Expression Level of Membrane Markers CD5, CD19, and CD20 in B Cells after UV-Irradiation and Incubation in the Presence of Autologous Plasma

Abstract Background: Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by abnormal immune cell activation. This study aimed to investigate differentially expressed long non-coding RNA (lncRNA) in peripheral blood mononuclear cells (PBMCs) in patients with pSS to identify lncRNAs that affect pSS pathogenesis. Methods: Total RNA was extrated from PBMCs of 30 patients with pSS and 15 healthy persons. Transcriptome sequencing was used to screen differentially expressed lncRNAs and mRNAs in 8 RNA samples from the discovery cohort. The differentially expressed mRNAs underwent functional enrichment analysis. A protein interaction relationship (PPI) and ceRNA network was constructed. Real-time PCR was used to validate screened lncRNAs in all 45 RNA samples. Results: 1180 lncRNAs and 640 mRNAs were differentially expressed in pSS patients (fold change > 2 in healthy persons). The PPI network was constructed with 640 mRNAs and a ceRNA network with four key lncRNAs (GABPB1-AS1, PSMA3-AS1, LINC00847 and SNHG1). RT-PCR revealed that GABPB1-AS1 and PSMA3-AS1 were significantly upregulated 3.0-and 1.4-fold in the pSS group, respectively. The GABPB1-AS1 expression level was positively correlated with the percentage of B cells and IgG levels. Conclusions: GABPB1-AS1 was significently upregulated in pSS patients, and its expression level is positively correlated with the percentage of B cells and IgG levels. GABPB1-AS1 may be involved in the pathogenesis of pSS.

Download Full-text

Immune Cell Landscape in Gastric Cancer

BioMed Research International ◽

10.1155/2021/1930706 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Yang Yang ◽

Wei He ◽

Zi-rui Wang ◽

Yu-jiao Wang ◽

Lan-lan Li ◽

...

Keyword(s):

Gastric Cancer ◽

B Cells ◽

Immune Cells ◽

Immune Cell ◽

Univariate Analysis ◽

Molecular Therapy ◽

Expression Level ◽

Regulatory Factors ◽

Normal Tissues ◽

The Relationship

Background. The tumor-infiltrating immune cells are closely associated with the prognosis of gastric cancer (GC). This article is aimed at determining the composition change of immune cells and immune regulatory factors in GC and normal tissues, depicting their prognosis value in GC, and revealing the relationship between them and GC clinical parameters. Methods. We used CIBERSORT to calculate the proportion of 22 immune cells in the GC or normal tissues; a t -test was applied to assess the expression difference of immune cells and immune regulatory factors in normal and GC tissues. The relationship of the immune cells, immune regulatory factors, and GC patients’ clinical characteristics was assessed by univariate analysis. Results. In this study, we found that the proportion of macrophages increased, while plasma cells and monocytes decreased in GC tissues. In these immune fractions, Tregs and naïve B cells were found to be correlated with GC patients’ prognosis. Interestingly, the expression of immune regulatory factors was ambiguous with their classical function in GC tissues. For example, TIM-3, FOXP3, and CMTM6 were overexpressed, while CD27 and PD-1 were underexpressed in GC tissues. We also found that IDO1, PD-1, TIGIT, and TIM-3 were highly expressed in high-grade GC tissues, the HERC2 expression level was related to patients’ gender, and the TIGIT expression level was sensitive to targeted therapy. Furthermore, our results suggested that the infiltration of Tregs and naive B cells was strongly correlated with the T stage, radiation therapy, targeted molecular therapy, and the expression levels of TIM-3 and FOXP3 in GC. Conclusion. The expression pattern of tumor-infiltrating immune cells and immune regulatory factors was systematically depicted in the GC tumor microenvironment, indicating that individualized treatment based on the tumor-infiltrating immune cells and immune regulatory factors may be beneficial to GC patients.

