Percentage Of Peripheral Blood Research Articles

Correlation of gasdermin B staining patterns with prognosis, progression, and immune response in colorectal cancer

BackgroundPyroptosis is a type of programmed cell death mediated by the gasdermin family. Gasdermin B (GSDMB), as a member of gasdermin family, can promote the occurrence of cell pyroptosis. However, the correlations of the GSDMB expression in colorectal cancer with clinicopathological predictors, immune microenvironment, and prognosis are unclear.MethodsSpecimens from 267 colorectal cancer cases were analyzed by immunohistochemistry to determine GSDMB expression, CD3+, CD4+, and CD8+ T lymphocytes, CD20+ B lymphocytes, CD68+ macrophages, and S100A8+ immune cells. GSDMB expression in cancer cells was scored in the membrane, cytoplasm, and nucleus respectively. GSDMB+ immune cell density was calculated. Univariate and multivariate survival analyses were performed. The association of GSDMB expression with other clinicopathological variables and immune cells were also analyzed. Double immunofluorescence was used to identify the nature of GSDMB+ immune cells. Cytotoxicity assays and sensitivity assays were performed to detect the sensitivity of cells to 5-fluorouracil.ResultsMultivariate survival analysis showed that cytoplasmic GSDMB expression was an independent favorable prognostic indicator. Patients with positive cytoplasmic or nuclear GSDMB expression would benefit from 5-fluorouracil based chemotherapy. The assays in vitro showed that high GSDMB expression enhanced the sensitivity of colorectal cancer cells to 5-fluorouracil. Patients with positive membranous or nuclear GSDMB expression had more abundant S100A8+ immune cells in the tumor invasive front. Positive nuclear GSDMB expression indicated more CD68+ macrophages in the tumor microenvironment. Moreover, GSDMB+ immune cell density in the stroma was associated with a higher neutrophil percentage but a lower lymphocyte counts and monocyte percentage in peripheral blood. Furthermore, the results of double immunofluorescence showed that GSDMB co-expressed with CD68 or S100A8 in stroma cells.ConclusionThe GSDMB staining patterns are linked to its role in cancer progression, the immune microenvironment, systemic inflammatory response, chemotherapeutic efficacy, and prognosis. Colorectal cancer cells with high GSDMB expression are more sensitive to 5-fluorouracil. However, GSDMB expression in immune cells has different effects on cancer progression from that in cancer cells.

CD38 gene polymorphism rs1130169 contribution to the increased gene expression and risk of colorectal cancer (pilot study).

Genetic variations in immune signaling genes may have regulatory effect on phenotypic heterogeneity of immune cells and immune functions, hence promoting tumor growth. We compared the frequencies of potentially functional CD38 gene single nucleotide polymorphisms rs1130169 (T > C) in 86 healthy controls and 90 colorectal cancer (CRC) cases to assess their association with cancer risk and CD38 gene expression. The association between allele C rs1130169 and CRC risk was observed. Allele C was also significantly correlated with an increased CD38 mRNA level and CD38 positive cell percentages in peripheral blood of healthy controls that could be a possible explanation for CRC risk in C allele carriers. In peripheral blood of CRC patients CD38 mRNA and serum soluble CD38 protein levels significantly differed from those in healthy controls. Calculation of the CD38 full-length and with the third exon deletion mRNA ratio in corresponding samples showed that the mRNA isoform ratio was significantly higher in CRC cases than in controls. It suggests that alternative splicing regulates elevation of CD38 full-length mRNA level in peripheral blood of CRC patients. We also have observed higher expression levels of CD38 full-length mRNA in peripheral blood of CRC patients with lymph node metastases compared to patients without metastases. This study indicated biological significance of rs1130169 variations that can alter differences in CRC risk by regulating CD38 gene expression.

Prognostic value of peripheral blood and bone marrow infiltration assessed by flow cytometry in dogs with de novo nodal peripheral T-cell lymphoma receiving alkylating-rich chemotherapy

Peripheral T-cell lymphoma (PTCL) is highly aggressive in dogs and demonstrates a poor response to traditional chemotherapy. The aim of this retrospective study was to assess the prognostic significance of peripheral blood (PB) and bone marrow (BM) infiltration evaluated by flow cytometry (FC) in dogs with treatment-naïve and histologically confirmed PTCL. To be included, dogs had to undergo complete staging, including FC on lymph nodes, PB and BM samples. Additionally, dogs had to receive an alkylating-rich protocol and have a complete follow-up. Treatment response was evaluated based on RECIST criteria at each chemotherapy session, and the end-staging was conducted at the completion of treatment. Endpoints were time to progression (TTP) and lymphoma-specific survival (LSS). The relationship between TTP/LSS and the percentage of PB and BM infiltration, categorized as > 1%, > 3%, > 5%, > 10%, > 15% and > 20% was investigated.Fifty dogs were included: based on imaging and FC, 78.0% had stage 5 disease, 14.0% had stage 4, 6.0% had stage 3 and 2.0% had stage 1. By multivariable analysis, the CD4-negative phenotype was the only factor associated with a shorter TTP (P = 0.049), while BM infiltration was significantly associated with LSS (P = 0.037). Dogs with BM infiltration > 5% had shorter median LSS (114 days; 95%CI: 0–240) compared to dogs with BM infiltration ≤ 5% (178 days; 95%CI: 145–211). Lack of complete response (P = 0.039) and administration of corticosteroids before chemotherapy (P = 0.026) also significantly worsened LSS. BM flow cytometric evaluation could be considered an essential part of staging work-up for dogs with PTCL and has prognostic relevance.

