CD20 Antigen Research Articles

Atypical Chronic Lymphocytic Leukemia-The Current Status.

A diagnosis of typical chronic lymphocytic leukemia (CLL) requires the presence of ≥5000 clonal B-lymphocytes/μL, the coexistence of CD19, CD20, CD5, and CD23, the restriction of light chain immunoglobulin, and the lack of expression of antigens CD22 and CD79b. Atypical CLL (aCLL) can be distinguished from typical CLL morphologically and immunophenotypically. Morphologically atypical CLL cells have been defined mainly as large, atypical forms, prolymphocytes, or cleaved cells. However, current aCLL diagnostics rely more on immunophenotypic characteristics rather than atypical morphology. Immunophenotypically, atypical CLL differs from classic CLL in the lack of expression of one or fewer surface antigens, most commonly CD5 and CD23, and the patient does not meet the criteria for a diagnosis of any other B-cell lymphoid malignancy. Morphologically atypical CLL has more aggressive clinical behavior and worse prognosis than classic CLL. Patients with aCLL are more likely to display markers associated with poor prognosis, including trisomy 12, unmutated IGVH, and CD38 expression, compared with classic CLL. However, no standard or commonly accepted criteria exist for differentiating aCLL from classic CLL and the clinical significance of aCLL is still under debate. This review summarizes the current state of knowledge on the morphological, immunophenotypic, and genetic abnormalities of aCLL.

Effectiveness and safety of CD22 and CD19 dual-targeting chimeric antigen receptor T-cell therapy in patients with relapsed or refractory B-cell malignancies: A meta-analysis.

The efficacy of CD22 or CD19 chimeric antigen receptor T (CAR-T) cells in the management of acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) was observed. Because antigen loss and lack of CAR-T-cell persistence are the leading causes of progressive disease following single-antigen targeting, we evaluated CD22/CD19 dual-targeting CAR-T-cell therapy efficacy and safety in relapsed/refractory B-cell malignancies. The Web of Science, PubMed, Cochrane, and Embase databases were searched until July 2022. Patients confirmed with any relapsed/refractory B-cell hematological malignancies were included regardless of age, gender, or ethnicity, receiving CD22 and CD19-dual-targeting CAR-T-cell therapy. The studies conducted on patients with coexisting other cancer were excluded. We used random-effect models to explore the outcome, and heterogeneity was investigated by subgroup analysis. Fourteen studies (405 patients) were included. The pooled overall response (OR) and complete remission (CR) were 97% and 93%, respectively, for ALL patients. The 1-year proportions of overall survival (OS) and progression-free survival (PFS) were 70% and 49%, respectively. For NHL, OR occurred in 85% of patients, and 57% experienced CR. The results illustrated that the 1-year OS and 1-year PFS were 77% and 65%, respectively. The subgroup analysis showed that the dual-targeting modality achieved higher CR in the following cases: coadministration of CD22/CD19-CAR-T cells and third-generation CAR-T cells combined with ASCT and BEAM pretreatment. The ALL and NHL groups seemed similar in treatment-related toxicity: all grade cytokine release syndrome (CRS), severe CRS, and neurotoxicity occurred in 86%, 7%, and 12% of patients, respectively. Our meta-analysis demonstrated that the CD22/CD19 dual-targeting CAR-T-cell strategy has high efficiency with tolerable adverse effects in B-cell malignancies.

The Derivation and Comparative Characterization of Mesenchymal Stem Cell Lines, Isolated from Human Pulp of a Deciduous Tooth of Children of Different Sexes

Two new non-immortalized fibroblast-like cell lines isolated from the pulp of Deciduous Tooth of Children of Different Sexes, named MSC-DP-1 and MSC-DP-2, were derived and characterized. In order to confirm the status of mesenchymal stem cells, a comparative analysis of a some characteristics in these lines was carried out at early and late passages. In the process of long-term cultivation, significant interline differences were revealed in the nature of replicative senescence (RS) and in growth characteristics. The MSC-DP-1 line was characterized by a later entry into the active stage of RS and more active proliferation compared to the MSC-DP-2 line. Karyotypic analysis showed that both lines have a normal human diploid karyotype at early passages. At the late (18th passage), in the stage of active PS, the MSC-DP-2 line also has a normal karyotype. And the MSC-DP-1 line, which enters the active stage of RS much later (at passage 42), has an abnormal karyotype with a large number of clonal chromosomal rearrangements. Both lines showed a high proportion of cells carrying antigens characteristic of human MSCs: CD44, CD73, CD90, CD105, HLA-ABC, and a low frequency of cells with CD34, CD45, and HLA-DR antigens. The cells of the derived lines at an early passage have the ability to differentiate in adipogenic, osteogenic and chondrogenic directions. But the MSC-DP-2 line shows weaker differentiation in the adipogenic direction, than the MSC-DP-1 line. In the process of RS, a significant weakening of adipogenic differentiation takes place in the MSC-DP-1 line, and it disappears in the MSC-DP-2 line. The activity of other differentiations does not change during RS. In general, the derived results confirm the status of MSCs for the derived lines and indicate interline differences in the RS process. However, the derived differences between these lines, as well as the comparison with the previously derived line MSC-DP do not indicate their gender nature. Apparently, they are associated with the genetic characteristics of different donors.