Download Full-text

Role of Ts-RNAs in CLL

Blood ◽

10.1182/blood.v128.22.2016.2016 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2016-2016

Author(s):

Veronica Balatti ◽

Alexey Palamarchuk ◽

Lara Rizzotto ◽

Dario Veneziano ◽

Giovanni Nigita ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Research Funding ◽

Prognostic Markers ◽

Clinical Course ◽

Differentially Expressed ◽

Expression Level ◽

Transcriptional Level ◽

Indolent Disease ◽

Aggressive Clinical Course

Abstract Chronic lymphocytic leukemia (CLL) is the most common human leukemia. This disease occurs in two forms, aggressive and indolent, both characterized by clonal expansion of CD5+ B-lymphocytes. Hallmarks of aggressive CLL are the high expression level of ZAP-70, the unmutated sequence of IgVH genes, and activation of the TCL1 oncogene. While investigating whether the expression of miR-3676/4521 cluster in CLL is correlated with TCL1 regulation and ZAP-70 methylation in aggressive CLLs, we found that these two microRNAs are associated with tRNA sequences and that this region produces two small RNAs, members of a novel class of small non-coding RNAs (tsRNAs). Ts-3676/4521 cluster is located on the short arm of chromosome 17 and is co-deleted with p53 in 17p deleted CLL patients. We previously demonstrated that ts-3676 and ts-4521 are down-regulated and mutated in all CLL types. Furthermore, ts-3676 targets TCL1 3'UTR, indicating that tsRNAs can interfere with gene expression at a post-transcriptional level in a micoRNA-like manner. However, tsRNAs do not share the biogenesis mechanism of microRNAs but are cleaved from the 3' ends of pre-tRNAs by the endonuclease RNase Z and represent unique sequences starting at the 3' ends of the tRNAs and ending at a sequence of 4 consecutive T nucleotides. Thus tsRNAs are single stranded RNAs, more similar to piRNAs than miRNAs. Since piRNAs interact with Piwi proteins to affect gene methylation, we hypothesized that tsRNAs could also interact with Piwi proteins. By performing a series of RNA immunoprecipitation experiments, we verified the interaction of ts-3676 and ts-4521 with Piwi proteins, suggesting that these molecules could be involved in gene promoter methylation, hence affecting gene expression at a pre-transcriptional level in a piRNA-like manner. Since ts-3676 and ts-4521 are deregulated in CLLs, we studied if other tsRNAs are differentially regulated CLL. We retrieved the tsRNAs sequences from 3' ends of tRNA genes, obtaining a total of 120 tsRNA sequences, and designed a tsRNA microarray chip to investigate signatures of tsRNAs that can distinguish different classes of CLLs. We hybridized total RNAs extracted from 23 CLL samples (11 indolent, 12 aggressive) and 8 CD19+ B cells of healthy donors and found 17 tsRNA differentially expressed in these cohorts of samples. 9 tsRNAs are differentially expressed in aggressive CLL vs. normal B-cells, 10 tsRNAs are differentially expressed in indolent CLL vs. normal B-cells, 15 tsRNAs are differentially expressed in indolent vs. aggressive CLL. In these signatures, we identified ts-46 and ts-47 strongly downregulated in aggressive CLL, suggesting these ts-RNAs as potential tumor-suppressors. Finally, we found that ts-3676 and ts-4521 have a prognostic value for particular subgroups of CLLs. Patients with low ZAP-70 expression and mutated IgVH generally show indolent disease, while patients with high ZAP-70 expression and un-mutated IgVH show aggressive disease. However, a relatively small cohort of patients shows aggressive prognostic markers but clinically indolent disease. Our analysis revealed that expression of ts-3676 is lower in aggressive CLLs vs indolent CLLs, although, we did not test these tsRNAs in cohort of patients with aggressive prognostic markers but indolent disease. Currently there is no good marker to discriminate these patients from aggressive CLLs. Thus, we selected 8 patients with unfavorable prognostic markers but indolent disease and 12 patients with both aggressive clinical course and unfavorable prognosis markers, to test for differences in the expression level of ts-3676 and ts-4521. We found that in patients with unfavorable prognostic markers but indolent disease, expression of ts-3676 and ts-4521 was almost double of that in patients displaying both aggressive clinical course and markers. In conclusion, we provided an evidence that tsRNAs deregulation can affect gene expression by interfering with the epigenetic machinery and/or with the post-transcriptional stability of target genes. We also identified the first tsRNAs important in CLL as new markers and/or targets for therapy: ts-3676, ts-4521, ts-46 and ts-47. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau.