Phase I First-in-Human Study on the Feasibility, Safety, and Antitumor Activity of CD4-Redirected Chimeric Antigen Receptor (CAR) T-Cell Therapy in Patients with CD4+ T-Cell Malignancies (TCM)

Background TCM are aggressive diseases with poor outcomes. Translating the CAR T-cell therapy success from B-cell diseases to TCM has proven challenging. CD4 is an attractive therapeutic target, owing to its restricted expression on normal tissues. In this first-in-human phase I study, we investigate the autologous third generation CD4-redirected CAR T-cell safety, tolerability, manufacturing feasibility, trafficking and preliminary efficacy in patients with R/R CD4+ TCM who failed standard therapies. Methods The investigational construct is engineered with a single-chain variable fragment (ScFV) and dual co-stimulators (CD28; 4-1BB), fused to CD3zeta and CD8 leader sequence, packaged in lentivirus and transducer into T-cells. Patients received conditioning therapy with fludarabine and cyclophosphamide. CD4CAR product is administered in a 3+3 dose-escalation scheme. Dose-limiting toxicities (DLT) were monitored during the initial 42-days post-treatment. Treatment-emergent adverse events (AE) were graded by CTCAE v5.0 Results Three patients with median age of 63 years (range, 18-68) were enrolled and treated at DL1 (2.0x10^5/kg), including 2 (66%) women and 2 (66%) African-American. Median number of prior therapies was 3 (range, 2-4) (Figure). AEs included grade 3-4 hematologic toxicity in 3 (100%) patients, all present before enrollment. There was no protocol defined DLTs. All grade ≥3 lymphopenia reverted to grade 2 by day 30 and no related infections occurred between CAR infusion and hematopoietic stem cell transplantation (HSCT). Since infusion, the CAR T-cells percentage in peripheral blood had continued to expand. CD4CAR T-cells were detectable in all patients for at least 28 days post-infusion, meeting the primary endpoint, and on D111 in one patient. CD4CAR expansion was reflected on by a decrease in CD4/CD8 ratio and flow cytometry using ScFV Fab2 specific antibodies (Figure). Cytokine response analysis CD4CAR was associated with variable but significant production that seems to correlate with clinical responses (Figure; More data at meeting). No cytokine-mediated organ toxicities were observed. Bone marrow and peripheral blood flow cytometry confirmed complete remission (CR) in 2 patients at day 30 (PTCL and T-ALL). Patient 3 (mycosis fungoides) achieved hematological CR with stable skin lesions. Post-treatment day 30 skin biopsy demonstrated persistent disease with marked loss of CD4. Conclusions CD4CAR T-cell therapy is feasible in patients with R/R CD4+ TCM. 2/3 patients achieved CR and the third achieved hematological CR with stable skin disease. Toxicities were manageable without DLT upon completion of cohort 1. The cytokine response suggests immune activation and tumor recognition by CD4CAR T-cells. Dose escalation will proceed. NCT03829540

Clinical Features and Outcomes in Large Granular Lymphocyte Leukemia-Associated Pure Red Cell Aplasia with STAT3 Mutation

Background Large granular lymphocyte leukemia (LGLL) is a rare disease frequently complicated with pure red cell aplasia (PRCA) [1, 2]. STAT3 mutations have been describled in 30-40% of LGLL patients [3]. The aim of this work was to evaluate whether STAT3 mutations might be associated with specific clinical features and outcomes in LGLL-associated PRCA. Methods and results From January 2016 to July 2023, 81 patients with LGLL-associated PRCA were enrolled in the China Eastern Cooperation Group for Anemia (CECGA) database (ChiCTR2100043485). As an initial step in the STAT3 mutational analysis, we screened all patients with Sanger sequencing or Next-generation sequencing (NGS). The initial dose of CsA was 3-5mg/kg/day, and the serum concentration was adjusted to be 150-200ng/mL based on adverse reactions. After 12 months of maintenance, the dosage was slowly reduced. Cyclophosphamide combined with prednisone (CP) regimen was used as a salvage therapy for patients who failed to CsA. The initial dose of cyclophosphamide and prednisone were 100mg/day and 0.5-1mg/kg/day, respectively. Among the 81 cases, 26% of them were positive for STAT3 mutations. The clinical characteristics of patients with or without STAT3 mutation were summarized in Table 1. Patients with STAT3 mutations had a higher reticulocyte percentage (0.88% vs 0.28%, P=0.039) and red cell distribution width-coefficient of variation (18.8% vs 15.8%, P=0.008) than patients without STAT3 mutations. Y640F mutation were found in 9 of 21 cases. Subgroup analysis showed that patients with Y640F mutation were associated with younger age (44 vs 65 years old, P=0.007) and higer lymphocyte percentage in peripheral blood (63.7% vs 34.4%, P=0.033). Deep sequencing of rearranged T-cell receptor Vβ complementarity-determining region 3 by NGS was conducted in 61 patients. The predominant V-gene of LGLL clonotypes belonged to the TRBV06 family gene was detected in 12 (25%) patients. The expression of TRBV06 family gene was a litter lower in patients with STAT3 mutation than non-mutant groups (8% vs 31%, P=0.189). The complete response rate (CRR) [31% (5/16) vs 33% (19/58), P=0.909] and overall response rate (ORR) [56% (9/16) vs 50% (29/58), P=0.658] of cyclosporine (CsA) treatment were similar in patients with STAT3 mutations or not. In STAT3 mutant group, the CRR [54% (7/13) vs 31% (5/16), P=0.274] and ORR [85% (11/13) vs 56% (9/16), P=0.130] of CP regimen tended to be better than CsA . 6 patients (67%) relapsed among the 9 patients who responded to CsA in STAT3 mutant group. In patients without STAT3 mutation, 19 of 29 (66%) CsA-responders relapsed. There was no siginificant difference in the relapse-free survival between the two group (Figure 1). Discussion/Conclusions In summary, STAT3 mutation was frequently recognized in LGLL-associated PRCA, and the hotspot site was Y640F. Patients with STAT3 mutations responded to CsA as well as those without the mutation. CP regimen could be used as a salvage therapy for patients who failed to CsA. Reference 1. Balasubramanian SK, Sadaps M, Thota S, et al. Rational management approach to pure red cell aplasia. Haematologica. 2018, 103(2): 221-230. 2. Wu X, Cheng L, Liu X, et al. Clinical characteristics and outcomes of 100 adult patients with pure red cell aplasia. Ann Hematol. 2022, 101(7):1493-1498. 3. Barilà G, Teramo A, Calabretto G, et al. Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients. Leukemia. 2020, 34: 1116-1124.

