[Immune Reconstitution after BTKi Treatment in Chronic Lymphocytic Leukemia].

OBJECTIVE: To analyze the immune reconstitution after BTKi treatment in patients with chronic lymphocytic leukemia (CLL). METHODS: The clinical and laboratorial data of 59 CLL patients admitted from January 2017 to March 2022 in Fujian Medical University Union Hospital were collected and analyzed retrospectively. RESULTS: The median age of 59 CLL patients was 60.5(36-78). After one year of BTKi treatment, the CLL clones (CD5 +/CD19 +) of 51 cases (86.4%) were significantly reduced, in which the number of cloned-B cells decreased significantly from (46±6.1)×109/L to (2.3±0.4)×109/L (P =0.0013). But there was no significant change in the number of non-cloned B cells (CD19 + minus CD5 +/CD19 +). After BTKi treatment, IgA increased significantly from (0.75±0.09)g/L to (1.31±0.1)g/L (P <0.001), while IgG and IgM decreased from (8.1±0.2)g/L and (0.52±0.6)g/L to (7.1±0.1)g/L and (0.47±0.1)g/L, respectively (P <0.001, P =0.002). BTKi treatment resulted in a significant change in T cell subpopulation of CLL patients, which manifested as both a decrease in total number of T cells from (2.1±0.1)×109/L to (1.6±0.4)×109/L and NK/T cells from (0.11±0.1)×109/L to (0.07±0.01)×109/L (P =0.042, P =0.038), both an increase in number of CD4 + cells from (0.15±6.1)×109/L to (0.19±0.4)×109/L and CD8 + cells from (0.27±0.01)×109/L to (0.41±0.08)×109/L (both P <0.001). BTKi treatment also up-regulated the expression of interleukin (IL)-2 while down-regulated IL-4 and interferon (IFN)-γ. However, the expression of IL-6, IL-10, and tumor necrosis factor (TNF)-α did not change significantly. BTKi treatment could also restored the diversity of TCR and BCR in CLL patients, especially obviously in those patients with complete remission (CR) than those with partial remission (PR). Before and after BTKi treatment, Shannon index of TCR in patients with CR was 0.02±0.008 and 0.14±0.001 (P <0.001), while in patients with PR was 0.01±0.03 and 0.05±0.02 (P >0.05), respectively. Shannon index of BCR in patients with CR was 0.19±0.003 and 0.33±0.15 (P <0.001), while in patients with PR was 0.15±0.009 and 0.23±0.18 (P <0.05), respectively. CONCLUSIONS: BTKi treatment can shrink the clone size in CLL patients, promote the expression of IgA, increase the number of functional T cells, and regulate the secretion of cytokines such as IL-2, IL-4, and IFN-γ. BTKi also promote the recovery of diversity of TCR and BCR. BTKi treatment contributes to the reconstitution of immune function in CLL patients.

The novel HLA-DQB1*04:93 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-DQB1*04:93 differs from HLA-DQB1*04:01:01:01 by one nucleotide in exon 2.

SENP2 exerts an anti­tumor effect on chronic lymphocytic leukemia cells through the inhibition of the Notch and NF­κB signaling pathways.

Chronic lymphocytic leukemia (CLL) is one of the most often diagnosed hematological malignant tumors in the Western world and a type of inert B­cell lymphoma that commonly attacks the elderly. Small ubiquitin related modifier (SUMO)­specific protease 2 (SENP2) can act as a suppressor in various types of cancer by regulating the stability of ß­catenin to affect the Notch signaling pathway; however, it has a low expression level in CLL cells. In this study, we firstly used western blot analysis and RT­qPCR to detect the protein and mRNA expression levels of SENP2 in the peripheral blood of patients with CLL and healthy volunteers. Secondly, we overexpressed or knocked down the expression of SENP2 in CLL cells and then determined the cell invasive and chemotactic ability in a Transwell assay and chemotaxis assay. We examined the sensitivity of the cells to cytarabine and dexamethasone via a CCK­8 assay and determined the cell apoptotic condition and the expression of the Notch signaling pathway using flow cytometry and western blot analysis. The results demonstrated that the patients with CLL had relatively low expression levels of SENP2. The overexpression of SENP2 in the CLL cells decreased their invasive and proliferative ability, as well as their chemotactic response and enhanced their sensitivity to cytarabine and dexamethasone, while it promoted cell apoptosis. The silencing of SENP2 in the CLL cells generally produced the opposite results. We thus hypothesized that the overexpression of SENP2 downregulated ß­catenin expression, thus inhibiting the Notch signaling pathway in CLL cells. Moreover, the nuclear factor (NF)­κB signaling pathway was also regulated by the overexpression of SENP2. On the whole, the findings of this study indicate tha SENP2 can act as a tumor suppressor in CLL cells, and may thus prove to be a novel target for CLL treatment in clinical practice.

