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Effective treatment and prevention of infections challenge management of patients with chronic lymphicytic leukemia (CLL). The COVID-19 pandemic resulted in the reduction of outpatient hospital visits as a part of non-pharmaceutical interventions that could affect the incidence of infectious complications. Study enrolled patients with CLL receiving ibrutinib or/and venetoclax who were observed at the Moscow City Centre of Hematology from 01 April 2017 to 31 March 2021. We found a reduction in the incidence of infectious episodes after the implementation of the lockdown in Moscow in 01 April 2020, when compared to data on the year prior to the lockdown (p < 0.0001), as well as when compared to the predictive model (p = 0.02), and based on individual infection profiles using cumulative sums (p < 0.0001). Bacterial infections had 4.44-fold decrease, bacterial in combination with undefined infections had 4.89-fold decrease, viral infections had unsignificant changes. The decrease in the number of outpatient visits coincides with the time of the lockdown could be a likely factor, explaining a decline in the incidence of infection. Patients were clustered according incidence and severity of infectious episodes for subgroup mortality assessment. No differences in overall survival due to COVID-19 were observed. Typical respiratory infections, bacterial and undefined, the transmission of which may be affected by patient-to-patient contact in the settings of out-patient health care visits were decreased, possibly due to SARS-CoV-2 restrictive measures. A positive correlation between outpatient visits and the incidence of bronchial and upper respiratory tract infection points at the role of hospital-acquired infection and attests to the necessity of reorganizing care for all patients with CLL., (© 2023 John Wiley & Sons Ltd.)

The clinical course of the new coronavirus disease 2019 (COVID-19) has shown that patients with chronic lymphocytic leukemia (CLL) are characterized by a high mortality rate, poor response to standard treatment, and low virus-specific antibody response after recovery and/or vaccination. To date, there are no data on the safety and efficacy of the combined vector vaccine Sputnik V in patients with CLL. Here, we analyzed and compared the magnitudes of the antibody and T cell responses after vaccination with the Sputnik V vaccine among healthy donors and individuals with CLL with different statuses of preexposure to coronavirus. We found that vaccination of the COVID-19-recovered individuals resulted in the boosting of pre-existing immune responses in both healthy donors and CLL patients. However, the COVID-19-naïve CLL patients demonstrated a considerably lower antibody response than the healthy donors, although they developed a robust T cell response. Regardless of the previous infection, the individuals over 70 years old demonstrated a decreased response to vaccination, as did those receiving anti-CD20 therapy. In summary, we showed that Sputnik V, like other vaccines, did not induce a robust antibody response in individuals with CLL; however, it provided for the development of a significant anti-COVID-19 T cell response.

We performed a comparative analysis of the pharmacological activity of the hybrid organotin compound bis(3,5-di-tert-butyl-4-hydroxyphenylthiolate)dimethylol (Me-3) administered in different modes to mice with transplanted melanoma B16 to identify the most effective dosage regimen. Three modes of administration were used: preventive (before transplantation of tumor cells), classical according to Z. P. Sof'ina (48 h after transplantation of tumor cells), and delayed (7 days after transplantation of tumor cells, after formation of nodules of the primary tumor node). Compound Me-3 was administered at a total dose of 375 mg/kg intraperitoneally once a day for 5 days. The classical mode of administration was identified as the most effective, which indicates the preventive antimetastatic activity of Me-3 on the model of the transplanted mouse tumor melanoma B16., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Aim: The primary objective of the interim analysis of the MULTISPECT study was to evaluate the short-term efficacy of the treatment and long-term outcomes in cohorts of primary and pretreated patients with multiple myeloma (MM) receiving treatment in actual clinical practice in various regions of the Russian Federation. Secondary objectives were a description of the main characteristics of patients; analysis of the most commonly used therapy regimens of the 1st and later lines and the sequence of their changes; evaluation of the response to therapy. Additional objectives included evaluation of the effect of the new COVID-19 coronavirus infection on the course of MM in patients., Materials and Methods: The study is an observational retrospective-prospective multicenter cohort study. For its implementation, a structured database of patients with MM was used, provided by hematologists of the centers affiliated for the study., Results: The study included 1,294 patients (cohort 1 806, cohort 2 488). In both cohorts, patients aged 6069 years were in the majority. 3 lines of therapy (L1, L2, L3) were used for cohort 1; in cohort 2, the 4th line of therapy was also used in 2 patients. The therapy regimens were analyzed for 290 (22.41%) of all patients in the study. Responses to therapy were analyzed for 214 patients of cohort 1 and 109 patients of cohort 2. Autologous and allogeneic hematopoietic stem cell transplantations were carried out for a limited proportion of patients in both cohorts. At the end of the study and upon presentation of its results, the status of patients was the following: 96% of patients in cohort 1 and 89% in cohort 2 were alive. The therapy regimens in both cohorts were characterized by variability. The most commonly used regimens in each of the lines of therapy have been identified. The most used therapy regimen in patients with MM of both cohorts was the VCD-regime. Rd-regime in cohort 1 and RD-regime in cohort 2 were the second most frequent used regimens. In patients of both cohorts, the therapy regimens including Bortezomib were most often used., Conclusion: The variety of therapy regimens used to treat MM in actual clinical practice may be due to the factors of availability of new medicines and updated recommendations for the treatment of the disease. Further, in the context of this study, a more detailed analysis of the efficacy of certain therapy regimens in the 1st and later lines on progression free survival and overall survival of MM patients should be carried out.

