Objective To investigate the differences of type 2 innate lymphocytes (ILC2) and interlukin 9 (IL-9) between chronic lymphocytic leukemia (CLL) patients and healthy controls, and to understand the effects of ILC2 on the function of regulatory T cells (Tregs), CD8⁺ T cells and CLL cells through IL-9. Methods Flow cytometry was used to detect the levels of ILC2 and Tregs in the peripheral blood of 45 newly diagnosed CLL patients and 24 healthy controls, and the expressions of granzyme B and perforin in CD8⁺ T cells in the peripheral blood of 28 patients and 15 healthy controls; ELISA was used to detect the level of IL-9 in the serum. ILC2 of patients and healthy controls was sorted by immunomagnetic beads and cultured separately, and the level of IL-9 in the culture supernatant was measured by ELISA. ILC2 sorted from CLL patients and healthy control-derived peripheral blood mononuclear cells(PBMCs) were co-cultured with the B cell leukemia MEC-1 cells, one group was supplemented with IL-9 antibody and the other group was not. After 72 hours of culture, the ratio of Tregs, programmed death 1 (PD-1), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), cytotoxic T lymphocyte antigen 4 (CTLA-4) on Tregs, granzyme B and perforin in CD8⁺ T cells were measured by flow cytometry, IL-9 level of the culture supernatant was measured by ELISA, the apoptosis of MEC-1 cells was measured by Annexin V-PI. Results Compared with the healthy control group, the levels of ILC2, Tregs and IL-9 in the CLL group increased significantly. The levels of granzyme B and perforin in CD8⁺ T cells were positively correlated in the peripheral blood of CLL patients. Compared with the healthy control group, IL-9 levels in the supernatant of sorted ILC2 from CLL patients increased. In the anti-IL9 antibody group, the level of PD-1 and TIGIT on Tregs decreased, and the level of granzyme B in CD8⁺ T cells increased significantly. The level of IL-9 in the anti-IL9 antibody group decreased statistically. And MEC-1 cells showed increased early apoptotic rate in the anti-IL9 antibody group statistically. Conclusion In CLL, ILC2 affects CD8⁺ T cells and Tregs through IL-9, which weakens the anti-tumor effect of CD8⁺ T cells, enhances the immunosuppressive effect of Tregs, and plays a role in the occurrence and development of CLL disease.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; CD8-Positive T-Lymphocytes/immunology* ; T-Lymphocytes, Regulatory/immunology* ; Middle Aged ; Male ; Female ; Interleukin-9/blood* ; Aged ; Granzymes/metabolism* ; Perforin/metabolism* ; Immunity, Innate ; Adult ; Lymphocytes/immunology*

OBJECTIVE: To investigate the expression of co-inhibitory molecules TIGIT/CD155 and PD-1 on CD4⁺T cells and Treg cells in peripheral blood of patients with chronic lymphocytic leukemia (CLL) and analyze their clinical significance. METHODS: The expression of PD-1 and TIGIT on CD4⁺T cells and Treg cells was detected by flow cytometry in 40 CLL patients and 20 healthy controls. Additionally, the expression of CD155 on peripheral blood B cells and DC cells of the enrolled subjects was detected. RESULTS: The proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells and CD155⁺DC cells in peripheral blood of CLL patients were significantly higher than those of healthy controls ( P < 0.05). The proportions of PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells in CLL patients were significantly higher than those of PD-1⁺TIGIT^-CD4⁺T cells and PD-1⁺TIGIT^-Treg cells, respectively ( P < 0.05). Both PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells were positively correlated with the level of CD155⁺DC cells (r =0.742, r =0.766). With the progression of Binet stage, the proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells, and CD155⁺DC cells gradually increased ( P < 0.05), and the aforementioned three types cells were all increased in patients with CD38≥30%, IGVH unmutated, or poor prognosis due to chromosomal abnormalities ( P < 0.05). CONCLUSION Co-inhibitory molecules PD-1 and TIGIT may be involved in immunodepletion in patients with advanced CLL, which has clinical prognostic value. Dual inhibitor molecular targeted therapy provides a new direction for the individualized treatment of CLL.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; Receptors, Immunologic/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; T-Lymphocytes, Regulatory/metabolism* ; Receptors, Virus/metabolism* ; CD4-Positive T-Lymphocytes/metabolism* ; Male ; Female ; Middle Aged ; Flow Cytometry ; Clinical Relevance