1187 A Study of Outcomes in Patients with Gastrointestinal Tract Lymphomas Who Require Inadvertent Surgery

Abstract Aim Patients may present with acute surgical symptoms from their lymphoma. We aimed to study postoperative outcomes and 3-year overall survival in patients who had inadvertent surgery as primary management of their gastrointestinal tract lymphomas. Method Suitable patients were identified using electronic records from January 2013 to December 2022. Demographics, clinicopathological characteristics and survival outcomes of these patients were collected retrospectively. Results A total of 25 patients were identified. The most common clinical symptom was abdominal pain (n = 18, 72%) with demonstration of an obstruction (n = 16, 64%) or perforation (n = 4, 16%) by a mass on CT scan. Small bowel was the most common site, followed by colon and stomach (55%, 24% and 21% respectively) Diffuse large B-cell lymphoma was the most common subtype (66.7%). Immunohistochemistry revealed B-lymphocyte antigen CD20 to be positive in most patients (n = 17, 68%) and Ki 67% to be high (>70%) in a third (n = 8, 32%). Upon discharge, two thirds of patients were well enough to have systemic chemotherapy (n = 19, 76%). Two patients died from surgical complications while the rest succumbed to disease progression (n = 10, 76%). The overall 3-year survival rate was 56%. Conclusions Patients with gastrointestinal tract lymphoma who do not die in the postoperative period after inadvertent surgery are likely to be alive at 3 years with the use of systemic chemotherapy.

CAR-T cells dual-target CD123 and NKG2DLs to eradicate AML cells and selectively target immunosuppressive cells

ABSTRACT Chimeric antigen receptor (CAR)-T cells have not made significant progress in the treatment of acute myeloid leukemia (AML) in earlyclinical studies. This lack of progress could be attributed in part to the immunosuppressive microenvironment of AML, such as monocyte-like myeloid-derived suppressor cells (M-MDSCs) and alternatively activated macrophages (M2 cells), which can inhibit the antitumor activity of CAR-T cells. Furthermore, AML cells are usually heterogeneous, and single-target CAR-T cells may not be able to eliminate all AML cells, leading to disease relapse. CD123 and NKG2D ligands (NKG2DLs) are commonly used targets for CAR-T therapy of AML, and M-MDSCs and M2 cells express both antigens. We developed dual-targeted CAR-T (123NL CAR-T) cells targeting CD123 and NKG2DL by various structural optimization screens. Our study reveals that 123NL CAR-T cells eradicate AML cells and selectively target immunosuppressive cells. A highly compact marker/suicide gene, RQR8, which binds targeting epitopes of CD34 and CD20 antigens, was also incorporated in front of the CAR structure. The binding of Rituximab to RQR8 leads to the elimination of 123NL CAR-T cells and cessation of their cytotoxicity. In conclusion, we successfully developed dual effects of 123NL CAR-T cells against tumor cells and immunosuppressive cells, which can avoid target escape and resist the effects of immunosuppressive microenvironment.

Analysis of the Selection Signal of the Tibetan Black Chicken Genome Based on Whole-Genome Sequencing.