Acute Myeloid Leukemia with Inv(3) or t(3;3): A Clinical and Cytogenetic Characterization of 40 Patients

Background: Acute myeloid leukemia (AML) with inv(3) or t(3;3) is an extremely aggressive and rare subtype of AML. Leukemogenesis in inv(3) AML is in part due to juxtaposition of the GATA2 enhancer in close proximity to the MECOM/EVI1 promoter; as the result, the fusion drives EVI1 overexpression and uncontrolled proliferation of myeloid progenitors. Given its rarity, published clinical outcome data are limited. To improve our understanding of the clinical outcomes of inv(3) AML, we conducted a retrospective study to describe the clinical features of a cohort of newly diagnosed inv(3)/t(3;3) AML patients treated at Stanford Cancer Center between 2002 and 2023. Methods: Data were retrospectively collected from databases associated with the Stanford Health Care electronic health record (EHR), and diagnosis dates came from the Stanford Cancer Registry. Inv(3)/t(3;3) AML diagnosis was defined according to WHO criteria as the presence of ≥20% blasts in the bone marrow (BM) or peripheral blood (PB) and the presence of inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2) on cytogenetic analysis at initial diagnosis. Response to first induction, salvage, and consolidation therapy as well as allogeneic hematopoietic stem cell transplant (alloHSCT) were captured. CR was defined as the presence of <5% of blasts in the BM evaluation by morphological evaluation after each line of treatment. Overall survival (OS) was defined as time from diagnosis to death from any cause. A descriptive study of the main demographic, clinical, hematologic, and cytogenetic variables of patients who received intensive and non-intensive therapy was performed. The analysis was performed using R 4.3.1. A p value of < 0.05 was defined to be statistically significant. Results: A total of 40 patients met inclusion criteria including 68% (n=27) with de novo AML, 25% (n=10) with secondary AML from an antecedent hematological disorder and 7.5% (n=3) therapy-related AML. Clinical characteristics of the patients are summarized in Figure 1A. The median age at time of diagnosis was 56 (range, 19-78). There was a slight female predominance, with 58% (n=23) female and 42% (n=17) males. Ninety-three percent of patients had an ECOG score ≤1 (n=37). Median leukocyte, hemoglobin, and platelet counts were 4.5 K/uL, [1.3-761], 9.4 g/dL, [3.4-13.6], and 98 K/uL [9-592], respectively. The median blast percentage in BM and PB was 49% [13-98] and 30% [0-95], respectively. Inv(3)/t(3;3) was the only cytogenetic aberration in 10 patients (25%) and chromosome (chr) 7 abnormalities were the most common additional aberration, present in 22 patients (55%). CR after the first induction therapy was achieved in 9 patients (22%); 6 patients (15%) died during or shortly after induction therapy, and the remaining 25 (63%) were resistant. Salvage therapies for patients included re-induction with 7+3, FLAG-Ida, mitoxantrone-containing regimens, clofarabine-based protocols, or combination of hypomethylating agents with venetoclax. Seventeen (43%) patients underwent alloHSCT (10 in CR and 7 with active disease). When comparing intensive vs non-intensive induction regimen, no statistical significance was identified for complete remission (CR) rate (p= 1.00), overall survival (OS) rate (p=0.65) or relapse-free survival (RFS) rate (p=0.86). Similarly, no statistically significant prognostic factors (age, WBC, BM blast %, platelet count, hemoglobin, ANC, or time to transplant) for OS were identified. Interestingly, chr 7 abnormalities in inv(3) patients conferred a worse prognosis in this cohort (OS 41% vs. 59%, p= 0.0064, Figure 1B). This association was confirmed using a multiple Cox proportional hazards regression adjusting for age and blast percentage. Moreover, while chr 7 abnormality was also associated with a lower response rate (CR 14% vs. 40%), it did not reach statistical significance (p=0.12). Conclusions: In summary, de novo AML inv(3)/t(3;3) is a rare and aggressive subtype of AML conferring extremely poor prognosis. There appears to be an increase of incidence in females affecting a slightly younger demographic. Consistent with previous studies, the presence of chr 7 abnormalities was associated with even worse outcomes, which interestingly also drive EVI1 overexpression. The exact link between 3q26 loci rearrangements and chr 7 abnormalities warrants further investigation.