Download Full-text

Sequence Level Analysis of Hodgkin Lymphoma Clonotypes Detected in Peripheral Blood Using a Next-Generation Sequencing Approach

Blood ◽

10.1182/blood.v124.21.1610.1610 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1610-1610

Author(s):

Yasuhiro Oki ◽

Sattva S. Neelapu ◽

Michelle Fanale ◽

Larry W. Kwak ◽

Luis Fayad ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Expression Level ◽

Employment Equity ◽

Tumor Biopsy ◽

Cellular Compartment ◽

Equity Ownership ◽

Level Analysis

Abstract Background: Classical Hodgkin lymphoma (CHL) has been established as B-cell malignancy characterized by a clonal expansion of pathognomonic Reed Sternberg cells (Marafioti et al. Blood 2000). A previous report suggests that clonotypic B-cells may be present in the blood of patients with CHL; however, the relationship between these circulating clonotypic B-cells and CHL is unclear. We utilized the LymphoSIGHT™ method, a next-generation sequencing approach, to detect lymphoma-specific clonotypes in peripheral blood in patients with CHL at diagnosis or disease recurrence as well as in follow-up blood samples. This method has the sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood, and has been applied to minimal residual disease (MRD) detection in multiple B-cell malignancies. We evaluated the extent of somatic hypermutation in the lymphoma clonotypes, and performed sequence and expression level analyses of the lymphoma clonotype repertoire. Methods: Frozen primary tumor biopsy samples were first analyzed for clonality at the immunoglobulin heavy chain (IGH) and kappa chain (IGK) loci using the LymphoSIGHT method. Rearranged immunoglobulin gene segments (IGH-VDJ, IGH-DJ and IGK) in the genomic DNA and/or RNA were amplified with locus-specific primer sets, sequenced, and analyzed using standardized algorithms for clonotype determination. Clonotypes with a frequency >5% in the B-cell repertoire of the tumor biopsy were considered to represent tumor clonotypes. IGH-VDJ clonotypes with a frequency >2% in DNA were deemed to be cancer-specific if the clonotype was not present in the RNA. Such clonotypes were then quantitated in serum and peripheral blood mononuclear cells (PBMC), and DNA sequence and/or RNA expression level analysis was performed. Results: A total of 17 CHL patients were analyzed. A high-frequency clonal rearrangement was identified for at least one receptor (IGH-VDJ, IGH-DJ and IGK) in 12 of 17 cases (71%). Lymphoma-specific clonotypes were detected in blood samples from 8 of 11 patients (73%). Notably, a lymphoma-specific clonotype was detected in the serum compartment in 8 of 9 cases (89%) tested (Figure 1A), while it was detected in PBMC only in 3 of 9 cases (33%) tested (Figure 1B). Follow-up samples obtained from three patients in remission were negative for the tumor-specific clonotype in both the serum and cellular compartments. We conducted sequence and expression level analysis of each IGH-VDJ clonal rearrangement. We calculated the number of somatic mutations in each lymphoma-specific clonotype compared to the germline sequence in the interrogated region. In the ten patients with IGH-VDJ clonal rearrangements, we observed a median of 14 somatic mutations (range 0 to 27). This confirms that HRS cells correspond in their developmental stage to germinal or post-germinal center B-cells. While IGH-VDJ clonotypes were observed frequently in DNA obtained at diagnosis, IGH-VDJ clonotypes were not detected in the RNA from the same sample. We evaluated the relationship between the presence of lymphoma-specific clonotypes in the cellular compartment at diagnosis and eventual progression. All three untreated patients that were positive at baseline in the cellular compartment experienced relapse or progression (at 3, 11 and 17 months). In contrast, zero of 5 patients without detectable lymphoma-specific clonotypes in their cellular compartment at baseline experienced relapse (follow up duration 23-45 months, log-rank test p=0.004). Conclusions: This is the first clinical assay that can be used to detect and monitor MRD in CHL. Lymphoma-specific sequences can be identified in serum in 80% of cases. Our preliminary analysis suggests that the presence of lymphoma-specific clonotypes in PBMCs may indicate high risk for recurrence. This study demonstrates proof-of-principle and underscores the promise of a new methodology to measure disease burden and provide prognostic information from a blood test in patients with CHL. Figure 1. Lymphoma clonotype levels in A) cell-free plasma and B) PBMC samples are shown for the different patients. Figure 1. Lymphoma clonotype levels in A) cell-free plasma and B) PBMC samples are shown for the different patients. Figure 2 Figure 2. Disclosures Klinger: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Kong:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Differences in the C13orf25 miRNA Cluster in Non-Hodgkin Lymphoma and Normal B-Cell Subtypes.