We describe a retrospective cohort, 156 patients with chronic lymphocytic leukemia (CLL) diagnosed with COVID-19, analyze factors associated with a severe disease course and the effects of various treatment regimens. Anti-SARS-CoV-2 IgG and IgM levels are significantly lower. Patients with CLL are more likely to have a severe course of COVID-19, with IL-6 levels acting as a consistent biomarker of disease severity. Ten patients had recurrent episodes, fatality rate of 20%. Overall survival did not differ between patients receiving ibrutinib monotherapy and anti-CD20 antibodies ± chemotherapy. It seems that the immunodeficiency inherent to CLL influences outcomes to a larger degree than does the treatment. Glucocorticoids are not associated with significant OS improvement whereas anti-cytokine compounds usage seemed to be beneficial in patients with mild pulmonary involvement. Our data attest to the necessity of reorganizing health care for patients with CLL. Early administration of effective antiviral compounds and tailored vaccination protocols are warranted.

The central element of the "metastatic organotropism" is a shift of the pro/antioxidant balance in cells and activation of oxidative stress and protective antioxidant systems. We studied the effects of bis(3,5-di-tert-butyl-4-hydroxyphenylthiolate)dimethylol (Me-3) in the maximum effective and toxic total doses on the level of markers of oxidative stress and antioxidant protection in the liver of mice with melanoma B16 before the appearance of macroscopic metastases. In 48 h after tumor inoculation, Me-3 was administered intraperitoneally once a day for 5 days in total doses of 375 and 500 mg/kg according to the classical method. Administration of the hybrid organotin compound Me-3 produced different effects on the pro/antioxidant state of the microenvironment of liver tissue as the target of melanoma B16 metastasis. The results suggest that inversion of the anti/prooxidant profile of Me-3 is determined by its dose., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Immunoglobulin heavy chain variable region (IGHV) repertoire narrowing could be an evidence for the involvement of a limited set of antigens in the development of lymphomas. For chronic lymphocytic leukemia (CLL) the existence of more than 200 subgroups of tumor IGHV antigen-binding sites, so called "stereotypical" antigen receptors (SAR) has been shown. For others lymphomas the possibility of SARs is also suggested. The aim of this study is to compare the tumor IGHVs and possible SARs in various B-cell malignancies in Russia and other countries., Materials and Methods: The study included samples of 1800 CLL patients, 52 patients with mantle cell lymphoma, 48 patients with hairy cell lymphoma and 37 patients with splenic marginal cell lymphoma. The nucleotide sequences of the IGHV genes were determined according to ERIC protocol., Results: In CLL most common IGHV genes were IGHV1-69, IGHV1-2, IGHV3-30 and IGHV4-34. The most common SARs were CLL#1, CLL#6, CLL#2, CLL#3. In MCL the most common genes were IGHV4-34, IGHV3-21, IGHV3-23. In 5 MCL patients CDR3 sequences were identified matching definitions of a stereotyped. In the half of SMZL patients was identified gene IGHV1-2. Other IGHV genes were much less common. Two pairs of SMZL patients have motives similar to each other. In HCL IGHV repertoire was the most variable, no trends for antigen receptor stereotypy were observed. It was found that SARs are highly disease-specific both at the level of nucleotide and amino acid sequences., Conclusion: Our results suggest that antigens crucial for the pathogenesis of B-cell malignancies could be disease-specific. Further studies on extended samples of non-CLL patients concerning the role of SARs in pathogenesis of these diseases may also contribute to the development of new diagnostic and prognostic markers., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chronic lymphocytic leukemia (CLL) is a malignant lymphoproliferative disease characterized by the accumulation of immature monoclonal B lymphocytes in blood cells, bone marrow, spleen and lymph nodes. This is the most common type of leukemia among the Caucasoid race. When CLL skin lesions occur in about 25% of patients, they are extremely diverse. These lesions can be divided into specific, including infiltration of the skin by leukemic cells and the skin form of Richter's syndrome, secondary skin tumors, nonspecific lesions and associated skin diseases.Leukemic infiltration of the skin in patients with leukemia is called specific skin lesions (SSL). Many authors associate the unfavorable prognosis with the transformation of CLL with specific infiltration of the skin into Richter syndrome, as well as the appearance of SSL before the diagnosis of CLL. The risk of developing various cancer pathologies in patients with CLL is three times higher than in healthy people identical in sex and age. It was found that the risk of skin cancer in these patients is eight times higher than in the healthy population. The most common secondary skin tumors in CLL are basal-cell carcinoma, squamous-cell carcinoma, melanoma, and Merkel tumor.Nonspecific skin changes are extremely diverse and occur in patients with CLL in 30-50% of cases. The most common secondary changes in the skin in CLL are those of infectious nature. There are also increased reactions to insect bites, generalized itching, exfoliative erythroderma, nodular erythema, paraneoplastic pemphigoid, bullous pemphigoid, drug eruption. Concomitant dermatoses in these patients are more severe and often torpid to the previously conducted therapy. There is no doubt that together with the clarification of the etiology and pathogenesis of CLL, particular issues related to the study of clinical and morphological changes in individual organs and systems, in particular the skin, formed at various stages of the development of this disease should be studied in detail. This can not only expand and clarify our understanding of this pathology, but also can help to clarify the essence of the disease.

