apoptosis blocking and inducing. This way regulates proliferation efficiency indexes P/D that reflect Ph+ cells proliferating potential and performs cycle completion for proliferation and differentiation. The second way of apoptosis regulation starts from proliferation stage and performs for 1/2/1 alternations under diminished content of neutrophils and a little increase under next maturation. It leads to resistant depressed apoptosis levels that, at maximal points, are 3-8 times lower than those under alternation 2/1/2. Resistant apoptosis blocking is observed in the Ph+ cells with prolong proliferation or maturation stages, when blasts and myelosytes are accumulated under enhanced bcr/abl and bcl2 > box gene expression and remain under next maturation. Stable apoptosis blocking is accompanied by increasing amounts of blasts and myelocytes and enhancing bcr/abl and bcl 2 > bax expression. This is observed under CML progression. Ph+ cells cultivation may be useful for more distinct diagnostics of CML phases of individual CML patients and optimization of the treatment.

[Clinical statistics and effectiveness of different treatments of idiopathic thrombocytopenic purpura].

AIM: To ascertain efficacy of modern treatment methods in idiopathic thrombocytopenic purpura (ITP), to characterize basic indications for administration of these methods; perspective trends in ITP therapy. MATERIAL AND METHODS: The article presents 20-year experience in observation and treatment of 1000 ITP patients gained from 1988 by the department of standardization of blood disease treatment methods on the basis of a consultative and outpatient department of Hematological Research Center. The results were assessed by conventional ITP treatment lines. RESULTS: First-line therapy--glucocorticoids--provided remission in 70% patients, long-term (60 months and longer) in 14%, prednisolone resistance was in 19% patients.Intravenous immunoglobulin provided a rapid hemostatic effect (in 1-2 days) in all the patients. A positive response to treatment was seen in 86% patients but it was short-term (for a year and longer in 27%, resistance to the drug--14%). Second line treatment--splenectomy--is the most effective treatment: 80% remission, 32% patients had remission for longer than 60 months. Resistance occurred in 6% patients. Ineffectiveness of treatment in 20% patients stimulated the search for new pathogenetic treatment among synthetic analogues of thrombopoietin. Clinical trials proved high efficacy of two of them--eltrombopag and romiplostim (86-87% response), possible maintenance of remission in long-term interrupted administration of low doses. CONCLUSION: Modern ITP treatment allows a complete management of hemorrhagic syndrome and deep thrombocytopeny in 80% patients with provision of good quality of life and ability to work. Introduction into clinical practice of thrombopoietin analogues improves treatment results including in 20% patients resistant to all treatments in the absence of marked side effects even in long-term 3-year administration of such medication.

[The detection of minimal residual disease in patients with chronic B-cell lymphatic leukemia using patient-specified polymerase chain reaction].

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.

[Hereditary spherocytic hemolytic anemia in an adult with the formation of ectopic foci of extramedullary hemopoiesis in the chest].

The paper describes a rare case of formation of paravertebral extramedullary hemopoietic foci in microspherocytic anemia or Minkovsky-Shoffar disease in an adult. Therapeutic splenectomy has led to regression of extramedullary hemopoietic foci, which supports that there is a direct relationship of the above formations to the specific features of the etiology and pathogenesis of microspherocytic anemia.

[Trisomy of chromosome 8 in Ph-negative cells of the bone marrow in patients with chronic myeloid leukemia treated with inhibitors of BCR-ABL tyrosine kinases].