[Recent Research Advances on Signaling Pathway in B-cell Malignancy - Review].

B-cell malignancy, a kind of malignant tumor of blood system, is characterized by heterogeneity and complexity of the pathogenesis. In this review, the recent advances of studies on B-cell malignancy and signaling pathways are briefly summarized. This review focuses on the role of signaling pathways, especially the NF-κ B, Wnt, PI3K/Akt, Notch and JAK/STAT signaling pathway in the occurrence and development of B-cell malignancy, and discussed the influence of signaling pathways on the invasiveness and drug resistance of B-cell malignancy, and the clinical application of signaling pathway inhibitors.

[Expression of Notch Gene and Its Role of Anti-apoptosis and Drug-resistance of Cells in Chronic Lymphocytic Leukemia].

OBJECTIVE: To investigate the expression of Notch gene in chronic lymphocytic leukemia cells and to explore the change of Notch protein after the therapy with cytosine arabinoside or dexmethasone, and the mechanism of Notch mediated anti-apoptosis and drug-resistance in chronic lymphocytic leukemia cells. METHODS: The mononuclear cells from bone marrow or peripheral blood of chronic lymphocytic leukemia patients (24 cases) and healthy donors (14 cases) were collected, then the expression of Notch gene, BCL-2, as well as NF-κB gene were detected by real-time fluorescent quantitative PCR (qRT-PCR) at the level of transcription. The change of Notch protein in L1210 cell lines after therapy with cytosine arabinoside and dexmethasone was determined by Western blot. RESULTS: mRNA expression levels of Notch1, Notch2, BCL-2 and NF-κB gene in CLL group were significantly higher than those in healthy control group (0.8556 ± 0.8726 vs 0.6731 ± 0.5334, P = 0.0182; 1.2273 ± 0.8207 vs 0.6577 ± 0.6424, P < 0.0001; 8.0960 ± 7.5661 vs 0.5969 ± 0.4976, P < 0.0001; 1.0966 ± 0.6925 vs 0.5373 ± 0.7180, P < 0.0001, respectively), but no significant difference was found between Notch3 and Notch4 gene (1.1914 ± 2.4219 vs 0.8713 ± 0.7937, P = 0.3427; 0.8174 ± 1.0869 vs 0.9752 ± 1.3446, P = 0.2402, respectively). Notch1 protein expression in L1210 cells were significantly decreased after treating with cytosine arabinoside of low and middle concentrations, but increased after treating with cytosine arabinoside of high concentration or prolonging time of cytosine arabinoside of middle con-centration. Notch1 protein expression in L1210 cells dereased after treating with dexamethasone, but did not be changed with the different concentrations and different times of dexmethason. CONCLUSION: The transcription level of Notch gene in CLL patients significantly higher than that in normal controls. The Notch1 protein expression is down-regulated in process of inhibiting L1210 cell proliferation by Ara-C and dexmethason. Notch signaling pathway may mediated anti-apoptosis and drug resistance of CLL cells. Notch molecule possibly plays an important role in the anti-apoptosis and drug-resistance of CLL cells.

Constitutive activation of NF-κB signaling by NOTCH1 mutations in chronic lymphocytic leukemia.

