Prevalence of STAT3 mutations in patients with rheumatoid arthritis-associated T-cell large granular lymphocytic leukaemia and Felty syndrome.

OBJECTIVES: Neutropenia is a key presentation of Felty syndrome (FS) and rheumatoid arthritis (RA)-associated T-cell large granular lymphocytic (T-LGL) leukaemia. Clonal rearrangement of T-cell receptor (TCR) gene supports the diagnosis of T-LGL leukaemia but not FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) gene are highly specific for T-LGL leukaemia, but their prevalence in FS remains poorly clarified. METHODS: The study included 100 patients with RA and unexplained neutropenia. TCR rearrangements were examined in blood (100 cases), bone marrow (47 cases), and spleen (12 cases) using the BIOMED-2 protocol. Patients were stratified into RA-associated T-LGL leukaemia cohort if a clonal TCR rearrangement was identified in any of the tested patient samples, and into FS cohort in other cases. Mutations in the STAT3 were examined using next-generation sequencing (NGS) technology in blood (100 cases), bone marrow (37 cases), and spleen (7 cases). RESULTS: STAT3 mutations were identified in 71% (49/69) patients with RA-associated T-LGL leukaemia and in 10% (3/31) patients with FS (p=4.7×10-8). Three samples from the RA-associated T-LGL leukaemia cohort and 5 samples from the FS cohort had STAT3 mutations in the absence of clonal TCR rearrangement. CONCLUSIONS: The results suggest that STAT3 mutations are significantly less common in FS than in RA-associated T-LGL leukaemia. Moreover, NGS can detect clones undetectable by fragment analysis. We speculate that in patients with RA and neutropenia, the detection of STAT3 mutations can point to T-LGL leukaemia even in the absence of clonal TCR rearrangement.

Loss of Heterozygosity and Mutations in the RAS-ERK Pathway Genes in Tumor Cells of Various Loci in Multiple Myeloma.

Multiple myeloma (MM) is a disease characterized by spatiotemporal heterogeneity of tumor clones. Different genetic aberrations can be observed simultaneously in tumor cells from different loci, and as the disease progresses, new subclones may appear. The role of liquid biopsy, which is based on the analysis of tumor DNA circulating in the blood plasma, continues to be explored in MM. Here, we present an analysis of the STR profiles and mutation status of the KRAS, NRAS, and BRAF genes, evaluated in plasma free circulating tumor DNA (ctDNA), CD138+ bone marrow cells, and plasmacytomas. The prospective single-center study included 97 patients, with a median age of 55 years. Of these, 94 had newly diagnosed symptomatic MM, and three had primary plasma cell leukemia. It should be noted that if mutations were detected only in ctDNA, "non-classical" codons were more often affected. A variety of adverse laboratory and clinical factors have been associated with the detection of rare KRAS or NRAS gene mutations in bone marrow or ctDNA, suggesting that these mutations may be factors of an unfavorable prognosis for MM. Liquid biopsy studies provide undeniable fundamental information about tumor heterogeneity and clonal evolution in MM. Moreover, we focus on using liquid biopsy to identify new high-risk factors for MM.

Biallelic Loss of 7q34 (TRB) and 9p21.3 (CDKN2A/2B) in Adult Ph-Negative Acute T-Lymphoblastic Leukemia.

Tumor cells of acute lymphoblastic leukemia (ALL) may have various genetic abnormalities. Some of them lead to a complete loss of certain genes. Our aim was to reveal biallelic deletions of genes in Ph-negative T-ALL. Chromosomal microarray analysis (CMA) was performed for 47 patients with de novo Ph-negative T-ALL, who received treatment according to RALL-2016m clinical protocol at the National Medical Research Center for Hematology (Moscow, Russia) from 2017 to 2023. Out of forty-seven patients, only three had normal molecular karyotype. The other 44 patients had multiple gains, losses, and copy neutral losses of heterozygosity. Biallelic losses were found in 14 patients (30%). In ten patients (21%), a biallelic deletion of 9p21.3 involved a different number of genes, however CDKN2A gene loss was noted in all ten cases. For seven patients (15%), a biallelic deletion of 7q34 was found, including two genes-PRSS1, PRSS2 located within the T-cell receptor beta (TRB) locus. A clonal rearrangement of the TRB gene was revealed in 6 out of 7 cases with 7q34 biallelic loss. Both biallelic deletions can be considered favorable prognostic factors, with an association with 9p21 being statistically significant (p = 0.01) and a trend for 7q34 (p = 0.12) being observed.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

DNA Copy Number Alterations and Copy Neutral Loss of Heterozygosity in Adult Ph-Negative Acute B-Lymphoblastic Leukemia: Focus on the Genes Involved.