AIM: To analyse clinical implications of chromosome 8 trisomy in Ph-negative cells of the bone marrow in patients with chronic myeloid leukemia (CML) treated with inhibitors of tyrosinkinases (ITK). MATERIAL AND METHODS: A total of 386 patients with CML (chronic phase--288, acceleration phase--77) received imatinib (400-800 mg/day). Because of resistance and/or intolerance some patients were switched to ITK II (nilotinib, dasatinib, bozutinib). This study included 8 CML patients (7 in a chronic phase, 1 in acceleration phase) treated with BCR-ABL ITK inhibitors of the first (imatinib) and the second line (ITK-II). The standard cytogenetic examination, on demand--investigation of the interphase nuclei with FISH, in some cases morphological, cytochemical and histological examinations of the bone marrow were made. RESULTS: The existence of a Ph-negative clone with trisomy of chromosome 8 had no negative effect on the course of the disease. The patients showed a stable hematological and cytogenetic response and no need in changing treatment policy. In long-term follow-up Ph-negative clone with trisomy of the chromosome 8 persisted without a clear trend to rise in most patients. CONCLUSION: Detection of a Ph-negative clone with chromosome 8 trisomy at early stages suggests parallel existence of Ph-positive and Ph-negative clones. None of the patients had myelodisplasia.

[Prognostic significance of immunoglobulin variable region gene mutations in B-CLL patients treated with combination therapy fludarabine plus cyclophosphamide].

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.

[Prognosis factors in imatinib mesilate therapy in patients with a chronic phase of Ph-positive chronic myeloid leukemia: data from a multicenter non-randomized trial in Russia].

AIM: To reveal prognostically significant factors affecting efficacy of glivek therapy in untreated (duration of the disease < or = 6 months) and pretreated (duration of the disease > 6 months) patients with chronic myeloid leukemia (CML) in a chronic phase. MATERIAL AND METHODS: A total of 338 patients (64 untreated and 274 pretreated) with a chronic-phase CML on glivek therapy entered the trial. RESULTS: Five-year survival on glivek was high (89, 98 and 88% in untreated and pretreated patients, respectively). Incidence of transformation in the acceleration phase and blast crisis was low both in untreated and pretreated patients (1.6 and 11%, respectively) and correlated with the rate of a complete cytogenetic response (CCR). Untreated patients had no factors affecting treatment efficacy negatively, CCR probability was 96%. Blastemia, thrombocytosis and splenomegaly reduced CCR probability significantly in pretreated patients. Slow reduction of the tumor mass, late achievement of a complete hematological response and a cytogenetic response decreased probability of CCR. CONCLUSION: Glivek is a drug of choice for patients with chronic-phase CML. High probability of CCR both in untreated and pretreated patients lowers the risk of the disease transformation into the phase of acceleration/blast crisis and raises overall survival in both groups.

[The role of morphological characteristics of lymph nodes in differential diagnosis of reactive lymphadenopathies].

AIM: To analyse causes of reactive lymphadenitis regarding histological changes in the lymph node, of diagnostic errors; to determine frequency of establishment of nosological diagnosis. MATERIAL AND METHODS: A retrospective analysis of diagnostic examination of 375 patients with reactive lymphadenitis observed from 1992 to 2000 was made. RESULTS: For differential diagnosis 6 groups of reactive lymphadenopathies with different histological picture were identified: follicular hyperplasia, paracortical hyperplasia, granulomatous lymphadenitis, granulomatous pyonecrotic lymphadenitis, sinus histiocytosis as a leading sign and node necrosis as a leading sign. The analysis of making nosological diagnosis for each group and of diagnostic errors was conducted. CONCLUSION: Characteristics of lymph node changes in reactive lymphadenitis is of great importance. Better cooperation between histologists and clinicians must improve efficacy of reactive lymphadenitis diagnosis.

[Cytogenetic response as a marker of efficacy of chronic myeloid leukemia therapy with a BCR-ABL thyrosine kinase inhibitor glivek].