The Tibetan chicken has adapted well to high altitudes genetically after its long-term habitation in the plateau. In this study, we analyzed the selection signal of Tibetan black chickens (TBCs) and discovered genes associated with the characteristics of germplasm. Whole-genome sequencing (WGS) was used to identify the single-nucleotide polymorphism (SNP) markers and genetic structures in the genome of Tibetan black chickens. Further, we performed a comparative population genomics analysis between the genomic data obtained in this present study and the genomic data for five wild red jungle fowls (RJFs) accessed from the NCBI database (GenBank accession number PRJNA241474). Thereafter, the Fst and Pi selections were used to identify genes under positive selection in the Tibetan black chicken genome. A total of 9,490,690 SNPs were identified in the Tibetan black chickens. In addition, the results from the gene ontology (GO) analysis showed that 732 genes of TBCs were enriched in a total of 210 GO terms with specific molecular functions such as regulation of cellular catabolic process, the MAPK signaling pathway, regulation of ion transport, growth, morphogenesis and lung alveolus development which may provide a better mechanism to facilitate oxygen transport and utilization in TBCs. Moreover, the results from the KEGG analysis showed that 732 genes of the TBCs were significantly enriched in the calcium signaling pathway, circadian entrainment (ADCY1, GNG7 and PER3), oxytocin signaling pathway and pathways of multiple neurodegeneration diseases. In addition, the CD86 antigen (CD86) was identified as a gene associated with the immune response in chickens. It was also revealed that genes such as TRIT1, HPCAL4, NT5C1A and HEYL were discovered under selection in Tibetan black chickens on chromosome 23. These genes may be related to the local adaptive characteristics of Tibetan black chickens, for instance, NT5C1A and HEYL may be involved in the high-altitude adaption of oxygen delivery in Tibetan black chickens. In summary, we found that selection mainly affects the disease resistance and cold acclimatization of Tibetan black chickens. Hence, these results may provide important genetic information for the evolution and breeding of Tibetan black chickens.

The Value of Neutrophil Gelatinase-Associated Lipocalin Receptor as a Novel Partner of CD38 in Chronic Lymphocytic Leukemia: From an Adverse Prognostic Factor to a Potential Pharmacological Target?

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of neoplastic B lymphocytes that escape death, and correlates with the expression of negative prognostic markers such as the CD38 antigen. Although certain new drugs approved by the US Food and Drug Administration improve the clinical outcome of CLL patients, drug resistance and disease relapse still occur. Like CD38, neutrophil gelatinase-associated lipocalin receptor (NGAL-R) is frequently overexpressed in CLL cells. Here, we evaluated the concomitant surface expression of NGAL-R and CD38 in leukemic blood cells from 52 CLL patients (37 untreated, 8 in clinical remission, and 7 relapsed). We provide evidence of a positive correlation between NGAL-R and CD38 levels both in the interpatient cohorts (p < 0.0001) and in individual patients, indicating a constitutive association of NGAL-R and CD38 at the cell level. Patients with progressing CLL showed a time-dependent increase in NGAL-R/CD38 levels. In treated CLL patients who achieved clinical remission, NGAL-R/CD38 levels were decreased, and were significantly lower than in the untreated and relapsed groups (p < 0.02). As NGAL-R and CD38 participate in CLL cell survival, envisioning their simultaneous inhibition with bispecific NGAL-R/CD38 antibodies capable of inducing leukemic cell death might provide therapeutic benefit for CLL patients.

Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices.

Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are "living cell" therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

Mechanisms and Clinical Research Progress of Rituximab in the Treatment of Adult Minimal Change Disease

Introduction: Minimal change disease (MCD) is a common subtype of primary nephrotic syndrome in adults. The pathogenesis of MCD is still not well understood, but some studies suggest that MCD is a T cell-mediated disease related to podocyte dysfunction. Previous research has also indicated the crucial role of B cells in the pathogenesis of MCD. Rituximab (RTX) is a recombinant chimeric mouse/human antibody targeting CD20 antigen. In recent years, RTX has been increasingly used in adult MCD patients. Methodology: We searched the PubMed database using the keywords “Minimal change disease”, “Nephrotic syndrome”, and “Rituximab” and obtained a total of 140 articles. We will now provide a literature review based on these 140 articles, according to our research topic. Discussion: This article provides an overview of the mechanisms and clinical research progress of RTX in the treatment of adult MCD. We have also discussed the current treatment methods for MCD, exploring the potential of using RTX as a first-line therapy for refractory adult MCD. Conclusion: MCD is a common pathological type of nephrotic syndrome, and the exact mechanisms are still not fully understood. Although RTX as a treatment of adult MCD has shown promising clinical results in patients with refractory adult MCD, the safety and efficacy of RTX still lack high-quality clinical evidence. Further research is needed to explore the pathogenesis of MCD and the RTX treatment for MCD.

Identifying Suitable Targets for Alzheimer's Disease and Other Eight Common Neurological Disorders Using the Human Plasma Proteome: A Mendelian Randomization Study.