The landscape of chromosomal aberrations in the tumor cells of the patients with B-ALL is diverse and can influence the outcome of the disease. Molecular karyotyping at the onset of the disease using chromosomal microarray (CMA) is advisable to identify additional molecular factors associated with the prognosis of the disease. Molecular karyotyping data for 36 patients with Ph-negative B-ALL who received therapy according to the ALL-2016 protocol are presented. We analyzed copy number alterations and their prognostic significance for CDKN2A/B, DMRTA, DOCK8, TP53, SMARCA2, PAX5, XPA, FOXE1, HEMGN, USP45, RUNX1, NF1, IGF2BP1, ERG, TMPRSS2, CRLF2, FGFR3, FLNB, IKZF1, RUNX2, ARID1B, CIP2A, PIK3CA, ATM, RB1, BIRC3, MYC, IKZF3, ETV6, ZNF384, PTPRJ, CCL20, PAX3, MTCH2, TCF3, IKZF2, BTG1, BTG2, RAG1, RAG2, ELK3, SH2B3, EP300, MAP2K2, EBI3, MEF2D, MEF2C, CEBPA, and TBLXR1 genes, choosing t(4;11) and t(7;14) as reference events. Of the 36 patients, only 5 (13.8%) had a normal molecular karyotype, and 31 (86.2%) were found to have various molecular karyotype abnormalities-104 deletions, 90 duplications or amplifications, 29 cases of cnLOH and 7 biallelic/homozygous deletions. We found that 11q22-23 duplication involving the BIRC3, ATM and MLL genes was the most adverse prognostic event in the study cohort.

Clinical Study of the Relationship between Sjögren Syndrome and T-Cell Large Granular Lymphocytic Leukemia: Single-Center Experience.

The relationship between Sjögren syndrome (SS) and T-cell large granular lymphocytic (T-LGL) leukemia remains unclear. In this paper, we report for the first time a large case series of 21 patients with primary and secondary SS associated with T-LGL leukemia. Our results suggest the importance of considering T-LGL leukemia in the diagnostic evaluation of SS patients, particularly when neutropenia occurs. We also postulate that elevated antinuclear antibody titers in patients with T-LGL leukemia indicate the need for the clinical assessment of SS. To assess whether SS affects the frequency of the signal transducer and activator of transcription 3 (STAT3) gene mutations in T-LGL leukemia, we examined STAT3 mutations by next-generation sequencing in two cohorts of patients: with SS-associated T-LGL leukemia and T-LGL leukemia in the setting of rheumatic diseases but without SS. While our results suggest that SS, per se, is not associated with an increased frequency of STAT3 mutations in T-LGL leukemia, further studies are needed to better assess the role of the STAT pathway in the development of concomitant SS and T-LGL leukemia.

Analysis of a single-institution cohort of patients with Felty's syndrome and T-cell large granular lymphocytic leukemia in the setting of rheumatoid arthritis.