NOTCH1 mutations occur in approximately 10% of patients with chronic lymphocytic leukemia (CLL). However, the relationship between the genetic aberrations and tumor cell drug resistance or disease progression remains unclear. Frameshift deletions were detected by gene sequencing in the NOTCH1 PEST domain in three naive CLL patients. These mutations were associated with chromosomal abnormalities including trisomy 12 or 13q deletion. Of note, one of the patients developed Richter's transformation during FCR treatment. Immunofluorescent and western blot analyses revealed a markedly higher intracellular domain of NOTCH (ICN) expression in the mutated cells compared with their unmutated counterparts and normal CD19+ B lymphocytes (P<0.01 and P<0.001, respectively). In addition, strong DNA-κB binding activities were observed in the mutant cells by gel shift assays. RT-PCR analysis revealed elevated RelA mRNA expression in the mutant cells, while RelB levels were variable. Reduced levels of RelA and RelB mRNA were observed in unmutated CLL and normal B cells. Compared to unmutated CLL and normal B cells, increased apoptosis occurred in the mutant cells in the presence of GSI (ICN inhibitor) and PDTC (NF-κB inhibitor), particularly under the synergistic effects of the two drugs (P=0.03). Moreover, IKKα and IKKß, the active components in the NF-κB pathway, were markedly inhibited following prolonged treatment with GSI and PDTC. These results suggested that NOTCH1 mutations constitutively activate the NF-κB signaling pathway in CLL, which is likely related to ICN overexpression, indicating NOTCH1 and NF-κB as potential therapeutic targets in the treatment of CLL.

[Notch signal pathway and chronic lymphocytic leukemia].

Chronic lymphocytic leukemia (CLL), an indolent B-cell malignancy, is characterized by heterogeneity of the clinical course. Notch signaling pathway is an evolutionarily conserved signaling pathway and involved in the normal regulation of cell survival, proliferation, differentiation, apoptosis and other physiological processes. In recent years, more and more researchers study the relationship between Notch signaling pathway and chronic lymphocytic leukemia and have found that Notch molecules present in CLL cells with high expression or mutation, which associated with the prognosis, anti-apoptosis, drug-resistance and so on. In this article, the recent advances of studies on CLL and Notch pathway, including the expression level of Notch molecules in CLL cells, the anti-apoptosis and drug-resistance of Notch molecules in CLL cells, the mutation of Notch molecules in CLL cells, the relation of Notch molecules with CLL prognosis and the application prospect of Notch molecule inhibitors are reviewed.

[Clinical analysis on 40 patients with chronic lymphocytic leukemia].

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum ß2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum ß2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.

[A case of Richter syndrome transformed from chronic lymphocytic leukemia with karyotype aberration of trisomy 12].

This study was aimed to investigate the relationship between Richter's syndrome (RS) transformation and clinical characteristics as well as karyotype of patient with chronic lymphocytic leukemia (CLL). By the follow-up of a patient with CLL, the clinical characteristics, karyotype, treatment pattern and its effect, as well as disease progression were monitored regularly with serological test, flow cytometry and FISH technique. The results indicated that the patient typically presented with history of CLL at initial diagnosis, with expression of CD5(+), CD19(+) and CD23(+), Binet stage C, as well as karyotype aberration of trisomy 12, and poorly responded to 4 cycles of standard chemotherapy of FCR regimen. The disease progression was confirmed at 5 months with the symptoms of fever in the absence of infection, elevated lactate dehydrogenase level and rapidly enlarging lymphnodes which showed typically diffuse large B cell lymphoma by the biopsy. It is concluded that karyotype aberration of trisomy 12 is one of the risk factors for RS transformation, and treatment pattern of the patient with CLL may be associated with the transformation of RS.

Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.