Background: Therapy with irreversible Bruton's tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) is associated with bleeding., Objectives: To propose the predictive markers of such bleeding, as well as mechanisms responsible for decreased bleeding at later therapy stages., Patients/methods: We investigate platelet functional activity in 50 CLL and 16 MCL patients on ibrutinib using flow cytometry and light transmission aggregometry., Results: Prior to treatment, both patient groups had decreased platelet counts; impaired aggregation with adenosine diphosphate (ADP); and decreased binding of CD62P, PAC1, and annexin V upon stimulation. Bleeding in patients treated with ibrutinib was observed in 28 (56%) CLL patients, who had decreased aggregation with ADP and platelet count before therapy. Their platelet count on therapy did not change, platelet aggregation with ADP steadily improved, and aggregation with collagen first decreased and then increased in anticorrellation with bleeding. Bleeding in MCL was observed in 10 (62%) patients, who had decreased dense granule release before therapy. ADP and ristocetin induced platelet aggregation in ibrutinib-treated MCL patients increased on therapy, while collagen-induced aggregation evolved similarly to CLL patients., Conclusions: Our results suggest that ibrutinib-dependent bleeding in CLL patients involves three mechanisms: decreased platelet count (the most important discriminator between bleeding and non-bleeding patients), impaired platelet response to ADP caused by CLL, and inhibition by ibrutinib. Initially, ibrutinib shifts the balance to bleeding, but then it is restored because of the improved response to ADP., (© 2020 International Society on Thrombosis and Haemostasis.)

Monoclonal gammopathy of renal significance (MGRS) is a new nosology in modern nephrology and oncohematology. MGRS is defined as kidney injury due to nephrotoxic monoclonal immunoglobulin produced by the B-cell line clone which does not reach the hematological criteria for specific treatment initiation. Monoclonal proteins pathological effects on kidney parenchyma result in irreversible decline of kidney function till the end stage renal disease that in line with the position of International Consensus of hematologists and nephrologists determinates critical necessity for clone specific treatment in patients with MGRS despite the absence of hematological indications for treatment initiation. Main challenge of MGRS in Russian Federation is an inaccessibility of an in-time diagnostic and appropriate treatment for the great majority of patients due to the following reasons: 1) limited knowledge about the MGRS among hematologists and nephrologists; 2) lack of necessary diagnostic resources in most health-care facilities; 3) lack of approved clinical recommendations and medical economic standards for treatment of this pathological entity. Consensus document comprises the opinion of experts leading nephrologists and hematologists of Russian Federation on the problem of MGRS including the incoherence in nosology classification, diagnostics approach and rationale for clone specific treatment. Consensus document is based on conclusions and agreements reached during the conference of leading nephrologists and hematologists of Russia which was held in the framework of symposia Plasma cell dyscrasias and lymphoproliferative diseases: modern approaches to therapy, 1516 of March 2019, Pavlov First Saint Petersburg State Medical University. The present Consensus is intended to define the principal practical steps to resolve the problem of MGRS in Russian Federation that are summarized as final clauses.