T-regulatory Cells in Acute Leukemia and their Relation with Induction Outcome

Abstract Background: The leukemic microenvironment has become an area of interest for researchers in recent times. Nonmalignant T and B lymphocytes form a significant part of leukemic microenvironment and are postulated to have an effect on its progression and behavior. These lymphocytes are constantly interacting with blasts and are actively involved in innate and acquired immune responses against these blasts. We decided to look at the role of T-regulatory lymphocytes in tumor surveillance and treatment outcome. Aim and Objectives: The aims and objectives of the study are to evaluate the role of T-regulatory lymphocytes in acute leukemia cases and its correlation with treatment outcome. Materials and Methods: We evaluated consecutive 55 acute leukemia cases to investigate the prognostic effect of T-regulatory cells (T-regs) in acute leukemia. The study period was between January 2021 and December 2021. All patients whose complete records were available till the completion of induction were included in the study. The immunophenotype used to characterize and evaluate T-regs was CD4+CD25+ T lymphocytes. For comparison, a total of 10 healthy controls were included in the study. Clinicohematological parameters including fever, lymphadenopathy, hepatosplenomegaly, extramedullary involvement, day 1 total leukocyte count, and blast percentage in peripheral blood and bone marrow were studied and documented to establish any possible correlation with T-regs percentage and treatment outcome. The treatment outcome evaluated in the study was complete remission postfirst induction. Results: As compared to those of healthy participants, the frequencies of CD4+CD25+ T-regs in the peripheral blood of acute leukemia patients were significantly increased (1.5% [range: 0.50%–4.3%] vs. 0.58% [range: 0.40%–1.05%], P = 0.0001). Furthermore, the frequencies of CD4+CD25+ T-regs in the peripheral blood of acute leukemia patients who did not achieve remission were significantly increased as compared to those who achieved remission (2.9% [range: 0.20%–4.3%] vs. 1.3% [range: 0.30%–2.9%], P = 0.002). No significant correlation was noted in the T-reg cells and parameters such as fever, lymphadenopathy, hepatosplenomegaly, extramedullary involvement, day 1 total leukocyte count, and blast percentage. Conclusion: This study suggests the possibility of using T-regs percentages as a prognostic indicator of acute leukemia and possible role of anti-T-regulatory immunomodulators in improving the outcome of disease.

Blood regulatory T cells in inflammatory bowel disease, a systematic review, and meta-analysis.

Inflammatory bowel disease (IBD) is an autoimmune disease involving various parts of the gastrointestinal (GI) tract, which includes Crohn's disease (CD) and ulcerative colitis (UC). Due to the contradictory results regarding the percentage of peripheral blood (PB) regulatory T cells (Tregs) in IBD patients, this meta-analysis aimed to determine the Tregs frequency in IBD patients. We searched PubMed, Web of Science, SCOPUS, and Google Scholar databases for relevant observational articles that analyzed and reported the frequency of PB Tregs in IBD patients and healthy control groups. After choosing the related articles by two reviewers, the data regarding the definition of Tregs and their frequencies in different groups were recorded. In 22 studies, the results showed a nonsignificant difference in the frequency of PB Tregs between IBD cases and control subjects (SMD: -0.27, 95% CI: -0.78, 0.23). However, the frequency of CD4+CD25+CD127- (SMD: -0.89, 95% CI: -1.52, -0.26) and CD4+CD25+FoxP3+ (SMD: -1.32, 95% CI: -2.37, -0.26) Tregs were significantly lower in IBD cases, compared to healthy subjects. Also, UC cases and active IBD cases showed a significantly lower frequency of Treg cells, compared to controls and remission IBD cases, respectively (SMD: -0.68, 95% CI: -1.24, -0.11 and SMD: -0.60, 95% CI: -0.93, -0.27). Our study highlighted a probable decrease of Tregs in IBD patients, especially the patients with active states of the disease. The decrease of Treg cells might cause an imbalance in the immune system and the over-activation of auto-immune responses against the digestive tract.

Neoadjuvant Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) and chemotherapy versus placebo plus chemotherapy in patients with HER2-negative breast cancer: a randomized, controlled, double-blind trial.

According to preclinical experiments, Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) exerts antiproliferative effects against breast cancer cells. It has been approved by the State Food and Drug Administration in China for complementary cancer treatment, and its safety has been confirmed in previous clinical trials. The present randomized, controlled, double-blind clinical trial was conducted to investigate the efficacy and safety of neoadjuvant PA-MSHA and placebo with chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Eligible patients aged 18 years or older with previously untreated HER2-negative stage II-III breast cancer were enrolled and randomly assigned at a 1:1 ratio to receive neoadjuvant chemotherapy with PA-MSHA or a placebo. The Response Evaluation Criteria in Solid Tumors (RECIST) was used to assess clinical response every 2 cycles. The primary endpoint was the objective response rate (ORR) based on the clinical response following neoadjuvant chemotherapy. A total of 75 patients were randomly assigned to either the PA-MSHA group (37 patients) or the control group (38 patients). The ORR was found to be significantly higher in the PA-MSHA group compared with the control group [86.5% versus 60.5%; rate difference 26.0; 95% confidence interval (CI): 5.9-43.5%; P=0.011]. The pathological complete response (pCR) and survival outcomes did not differ significantly between the 2 groups. Patients with immune-related adverse events (irAEs) appeared to benefit from the PA-MSHA treatment, with greater disease-free, relapse-free, and overall survival. The application of PA-MSHA to neoadjuvant chemotherapy did not increase the incidence of severe adverse events. Moreover, the addition of PA-MSHA increased serum interferon-γ levels and the percentage of peripheral blood T cells, CD8+/CD4+ T cells, CD8+CD28+ T cells, and natural killer (NK) cells, and decreased serum interleukin 4 levels. The addition of PA-MSHA to neoadjuvant chemotherapy is an effective alternative regimen for HER2-negative breast cancer. Patients with irAEs caused by PA-MSHA may obtain more benefits from this treatment. Chinese Clinical Trial Registry ChiCTR-TRC-10000794.