OBJECTIVE: To investigate the expression of CD19/CD73 in chronic lymphocytic leukemia (CLL) and its correlation with clinical features. METHODS: The clinical data of 60 CLL patients and 40 healthy volunteers (control group) from January 2022 to November 2023 were retrospectively analyzed. The levels of CD19 and CD73 in peripheral blood of CLL patients were measured by flow cytometry. Kaplan-Meier method was used for survival analysis. RESULTS: The hemoglobin (Hb) and CD19/CD73 levels in CLL group were significantly lower than those in control group, while CD19, CD73 and β₂-MG were significantly higher (all P <0.001). According to ROC curve analysis, the AUC value of CD19/CD73 for CLL diagnosis was 0.980 (95%CI : 0.949-1.000, P <0.05), the specificity was 92.50%, and the sensitivity was 98.30%. The CD19/CD73 level of CLL patients with splenomegaly was significantly lower than those without splenomegaly (P <0.01). There was no significant correlation between CD19/CD73 and Hb in CLL patients ( r =0.056, P >0.05). CD19/CD73 was positively correlated with β₂-MG ( r =0.837, 95%CI : 0.740 2-0.899 6, P <0.01). According to the median value (12.84) of CD19/CD73, the patients were divided into high and low expression groups. Kaplan-Meier survival analysis showed that the overall survival rate and progression-free survival rate at 18^th month in the low expression group were 87.08% and 93.25%, while those in the high expression group were 96.41% and 99.90%, respectively (both P <0.05). CONCLUSION The level of CD19/CD73 is low in CLL patients, which can be used as an auxiliary index for clinical diagnosis of CLL. CD19/CD73 is closely related to splenomegaly in CLL patients. Low expression of CD19/CD73 predicts poor prognosis.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/metabolism* ; 5'-Nucleotidase/metabolism* ; Antigens, CD19/metabolism* ; Retrospective Studies ; Male ; Female ; Prognosis ; Middle Aged ; Aged ; Adult ; GPI-Linked Proteins

Abnormal cell apoptosis is closely related to the occurrence of hematologic tumors, B-cell lymphoma-2 (BCL-2), as a key anti-apoptotic protein in intrinsic programmed cell death, has become a hot spot in the treatment of hematologic tumors in recent years. Venetoclax is an oral small-molecule selective BCL-2 inhibitor approved by the Food and Drug Administration (FDA） for the treatment of chronic lymphocytic leukemia (CLL) patients or small lymphocytic lymphoma (SLL) patients and for the treatment of elderly acute myeloid leukemia (AML) patients that is not suitable for aggressive chemotherapy. In addition, it also showed a promising clinical application in treatment of non-Hodgkin's lymphoma (NHL) patients, which is a new expansion of the clinical indications for venetoclax. In this review, the role of BCL-2 protein family played in the regulation of NHL cell apoptosis, the development of BCL-2 inhibitors and the recent research progress of venetoclax in the treatment of NHL are reviewed.
Aged ; Antineoplastic Agents/therapeutic use* ; Apoptosis Regulatory Proteins ; Bridged Bicyclo Compounds, Heterocyclic/therapeutic use* ; Hematologic Neoplasms/drug therapy* ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy* ; Lymphoma, B-Cell ; Lymphoma, Non-Hodgkin/drug therapy* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Sulfonamides