AIM: Clinical practice with the drug glivek (imatinibe mesilate, ST1571) blocking activity of oncoprotein p210 shows that a cytogenetic response can be reached in 50-60% of patients with chronic myeloid leukemia (CML), in a late chronic phase (CP) in resistance to or intolerance of interferon alpha (IF-alpha) and in 24-43% of patients in the acceleration phase (AP). This study aimed at assessment of the rate and stability of a cytogenetic response (CR) and long-term results of survival in CML patients on glivek. MATERIAL AND METHODS: Glivek was given to 195 CML patients (median of the treatment duration was 42 months, 1-156 months, of the patients' age--46 years). 79 patients were in CP, 116--in AP. The doses were 400 mg/day and 116 mg/day, respectively. Karyotype was studied before the treatment and later after each 6 months. RESULTS: A considerable CR was achieved in 57% patients in CP and 44%--in AP. Of them complete CR was obtained in 48 and 35%, respectively. Marked CR is a favourable prognostic factor. Survival of patients with marked CR in CP (97% 0 and AP (89%) was significantly higher than without CR (58 and 47%, respectively, p < 0.05). Marked CR persisted in 95% cases in both phases of CML. In complete CR, a repeated study of karyotype revealed residual number of Ph+ cells both in CP and AP in 86% patients. This demonstrates necessity to take glivek continuously in achievement of a complete CR by karyotypic test. Glivek inhibits the disease progression, lowers annual lethality. 42-month (median of glivek treatment duration) overall survival reached 91 and 59% in CP and AP, respectively. CONCLUSION: CR is an integral index prognosticating CML course. Survival rose significantly in patients with marked CR both in CP and AP of CML. Marked CR is persistent in continuous glivek therapy. The rate of a CR depends much on the disease stage.

Effects of cytapheresis on cell cycle distribution of peripheral blood and bone marrow cells in some hematological malignancies.

Feulgen-DNA cytophotometry and 3H-thymidine autoradiography were used to evaluate therapeutic cytapheresis effects on cell cycle distributions of peripheral blood leukemic cells in 15 acute leukemia, 5 CML and 8 CLL patients and of bone marrow erythroblasts in 18 PV patients. Both individual cytapheresis procedures and a long-term program of cytapheresis altered cell cycle distributions and biochemistry which may be summarized as follows: An increase in percentage of cells in S-phase (acute leukemia, CML); a decrease in the differences between values of S-cell number obtained by cytophotometry and autoradiography (acute leukemia, CML, PV); a decrease in some cases in enlarged fraction of cells in G2 (blast crisis of CML, PV); an activation of G0----G1 transition and an increase in the PAS-positive lymphocyte fraction in CLL patients. An apparent increase of blasts in S-phase was found after a pheresis in acute leukemia patients who responded to subsequent chemotherapy. In "nonresponders", a pheresis had no effect on cell cycle distributions. The results of the study demonstrate that despite the wide variability of individual responses, cytapheresis activates cell proliferation and metabolic processes in patients with hematological malignancies.

[Clonogenic cultivation of bone marrow fibroblast precursors in human myeloproliferative diseases]. / Klonogennoe kul'tivirovanie predshestvennikov kostno-mozgovykh fibroblastov pri mieloproliferativnykh zabolevaniiakh cheloveka.

Techniques of clonogenic cultivation with the application of xenogenous feeder (rabbit irradiated bone marrow) were used to study a number of bone marrow colony-forming cells (CFU-F) in 70 patients. A significant increase of CFU-F is observed in chronic myelocytic leukemia and in hepatosplenomegalies of non-leukemic origin CFU-F decreases considerably in the cases of myelofibrosis. Trypsinisation of the bone marrow taken from the cases of myelofibrosis results in a sharp CFU-F increase.

[Immunodepressive effect of measles and parotitis virus in vitro]. / Immunodepressivnoe deistvie virusov kori i parotita in vitro.

Mechanisms of suppressive effect of measles and parotitis virus on lymphocyte proliferation and cytolytic activity of natural killers are studied in vitro. Both viruses exert a weak mitogenic effect on human blood lymphocytes and at the same time suppress their response to phytohemagglutinin stimulation in vitro. Suppressive effect of measles virus is more expressed than of parotitis virus. Moreover, measles virus suppresses lysis of human blood killer target cells. The suppressive effect manifests as early as in 1 h and increases after 3 h of natural killer cell contact with measles virus.

[Herpes simplex virus-associated antigen in leukemic cells]. / Antigen, assotsiirovannyi s virusom prostogo gerpesa v leikoznykh kletkakh.