Neurological disorders, such as Alzheimer's disease (AD), comprise a major cause of health-related disabilities in human. However, biomarkers towards pathogenesis or novel targets are still limited. To identify the causality between plasma proteins and the risk of AD and other eight common neurological diseases using a Mendelian randomization (MR) study. Exposure data were obtained from a genome-wide association study (GWAS) of 2,994 plasma proteins in 3,301 healthy adults, and outcome datasets included GWAS summary statistics of nine neurological disorders. Inverse variance-weighted MR method as the primary analysis was used to estimate causal effects. Higher genetically proxied plasma myeloid cell surface antigen CD33 level was found to be associated with increased risk of AD (odds ratio [OR] 1.079, 95% confidence interval [CI] 1.047-1.112, p = 8.39×10-7). We also discovered the causality between genetically proxied elevated prolactin and higher risk of epilepsy (OR = 1.068, 95% CI = 1.034-1.102; p = 5.46×10-5). Negative associations were identified between cyclin-dependent kinase 8 and ischemic stroke (OR = 0.927, 95% CI = 0.896-0.959, p = 9.32×10-6), between neuralized E3 ubiquitin-protein ligase 1 and migraine (OR = 0.914, 95% CI = 0.878-0.952, p = 1.48×10-5), and between Fc receptor-like protein 4 and multiple sclerosis (MS) (OR = 0.929, 95% CI = 0.897-0.963, p = 4.27×10-5). The findings identified MR-level protein-disease associations for AD, epilepsy, ischemic stroke, migraine, and MS.

The Relationship Between Lymphocyte Subtypes with Clinicopathological Features and Prognosis of Prostate Cancer in Patients Undergoing Radical Prostatectomy.

The immune system has an essential role in the development of cancer by showing both anti-tumor and pro-tumor activities. Understanding the immune func- tion of patients with malignancy is of clinical importance for the evaluation, treatment, and prognosis of the disease. We aimed to evaluate lymphocyte subtypes in peripheral blood samples of prostate cancer patients and their relationship with clinicopathologi- cal features and prognosis. One hundred thirty-seven patients who underwent open radical prosta- tectomy were included in our study. The percentages of CD3+T lymphocyte, CD19+ B lymphocyte, CD16/56 natural killer cells, CD4+ helper T lymphocyte, CD8+ cytotoxic T lymphocyte, and CD45 total lymphocyte were evaluated for each patient using the blood sample taken into a hemogram tube before surgery. The pathological stage was T2 for 64 of the cases and T3 for 73. The mean follow-up period of the patients was 12.81 ± 6.20 months. The CD3+/CD4+ counts of the patients with pathological stage T2 were found to be statistically significantly higher than stage T3. There was a statistically significant negative correlation between the prostate-specific antigen levels and CD3+/CD4+ percentages of the patients. There was no statistical significance between the percentages of lymphocyte subtypes and the presence of surgical margin, biochemical recurrence, adjuvant therapy, and cancer upgrade. We consider that the increase in the pathological stage and prostate-spe- cific antigen value and the decrease in the number of CD4+ T lymphocyte subtypes may be prognostic markers in prostate cancer patients.

Selection of a novel cell-internalizing RNA aptamer specific for CD22 antigen in B cell acute lymphoblastic leukemia

Despite improvements in B cell acute lymphoblastic leukemia (B-ALL) treatment, a significant number of patients experience relapse of the disease, resulting in poor prognosis and high mortality. One of the drawbacks of current B-ALL treatments is the high toxicity associated with the non-specificity of chemotherapeutic drugs. Targeted therapy is an appealing strategy to treat B-ALL to mitigate these toxic off-target effects. One such target is the B cell surface protein CD22. The restricted expression of CD22 on the B-cell lineage and its ligand-induced internalizing properties make it an attractive target in cases of B cell malignancies. To target B-ALL and the CD22 protein, we performed cell internalization SELEX (Systematic Evolution of Ligands by EXponential enrichment) followed by molecular docking to identify internalizing aptamers specific for B-ALL cells that bind the CD22 cell-surface receptor. We identified two RNA aptamers, B-ALL1 and B-ALL2, that target human malignant B cells, with B-ALL1 the first documented RNA aptamer interacting with the CD22 antigen. These B-ALL-specific aptamers represent an important first step toward developing novel targeted therapies for B cell malignancy treatments.