BACKGROUND: Classification of the pulmonary neuroendocrine tumor (pNET) categories is a step-wise process identified by the presence of necrosis and number of mitoses per 2 mm. In neuroendocrine tumor pathology, Ki-67 was first described as a prognostic factor in the pancreas and incorporated into the grading system of digestive tract neuroendocrine neoplasms in the 2010 WHO classification. However, the significance of Ki-67 in pNETs was still a controversial issue. This study was to investigate the potentially diagnostic value of Ki-67 in pNETs. METHODS: We retrieved 159 surgical specimens of pNETs, including 35 typical carcinoids (TCs), 2 atypical carcinoid (ACs), 28 large-cell neuroendocrine carcinomas (LCNECs), 94 small-cell lung cancers (SCLCs). Manual conventional method (MCM) and computer-assisted image analysis method (CIAM) were used to calculate the Ki-67 proliferative index. In CIAM, 6 equivalent fields (500 × 500 μm) at 10× magnification were manually annotated for digital image analysis. RESULTS: The Ki-67 index among the 4 groups with ranges of 0.38% to 12.66% for TC, 4.34% to 29.48% for AC, 30.67% to 93.74% for LCNEC, and 40.71% to 96.87% for SCLC. The cutoff value of Ki-67 index to distinguish low grade with high grade was 30.07%. For the univariate survival analyses in pNETs, both the overall survival and progression-free survival correlated with Ki-67 index. In addition, the Ki-67 index performed by CIAM was proved to be of great positive correlation with MCM. CONCLUSIONS Ki-67 index counted by CIAM is a reliable method and can be a useful adjunct to classify the low- and high-grade NETs.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Neuroendocrine ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Leukemia, Lymphocytic, Chronic, B-Cell ; metabolism ; pathology ; Male ; Middle Aged ; Neuroendocrine Tumors ; metabolism ; pathology ; Prognosis ; World Health Organization

<b>BACKGROUNDb>The established clinical staging systems (Rai/Binet) of chronic lymphocytic leukemia (CLL) cannot accurately predict the appropriate treatment of patients in the earlier stages. In the past two decades, several prognostic factors have been identified to predict the outcome of patients with CLL, but only a few studies investigated more markers together. To predict the time to first treatment (TTFT) in patients of early stages, we evaluated the prognostic role of conventional markers as well as cytogenetic abnormalities and combined them together in a new prognostic scoring system, the CLL prognostic index (CLL-PI).

<b>METHODSb>Taking advantage of a population of 406 untreated Chinese patients with CLL at early and advanced stage of disease, we identified the strongest prognostic markers of TTFT and, subsequently, in a cohort of 173 patients who had complete data for all 3 variables, we integrated the data of traditional staging system, cytogenetic aberrations, and mutational status of immunoglobulin heavy chain variable region (IGHV) in CLL-PI. The median follow-up time was 45 months and the end point was TTFT.

<b>RESULTSb>The median TTFT was 38 months and the 5-year overall survival was 80%. According to univariate analysis, patients of advanced Rai stages (P < 0.001) or with 11q- (P = 0.002), 17p- (P < 0.001), unmutated IGHV (P < 0.001), negative 13q- (P = 0.007) and elevated lactate dehydrogenase levels (P = 0.001) tended to have a significantly shorter TTFT. And subsequently, based on multivariate Cox regression analysis, three independent factors for TTFT were identified: advanced clinical stage (P = 0.002), 17p- (P = 0.050) and unmutated IGHV (P = 0.049). Applying weighted grading of these independent factors, a CLL-PI was constructed based on regression parameters, which could categorize four different risk groups (low risk [score 0], intermediate low [score 1], intermediate high [score 2] and high risk [score 3-6]) with significantly different TTFT (median TTFT of not reached (NR), 65.0 months, 36.0 months and 19.0 months, respectively, P < 0.001).

<b>CONCLUSIONSb>This study developed a weighted, integrated CLL-PI prognostic system of CLL patients which combines the critical genetic prognostic markers with traditional clinical stage. This novel modified PI system could be used to discriminate among groups and may help predict the TTFT and prognosis of patients with CLL.