Dysregulation of circulating CD4 + CXCR5 + PD-1+ T cells in diabetic retinopathy

AimsWe aimed to determine an association between follicular helper T (Tfh) cells and Bcl-6 and CXCL13 levels and determine the role of Tfh cells, Bcl-6, and CXCL13 serum levels in the pathogenesis of diabetic retinopathy (DR) since Tfh cells have an important role in type 1 diabetes; however, their role in type 2 diabetes-related DR requires exploration. MethodsBlood samples were collected from 24 patients with non-proliferative diabetic retinopathy (NPDR), 20 with proliferative diabetic retinopathy (PDR), and 18 age- and sex-matched healthy volunteers. Flow cytometry detected CD4 + CXCR5 + PD1+ Tfh cells. Serum Bcl-6 and CXCL13 levels were determined using enzyme-linked immunosorbent assay. ResultsCD4 + CXCR5 + PD-1+ Tfh cell percentages in peripheral blood and serum levels of Bcl-6 and CXCL13 in the non-proliferative DR (NPDR) and proliferative DR (PDR) groups' were significantly higher than those in healthy individuals. The proportion of Tfh cells in DR patients' peripheral blood positively correlated with Bcl-6 and CXCL13 serum levels, DR course severity, Fasting blood glucose, glycosylated hemoglobin and body mass index. ConclusionsThe increased circulating Tfh cells, serum Bcl-6 levels, and CXCL13 levels of DR patients with type 2 diabetes suggested that circulating Tfh cells and the germinal center response may have a role in the occurrence and development of DR.

The relationships of CD8+ T cell subsets in RA patients with disease activity and clinical parameters.

CD8+T cells are plentiful in rheumatoid arthritis (RA) and have a important role in it's pathogenesis. Many subsets have been identified in CD8+T cells, however, the relationship between CD8+T subpopulations and disease activity of RA is poorly defined. Here we detected different CD8+T cell subsets in peripheral blood and examined their relationships with clinical features and serological parameters in RA. CD8+T cell phenotypes and percentages in peripheral blood were determined by flow cytometry in 39 patients with RA. The clinical characteristics and serological parameters of RA patients were collected and DAS28-ESR was measured as indicator of disease activity. Linear regression was performed to assess the correlation of CD8+T cell subsets with RA clinical variables. Naive CD8+T cells were significantly and negatively correlated with RA disease activity indicator DAS28-ESR(r2=0.1027, p=0.0468), erythrocyte sedimentation rate (ESR)(r2=0.1891, p=0.0057), clinical disease activity index(CDAI)(r2=0.1474, p=0.0158), simplified disease activity index(SDAI)(r2=0.1465, p=0.0255), and duration(r2=0.1247, p=0.0274). And the percent of naive CD8+T cells were obviously decreased in RA with high disease activity when compared with RA in low disease activity(p<0.01). In addition, Our results indicated significant positive correlations between CD8+CD28- T cells and DAS28-ESR(r2=0.1881, p=0.0058), ESR(r2=0.2279, p=0.0021), c reaction protein (CRP)(r2=0.2203, p=0.0051), CDAI (r2=0.1778, p=0.0075), SDAI (r2=0.2618, p=0.0020), rheumatoid factor(RF)(r2=0.1823, p=0.0067), age(r2=0.1968, p=0.0047), as well as similar positive correlations between CD8+CD27- T cells and DAS28-ESR(r2=0.1661, p=0.01), ESR(r2=0.1586, p=0.012), CRP(r2=0.1778, p=0.013), CDAI (r2=0.1622, p=0.0110), SDAI(r2=0.2316, p=0.0040), RF(r2=0.2097, p=0.0034), age(r2=0.1932, p=0.0051). Furthermore, interesting results showed observable positive correlations between activated CD8+T cells and total cholesterol(TC)(r2=0.2757, p=0.0007), triglyceride(TG)(r2=0.2886, p=0.0005), low density lipoprotein(LDL-C)(r2=0.09643, p=0.0264) and Krebs yon denlungen-6(KL-6)(r2=0.4171, p=0.0002). And TCRγδ+CD8+T cells were also found positively related with total cholesterol(TC)(r2=0.5015, p<0.0001), triglyceride(TG)(r2=0.2031, p=0.0045), and KL-6(r2=0.2122, p=0.0136). Our results suggest that naive CD8+T cells, CD8+CD28- T cells, and CD8+CD27- T cells are obviously correlated with inflammation and disease activity of RA. While activated CD8+T cells and TCRγδ+CD8+T cells may involve in lipidmetabolism and lung fibrosis of RA. These CD8+T cell subsets may be new biomarkers and targets for RA disease evaluation, therapeutic target-selecting, curative effects and prognoses assessment.

Role of large unstained cells in predicting successful stem cell collection in autologous stem cell transplantation

IntroductionSufficient stem cell collection is mandatory for Autologous stem cell transplantation (ASCT). Peripheral CD34+ stem CD34 + stem cell counting by flow cytometry is the gold standard method in both the predicting and timing of successful stem cell collection. Large unstained cells (LUC) are large peroxidase-negative cells that are displayed on certain automatic cell counters and present large lymphocytes, virocytes, blasts, abnormal cells and hematopoietic stem cells. In this study, we evaluated the role of LUC parameters in the timing and prediction of successful stem cell collection. MethodsPatients with a diagnosis of multiple myeloma, lymphoma and testis tumor who proceed to ASCT were included in this study. Preapheresis LUC parameters were analyzed with Siemens ADVIA® 2120i system., Kruskal Wallis, Mann-Whitney U, Spearman Rho and receiver–operator curve (ROC) tests were used for analyses. ResultsNinety patients were evaluated. Peripheral CD34 + cell count was positively correlated with both LUC count (p = 0014) and LUC percentage (p = 0,01). LUC percentage in peripheral blood was positively correlated with mobilized stem cell count in the yield (p = 0.003). We found a LUC count of > 0.485 × 109/L as a cut-off value for detecting > 20 × 106/L CD34 +cells in the peripheral blood with a sensitivity of 64.6% and specificity of 75%. We defined > 2.15% as a cut-off value for LUC percentage to collect > 5 × 106/kg of stem cells with a sensitivity of 64% and specificity of 63%. Additionally, total nucleated cell (TNC) count was negatively correlated with LUC percentage (p = 0.014) and positively correlated with LUC count (p = 0.001). ConclusionLUC parameters are readily available, simple and cheap tools that can be useful in both timing of CD34 count by flow cytometry in peripheral blood and in the prediction of successful mobilization. LUCs can also be an indicator of graft composition.