Immunological methods (CFT, indirect IF) detected herpes simplex virus-associated antigen in leukocytes of 21 out of 56 leukemia patients. The antigen was more frequently found in leukocytes of patients with chronic lympho- and myeloleukemias, less frequently in acute forms of these diseases. No antigen was detectable in leukocytes of normal donors.

[The immune status of children in foci of measles infection studied by an immunoenzyme method]. / Izuchenie immunnogo statusa detei v ochagakh korevoi infektsii metodom IFA.

Children immunized with live measles vaccine in the foci of measles infection varying in intensity (1-9 cases per focus) have been subjected by two methods: the hemagglutination inhibition (HAI) test and the enzyme immunoassay (EIA). As shown in this study, in most cases (98% of all blood serum samples) the correlation between the results of the HAI test and EIA is not high (r = 0.5), which is linked with the detection of a wider spectrum of antibodies in EIA. The percentage of seronegative children detected by these two methods was practically the same (4.05 and 4.4, respectively). The analysis of the results obtained in this study indicates that EIA is a more informative and sensitive method, which confirms the effectiveness of its use for the determination of the level of collective immunity.

[Therapeutic efficacy of imatinib mesylate (glivec) in chronic phase of myeloid leukemia]. / Effektivnost' terapii imatiniba mezilatom (glivekom) v khronicheskoi faze mieloleikoza.

AIM: To evaluate efficacy and tolerance of glivek in chronic myeloid leukemia (CML) in patients who failed interferon-alpha (If-a) preparations. MATERIAL AND METHODS: 79 patients in a chronic phase of Ph + CML with hematological and cytogenetic resistance or intolerance of If-a. The response to glivec was assessed by achievement of a complete hematological remission and the cytogenetic effect (the degree of reduction of cell clone Ph+ in bone marrow). Tolerance and safety of the drug was studied by monthly standard clinicohematological tests. RESULTS: Not only a hematological remission (92.4%), but also partial (46.8%) or complete (27.8%) elimination of BCR-ABL +/- cells were achieved after 12 months of the treatment. Glivec was well tolerated. Hematological toxicity primarily as neutropenia and thrombocytopenia were observed in 54.4 and 42% patients, respectively. Neutropenia of the third degree which made impossible to continue the treatment was observed in 29.1% patients; throbocytopenia of the third degree was registered in 16.5% patients. Among most frequent non-hematological side effects there were moderate edema, nausea, leg muscle convulsions, weight gain, arthralgias, skin eruption. All the complications were transient, were managed in all cases with only a short-time discontinuation of glivec therapy. CONCLUSION: High activity of glivec at early stages of CML allows using this drug as a first-line therapy in patients with CML.

[A comparison of the forms of chronic lympholeukemia in relation to the mutational status of the genes of the immunoglobulin variable region]. / Sravnenie form khronicheskogo limfoleikoza v zavisimosti ot mutatsionnogo statusa genov variabel'nogo regiona immunoglobulinov.

AIM: To compare forms of chronic lymphoid leukemia (CLL) regarding mutational status of immunoglobulin variable genes. MATERIAL AND METHODS: We have compared clinical, prognostic and immunophenotypic data obtained on 25 cases with different mutational status of IgV genes. There were 10 patients in the mutated group (median age 49.2 years, male to female ratio = 7:3), and 15 patients in unmutated group (median age 46.5, M:F = 13:2). RESULTS: Statistically significant differences were noted in overall survival and CD38 expression. 5-year overall survival in unmutated group was 35%, in mutated group 80% (p = 0.07). In unmutated group CD38 was expressed on more than 50% of cells in 7 out of 14 patients, while in the mutated group in 0 of 8 patients (p = 0.007). We noted high frequency of VH1-69 gene usage in unmutated group (7 of 15 patients), while in mutated group it was used in only 1 case of 10. CONCLUSION: We confirm the differences between groups of CLL with different mutational status of IgV genes. Highly restricted usage of VH-genes and CD38 expression possibly suggest that unmutated group also arises from antigen driven cells.

[Lymphocytoma of the spleen--a separate nosological form requiring a specific management procedure]. / Limfotsitoma selezenki--otdel'naia nozologicheskaia forma, trebuiushchaia spetsificheskoi taktiki vedeniia.