Adult ; Aged ; Aged, 80 and over ; China ; Chromosome Aberrations ; Chromosomes, Human, Pair 17 ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; metabolism ; In Situ Hybridization, Fluorescence ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Prognosis

OBJECTIVE: To study the pathologic characteristics of bone marrow for CD5 positive small B cell lymphoma (SBL).
 METHODS: The pathologic profiles of 92 patients with CD5 positive SBL were retrospectively analyzed. The morphologic and immunophenotypic features were analyzed by flow cytometry and immunohistochemistry. IgH/CCND1 was examined by fluorescence in situ hybridization (FISH).
 RESULTS: A total of 92 patients with CD5 positive SBL were enrolled in this study, including 56 (60.9%) chronic lymphocytic leukemia /small lymphocytic lymphoma (CLL/SLL), 23 (25.0%) mantle cell lymphoma (MCL) and 13 other SBL (14.1%). Among the 13 other cases, 5, 4 and 4 cases were follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL) and splenic marginal zone lymphoma (SMZL), respectively. The frequency of patterns for bone marrow infiltration was as follow: diffuse pattern (19/92), mixed pattern (15/92), nodular pattern (9/92), interstitial pattern (8/92), and intrasinusodial pattern (2/92). All patients expressed CD19, CD20 and CD5. According to the immunophenotypic score system, all the CLL patients had 4-5 scores, while SMCL and other SBL patients had less than 3 scores. For the other SBL patients, 5 FL expressed CD10, while 3 FL, 1 LPL and 3 SMZL expressed CD23. There was a significant difference in the expression of CD23, sIgM, FMC7, CD11C and CD22 between the CLL and MCL groups (P<0.01). All 23 MCL patients expressed cyclin D1 and showed IgH/CCND1 gene translocation by FISH detection.
 CONCLUSION CD5 positive SBL includes a variety of types of lymphoma. Patterns of bone marrow for CD5 positive SBL are diversity. Immunophenotypic analysis by flow cytometry is essential in the diagnosis and differential diagnosis of CD5 positive SBL, especially for CLL.
Bone Marrow ; pathology ; CD5 Antigens ; metabolism ; Diagnosis, Differential ; Flow Cytometry ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; Lymphoma, B-Cell ; diagnosis ; Lymphoma, Follicular ; diagnosis ; Lymphoma, Mantle-Cell ; diagnosis ; Oncogene Proteins, Fusion ; metabolism ; Retrospective Studies ; Splenic Neoplasms ; diagnosis

<b>OBJECTIVEb>To investigate the feasibility of CyclinD1/IgH detection by FISH in diferential diagnosis between mantle cell lymphoma (MCL) and chronic lymphocytic leukeamia (CLL).

<b>METHODSb>The FISH detection was performed for CyclinD1/IgH fusion gene. A comprehensive analysis was carried out for clinical features, such as age, sex , WBC count and lymphocyte count, the bone marrow morphology and immunohistochemical staining were carried for CyclinD1/IgH.

<b>RESULTSb>It is often difficult to distinguish MCL from CLL by bone marrow morphology, when the cell morphology was not typical; there was no difference in age, sex, WBC count and lymphocyte count between MCL and CLL groups; 9 out of 52 patients were diagnosed as MCL, and the direction of CyclinD1/IgH by FISH was positive in 7 of 9 MCL, while 3 of the 7 patients were negative by immunohistochemical staining for CyclinD1.

<b>CONCLUSIONb>Detection of CyclinD1/IgH by FISH can be used as a specific and feasible method for differential diagnosis of mantle cell lymphoma from chronic lymphocytic leukeamia.

Bone Marrow ; pathology ; Diagnosis, Differential ; Humans ; Immunophenotyping ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; metabolism ; Lymphoma, Mantle-Cell ; diagnosis ; metabolism ; Oncogene Proteins, Fusion ; metabolism

<b>OBJECTIVEb>To investigate the function of alternative NF-κB activity in B-cell chronic lymphocytic leukemia cells (B-CLL).

<b>METHODSb>The mRNA expression of individual NF-κB subunits in CD5⁺CD19⁺ cells (CLL B-cells) from bone marrow (BM) of 56 patients with B-CLL was analyzed by quantitative RT-PCR. An ELISA-based NF-κB family transcription factor activity assay was performed to quantify the κB DNA-binding activity in nuclear extracts from CLL B-cells. Cell death of CLL-B cells was determined by PI staining, RelA and RelB expression at protein level of CLL B-cells by Western blot analyses.