Blood ◽

10.1182/blood.v110.11.3586.3586 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3586-3586

Author(s):

Jan-Lukas Robertus ◽

Miao Wang ◽

Geert Harms ◽

Tjasso Blokzijl ◽

Rikst Nynke Schakel ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Hodgkin Lymphoma ◽

Cell Lymphoma ◽

Expression Patterns ◽

Expression Level ◽

Expression Levels ◽

Qrt Pcr ◽

Non Hodgkin Lymphoma ◽

B Cell Subsets

Abstract Introduction Amplification of chromosome 13q31 is a frequent occurrence in lymphoma and solid tumors. The C13orf25 gene at 13q31.3 is the primary miRNA transcript for seven miRNAs. This specific cluster of miRNAs is sequentially related to the homologous miR-106a-92 cluster on chromosome X and the miR-106b-25 cluster on chromosome 7. The miR17-92 cluster has been shown to be over expressed in various non-Hodgkin Lymphoma (NHL). As a specific group of miRNAs which are derived from the same primary miRNA transcript, each miRNA in the cluster may be expected to show a similar expression level. However expression patterns vary markedly in different cancers. Aim of study To compare the expression pattern of the C13orf25 gene and the miRNAs in normal and malignant B-cells. Methods and Materials 51 cases of Ann Abour stage I and II primary diffuse large B-cell lymphoma (DLBCL) were collected together with 29 cases of B-CLL. All samples were examined for expression of miRNA miR-18a, -19, -20a, 17-3p, -17-5p and -92 and the C13orf 25 gene. Expression levels of the mature miRNAs were determined by qRT-PCR using Taqman miRNA assays. The level of C13orf25 was also determined by qRT-PCR using random primers and a Sybergreen probe. Results were compared by using 2−ΔCt and 2−ΔΔCt. Normal B-cell subpopulations were isolated from fresh tonsils obtained during routine pediatric tonsillectomies. B-cell subsets were stained accordingly and sorted by FACS. Results Comparison of the miRNA pattern (2−ΔCt) revealed that DLBCL have the highest expression level of miR-19b and that B-CLL shows a relative high expression level of miR-92. Normal naïve, germinal center and memory B-cells all show a similar expression pattern with miR-92 having the highest expression level. Remarkably a low expression level of miR-19b is seen in all three B-cell populations in contrast to DLBCL. Comparing the relative expression levels (2−ΔΔCt) of both NHL for each individual miRNA shows that B-CLL has a significantly higher level of expression for miR-19a. In DLBCL miR-17-5p, miR-18a and miR-20a have the highest expression levels. Currently 20 Mantle cell lymphoma are also being analyzed for the C13orf25 miRNAs cluster. Conclusion Our results show that both of the NHL B-cell malignancies and the B-cell subsets have different expression patterns of the individual levels of the 7 miRNAs contained in the same C13orf25 cluster. The relative expression levels of certain miRNAs from the same primary transcript are different in various NHL. Although there are no marked differences in the normal B-cell subsets for the C13orf25 cluster, further profiling revealed various different expression levels of other miRNAs. These findings may point towards a difference in processing efficiency or stability of miRNAs in the C13orf25 cluster leading to differences in pathogenesis specific for each malignancy even in cells of similar origin and differentiation stage.