T-cell large granular lymphocytic leukemia (T-LGLL) is a lymphoproliferative disorder characterized by a persistent increase in the number of large granular lymphocytes (LGLs), neutropenia, and splenomegaly. Clinical manifestations of T-LGLL in the setting of rheumatoid arthritis (RA) are often identical to those in which one would suspect Felty's syndrome (FS). These disorders are distinguished by the presence of T-cell clonality, which is present in T-LGLL but not in FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) and 5b (STAT5b) genes can be used as molecular markers of T-LGLL, but their prevalence in FS is unknown.Eighty-one patients with RA and unexplained neutropenia or/and an increase in the number of LGLs above 2 × 109/L were stratified into RA-associated T-LGLL (N = 56) or FS (N = 25) groups based on the presence or absence of T-cell clonality. STAT3 and STAT5b gene mutations were assessed in each group by means of allele-specific polymerase chain reaction assays. Clinical, immunological, laboratory data and the results of immunophenotyping of blood and bone marrow lymphocytes were also evaluated.Mutations of the STAT3 gene and an increase in the number of LGLs above 2 × 109/L were detected in RA-associated T-LGLL, but not in FS (39% vs 0% and 21% vs 0%, respectively). Mutations in the STAT5b gene were not observed in either group. Expression of CD57, CD16, and CD5-/dim on CD3+CD8+ T-lymphocytes was observed in both RA-associated T-LGLL and FS.STAT3 gene mutations or LGL counts over 2 × 109/L in RA patients are indicative of T-LGLL.

Clonal relationship of marginal zone lymphoma and diffuse large B-cell lymphoma in Sjogren's syndrome patients: case series study and review of the literature.

The occurrence of diffuse large B-cell lymphoma (DLBCL) in the course of Sjogren's syndrome (SS) is considered to be equally related either to the development of DLBCL de novo or to the transformation from marginal zone lymphoma (MZL). However, the question of possible clonal relationship between MZL and DLBCL in the group of SS patients remains open. Here we present the data concerning 194 patients with lymphoma complicated SS followed up at Nasonova Research Institute of Rheumatology during the last 22 years. Molecular analysis of tumor cells was performed for 6 SS patients who had developed both MZL and DLBCL. To assess clonal relationship between each of the tumor pairs immunoglobulin heavy chain (IGH) gene rearrangements were identified according BIOMED-2 protocol by means of multiplex polymerase chain reaction followed by GeneScan fragment analysis. Despite different localization MZL and DLBCL were clonally related in five tumor pairs. The median time to transformation was 11 months (range 0-78 months). MZL and DLBCL were clonally related in most cases from our cohort of SS patients. No statistically significant difference in survival between patients with DLBCL transformed from MZL and patients with de novo DLBCL was found in the cohort of SS patients investigated.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

COVID-19 and Cancer Detection in Russia.

Overdiagnosis, associated with mass testing in healthy populations, is a significant issue for breast, prostate, renal, and thyroid cancers. During the lockdowns caused by the COVID-19 pandemic, the intensity of cancer screening was expected to go down. In this study, we analyzed the impact of the expected reduction in screening intensity on morbidity and mortality from certain malignancies. Cumulative data from the Russian National Cancer Registry available from 2000 to 2022 were analyzed. It was noted that there has been no noticeable effect of the COVID-19 lockdowns on mortality rates from breast, prostate, renal, or thyroid cancers. At the same time, the detectable incidence decreased markedly in 2020 at the time of the lockdowns and then returned to pre-pandemic levels in 2022. At the moment, there is no sufficient reason to believe that skipping screening tests in 2020 could have any impact on breast, prostate, renal, or thyroid cancer mortality two years later (2022). The data presented further confirm that the overdiagnosis of these types of malignancies is caused by widespread screening among a generally healthy population.

Aleukemic variant of T-cell large granular lymphocyte leukemia in patients with rheumatoid arthritis - diagnostically challenging subtype.

INTRODUCTION: The typical clinical manifestations of T-cell large granular lymphocyte (T-LGL) leukemia are an increase in the number of large granular lymphocytes (LGLs) in the blood > 2000 cells/µL, neutropenia, and splenomegaly. In rare cases of so-called 'aleukemic' T-LGL leukemia, the number of LGLs is <400-500 cells/µL. In patients with rheumatoid arthritis (RA), distinguishing T-LGL leukemia with low tumor burden in the blood and bone marrow from Felty syndrome (FS) poses diagnostic challenges. AREAS COVERED: This review aimed to describe the basic characteristics and variants of aleukemic T-LGL leukemia, with a special focus on aleukemic T-LGL leukemia with massive splenomegaly (splenic variant of T-LGL leukemia) and differential diagnosis of such cases with hepatosplenic T-cell lymphoma. The significance of mutations in the signal transducer and activator of transcription 3 (STAT3) gene for distinguishing aleukemic RA-associated T-LGL leukemia from FS is discussed, along with the evolution of the T-LGL leukemia diagnostic criteria. PubMed database was used to search for the most relevant literature. EXPERT OPINION: Evaluation of STAT3 mutations in the blood and bone marrow using next-generation sequencing, as well as a comprehensive spleen study, may be necessary to establish a diagnosis of aleukemic RA-associated T-LGL leukemia.