Bictegravir, emtricitabine, and tenofovir alafenamide versus dolutegravir, emtricitabine, and tenofovir disoproxil fumarate for initial treatment of HIV-1 and hepatitis B coinfection (ALLIANCE): a double-blind, multicentre, randomised controlled, phase 3 non-inferiority trial

For most adults with HIV-1 and hepatitis B virus (HBV) coinfection, initial recommended treatment is a tenofovir-containing antiretroviral regimen, but no randomised studies have compared tenofovir disoproxil fumarate with tenofovir alafenamide. We aimed to investigate whether bictegravir, emtricitabine, and tenofovir alafenamide is non-inferior to dolutegravir, emtricitabine, and tenofovir disoproxil fumarate for viral suppression in individuals with HIV-1 and HBV coinfection at 48 and 96 weeks. We did this randomised, double-blind, active-controlled, phase 3, non-inferiority trial at 46 outpatient centres in China, Dominican Republic, Hong Kong, Japan, Malaysia, South Korea, Spain, Taiwan, Thailand, Turkey, and the USA. Eligible participants were treatment-naive adults (aged ≥18 years) with plasma HIV-1 RNA of at least 500 copies per mL and plasma HBV DNA of at least 2000 IU/mL. Participants were randomly assigned (1:1) to receive daily oral bictegravir 50 mg, emtricitabine 200 mg, and tenofovir alafenamide 25 mg, or dolutegravir 50 mg, emtricitabine 200 mg, and tenofovir disoproxil fumarate 300 mg, each with corresponding matching placebo. Randomisation was stratified by hepatitis B e antigen (HBeAg) status (positive vs negative), HBV DNA (<8 vs ≥8 log10 IU/mL), and CD4 count (<50 vs ≥50 cells per μL) at screening. All investigators, participants, and staff providing treatment, assessing outcomes, and collecting data were masked to study treatment for 96 weeks. Coprimary endpoints were the proportion of participants with plasma HIV-1 RNA less than 50 copies per mL (defined by the US Food and Drug Administration snapshot algorithm) and plasma HBV DNA less than 29 IU/mL (using the missing-equals-failure approach) at week 48, with a prespecified non-inferiority margin of -12%. Coprimary endpoints were assessed in the full analysis set, which included all randomly assigned participants who received at least one dose of study drug and had at least one post-baseline HIV-1 RNA or HBV DNA result while on study drug. Safety endpoints were assessed in all randomly assigned participants who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, NCT03547908. Between May 30, 2018 and March 16, 2021, 381 participants were screened, of whom 243 initiated treatment (121 in the receive bictegravir, emtricitabine, and tenofovir alafenamide group; 122 in the dolutegravir, emtricitabine, and tenofovir disoproxil fumarate group). At week 48, both endpoints met the criteria for non-inferiority: 113 (95%) of 119 participants in the bictegravir, emtricitabine, and tenofovir alafenamide group and 111 (91%) of 122 participants in the dolutegravir, emtricitabine, and tenofovir disoproxil fumarate group had HIV-1 RNA less than 50 copies per mL (difference 4·1, 95% CI -2·5 to 10·8; p=0·21), and 75 (63%) of 119 participants in the bictegravir, emtricitabine, and tenofovir alafenamide group versus 53 (43%) of 122 participants in the dolutegravir, emtricitabine, and tenofovir disoproxil fumarate group had HBV DNA suppression (difference 16·6, 5·9 to 27·3; nominal p=0·0023). Drug-related adverse events up to week 96 occurred in 35 (29%) of 121 participants in the bictegravir, emtricitabine, and tenofovir alafenamide group and 34 (28%) of 122 participants in the dolutegravir, emtricitabine, and tenofovir disoproxil fumarate group. One (1%) of 121 participants in the bictegravir, emtricitabine, and tenofovir alafenamide group reported a serious adverse event (cryptococcal meningitis attributed to immune reconstitution inflammatory syndrome) that was deemed to be treatment-related. Coformulated bictegravir, emtricitabine, and tenofovir alafenamide is an effective therapy for adults with HIV-1 and HBV coinfection starting antiviral therapy. Gilead Sciences.

Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR Tcells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR Tcell activity to prevent incoming graft rejection. Transient expression of an anti-CD117 CAR by mRNA conferred Tcells the ability to eliminate CD117+ targets invitro and invivo. As an alternative approach, we used a Sleeping Beauty transposon vector for the generation of CAR Tcells incorporating an inducible Caspase 9 safety switch. Stable CAR expression was associated with high proportion of T memory stem cells, low levels of exhaustion markers, and potent cellular cytotoxicity. Anti-CD117 CAR Tcells mediated depletion of leukemic cells and healthy hematopoietic stem cells in NSG mice reconstituted with human leukemia or CD34+ cord blood cells, respectively, and could be terminated invivo. The use of a non-viral technology to control CAR Tcell pharmacokinetic properties is attractive for a first-in-human study in patients with acute myeloid leukemia prior to hematopoietic stem cell transplantation.

CD26 CAR-T cells have attenuated mitochondrial and glycolytic metabolic profiling

BackgroundMultiple targets of chimeric antigen receptor T cells (CAR-T cells) are shared expressed by tumor cells and T cells, these self-antigens may stimulate CAR-T cells continuously during the expansion. Persistent exposure to antigens is considered to cause metabolic reprogramming of T cells and the metabolic profiling is critical in determining the cell fate and effector function of CAR-T cells. However, whether the stimulation of self-antigens during CAR-T cell generation could remodel the metabolic profiling is unclear. In this study, we aim to investigate the metabolic characteristics of CD26 CAR-T cells, which expressed CD26 antigens themselves.MethodsThe mitochondrial biogenesis of CD26 and CD19 CAR-T cells during expansion was evaluated by the mitochondrial content, mitochondrial DNA copy numbers and genes involved in mitochondrial regulation. The metabolic profiling was investigated by the ATP production, mitochondrial quality and the expression of metabolism-related genes. Furthermore, we assessed the phenotypes of CAR-T cells through memory-related markers.ResultsWe reported that CD26 CAR-T cells had elevated mitochondrial biogenesis, ATP production and oxidative phosphorylation at early expansion stage. However, the mitochondrial biogenesis, mitochondrial quality, oxidative phosphorylation and glycolytic activity were all weakened at later expansion stage. On the contrary, CD19 CAR-T cells did not exhibit such characteristics.ConclusionCD26 CAR-T cells showed distinctive metabolic profiling during expansion that was extremely unfavorable to cell persistence and function. These findings may provide new insights for the optimization of CD26 CAR-T cells in terms of metabolism.

Update on diagnosis and treatment of Chronic Lymphocytic Leukemia (CLL)

commonest leukemia found in adults western countries, affects more than 200000 people and is associated with approximately 4410 death in the USA annually. CLL is characterized by a progressive proliferation and accumulation of mature yet functionally incompetent lymphocytes. The disease typically occurs in elderly patients and has a highly variable clinical course. Leukemic transformation is initiated by specific genomic alterations that impair apoptosis of clonal B-cells. Diagnosis: The diagnosis of chronic lymphocytic leukemia (CLL) requires the presence of more than or equal to 5 × 109/L B lymphocytes (5000/μL) in the peripheral blood for the duration of at least 3 months. Immunophenotyping of circulating B-lymphocytes, which identify a clonal B-cell population carrying the CD5 antigen, as well as typical B-cell markers are confirmatory test. Prognosis: The two similar clinical staging systems, Binet and Rai stages, create prognostic information by using results of physical examination and blood counts. Various molecular biology markers also have prognostic value. Mutations of the TP53 gene and x chromosome 17 chromosome (del [17p) predict resistance to chemoimmunotherapy and a shorter time to progression, with most targeted therapies. A comprehensive, international prognostic score (CLL-IPI) integrates genetic, biological and clinical variables to identify distinct risk groups of CLL patients. Therapy: Early-stage disease of CLL patients are followed up regularly, only patients with active or symptomatic disease or with advanced stages of Binet or Rai require treatment. When treatment is indicated, most patients with CLL have several options: a combination of obinutuzumab and venetoclax, ibrutinib monotherapy, or immunochemotherapy. In patients under 65 years of age who are physically fit (particularly if they have a mutated IGVH gene), immunochemotherapy with fludarabine, cyclophosphamide, and rituximab remains the standard of care because of its potential therapeutic potential. In case of relapse, the initial treatment can be repeated if the treatment-free period exceeds 3 years. And in case of early recurrence of the disease, treatment should be switched to an alternative regime. Patients with del(17p) or TP53 mutations are another high-risk group and should be treated with targeted drugs. Allogeneic SCT can be considered in patients with TP53 or del(17p) mutations or in patients on inhibitor therapy.

The “Mystery Cell Population” Residing in Murine Bone Marrow – A Missing Link Between Very Small Embryonic Like Stem Cells and Hematopoietic Stem Cells?