The ability of platelets to carry out their hemostatic function can be impaired in a wide range of inherited and acquired conditions: trauma, surgery, inflammation, pre-term birth, sepsis, hematological malignancies, solid tumors, chemotherapy, autoimmune disorders, and many others. Evaluation of this impairment is vitally important for research and clinical purposes. This problem is particularly pronounced in pediatric patients, where these conditions occur frequently, while blood volume and the choice of blood collection methods could be limited. Here we describe a simple flow cytometry-based screening method of comprehensive whole blood platelet function testing that was validated for a range of pediatric and adult samples (n = 31) in the hematology hospital setting including but not limited to: classic inherited platelet function disorders (Glanzmann's thrombasthenia; Bernard-Soulier, Wiscott-Aldrich, and Hermasky-Pudlak syndromes, MYH9-dependent thrombocytopenia), healthy and pre-term newborns, acute and chronic immune thrombocytopenia, chronic lympholeukemia, effects of therapy on platelet function, etc. The method output includes levels of forward and side scatter, levels of major adhesion and aggregation glycoproteins Ib and IIb-IIIa, active integrins' level based on PAC-1 binding, major alpha-granule component P-selectin, dense granule function based on mepacrine uptake and release, and procoagulant activity quantified as a percentage of annexin V-positive platelets. This analysis is performed for both resting and dual-agonist-stimulated platelets. Preanalytical and analytical variables are provided and discussed. Parameter distribution within the healthy donor population for adults (n = 72) and children (n = 17) is analyzed.

The authors summarized literature sources, concerning modern ideas about the role of nutritional therapy in the system of health-resort treatment. The authors also made a research on the basis of literature, concerning health-resort nutrition divided into two systems-- buffet system and special-order system. On the basis of sanatorium establishments of the Ministry of Defense authors summarized an experience of clinical nutrition in different age groups and categories of patients. On the basis of current regulatory acts were defined forms and methods of meal catering in the Russian Federation. The results of the study are a scientific supposition for substantiation of management decision-making process, which allows to increase an efficiency of health-resort treatment via nutrition therapy.

Identification of a single molecular trait that is determinant of common malignancies may serve as a powerful diagnostic supplement to cancer type-specific markers. Here, we report a DNA methylation mark that is characteristic of seven studied malignancies, namely cancers of lung, breast, prostate, pancreas, colorectum, glioblastoma and B cell chronic lymphocytic leukaemia (CLL) (n = 137). This mark was defined by substantial hypermethylation at the promoter and first exon of growth hormone secretagouge receptor (GHSR) through bisulfite pyrosequencing. The degree of aberrant methylation was capable of accurate discrimination between cancer and control samples. The highest sensitivity and specificity of cancer detection was achieved for cancers of pancreas, lung, breast and CLL yielding the area under the curve (AUC) values of 1.0000, 0.9952, 0.9800 and 0.9400, respectively. Narrowing to a single CpG site within the gene's promoter or four consecutive CpG units of the highest methylation levels within the first exon improved the detection power. GHSR hypermethylation was detected already at the early stage tumors. The accurate performance of this marker was further replicated in an independent set of pancreatic cancer and control samples (n = 78). These findings support the candidature of GHSR methylation as a highly accurate pan-cancer marker.

The authors showed the history of foundation and development of health resorts "Tarkhovsky" and "Priozersky", as well as an analysis of the treatment of patients with circulatory system diseases in these health resorts. It was found that the structure of patients fitted the profile of health resorts and in recent years has remained stable. Patients with circulatory system diseases accounted for 47.8%. The existing system of medical rehabilitation and restorative treatment of patients with diseases of the circulatory system in the health resorts "Tarkhovsky" and "Priozersky" ensures the implementation in full rehabilitation and recovery programs for all categories of contingent attached.

Background: Schnitzler syndrome (SS) is a rare clinical entity characterized by chronic recurrent urticarial rash, monoclonal IgM gammopathy, intermittent fever and other symptoms. In this report, we present the cases of two patients with SS: a male and a female aged 50 and 49 years, respectively. Both patients had hyperimmunoglobulinemia E and showed good response to elimination diet., Methods: The patients had chronic urticaria, IgM gammopathy and an elevation of the serum levels of inflammation markers. Total IgE levels were found to be high (2000 U/ml and 540 U/ml, respectively). No underlying causes for hyperimmunoglobulinemia E (allergy, parasites, etc.) were revealed. The first patient did not respond to the treatment with antihistamines, while the second one responded only to high doses. The response to prednisolone in the second patient was incomplete., Results: Following a strict elimination diet resulted in marked improvement in skin lesions in both patients. In one of our patients we observed a decrease in IgE and IgM levels after a 3 week diet. The systemic symptoms persisted and improved only after adding pefloxacin, followed by a 3-day empirical course of intravenous prednisone in the first patient and a course of plasmapheresis in the second one., Conclusion: The high serum levels of total IgE may be associated with chronic urticaria activity, severe disease course and a poor response to treatment with antihistamines, and may be considered a possible marker of a subset of patients with SS showing a good response to the restriction diet. In general, we can assume that elimination diet can have an influence on the skin lesions and other symptoms of SS as well as on total IgE and IgM levels, but such association, the underlying mechanisms and the reasons for excessive IgE synthesis should be investigated in further studies.