Download Full-text

Chromosomal Abnormalities in Bone Marrow Lymphocytes of MM Patients

Blood ◽

10.1182/blood.v116.21.4045.4045 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4045-4045

Author(s):

Carina S. Debes Marun ◽

Andrew Belch ◽

Linda M. Pilarski

Keyword(s):

Bone Marrow ◽

B Cells ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Disease Process ◽

Clonal Expansion ◽

Scanning System ◽

Autologous Plasma ◽

Immunodeficient Mice ◽

The Impact

Abstract Abstract 4045 Multiple Myeloma (MM) is characterized by accumulations of chromosomal abnormalities throughout the course of disease. Recurrent IgH translocations such as t(4;14) or deletion (del)13 occur during early stages of disease or perhaps as originating events. Other abnormalities appear to be acquired late in the disease process, after development of frank MM, including p53 deletion (Ch17) and amplification of CKS-1B (Ch1). We have previously identified drug resistant clonotypic B lymphocytes in MM that have malignant characteristics and can xenograft MM to immunodeficient mice. Using an automatic scanning system we scanned bone marrow (BM) cytospin slides stained with May-Grünwald Giemsa to identify lymphocytes and to determine whether or not they share the same chromosomal abnormalities that are found in autologous plasma cells (PC). For 200 MM patients, we performed interphase FISH using commercial probes, to detect deletion of chromosome 13, any translocations in 14q32, t(4;14)(p16;q32),t(11;14)(q13;q32), deletion of p53 and a custom probe to detect amplification of CKS-1B. For patients harboring a given chromosomal abnormality in their PC, we found the identical abnormalities in BM lymphocytes for 35% of patients having del13, 30% having t(4;14) and 23% having t(11;14). Likewise we detected amplification of locus 4p16, 11q13, 14q32, trisomy 1 and trisomy 17 in 45%, 15%, 30%, 37.5% and 44% respectively in the lymphocytes of patients demonstrating those same abnormalities in the PC. In a more limited series of experiments using immuno-FISH, we observed that BM lymphocytes with chromosomal abnormalities expressed CD20. In contrast, amplification of CKS-1B or p53deletion were undetectable in lymphocytes from patients having these abnormalities in their PC. We are currently analyzing the impact of abnormalities present in BM lymphocytes with regards to the clinical parameters and survival. An update of these results will be given at the meeting. Given the compelling evidence that B cells comprise a clinically important compartment of the MM clone, this work suggests that fundamental genetic events shared by all compartments of the MM clone include recurrent IgH translocations and Ch13 deletions. However, further clonal expansion occurring during disease progression and after relapse may be restricted to the PC population, characterized by acquisition of p53 deletions and CSK1B amplification. Furthermore in overt MM, clonal expansion may simultaneously occur on at least two levels, firstly that of the B cells which persistently give rise to PC, and secondly that of PC themselves as they continue clonal expansion in the absence of contributions from the B cells. Abnormalities such as del13 or IgH translocations characterize early events in the disease while p53del or amplification of CKS-1B may occur as progression events restricted to PC. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CLLU1 Gene Expression Level As a Prognostic Parameter in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.4603.4603 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4603-4603

Author(s):