Gamma-delta T-cell large granular lymphocytic leukemia in the setting of rheumatologic diseases.

Background: T-cell leukemia originating from large granular lymphocytes (T-LGL leukemia) is a rare lymphoid neoplasia characterized by clonal proliferation of large granular T lymphocytes expressing αß or γδ T-cell receptor (TCR) on the cell membrane. γδT-LGL leukemia, accounting for approximately 17% of all T-LGL leukemia cases, is associated with autoimmune diseases. However, the features of γδT-LGL leukemia in patients with rheumatologic diseases are still insufficiently characterized. Methods: In this retrospective study, 15 patients with rheumatologic disease-associated γδT-LGL leukemia were included. The patients were obtained from a single center from 2008 to 2023. Data related to clinical characteristics and rheumatologic diagnoses were collected. Immunophenotype evaluations as well as T-lymphocyte clonality (based on TCR-γ, TCR-ß, and TCR-δ gene rearrangements), and signal transducer and activator of transcription (STAT) three and STAT5B mutation analyses (by next-generation sequencing) were performed on blood, bone marrow, and spleen samples. Results: All but one patient had rheumatoid arthritis (RA). In 36% of patients, manifestations of γδT-LGL leukemia were present before or concurrently with clinical manifestations of RA. Splenomegaly was observed in 60% of patients and neutropenia (<1.5 × 109/L) was detected in 93% of cases. CD4-/CD8- and CD4-/CD8+ subtypes were detected in seven cases each. Mutations in STAT3 were detected in 80% of patients; however, STAT5B mutations were not detected. Evaluations of T-cell clonality and variant allele frequencies at STAT3 in the blood, bone marrow, and spleen tissue revealed an unusual variant of CD4-/CD8- γδT-LGL leukemia with predominant involvement of the spleen, involvement of the bone marrow to a less extent, and no tumor cells in peripheral blood. Conclusion: The mechanism by which γδT-LGL leukemia may induce the development of RA in some patients requires further investigation. Cases of RA-associated γδT-LGL leukemia with neutropenia and splenomegaly but no detectable tumor-associated lymphocytes in peripheral blood (the so-called splenic variant of T-LGL leukemia) are difficult to diagnose and may be misdiagnosed as Felty syndrome or hepatosplenic T-cell lymphoma.

Genetic Lesions in Russian CLL Patients with the Most Common Stereotyped Antigen Receptors.

Chronic lymphocytic leukemia (CLL) is one of the most common B-cell malignancies in Western countries. IGHV mutational status is the most important prognostic factor for this disease. CLL is characterized by an extreme narrowing of the IGHV genes repertoire and the existence of subgroups of quasi-identical stereotyped antigenic receptors (SAR). Some of these subgroups have already been identified as independent prognostic factors for CLL. Here, we report the frequencies of TP53, NOTCH1, and SF3B1 gene mutations and chromosomal aberrations assessed by NGS and FISH in 152 CLL patients with the most common SAR in Russia. We noted these lesions to be much more common in patients with certain SAR than average in CLL. The profile of these aberrations differs between the subgroups of SAR, despite the similarity of their structure. For most of these subgroups mutations prevailed in a single gene, except for CLL#5 with all three genes affected by mutations. It should be noted that our data concerning the mutation frequency in some SAR groups differ from that obtained previously, which could be due to the population differences between patient cohorts. The research in this area should be important for better understanding the pathogenesis of CLL and therapy optimization.

A simple and efficient method for DNA extraction from skin and paraffin-embedded tissues applicable to T-cell clonality assays.

PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.

STAT3 mutations in "gray-zone" cases of T-cell large granular lymphocytic leukemia associated with autoimmune rheumatic diseases.

A persistently increased T-cell large granular lymphocyte (T-LGL) count in the blood of more than 2 × 109/L for at least 6 months is necessary for a reliable diagnosis of T-LGL leukemia. In cases with LGL counts of approximately 0.5-2 × 109/L, a diagnosis of T-LGL leukemia can be made if clonal rearrangement of T-cell receptor (TCR) genes is present and if the patient shows typical manifestations of T-LGL leukemia, such as cytopenia, splenomegaly, or concomitant autoimmune disease. However, in cases with LGL counts of less than 0.5 × 109/L, the diagnosis of T-LGL leukemia is questionable (termed as "gray-zone" cases). Although mutations in signal transducer and activator of transcription 3 (STAT3) gene are the molecular hallmark of T-LGL leukemia, their diagnostic value in the "gray-zone" cases of T-LGL leukemia has not been evaluated - our study has been aimed to examine the prevalence of STAT3 mutations in these cases. Herein, we describe 25 patients with autoimmune rheumatic diseases, neutropenia, clonal rearrangement of TCR genes, and circulating LGL count of less than 0.5 × 109/L. Splenomegaly was observed in 19 (76%) patients. Mutations in the STAT3 were detected in 56% of patients using next-generation sequencing. Importantly, in 3 patients, no involvement of the blood and bone marrow by malignant LGLs was noted, but examination of splenic tissue revealed infiltration by clonal cytotoxic T-lymphocytes within the red pulp, with greater prominence in the cords. We suggest using the term "splenic variant of T-LGL leukemia" for such cases.

STR Profiling Reveals Tumor Genome Instability in Primary Mediastinal B-Cell Lymphoma.

Primary mediastinal B-cell lymphoma (PMBCL) is the only non-Hodgkin's lymphoma variant responding to immune checkpoint inhibitor (ICI) therapy, approximately in half of the cases; however, no molecular markers predicting a response to ICI therapy in PMBCL have been described so far. In this study, we assessed the incidence of the loss of heterozygosity (LOH), elevated microsatellite alteration at selected tetranucleotides (EMAST), and microsatellite instability (MSI) in the tumor genomes of 72 patients with PMBCL undergoing high-dose chemotherapy treatment at the National Research Center for Hematology (Moscow, Russia). Tumor DNA was isolated from biopsy samples taken at diagnosis. Control DNA was isolated from the blood of patients in complete remission or from buccal epithelium. STR-profiles for LOH and EMAST were assessed by PCR with COrDIS Plus multiplex kit (Gordiz Ltd., Moscow, Russia). LOH was detected in 37 of 72 patients (51.4%). EMAST was found in 40 patients (55.5%); 24 had a combination of EMAST with LOH. MSI-high was not found, while MSI-low was detected only in one patient. The association of certain genetic lesions with the clinical outcome in patients receiving treatment according to the standard clinical protocol R-Da-EPOCH-21 has been estimated (58 patients out of 72) and no associations with the worst overall or event-free survival were found.

Loss of Heterozygosity in the Tumor DNA of De Novo Diagnosed Patients Is Associated with Poor Outcome for B-ALL but Not for T-ALL.

Despite the introduction of new technologies in molecular diagnostics, one should not underestimate the traditional routine methods for studying tumor DNA. Here we present the evidence that short tandem repeat (STR) profiling of tumor DNA relative to DNA from healthy cells might identify chromosomal aberrations affecting therapy outcome. Tumor STR profiles of 87 adult patients with de novo Ph-negative ALL (40 B-ALL, 43 T-ALL, 4 mixed phenotype acute leukemia (MPAL)) treated according to the "RALL-2016" regimen were analyzed. DNA of tumor cells was isolated from patient bone marrow samples taken at diagnosis. Control DNA samples were taken from the buccal swab or the blood of patients in complete remission. Overall survival (OS) analysis was used to assess the independent impact of the LOH as a risk factor. Of the 87 patients, 21 were found with LOH in various STR loci (24%). For B-ALL patients, LOH (except 12p LOH) was an independent risk factor (OS hazard ratio 3.89, log-rank p-value 0.0395). In contrast, for T-ALL patients, the OS hazard ratio was 0.59 (log-rank p-value 0.62). LOH in particular STR loci measured at the onset of the disease could be used as a prognostic factor for poor outcome in B-ALL, but not in T-ALL.