The achievement of molecular remission is associated with increasing of survival of patients with chronic lymphatic leukemia. The important direction of research is seeking of parameters applicable to forecast of response to therapy. The purpose of the study was evaluating prognostic significance of indicator of minimal residual disease detected by technique of flow cytofluorometry of peripheral blood of patients with chronic lymphatic leukemia during therapy application. The sampling included 112 patients with chronic lymphatic leukemia aged from 43 to 82 years. All patients were given treatment consisted of 6 courses of immune chemotherapy combined with fludarabine with cyclophosphan and rituximab. The samples of peripheral blood were analyzed after 3 courses during therapy and after 6 courses after completion of treatment. The cells were analyzed using 5 and 6 color flow cytometry for the purpose of detection of immune phenotype associated with chronic lymphatic leukemia. The evaluation of minimal residual disease was implemented according international standardized protocol (Rawstron A.C. et al. 2007; 21 (5): 956-64). The minimal residual disease negative status was reached in 87 (78%) patients during evaluation of response after 6th course of treatment. The implementation of indicators of residual disease after 3 courses with fludarabine, cyclophosphan and rituximab permitted to sort out two groups of patients with chronic lymphatic leukemia i.e 67 patients with low (< 0.12%) level of minimal residual disease and 45 patients with high (> 0.12%) level of tumor cells. The rate of molecular remission after completion of treatment. in the given groups consisted 100% and 44% correspondingly. The study demonstrates possibilities of early immune phenotype evaluation of minimal residual disease to forecast differences in response to treatment in patients with chronic lymphatic leukemia that makes it possible to avoid undesirable toxicity of therapy or to choose method of consolidation.

The b-cell chronic lymphatic leukemia is the most common among all lymphatic proliferative diseases and is characterized by significant variability of its clinical course. The mutation status of genes of variable region of heavy chains of immunoglobulins (IgVH) is the most reliable prognostic factor forecasting time until beginning of treatment in case of b-cell chronic lymphatic leukemia. However its detection nowadays is inaccessible for routine diagnostics. Among surrogate markers of mutation status the indicator of expression of ZAP-70 by tumor cells estimated using flow cytofluorometry. However, in publications there are different guidelines concerning the technique of mentioned marker. To establish the optimal approach to evaluation of expression of ZAP-70 the peripheral blood samples of 5I patients with b-cell chronic lymphatic leukemia and 10 healthy persons were analyzed. The comparison with the results of detection of mutation status of IgVH-genes revealed the advantage of applying the technique of calculation of MFI ratio during interpretation of data of expression of ZAP-70 obtained with flow cytofluorometry. In this framework, the indicator of expression of ZAP-70 can be applied in assessing the course of disease and time until the beginning of treatment of b-cell chronic lymphatic leukemia.

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.

Polymorphisms in genes coding xenobiotic-metabolizing enzymes are considered as risk factors modifying susceptibility to cancer. We developed a biochip for the analysis of 18 mutations in 10 genes of metabolizing system: CYP1A1, CYP2D6, GSTT1, GSTM1, MTHFR, MTRR, NQO1, CYP2C9, CYP2C19, and NAT2. Using allele-specific hybridization on the biochip 76 T-cell non-Hodgkin's lymphoma (NHL) patients, 83 B-cell chronic lymphocytic leukemia (B-CLL) patients, and 177 healthy donors were tested. Polymorphic CYP1A1 alleles were more frequent in B-CLL patients relative to normal controls, for example, a combination of polymorphic variants 4887C > A, 4889A > G, and 6235T > C (OR = 1.76, 95% CI = 1.0-3.1). The GSTM1 null genotype was more frequent in NHL patients relative to controls (OR = 1.82, 95% CI = 1.1-3.1). The combination of unfavorable polymorphic CYP1A1 variants and GSTM1 null genotype was found more frequently in B-CLL patients relative to controls (OR = 2.52, 95% CI = 1.3-4.9). In addition, male B-CLL patients demonstrated a significantly increased occurrence of heterozygous and homozygous allele *2 of CYP2C9 gene (OR = 2.38, 95% CI = 1.1-5.2) as well as a combination of alleles *2 and *3 of the gene (OR = 2.09, 95% CI = 1.1-3.9). Thus, our findings show the association between polymorphic alleles of CYP1A1, GSTM1, and CYP2C9 genes and the risk to develop NHL or B-CLL. The developed biochip can be considered as a convenient analytical tool for research studies and predictive analysis in oncohematology., ((c) 2007 Wiley-Liss, Inc.)