Immunological risk factors for nonalcoholic fatty liver disease in patients with psoriatic arthritis: New predictive nomograms and natural killer cells.

ObjectiveTo search for the immunological risk factors of Psoriatic arthritis (PsA) combined with nonalcoholic fatty liver disease (NAFLD), development and assessment of predictive nomograms for NAFLD risk in patients with PsA, and to further explore the correlation between risk factors and dyslipidemia.MethdsA total of 127 patients with PsA (46 with NAFLD and 81 without NAFLD) were included in this retrospective study. The clinical and serological parameters of the patients were collected. The percentage and the absolute number of lymphocytes and CD4+T cells were determined by Flow cytometry. Univariate and multivariate binary logistic regression analysis was used to screen independent risk factors of PsA complicated with NAFLD in the model population, and a nomogram prediction model was developed and assessed.Results(1) Univariate and multivariate logistic regression analysis of the modeling population showed that the percentage of peripheral blood T helper 1 cells (Th1%) (OR=1.12, P=0.001), body mass index (BMI) (OR=1.22, P=0.005) and triglycerides (TG) (OR=4.78, P=0.003) were independent risk factors for NAFLD in patients with PsA, which were incorporated and established a nomogram prediction model. The model has good discrimination and calibration, and also has certain clinical application value. (2) The number of peripheral blood NK cells in PsA patients was significantly positively correlated with serum triglyceride (TG) (r=0.489, P<0.001), cholesterol (CHOL) (r=0.314, P=0.003) and low-density lipoprotein (LDL) (r=0.362, P=0.001) levels.ConclusionsOur study shows that the novel NAFLD nomogram could assess the risk of NAFLD in PsA patients with good efficiency. In addition, peripheral blood NK cell levels may be associated with dyslipidemia in patients with PsA.

Event-free Survival in Patients with Chronic Myeloid Leukemia Receiving Front-line Imatinib Mesylate

Objective: Chronic myeloid leukemia (CML) prognostication at the time of diagnosis is critical to determine the intensity of initial treatment. Event-free survival (EFS) has become a prominent concept of prognosis in the patients with chronic phase CML (CML-CP). The aim of this study is to assess the prognostic impact of bone marrow (BM) and peripheral blood (PB) cellular components, in correlation with the clinical parameters. Materials and Methods: One hundred forty-three patients with CML-CP on the front-line imatinib mesylate therapy were recruited into this study. Clinical and laboratory characteristics, therapeutic responses were recorded. Sokal, Euro/Hasford, The EUropean Treatment Outcome Study (EUTOS) and The EUTOS long-term survival (ELTS) scores were calculated for the studied patients. Results: Median follow-up time was 84 (IQR: 54-125) and median front-line therapeutic duration was 56 (IQR:23-89) months. Five-year EFS rate was 62.3% (95% CI: 53.9-70.7). The blast percentage in the BM, EUTOS scores, and basophil percentage in PB were related with the poor therapeutic outcomes in frontline therapy (p=0.002, p=0.002 and p=0.042, respectively). Although Sokal risk classification showed that the intermediate class had a higher event risk compared to the low-risk class (p=0.001), the predictive association disappeared in high-risk classes. Conclusion: EUTOS score system has better predictive capability for front-line imatinib therapy comparing with other indices. Higher blast percentage in BM and increased basophil percentage in PB are independent risk factors, adversely related with EFS in patients with CML.

Intestinal morphology and immune profiles are altered in piglets by early-weaning

This study was to investigate the developmental changes in intestinal morphology and immune profiles in suckling and weaning piglets. Seventy-two weaning piglets with equal initial body weight from 8 litters (Duroc × Landrace × Yorkshire, 9 piglets per litter) were selected. Thirty-two piglets in the suckling group were nursed by sows until they were 17, 21, 28, or 35 days of age. While the other forty piglets were weaned at 14 d of age, and then housed in the same farrowing cage without a sow and slaughtered until they were on d 0, 3, 7, 14 and 21 after weaning at d 14 of age (wd 0, 3, 7, 14, 21). Blood, jejunal mucosa, intraepithelial lymphocyte (IEL) and lamina propria T lymphocyte (LPL) were harvested from suckling piglets at d 14, 17, 21, 28 and 35 of age and weaning piglets on d 0, 3, 7, 14 and wd 0, wd 3, wd 7, wd 14 and wd 21). The results showed that compared with the wd 0, early weaning significantly declined the average daily gain of postweaning 0–7 (wd 0–7) (P < 0.05), and jejunal villus height on wd 3 (P < 0.05), as well as increased the jejunal crypt depth of piglets on wd 7, 14 and 21 (P < 0.05). And there were no significant differences in average daily gain and villus height after suckling (P > 0.05). The level of serum interferon-γ (IFN-γ) was increased on wd 7 and decreased on wd 21 (P < 0.05), while IFN-γ level in jejunal mucosa was enhanced at wd 3 in comparison with wd 0 (P < 0.05). And the serum interleukin-4 (IL-4) levels were increased at wd 14, wd 21, but the mucosa IL-4 concentration was strikingly increased on wd 7 (P < 0.05). Moreover, weaning led to the enhanced levels of interleukin-1b (IL-1b), interleukin-2 (IL-2) and soluble interleukin-2 receptor (sIL-2R) in serum (P < 0.05), as well as declined the levels of sIL-2R in jejunal mucosa at wd 3 (P < 0.05). In suckling piglets, the serum immunoglobulin A (IgA), IL-4 and IL-2 levels were increased with increasing age (P < 0.05), whereas the jejunal mucosa IL-1b and serum sIL-2R levels were lower (P < 0.05). Furthermore, significantly lower CD4 percentage in peripheral blood T lymphocyte subsets were found at wd 3 and wd 7 (P < 0.05), whereas the CD8 percentage in peripheral blood T lymphocyte subsets were enhanced on wd 3 and wd 7 than wd 0 (P < 0.05). Moreover, the weaning piglets at wd 3 had a lower CD4/CD8 ratio than wd 0 (P < 0.05). Additionally, we found that weaning decreased IgG, IL-4, IL-2 and IL-1b levels of IEL during 1-week post-weaning (P < 0.05). Similarly, the levels of IgA, IgG, IL-2 and sIL-2R in LPL medium were also declined from piglets postweaning 1 week (P < 0.05). Early weaning reduced the growth performance, damaged jejunal morphology, disrupted IFN-γ/IL-4, IL-2/sIL-2R and T lymphocyte balance, and impaired the IEL and LPL immune profiles of piglets.