Mustafa Sevinc ◽

Ahmet Emre Eskazan ◽

Aydin Karabulut ◽

Suzin Catal Tatonyan ◽

Emine Gulturk ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Clinical Course ◽

Lymphocytic Leukemia ◽

Expression Level ◽

Time To Treatment ◽

Prognostic Parameter ◽

Β2 Microglobulin ◽

Staging Systems ◽

The Impact

Abstract Abstract 4603 Background Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the Western world with an annual incidence of 5/100,000. The clinical course of the disease is highly variable; while some CLL patients experience a stable clinical course that will never affect their morbidity or mortality, some of them will eventually progress and require chemotherapy. In addition to the traditional prognostic markers (e.g. Rai and Binet staging systems), more recently, mutational status of the variable regions of the immunoglobulin heavy chains (IgVH), chromosomal aberrations, CD38 expression and Zeta-chain-associated protein kinase 70 (ZAP-70) expression are used to better determine the prognosis in CLL patients. A novel CLL-specific gene, CLL Up-regulated gene 1 (CLLU1) that uniquely overexpressed in CLL patients, was recently demonstrated. It has been shown that CLLU1 mRNA expression levels in CLL patients predict time to initiation of therapy as well as overall survival (OS), and CLLU1 is highly up-regulated in poor-risk groups. The aim of this study is to investigate the relationship between CLLU1 levels and well known prognostic parameters and, to determine the importance of CLLU1 gene on prognosis and clinical course in our CLL patients. Methods 116 (46 female, 70 male) CLL patients who consecutively visited our outpatient clinic between May 2009 and March 2010 were enrolled in the study. Median age was 60 years (range, 30–87 years). Blood samples were drawn from the patients for CLLU1 determination, and CLLU1 levels were determined by RT-PCR method. CLLU1 expression level was counted both in CLL patients and healthy B cells as the difference between CLLU1 and abl (taken as an house keeping gene) gene. Then, they are transformed as folds which is the ratio between CLLU1 level in CLL patients and that in healthy B cells. Patients with CLLU1 expression exceeding the CLLU1 expression of normal (CD19+) B-cells were taken as positive. Each patient was followed for at least one year for survival data. For the statistical analysis, student’s t-test, Mann-Whitney U test and Pearson correlation were used. p<0.05 was considered as statistical significant. The study was approved by the local research ethics committee, and written informed consent was obtained from the patients. Results There was no relationship between CLLU1 levels and, sex, age, modified RAI and BINET stages, lymphocyte counts and LDH levels at the time of diagnosis. Patients with nodular bone marrow infiltration had lower CLLU1 levels than patients with non-nodular infiltration (57.6 vs 498). Patients with high β2 microglobulin levels had higher CLLU1 levels than the ones with low β2 microglobulin levels (356.7 vs 13.6, p<0.05). ZAP-70 positive patients had higher CLLU1 levels than ZAP-70 negative patients (217.2 vs 10.2, p=0.007) (Figure 1). Among the patients with CD38 levels studied (n=53) CLLU1 levels were higher in patients with CD38 levels above the median value than patients with CD38 levels below the median value (438.4 vs 42.2). CLLU1 levels were higher in cases who needed treatment than cases without treatment. Patients with a shorter time to treatment had higher CLLU1 levels than patients with a longer time to treatment (p=0.028) (Figure 2). Conclusion With a limited number of patients we could demonstrate that CLLU1 levels correlated with β2 microglobulin levels and ZAP-70. Although there were similar findings also with CD38 levels; the association was not statistically significant due to the limited number of cases. Time to treatment was shorter in patients with CLLU1 levels above the median value than patients with CLLU1 levels below the median value. CLLU1 is a promising and specific new prognostic parameter in patients with CLL, and further studies in larger series are needed to define the impact of CLLU1 in the prognosis and clinical course of CLL patients. Disclosure This study was supported by Istanbul University Research Fund. Disclosures: No relevant conflicts of interest to declare.

Download Full-text