The paper presents new findings in favor of recognition of splenic lymphocytoma (SLC). This disease was characterized by A. I. Vorob'ev and M. D. Brilliant in 1982 in terms of detailed clinicomorphological features, prognosis and optimal treatment policy. The study included 52 patients (mean age 53 years) of which 36 were females and 16 males. They were followed up for 5.7 years, on the average. SLC manifested clinically by splenomegaly with minimally enlarged lymph nodes, morphologically by nodular lymphocytic proliferates in the spleen, bone marrow and liver, diffuse or diffuse-nodular proliferation in the lymph node. Peripheral blood contained middle-size lymphoid cells with round nuclei. SLC immunophenotype exhibits moderate or marked expression of CD22 and membrane immunoglobulins, the absence of CD5, CD23 and EM receptor, combination of CR1-/ CR2+. Paraprotein secretion was recorded in 49% of cases. There were frequent autoimmune reactions, especially against erythroid cells and platelets (42%). Optimal therapeutic policy is expectation and eventual splenectomy producing a persistent clinical effect in 94% of patients. In progressive disease long-term therapy with cyclophosphamide is recommended. Thus, SLC is a mature-cell lymphatic tumor growing as a rule in the spleen. Its prognosis in valid therapy is favourable.

[A benign form of chronic lympholeukemia]. / Dobrokachestvennaia forma khronicheskogo limfoleikoza.

According to the classification of chronic lymphocytic leukemia (CLL) proposed by A. I. Vorob'ev and M. D. Brilliant in 1983, benign CLL is a distinct form of CLL which is characterized by low level of absolute lymphocytosis, absent or mild peripheral lymphadenopathy, slow progression. No specific therapy is needed. The paper presents clinical, morphological and immunological analysis of 34 cases of benign CLL (17 males and 17 females, mean age 58 years). Patients were included in the study if they had lymphocyte count less than 30,000 and no significant growth of lymphoid tissue for at least 3 years. They were followed up from 3 to 24 years (11 years, on the average). The main features of benign CLL are the following: no "B" symptoms, no essential enlargement of the lymphoid organs, a stable low level of absolute lymphocytosis, low prolymphocyte count in the blood smear (0.95% +/- 0.2), nodular or nodular-interstitial proliferation in the bone marrow. We failed to find any cases with paraprotein secretion. There was immunophenotype typical for CLL in 91% of cases (CD19+, CD20+, CD23+, CD5+, EM+, CR1-/CR2+, sIg+(-)). None was positive for CD38 activation marker. One trisomy 12 cases was detected by FISH method. 8 patients died so far, but not because of the tumor progression or transformation, median survival was 22 years.

[Medico-social value of the organization and development of ambulatory hematological services]. / Mediko-sotsia'lnoe znachenie organizatsii i razvitiia ambulatornogo zvena gematologicheskoi pomoshchi.

The authors have validated socio-medical and economic advantages of dispensary treatment of patients with blood diseases. It has been documented that most patients with acute leukemia can betreated on ambulatory basis. The requirement of physicians-hematologists for outpatient treatment of children and adults has been calculated. The methods of treatment suggested can be used in the public health practice.

[Autolymphocytotoxins in the differential immunodiagnosis of chronic lympholeukemia and malignant non-Hodgkin's lymphoma at the leukemization stage]. / Autolimfotsitotoksiny v differentsial'noi immunodiagnostike khronicheskogo limfoleikoza i zlokachestvennoi nekhodzhkinskoi limfomy v stadii leikemizatsii.

The authors have investigated the possibility of using sera with cold auto-lymphocytotoxic antibodies for the differential diagnosis of chronic lymphocytic leukemia (CLL) and malignant non-Hodgkin's disease (MNHD) at the stage of leukemization. The new rapid immunologic method was approved in the diagnosis of 40 CLL and 52 MNHD patients. The high informative value of this method has permitted the authors to recommend its use as an additional procedure for the differential diagnosis of the above lympho-proliferative diseases.