BACKGROUND AND OBJECTIVES: Adult bone marrow (BM) is the major source of mesenchymal stem cells (MSC) for cell therapy. However, aspiration of BM involves invasive procedures. We isolated MSC from human full term umbilical cord tissues (UC). The biological characteristics of MSC derived from UC (UC-MSC) were further determined and compared with normal adult bone marrow-derived MSC (BM-MSC). DESIGN AND METHODS: MSC were isolated from UC by enzyme digestion and cultured in appropriate growth medium. The isolation efficiency, cell yield, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, multi-lineage differentiation capacity, cytokine spectrum as well as hematopoiesis-supportive function of UC-MSC were determined and compared with those of BM-MSC. RESULTS: MSC were successfully isolated from all 36 UC and six BM samples we collected for this study. The mean number of nucleated cells isolated from UC was 1yen106/cm and the yield of adherent cells was 8.6yen105/cm. UC-MSC shared most of the characteristic of BM-MSC, including fibroblastic-like morphology, immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials, and hematopoiesis-supportive function. The CFU-F frequency was higher in UC nucleated cells (1:1609 +/- 0.18) than in BM nucleated cells (1:35700 +/- 0.01) (p < 0.05). Furthermore, in comparison with BM-MSC, the UC-MSC had a higher proliferation capacity and lower levels of expression of CD106 and HLA-ABC (p < 0.05). Immunofluoresent and western blot assays revealed that UC-MSC had a higher percentage of neuron specific enolase-positive cells than had BM-MSC after neuronal induction. Finally, reverse transcriptase polymerase chain reaction analysis showed that UC-MSC had a cytokine spectrum very similar to that of BM-MSC, including expression of the mRNA of stem cell factor, leukemia inhibitor factor, macrophage-colony stimulating factor, Flt3-ligand, interleukin-6, vascular endothelial growth factor and stromal-derived factor-1, but UC-MCS additionally expressed mRNA of granulocyte macrophage and granulocyte colony-stimulating factors. After co-culture with CD34+ cord blood cells for 5 weeks, no significant difference in colony-forming cells was observed between the CD34+ cells/UC-MSC and CD34+ cells/BM-MSC co-cultures (p > 0.05). INTERPRETATION AND CONCLUSIONS: We have established a protocol to isolate abundant MSC from human umbilical cords with a 100% success rate. The comparative study indicates that UC is an excellent alternative to BM as a source of MSC for cell therapies.

Bone marrow stromal cells transduced with human hemangiopoietin gene support hematopoiesis in vitro.

BACKGROUND AND OBJECTIVES: The aim of this study was to construct a eukaryotic expression vector containing human hemangiopoietin (hHAPO) gene and express it in mouse bone marrow stromal cell line HESS-5, then support hematopoiesis in vitro with gene-modified HESS-5 (hHAPO-HESS-5). DESIGN AND METHODS: The polymerase chain reaction (PCR) products of HAPO were digested with BamHI and BgII. Then the HAPO gene segment obtained was again cloned into pIRES2-EGFP to construct recombinant eukaryotic expression vector HAPO-pIRES2-EGFP. The recombinant vector was identified by enzyme digestion analysis, PCR, and sequencing. HESS-5 cells were transformed by recombinant vector and positive clones were selected with G418. The expression of HAPO gene in the transformed cells was detected by studying EGFP expression, reverse transcription (RT)-PCR, and Western-blotting analysis. Support of human hematopoiesis by hHAPO-HESS-5 cells was evaluated in co-culture experiments with human CD34+ cells. RESULTS: Enzyme digestion analysis and sequencing showed that the target gene had been cloned into the recombinant vector. The expression of HAPO gene in the transformed stromal cells was demonstrated by fluoro-microscopy and RT-PCR analysis. HAPO protein was also detected in the supernatant of hHAPO-HESS-5 by Western blot analysis. As expected, stably transfected hHAPO-HESS-5 cells significantly increased in both relative and absolute numbers of CD34+ cells after 14 days of culture. The PKH26 study demonstrated that cell division was faster in CD34+ cells co-cultured with hHAPO-HESS-5 cells than in cells cocultured with vector-HESS-5 cells. The hHAPO-HESS-5 cells also supported human hematopoiesis in vitro more efficiently than did control vector-HESS-5 cells. INTERPRETATION AND CONCLUSIONS: A recombinant eukaryotic expression vector has been constructed and expressed successfully in transformed cells. The hHAPO-HESS-5 cells support rapid generation of primitive progenitor cells and maintain reconstituting ability of hematopoietic stem cells in vitro. Therefore, it would be possible to use stromal cells expressing HAPO gene as seed cells in the bone marrow transplantation.