Viral Load and Patterns of SARS-CoV-2 Dissemination to the Lungs, Mediastinal Lymph Nodes, and Spleen of Patients with COVID-19 Associated Lymphopenia.

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 109/L was observed in 23 of 36 (63.8%) patients. In 12 of 36 cases (33%) SARS-CoV-2 was found in lung tissues only with a median VL of 239 copies (range 18-1952) SARS-CoV-2 cDNA per 100 copies of ABL1. Histomorphological changes corresponding to bronchopneumonia and the proliferative phase of DAD were observed in these cases. SARS-CoV-2 dissemination into the lungs, lymph nodes, and spleen was detected in 23 of 36 patients (58.4%) and was associated with the exudative phase of DAD in most of these cases. The median VL in the lungs was 12,116 copies (range 810-250281), lymph nodes-832 copies (range 96-11586), and spleen-71.5 copies (range 0-2899). SARS-CoV-2 in all cases belonged to the 19A strain. A immunohistochemical study revealed SARS-CoV-2 proteins in pneumocytes, alveolar macrophages, and bronchiolar epithelial cells in lung tissue, sinus histiocytes of lymph nodes, as well as cells of the Billroth pulp cords and spleen capsule. SARS-CoV-2 particles were detected by transmission electron microscopy in the cytoplasm of the endothelial cell, macrophages, and lymphocytes. The infection of lymphocytes with SARS-CoV-2 that we discovered for the first time may indicate a possible link between lymphopenia and SARS-CoV-2-mediated cytotoxic effect.

The non-leukemic T cell large granular lymphocytic leukemia variant with marked splenomegaly and neutropenia in the setting of rheumatoid arthritis - Felty syndrome and hepatosplenic T cell lymphoma mask.

T cell large granular lymphocytic (T-LGL) leukemia is a rare type of mature T cell neoplasm. The typical features of T-LGL leukemia include an increased number of large granular lymphocytes in the peripheral blood, cytopenia (most commonly neutropenia), and mild-to-moderate splenomegaly. Up to 28% of patients with T-LGL leukemia have rheumatoid arthritis (RA). This study reports ten atypical cases (seven women and three men, median age 60.5 years) of RA-associated T-LGL leukemia presenting with lymphopenia, severe neutropenia, and marked splenomegaly. The weight of the spleens ranged from 892 to 2100 g (median 1100 g). Bone marrow histology and differential counts of bone marrow aspirates revealed no peculiarities in nine of ten cases. The red pulp of the spleen was expanded and showed moderate to strong infiltration by medium-sized slightly pleomorphic lymphocytes in nine cases and subtle infiltration in one. Although lymphocytic infiltration involved both cords and sinusoids, it was more apparent within the splenic cords. The white pulp was preserved and contained prominent germinal centers in eight patients and was atrophic in two patients. Immunohistochemically, malignant lymphocytes were CD3+, CD43+, and CD4- in all cases and TIA-1+ in nine out of ten. TCRαß positivity and TCRγδ positivity was observed in six and four cases out of ten, respectively. All ten patients had T cell clonality in the spleen tissue, but in three cases it was absent in both blood and bone marrow. STAT3 mutations in the spleen tissue were detected in three of ten cases. In all eight cases studied, neither isochromosome 7q nor trisomy 8 was detected in the spleen tissue. Cases of RA-associated T-LGL leukemia with low LGL count in the peripheral blood, neutropenia, and marked splenomegaly present a diagnostic challenge and can be misdiagnosed as Felty's syndrome or hepatosplenic T cell lymphoma.

Repertoire of Rearranged Immunoglobulin Heavy Chain Genes in Russian Patients With B-Cell Lymphoproliferative Diseases.

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.