Bone marrow (BM) contains not only hematopoietic stem cells (HSCs) but also some very rare, early development, small quiescent stem cells that, upon activation, may differentiate across germlines. These small cells, named very small embryonic like stem cells (VSELs), can undergo specification into several types of cells including HSCs. Interestingly, murine BM is also home to a “mystery” population of small CD45+ stem cells with many of the phenotypic characteristics attributed to resting HSCs. Since the size of the “mystery” population cells are between that of VSELs and HSCs, and because CD45− VSELs can be specified into CD45+ HSCs, we hypothesized that the quiescent CD45+ “mystery” population could be a missing developmental link between VSELs and HSCs. To support this hypothesis, we showed that VSELs first became enriched for HSCs after acquiring expression of the CD45 antigen already expressed on “mystery” stem cells. Moreover, VSELs freshly isolated from BM similar to the “mystery” population cells, are quiescent and do not reveal hematopoietic potential in in vitro and in vivo assays. However, we noticed that CD45+ “mystery” population cells, similar to CD45− VSELs, became specified into HSCs after co-culture over OP9 stroma. We also found that mRNA for Oct-4, a pluripotency marker that is highly expressed in VSELs, is also detectable in the “mystery” population cells, albeit at a much lower level. Finally, we determined that the “mystery” population cells specified over OP9 stroma support were able to engraft and establish hematopoietic chimerism in lethally irradiated recipients. Based on these results, we propose that the murine BM “mystery” population could be an intermediate population between BM-residing VSELs and HSCs already specified for lympho-hematopoietic lineages.Graphical

Results of the compassionate program of inotuzumab ozogamicin for adult patients with relapsed or refractory acute lymphoblastic leukemia in Spain.

The prognosis of relapsed B cell precursor acute lymphoblastic leukemia (B-ALL) is poor and few patients can be successfully rescued with conventional therapies. Inotuzumab ozogamicin (IO), an antibody against the CD22 antigen linked to calicheamicin, has been approved as a rescue treatment in relapsed/refractory (R/R) B-ALL. This was an observational, retrospective, multicenter study of adult patients included in the Spanish program of compassionate use of IO in centers from the PETHEMA group (Programa Español de Tratamientos en Hematología). Thirty-four patients with a median age of 43 years (range, 19-73) were included. Twenty patients (59%) were refractory to the last treatment, IO treatment was given as ≥3rd salvage treatment in 25 patients (73%) and 20 patients (59%) received allogeneic hematopoietic stem cell transplantation before IO treatment. After a median of 2 cycles of IO, 64% of patients achieved complete response (CR)/complete response with incomplete recovery. The median response duration, progression-free survival and overall survival (OS) were 4.7 (95%CI, 2.4-7.0 months), 3.5 (95%CI, 1.0-5.0 months) and 4 months (95%CI, 1.9-6.1 months) respectively, with better OS for patients with relapsed B-ALL versus refractory disease (10.4 vs. 2.5 months, respectively) (p = .01). There was a trend for better OS for patients with first CR duration >12 months (7.2 months [95%CI, 3.2-11.2] vs. 3 months [95% CI, 1.8-4.2] respectively) (p = .054). There was no sinusoidal obstruction syndrome (SOS) event during IO treatment, but three patients (9%) developed grade 3-4 SOS during alloHSCT after IO treatment. Our study showed slightly inferior outcomes of the pivotal trial probably due to poorer risk factors and late onset of IO therapy of recruited patients. Our results support early use of IO in relapsed/refractory ALL patients.

Disseminated Extranodal NK/T-Cell lymphoma, Presenting as an Orbital Mass.