Spectrum of immunophenotypic aberrancies in acute leukemia along with their correlation with adverse hematological parameters

BACKGROUND: The identification of aberrant phenotypes in acute leukemia is crucial for treatment monitoring and minimal residual disease analysis. Flowcytometric immunophenotyping in acute leukemia is an important tool for the detection of these aberrancies. There is wide variation in the incidence of aberrant antigens in acute leukemia in different studies and its correlation with prognostic markers.AIMS AND OBJECTIVES: The aim and objective of this study is to assess the frequency of aberrant markers in acute leukemia and determine any association with hematological parameters.MATERIALS AND METHODS: A total of 150 newly diagnosed cases of acute leukemia during the period January 2018–December 2020 were included. The flowcytometric immunophenotyping of all the above cases was done using BD FACS Canto II.RESULTS: Out of the total 150 cases of acute leukemia, 56.7% expressed aberrant phenotype. The proportional frequency of aberrant antigen expression in B-acute lymphoid leukemia (ALL), T-ALL and acute myeloid leukemia (AML) was 56.6%, 50%, and 58.5%, respectively. CD13, CD13, and CD7 were the most common aberrancies in B-ALL, T-ALL, and AML, respectively. Aberrant myeloid phenotype in B-ALL was associated with lower mean total leukocyte count (TLC) and blast percentage in peripheral blood as compared to the B-ALL with conventional phenotypes. Aberrant lymphoid phenotype in AML was associated with a higher TLC and greater blast percentage in peripheral blood than the AML with conventional phenotypes.CONCLUSION: Aberrant lymphoid phenotype in AML is associated with unfavorable hematological features. However, aberrant myeloid phenotype in B-ALL is not associated with adverse features.

Prognostički faktori rizika za ranu smrt kod bolesnika sa akutnom mijeloidnom leukemijom

Introduction: Early death is a known complication in the treatment of patients suffering from acute myeloid leukemia (AML). It has been defined as death occurring within 28 days of the initiation of induction chemotherapy. Aim: Determining the clinical characteristics of the patient, the risk factors, the frequency, and the most common causes of early death in AML patients. Materials and methods: This retrospective study included 248 patients with diagnosed AML. At diagnosis, the following demographic and clinical-laboratory characteristics were recorded: sex, age, general functional status, i.e., performance status according to the ECOG scale, complete blood count, LDH level in the blood, percentage of blasts in peripheral blood and bone marrow, fibrinogen, PT, aPTT, D-dimer, and BMI. Patients were treated with induction and reduction chemotherapy and palliative therapy. Statistical analysis was performed using the data from the discharge summaries taken from the registers of the Clinic for Hematology of the Clinical Center of Serbia. Results: Early death occurred in 53 (21.4%) patients. The prognostic risk factors for early death were the following: age (p = 0.047), ECOG ≥ 2 (p = 0.001), leukocyte count ≥ 30 x 109 /l (p = 0.022), LDH level ≥ 450 U/l (p = 0.022), the percentage of blasts in peripheral blood (p = 0.005) and the percentage of blasts in bone marrow (p = 0.003), PT (p &lt; 0.001), as well as the ISTH score (p = 0.018). The most common cause of early death in patients aged 40 - 65 years was sepsis, while in patients older than 65 years it was respiratory failure. Conclusion: The study showed that age, the leukocyte count, the percentage of peripheral blood and bone marrow blasts, the ECOG score, LDH, PT, and the ISTH score were significant prognostic risk factors of early death in patients with AML, and that the most common cause of early death in patients aged 40 - 65 years was sepsis, while in patients older than 65 years it was respiratory failure.