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.

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.

T-cell clonality estimation is important for the differential diagnosis between malignant and nonmalignant T-cell proliferation. Routinely used methods include polymerase chain reaction (PCR) analysis of T-cell receptor-gamma (TCR-gamma) gene rearrangements followed by Genescan analysis, polyacrylamide gel electrophoresis, or heteroduplex analysis to visualize amplification products. Here, we present a new method for the analysis after PCR of TCR-gamma rearrangements using hybridization on oligonucleotide microchip. A microchip was designed to contain specific probes for all functional variable (V) and joining (J) gene segments involved in rearrangements of the TCR-gamma locus. Fluorescently labeled fragments of rearranged gamma-chain from patients and donors were obtained in a multiplex nested PCR and hybridized with a microchip. The results were detected using a portable microchip analyzer. Samples from 49 patients with T-cell lymphomas or leukemias and 47 donors were analyzed for T-cell clonality by microchip and single-strand conformation polymorphism analysis, which served as a standard reference method. Comparison of two techniques showed full concordance of the results. The microchip-based approach also allowed the identification of V and J gene segments involved in the particular TCR-gamma rearrangement. The sensitivity of the method is sufficient to determine 10% of clonal cells in the sample.

Aim: To study a spectrum of histologic patterns in patients with paraneoplastic lymphadenopathies, to analyse repeating changes and the causes of diagnostic mistakes., Material and Methods: In a retrospective analysis of 457 patients observed in Hematology Research Center of the RF in 1994-2004, and diagnosed as having non-malignant lymphadenopathies, we identified 40 patients in whom the second or third biopsy showed lymphoma. Nineteen patients (47.5%) had Hodgkin's lymphomas, 11 (27.5%)--B-cell lymphomas and 10 (25%) T-cell lymphomas., Results: In patients subsequently diagnosed with Hodgkin's lymphoma there were 4 repeating histologic patterns in paraneoplastic lymph nodes: sinus histiocytosis (7 patients, 37%), paracortical reaction with numerous plasma cells and macrophages (7 patients, 37%), marked fibrotic changes (4 patients, 21%) and necrotizing lesions (3 patients, 16%). In 15 patients the second biopsy was performed within 4 weeks, while in 4 patients intervals between biopsies were 2, 4, 10 and 24 months. Eight patients (42%) had paraneoplastic changes in lymph nodes located not in the adjacent zones (in 7 patients above the diaphragm, and in 1 patient in both sides of the diaphragm). Amongst patients with B-cell lymphomas 7 had follicular lymphomas, 3--diffuse large B-cell lymphomas and 1--mantle cell lymphoma. In paraneoplastic lymph nodes there were 3 cases of sinus histiocytosis, 1--necrosis. In 5 patients with follicular lymphoma initially diagnosed as having follicular hyperplasia, retrospective analysis and immunohistochemistry showed partial involvement of lymph nodes with lymphoma. In two of them the presence of malignancy was clinically evident at the moment of the first biopsy, while three had a long history of lymphadenopathy (time to diagnostic biopsies were 5, 13 and 34 months). Amongst patients with T-cell lymphomas 5 had undetermined peripheral T-cell lymphomas, 2--anaplastic large cell lymphomas, 1--angioimmunoblastic lymphoma, 1--hepatolienal lymphoma and 1--Lennert's lymphoma. Median of time to the second biopsy was 4 months, range 1-48 months. Five patients (50%) had in the first lymph node sinus histiocytosis, 3 (30%)--paracortical hyperplasia, and 2--granulomatous lesions. Clonal rearrangements of gamma-chain T-cell receptor genes were found in 2 patients from 3 tested., Conclusion: Histologic patterns in lymph nodes not involved by lymphomas in patients with lymphomas are not random. While sinus histiocytosis and necrosis are universal findings, some patterns are disease specific. Paracortical hyperplasia is typical for T-cell lymphomas, prominent fibrosis--for Hodgkin's lymphoma. We believe that paraneoplastic changes in lymph nodes are caused by partial tumor involvement, cytokine reaction, or antitumor immune response. From practical point of view, finding of necrosis, prominent sinus histiocytosis, or prominent fibrosis of a lymph node in the absence of a history of chronic lymphadenitis or inflammation in the draining area should be considered as possible indication to second biopsy. Interpretation of such paraneoplastic phenomena as paracortical hyperplasia and formation of epithelioid-cell granulemas is not easy and must consider context of a clinical picture.