An 84-year-old male patient presented with decreased vision of 20/300 in the right eye, with additional painless upper eyelid fullness, hypoglobus, severe limitation of ocular motility, and conjunctival chemosis of the right eye for several months duration (panel A). He was referred after failing to respond to a trial of oral corticosteroids for presumed orbital myositis. MRI revealed an enlarged enhancing lacrimal gland (arrow), extraocular muscles, and mucosal thickening of the ethmoid sinus (arrowhead) on the right side (panel B). Lacrimal gland biopsy demonstrated medium-sized atypical lymphocytes with irregular nuclear contours, scant-to-moderate amounts of cytoplasm, and brisk mitotic figures (arrow) (panel C, hematoxylin-eosin, ×630), which expressed cytotoxic and natural killer-cell markers CD56 (panel D, ×400), perforin, and T-cell intracellular antigen 1, and were CD4-positive and CD8-negative. Neoplastic cells were positive for Epstein–Barr virus-encoded RNA by in situ hybridization (panel E, ×400). Clinical, morphologic, and immunophenotypic findings were compatible with extranodal natural killer/T-cell lymphoma (natural killer/T-cell lymphoma, nasal type). Optical coherence tomography revealed diffuse choroidal thickening with an undulating anterior border (sea-sick choroid) (arrow), compatible with choroidal involvement by lymphoma (panel F). Positron emission tomography scan showed significantly increased activity in the regional and distal lymph nodes and in multiple bones, compatible with disseminated disease. The patient succumbed to disease shortly after diagnosis. This case highlights the importance of prompt and accurate diagnosis of natural killer/T-cell lymphoma, nasal type, which can present with orbital and ocular involvement simulating an inflammatory process.FIG

P-352 Evaluation of CD25 and CD69 activation markers expression on peripheral blood cells subpopulations in endometriosis patients

Abstract Study question We aim to determine the percentages of CD25-positive and CD69-positive lymphocytes in peripheral blood of endometriosis (EMS) patients. Summary answer Higher percentages of lymphocytes expressing CD69 and CD25 markers in EMS and their correlation with the severity of the disease, indicate persistent activation of lymphocytes What is known already Exposure to antigens causes activation markers to appear on the surface of lymphocytes. Among them, we can distinguish early and late activation markers. The earliest one is CD69, which is expressed upon activation by the TCR receptor. CD69 plays a role in the proliferation and survival of activated T lymphocytes. CD25 is a moderate late activation marker, considered the most important marker of cellular response activation. The significance of CD69 and CD25 expression by T cells in endometriosis has not yet been determined. Increased CD69 expression on various lymphocyte subsets in peritoneal fluid in EMS has been reported. Study design, size, duration Within this prospective study between January 2016 and August 2018 immune diagnostics of the number of CD25-positive and CD69-positive lymphocytes in peripheral blood of EMS patients (firstly diagnosed during laparoscopy and confirmed histopatologically) and non-EMS controls were performed using flow cytometry analysis. Total number of 74 subjects were included in the study. All patients signed written informed consent before their enrollment in the study. Participants/materials, setting, methods We enrolled 54 subjects with previously untreated endometriosis and 20 healthy age-matched controls. Peripheral blood was collected from control patients to assess the immunophenotype of lymphocytes and measure expression of activation markers Cd25 and CD69 on several subtypes B and T lymphocytes. Diagnosis and assessment of the stage of the EMS was established during laparoscopy using rASRM score. Differences were considered statistically significant with a p &amp;lt; 0.05. Main results and the role of chance Significantly higher expression of the CD25 and CD69 antigen was found in both CD3+ T, CD4+ T and CD8+ T cells as well as CD19+ B cells. In each of the studied groups, a significance level of p &amp;lt; 0.001 was obtained. There was a weak positive correlation between the percentage of CD4+CD25+ T cells and the stage of endometriosis (R = 0.357; p = 0.008) and negative correlations between the percentage of endometriosis and the percentage of CD3+CD69+ T cells (R = −0.554; p &amp;lt; 0.001), T CD4+CD69 + (R = −0.554, p &amp;lt; 0.001) and T CD8+CD69 + (R = −0.553, p &amp;lt; 0.001). The expression of activation markers, CD25 and CD69 antigens in patients suffering from endometriosis with accompanying clinical symptoms was also evaluated. n the group of patients with endometriosis accompanied by infertility or pelvic pain, no statistical differences in the expression of CD25 and CD69 antigens were observed. The only clinical condition coexisting with statistically different expression of activation markers is adhesion disease accompanying endometriosis - a statistically significantly lower percentages of T CD3+CD69 + (p = 0.002), T CD4+CD69 + (p = 0.002) and T CD8+CD69 + (p = 0.002). Limitations, reasons for caution Small sample size is an important limitation of this study. In addition, evaluating immunological analysis only in serum samples does not let us drive any conclusions on the local changes of endometriosis lesions. Wider implications of the findings Clinical interpretation of changed expression of CD69 and CD25 in EMS could be useful in evaluating mechanisms of cellular activation, peripheral tolerance and immune imbalance involved in pathogenesis of endometriosis. Further studies to understand mechanisms underlying correlation between stage of the disease and expression of CD69 should be considered. Trial registration number not applicable