T Cell Engaging Bispecific Antibodies Produce Durable Response in Mesothelin-Positive Patient-Derived Xenograft Models of Pediatric AML

BackgroundImmunotherapy development in pediatric AML is lagging because of dearth of validated AML-specific targets. We showed recently that mesothelin (MSLN) is highly expressed on the leukemia cell surface in a subset of pediatric AML patients, and validated MSLN as a therapeutic target using antibody-drug conjugates directed against MSLN (Kaeding et al., Blood Adv, 5:2350-2361, 2021). Antibody single-chain variable region (scFv) sequences derived from amatuximab recognizing MSLN and from either blinatumomab or AMG330 targeting CD3 were used to engineer and express two MSLN/CD3-targeting BsAbs: MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG respectively. Both these antibodies demonstrated anti-leukemic activity in mice engrafted with MV4;11 cells engineered to overexpress MSLN, while they failed to show any effect in mice bearing MV4;11 cells without MSLN, confirming that these antibodies specifically targeted MSLN (Gopalakrishnapillai et al., Blood, 134:3925, 2019).MethodsMSLN cell surface expression was quantitated using BD Quantibrite PE Phycoreythrin Fluorescence Quantitation kit. 3x10 6 NTPL-146 cells were injected in NSG-B2m mice and 2x10 6 DF-5 cells were injected in NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when human cells were detectable in blood. The percentage of human chimerism in mouse peripheral blood was evaluated weekly by flow cytometry. Bipsecific antibodies were administered ip at 3 mg/Kg daily for six days. Human peripheral blood pan T cells from StemCell Technologies were injected iv (3x10 6 cells per mouse) to act as effector cells. Chemotherapy (DA) consisted of 3 doses of 1.5 mg/kg daunorubicin iv and 5 doses of 50 mg/kg cytarabine ip. Mice were monitored daily and euthanized when any of the experimental endpoints were met.ResultsIn this study, we evaluated the efficacy of two bispecific antibodies in two distinct patient-derived xenograft models of pediatric AML with endogenous MSLN expression quantitated at 6617 and 7414 MSLN antibodies bound per cell in NTPL-146 and DF-5 respectively. A Kaplan-Meier survival plot based on the time when each mouse reached experimental endpoint showed that 6/8 NTPL-146 engrafted mice receiving MSLN AMA-CD3 AMG and T cells survived disease-free until the end of the experiment at day 520 whereas all the mice in control groups had died by day 138 (Fig. 1a). The AML bone marrow load of MSLN AMA-CD3 AMG-treated mice was < 0.01% at 520 days, whereas the bone marrow load of mice from the other treatment groups was greater than 90% at the time of death, consistent with marrow failure as the proximal cause of death (Fig. 1b). These data show that treatment with MSLN AMA-CD3 AMG is curative in the vast majority of mice.Treatment with MSLN AMA-CD3 L2K and T cells increased the median survival by 109.5 days compared to untreated mice while treatment with MSLN AMA-CD3 AMG showed a complete remission in 6/8 mice (**P<0.001). Thus, the survival benefit of both MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG treated mice greatly exceeded the allogeneic effect T cells alone, which only showed a 22-day improvement in median survival. Either MSLN AMA-CD3 L2K or MSLN AMA-CD3 AMG treatment alone showed no improvement in survival compared to untreated mice.The efficacy of the potent BsAb MSLN AMA-CD3 AMG in comparison with chemotherapy (DA) was evaluated in DF-5. DA treatment, like T cell infusion, did not significantly change median survival compared to untreated mice, while BsAb MSLN AMA-CD3 AMG in the presence of human T cells was curative (Fig. 1c, **P<0.005). Mice treated with only T cells showed a rapid rise in AML cell percentage in peripheral blood until day 34 when they succumbed to disease, whereas the mice receiving T cells with BsAb had AML cell burden of less than 1% (Fig. 1d, *P<0.05). Mice treated with BsAbs showed greater expansion of allogeneic human T cells compared to mice receiving T cells alone (Fig. 1e, *P<0.05). Eight weeks post treatment initiation, the surviving mice were euthanized and AML burden in the bone marrow was evaluated. In contrast with mice receiving T cells alone, mice treated with T cells and BsAbs had no detectable AML cells (Fig. 1f, **P<0.005).ConclusionThese data validate the efficacy of MSLN-targeting BsAbs in PDX models with endogenous MSLN expression. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients. [Display omitted] DisclosuresGopalakrishnapillai: Geron: Research Funding. Correnti: Link Immunotherapeutics: Current Employment. Kaeding: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML.We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints.Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P<0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. [Display omitted] DisclosuresGopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

The emerging naturally reassortant strain of IBDV (genotype A2dB3) having segment A from Chinese novel variant strain and segment B from HLJ 0504-like very virulent strain showed enhanced pathogenicity to three-yellow chickens.

Novel variant infectious bursal disease virus (nvIBDV) is an emerging pathotype that can cause sub-clinical disease with severe, prolonged immunosuppression in young chickens. At present, two major pathotypes, including vvIBDV and nvIBDV, are prevailing in China. In this study, we propose that the nvIBDV is a new genotype (A2dB1b) and also first isolated and characterized a nvIBDV reassortant strain YL160304 (A2dB3) with segments A and B derived, respectively, from the nvIBDV and the HLJ-0504-like vvIBDV from yellow chickens in southern China. The YL160304 causes more extensive cytotropism and can infect specific-pathogen-free chicken embryos with severe subcutaneous hemorrhage. The pathogenicity of YL160304 to 4-week-old three-yellow chickens was determined and compared with those of the nvIBDV QZ191002 and the HLJ-0504-like vvIBDV NN1172. Weight gain was significantly reduced in all the challenged birds. No clinical signs and associated mortality were observed in the birds challenged with QZ191002, while the mortalities in the birds challenged with NN1172 and YL160304 were 30% (3/10) and 10% (1/10), respectively. At 7 days postchallenge, the bursa was severely damaged and the percentage of peripheral blood B lymphocyte (PBBL) decreased significantly in all the challenged birds and the quantity of the viral RNA detected in the bursa was in accordance with the results of the histomorphometry and the depletion of PBBL. This study not only confirmed the emerging epidemic of the novel variant and its reassortant strains, but also discovered that the naturally reassortant nvIBDV strain with the segment B of HLJ 0504-like vvIBDV can significantly enhance the pathogenicity to chickens.