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.

Aim: To study a relationship between cytogenetic disorders, clinicobiological characteristics and prognosis in chronic B-cell lymphoid leukemia (B-CLL)., Material and Methods: Cytogenetic examination of blood, bone marrow and lymph node cells from 135 patients (90 males and 45 females aged 23-84 years) with chronic B-CLL was made. The patients were followed up from 1 month to 25 years. Before the cytogenetic examination specific therapy was not given. B-CLL was staged by K. Rai, forms--by A.L. Vorobyev and M.D. Brilliant. All the patients have undergone standard cytogenetic examination, FISH with multicolor probe to loci with possible frequent aberrations (del3q14, del11q23, del17p13, trisomia 12), determination of CD38 antigen expression on circulating tumor cells. Mutation status of the genes of immunoglobulins variable region (IgVH) was defined in 61 patients., Results: Del13q14 was detected in 34 cases, del11q23--in 26, trisomia of chromosome 12--in 17 cases, del 17p13--in 8, absence of q-arm of chromosome 13--in 3 cases. 61 patients had no karyotype defects. Three prognostic groups of the patients were identified: favourable prognosis--patients without disorders of karyotype and one chromosomal aberration--del13q14; intermediate prognosis patients with dell1q23 and trisomia of chromosome 12; poor prognosis--patients with del17p13 and complex disorders of karyotype. CONCLUSION. Cytogenetic study help determine prognosis of B-CLL and detect patients in need of early therapy.

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.

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.

TNR/11q#1 is a polymorphic trinucleotide (GCC)n repeat located within the minimal region of the 11q deletion in chronic lymphocytic leukemia (CLL). It was recently shown that certain alleles of this repeat are associated with a worse prognosis in CLL patients. To investigate the role of TNR/11q#1 variants as risk-modifying factors in leukemogenesis, we conducted a case-control study on 113 acute lymphotic leukemia (ALL) patients, 82 CLL patients and 146 healthy controls of Russian origin. Comparison of allele and genotype distributions in the control, ALL and CLL groups, performed by Fisher's exact test with two-sized P-value, showed significant decrease in the presence of the GCC(6) allele in the ALL and CLL groups compared to controls. Moreover, 'rare' alleles GCC(7-8) and GCC(13-14) were significantly overrepresented in the ALL group versus controls. We found that CLL risk genotypes were those with both alleles containing more than 6 GCC repeats (P = 0,0212, odds ratio = 1,68 (95% CI, 1,121...2,531)). ALL risk genotypes include three allele combination variants: 1) both alleles containing more than 6 GCC repeats (P = 0,0019, odds ratio = 1,756 (95% CI 1,223...2,502)); 2) one of the alleles containing 7 or 8 repeats (P = 0,0155, odds ratio = 18,22 (95% CI 1,93...136.37)); 3) one of the alleles containing more than 12 repeats (P = 0,0209, Odds ratio = 2,599 (95% CI 1,161...5,815)). Association of certain alleles and genotypes of the TNR/11q#1 repeat with both acute and chronic lymphocytic leukemia suggests the presence of a cancer related gene, involved in a wide spectrum of neoplasia, in the vicinity of this repeat.

Aim: To determine the type and rate of secondary chromosomal aberrations and role in pathogenesis of mantle cell lymphoma (MCL)., Material and Methods: Standard cytogenetic examination (SCE) and fluorescent in situ hybridization (FISH) with tests for 11q22/ATM, 13q14, 17p13/p53, 9p21/p16 and chromosome 12 centromere were made in 28 patients., Results: Secondary chromosomal aberrations were detected in 22 patients. Chromosomal abnormalities occurring in more than 2 cases include deletions 6q15-q23 (53% cases); 11q22/ATM (50%); 9p21 (36%, in half the cases deletion 9p21 was biallele); 13q14 (32%); trisomy 3/3q, monosomy 20 and deletions/translocations 1q and 1p (27% and 20%) and deletion 17p13/p53 (18%). Trisomies 12/12q, 6 and 18/18q, monosomies 2 and 16, deletions/translocations 2p and 16p were found in 2 cases each. The FISH technique identified 9 chromosomal anomalies missed at SCE. Deletions 6q15-q23 were seen in patients with privalent lesions of lymph nodes. Deletions 11q, 13q14, 9p21 and 17p13 occur more frequently in transformation and the blastoid variant. Monosomy 20 was found only in patients with large cell transformations. This was the only cytogenetic defect characteristic for transformed cases, in contrast to denovoblastoid ones. Thus, deletions 6q, 11q23, 13q14 and 9p21 are typical for MCL. Deletions 9p21 and 17p13 are more characteristic for large cell variants of the tumor. Deletion 17p13 occurs at the terminal stage of the disease in rapidly growing tumor mass and resistance to chemotherapy. FISH technique is effective in detection of submicroscopic rearrangements especially small deletions. No significant differences were found between transformed and de novo blastoid cases. This shows that the blastoid variant is not a specific biological form, it is rather a less typical manifestation of the disease due to some preclinical cytogenetic disorders., Conclusion: Progression and transformation of MCL are related to mutation of proapoptotic genes and genes proteins of which are inhibitors of active complexes of D1 cycline. Specification of these mechanisms requires further investigations in patients with different clinicomorphological forms of the disease at various stages of the disease.

We report the primary characterization of a new gene KCNRG mapped at chromosome band 13q14.3. This gene includes three exons and has two alternatively spliced isoforms that are expressed in normal tissues and in some tumor cell lines. Protein KCNRG has high homology to tetramerization domain of voltage-gated K+ channels. Using the patch-clamp technique we determined that KCNRG suppresses K+ channel activity in human prostate cell line LNCaP. It is known that selective blockers of K+ channels suppress lymphocyte and LNCaP cell line proliferation. We suggest that KCNRG is a candidate for a B-cell chronic lymphocytic leukemia and prostate cancer tumor suppressor gene.

Aim: To distinguish T-cell lymphomas and reactive T-cell proliferation it is important to confirm the ability of T-cells to be cloned. Conventional histological and immunophenotypic methods fail to determine the ability of T-cells to be cloned. An experience in the use of detection of T-cell receptor gene gamma-chain (TCRy) rearrangement for determining T-cellular clonality is described., Material and Methods: Polymerase chain reaction (PCR) and single strand conformational polymorphism (SSCP) were used to determine T-cell clonality. Twenty healthy donors, 28 patients with T-lymphomas, and 26 patients with various non-T-cell lymphoproliferative disorders or reactive processes were studied., Results: T-cell monoclonality was detected in 23/28 (82%) T-cell lymphoma cases, whereas in all the samples from normal subjects a polyclonal pattern of rearrangements TCRy was found. The sensitivity of the method was estimated as 2.5%, 7%, and 10% was demonstrated for bone marrow, spleen, and peripheral blood, respectively., Conclusion: PCR-SSCP for TCRy was found to be a useful supplement to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphomas.

Aim: To assess factors of an unfavourable prognosis in a group of intermediate risk of B-cell chronic lymphoid leukemia (BCCLL)., Material and Methods: 206 BCCLL patients (mean age 55.5 years, male/female = 1.66) entered the study conducted by Hematological Research Center in 1992-2000., Results: Nine patients under 35 years of age did not survive 5 years except one female who achieved a complete remission on fludarabin. The type of bone marrow infiltration (diffuse vs interstitial and nodular), the time of lymphocyte count doubling (under or over 12 months) discriminate the patients by prognosis in the group of intermediate risk: medians of overall survival 65 months vs 148 months and 72 vs 133 months, respectively (p < 0.005 for both curves, log-rank criterion). Survival medians in groups with low (< 50% cells) and high (> 50% cells) expression of CD38+ cells in the group of intermediate BCCLL risk comprise 55 and 106 months (p = 0.005). The type of bone marrow infiltration and time of doubling of lymphocyte count overlap: > 70% patients with a diffuse type of bone marrow infiltration have the time of doubling under 12 months and vice versa while expression of CD38 do not overlap with these values. Combination of two signs (type of bone marrow infiltration and CD38 expression or time og lymphocyte count doubling and CD38 expression) allows more precise identification of prognostically unfavourable groups. Medians of survival for combination of the first two signs (two positive against two negative) comprise 51 months vs 169 months (p < 0.0001), for combination of the latter two signs 55 months vs 106 months was not reached (p < 0.001). Although most patients with a tumor form of BCCLL are referred to stage II, the prognosis in this form is much worse than in stage II, survival medians are 44 and 69 months, respectively (p < 0.05). A mutation status of the genes of a variable region of immunoglobulins enable identification of the group of patients with a relatively benign course of BCCLL (survival medians 61 and 289 months, p < 0.0001)., Conclusion: In patients under 35 years of age BCCLL runs unfavourably and seems to require intensive polychemotherapy. Usage of a combination of the signs (CD38, time of doubling of lymphocyte count and type of bone marrow infiltration) is a simple and reliable method of identification of prognostically different categories of patients in the group of an intermediate BCCLL risk. Prognosis in patients with a tumor form of BCCLL is unfavourable: medians of survival in patients with a tumor form and stage III-IV are comparable. Mutational status of the genes of immunoglobulin variable region may serve a marker of a long-term prognosis.