<b>RESULTSb>The expression levels of RelA, p50, RelB and p52 mRNA in CLL B-cells were all higher than that of normal B cells with statistical significance (P<0.05). RelA was activated in almost all the patients detected while RelB activity was induced in part of samples. The average RelA activity in CLL B-cells was increased compared to that in normal B cells while the average RelB activity was similar to that of normal B cells. When cultured in vitro for 24, 48 and 72 hours, the frequencies of cell death of CLL B-cells from RelA⁺/RelB⁻ group were(35.54±4.43)%,(50.92±8.44)%, and(49.24±8.16)%, respectively; that of the RelA⁺/RelB⁺ group were (20.65±2.37)%, (18.17±1.36)%, and (26.55±4.08)%, respectively. When the cells from RelA+/RelB⁻ group were co-cultured with bone marrow stromal cells (hBMSCs), the frequencies of cell death of CLL B-cells were decreased compared to that of the cells cultured alone, while the frequencies of cell death of RelA⁺/RelB⁻ CLL B-cells were higher than that of CLL B-cells from RelA⁺/RelB⁺ group when co-cultured with hBMSCs. RelA and RelB expression in CLL-B cells from the RelA⁺/RelB⁻ group was induced after co-cultured with hBMSCs for 48 h. RelB was reduced in the cytoplasm and increased in the nucleus in CLL-B cells from the RelA+/RelB+ group.

<b>CONCLUSIONb>The alternative NF-κB was indeed activated and presented heterogeneous in CLL B-cells from BM. Activation of RelB combined with RelA activity could provide the survival advantage to CLL B-cells from BM. Co-culture with hBMSCs could protect CLL-B cells through the induction of RelA and Rel B expressions.

Case-Control Studies ; Cell Nucleus ; metabolism ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; metabolism ; NF-kappa B p50 Subunit ; metabolism ; NF-kappa B p52 Subunit ; metabolism ; Transcription Factor RelA ; metabolism ; Transcription Factor RelB ; metabolism ; Tumor Cells, Cultured

This study was purpose to compare and analyze the chronic lymphocytic leukemia human bone marrow stromal cells (CLL-hBMSC) and normal hBMSC (N-hBMSC) so as to provide theoretical evidence for establishment of CLL-hBMSC interaction model to imitate CLL microenvironment. Mononuclear cells (MNC) were isolated from bone marrow of CLL patients and healthy donors and then were cultured, hBMSC were established by expanding for at least five passages. The mRNA expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), was analyzed by real-time PCR. The mRNA and protein expression of lymphotoxin beta receptor (LTβR) were determined by real-time PCR and Western blot, respectively. The individual NF-κB members at protein level of CLL-hBMSC and N-hBMSC were examined by Western blot. The effect of LTα1β2 on individual NF-κB family members at protein level in CLL-hBMSC and N-hBMSC was also examined by Western blot. The death of CLL cells was determined by flow cytometry with PI staining when cultured with or without CLL-hBMSC and N-hBMSC at different time points. The results showed that the hBMSC could be established successfully from bone marrow of CLL patients, which were similar to N-hBMSC. Adhesion molecules, such as VCAM-1 and ICAM-1, were found to be expressed at similar mRNA levels in CLL-hBMSC and N-hBMSC. LTβR expressions at mRNA and protein levels were comparable between CLL-hBMSC and N-hBMSC. The protein expression of the individual NF-κB family members could be detected in CLL-hBMSC and N-hBMSC with similar expression levels. LTα1β2 stimulation activated both the classical ( RelA/p50 ) and alternative ( RelB/p52 ) NF-κB complexes in CLL-hBMSC and N-hBMSC. The capacities of CLL-hBMSC and N-hBMSC to protect CLL cell survival were similar. It is concluded that there is no statistical difference between bone marrow from healthy donors and CLL patients in the efficiency of generating of hBMSC. LTβR-NF-κB signaling molecules are expressed and activated on hBMSC with a similar pattern.
Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; pathology ; Lymphotoxin beta Receptor ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism ; Transcription Factor RelB ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism