CD38 Expression Research Articles

Immunological Analysis of Prognostic Factors in Conversion Surgery Cases for Gastric Cancer.

In order to clarify the optimal strategy regarding conversion surgery (CS) for gastric cancer (GC) patients, we focused on clinicopathological findings, including immunological factors, related to the favorable prognosis in patients with stage IV GC who underwent CS. A total of 25 patients with Stage IV GC who underwent induction chemotherapy (IC) and CS at our hospital between 2010 and 2021 were enrolled in this study. Biopsy specimens before IC and surgical specimens were collected. Immunohistochemical staining was performed using programmed death-ligand 1 (PD-L1) antibody, translationally controlled tumor protein (TCTP) antibody, and CD20 antibody. Prognostic factors were investigated using clinicopathological factors as well as immunological factors such as PD-L1, TCTP, and CD20 expression. cN0, ycStage1-2, R0-1 surgery, D2 lymph node dissection, ypN0, and ypStage1-2 were significantly associated with favorable overall survival. Among patients who underwent R0/1 surgery, only histological type was a significant prognostic factor for recurrence-free survival. Low PD-L1 expression before IC and high TCTP expression after IC were significantly associated with favorable recurrence-free survival. In addition to clinical factors, high TCTP expression after IC was identified as a significant favorable prognostic factor, which could help in identifying candidates for CS in the future.

Characterization of patients with clonal mast cells in the bone marrow with clinical significance not otherwise specified.

Systemic mastocytosis (SM) diagnosis requires the presence of 3 minor criteria or 1 major and 1 minor criterion according to the WHO 2016 classification. The aim of this study was to characterize patients with 1 or 2 minor SM criteria including KIT 816 mutation and/or aberrant expression of CD2 and/or CD25 on bone marrow (BM) mast cells (MCs), but without MC activation syndrome (MCAS) criteria. We included eligible patients from two countries diagnosed between 2011 and 2021. These patients are reported herein as monoclonal MC with clinical significance (MMCS). MMCS patients were compared with 432 patients with indolent SM (ISM) and 51 with BM mastocytosis (BMM) from the CEREMAST database. Overall, 51 patients with MMCS were included. MMCS patients with (n=29) or without (n=22) KIT 816 mutation did not differ significantly with regard to the prevalence of anaphylaxis and basal tryptase level. Anaphylaxis, often in the context of hymenoptera venom allergy, was more frequent in MMCS than in ISM (78% vs 35%, respectively; p<0.001). Osteoporosis was similarly prevalent in MMCS and BMM (45% vs 32%, p=ns). The median baseline serum tryptase level was lower in MMCS compared with ISM or BMM (13 vs 26 vs 23ng/mL, respectively; p<0.001). Hereditary alpha-tryptasemia was similarly represented in MMCS and BMM (14.3% vs 19.7% respectively, p=ns). Clonal BMMCs may be associated with clinically relevant symptoms even if criteria for SM or MCAS are not fulfilled. These MMCS patients may require specific management and follow-up to capture potential transition to SM and/or MCAS. None.

A mechanistic, functional, and clinical perspective on targeting CD70 in cancer.

The oncoimmunology research has witnessed notable advancements in recent years. Reshaping the tumor microenvironment (TME) approach is an effective method to improve antitumor immune response. The T cell-mediated antitumor response is crucial for favorable therapeutic outcomes in several cancers. The United States Food and Drug Administration (FDA) has approved immune checkpoint inhibitors (ICIs) for targeting the immune checkpoint proteins (ICPs) expressed in various hematological and solid malignancies. The ICPs are T cell co-inhibitory molecules that block T cell activation and, thus, antitumor response. Currently, most of the FDA-approved ICIs are antagonistic antibodies of programmed death-ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In contrast to ICPs, the T cell costimulatory molecules are required for T cell activation, expansion, and effector function. However, the abrupt expression of these costimulatory molecules in tumors presents a concern for T cell-mediated antitumor response. One of the T cell costimulatory molecules, the cluster of differentiation 70 (CD70), has emerged as a druggable target in various hematological and solid malignancies due to its role in T cell effector function and immune evasion. The present review describes the expression of CD70, factors affecting the CD70 expression, the physiological and clinical relevance of CD70, and the current approaches to target CD70 in hematological and solid malignancies.

Innate and SARS-CoV-2 specific adaptive immune response kinetic in neutralizing monoclonal antibody successfully treated COVID-19 patients.

The impact of anti-Spike monoclonal antibody (mAbs) treatment on the immune response of COVID19-patients is poorly explored. In particular, a comparison of the immunological influence of different therapeutic regimens has not yet been performed. Aim of the study was to compare the kinetic of innate and adaptive immune response as well as the SARS-CoV-2 specific humoral and T cell response in two groups of SARS-CoV-2-infected patients treated with two different mAbs regimens: Bamlanivimab/Etesevimab (BAM/ETE) or Casirivimab/Imdevimab (CAS/IMD). SARS-CoV-2-infected patients (n=39) with mild/moderate disease were enrolled before (T0) and after 7days (T7) and 30day (T30) from mAbs infusion. Patients were divided in two groups on the basis of the mAb regimen: BAM/ETE (n=15) and CAS/IMD (n=24). The phenotype/function of immune cell subsets was evaluated by flow-cytometry and by ELISA. The Spike-specific T cell response (IFN-γ) and anti-Nucleocapside IgG were evaluated by chemiluminescence assay. SARS CoV-2 RNA in nasal swabs was evaluated by RT-PCR. Eleven out of the thirty-nine enrolled patients tested negative at T7, among which nine (81.8%) had been treated with CAS/IMD regimen. A comparable increase in CD4 and CD8 T cells was observed in both treatment groups. Moreover, a reduction of CD38 expression on T (CD4, CD8 and Vδ2) and on NK cells was observed in both groups, as well as a reduction overtime of the perforin expression in T (CD8, Vδ2) and in NK cells reaching significance only in CAS/IMD-treated patients. The SARS-CoV-2-specific T cells response increased at T7 in BAM/ETE-treated patients and at T30 in CAS/IND group. Of note, at T30 SARS-CoV2-specific T cells was higher in CAS/IMD than in BAM/ETE group. Furthermore, the titre of anti-N IgG increased overtime in both groups with a faster kinetic in CAS/IMD group. The spontaneous production of inflammatory cytokines by monocytes and neutrophils was similar the two mAb regimens, as well as the level of plasmatic IL-6. Finally, patients were also analysed according to sex. The male group showed a higher frequency of activated CD4 T cells, NKG2A-expressing CD8 T cells and perforin-expressing Vδ2 T cells compared to female group. Moreover, a higher specific T cell response at T30 was observed in the male compared to female group. In conclusion, these results show similar effects of both mAb regimens in restoring T and NK cell homeostasis and in reducing inflammation. In contrast, CAS/IMD allows a better humoral and cellular SARS-CoV2 specific immunization.

Abstract TP30: The Effect of Angiotensin II Administration Dose and Duration on The Brain CD38 Expression and Enzymatic Activity

Abstract TP30: The Effect of Angiotensin II Administration Dose and Duration on The Brain CD38 Expression and Enzymatic Activity

Upregulation of CD19 by low‐dose chidamide promotes CAR T cells functionality in B-cell non-Hodgkin lymphoma

B-cell non-Hodgkin lymphoma (B-NHL) is a highly heterogeneous group of lymphopoietic malignancies that account for 85% to 90% of all non-Hodgkin lymphomas. In recent years, CD19 Chimeric antigen receptor T (CAR T) cell immunotherapy has significantly improved the cure rate of B-NHL patients, but there are still some patients who cannot achieve remission after treatment, or relapse after remission. Therefore, it is of great importance to overcome the drug resistance of CD19 CAR T cells after B-NHL treatment and reduce the recurrence rate of CD19 CAR T cells after B-NHL treatment. We found that low concentrations of chidamide did not enhance the ability of CD19 CAR T cells to kill B-NHL cells during and after preparation. B-NHL cells pretreated with chidamide were more likely to be killed by CD19 CAR T cells. CD19 CAR T cells secreted more cytokines (IL-2, TNF-α, and IFN-γ) after co-culture with B-NHL cells pretreated with chidamide. At the same time, the expression of CD19 on B-NHL cell surface was increased by chidamide. In vivo experiments showed that infusion of CD19 CAR T cells after chidamide bridging intervention can enhance the therapeutic effect of B-NHL and prolong the overall survival of mice. This study provides a new direction and theoretical foundation for CD19 CAR T cell therapy in B-NHL.

Accumulation of CD38 in Hybrid Epithelial/Mesenchymal Cells Promotes Immune Remodeling and Metastasis in Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer. The epithelial-to-mesenchymal transition is a nonbinary process in the metastatic cascade that generates tumor cells with both epithelial and mesenchymal traits known as hybrid EM cells. Recent studies have elucidated the enhanced metastatic potential of cancers featuring the hybrid EM phenotype, highlighting the need to uncover molecular drivers and targetable vulnerabilities of the hybrid EM state. Here, we discovered that hybrid EM breast tumors are enriched in CD38, an immunosuppressive molecule associated with worse clinical outcomes in liquid malignancies. Altering CD38 expression in tumor cell impacted migratory, invasive, and metastatic capabilities of hybrid EM cells. Abrogation of CD38 expression stimulated an antitumor immune response, thereby preventing the generation of an immunosuppressive microenvironment in hybrid EM tumors. CD38 levels positively correlated with PD-L1 expression in samples from patients with TNBC. Moreover, targeting CD38 potentiated the activity of anti-PD-L1, eliciting strong antitumor immunity, with reduced tumor growth in hybrid EM models. Overall, this research exposes upregulation of CD38 as a specific survival strategy utilized by hybrid EM breast tumors to suppress immune cell activity and sustain metastasis, with strong implications in other carcinomas that have hybrid EM properties. Significance: Hybrid cells co-featuring epithelial and mesenchymal traits in triple-negative breast cancer express elevated levels of CD38 to induce immunosuppression and metastasis, indicating CD38 inhibition as potential strategy for treating breast cancer.

Natural killer cells occupy unique spatial neighborhoods in HER2- and HER2+ human breast cancers

Tumor-infiltrating lymphocytes are considered clinically beneficial in breast cancer, but the significance of natural killer (NK) cells is less well characterized. As increasing evidence has demonstrated that the spatial organization of immune cells in tumor microenvironments is a significant parameter for impacting disease progression as well as therapeutic responses, an improved understanding of tumor-infiltrating NK cells and their location within tumor contextures is needed to improve the design of effective NK cell-based therapies. In this study, we developed a multiplex immunohistochemistry (mIHC) antibody panel designed to quantitatively interrogate leukocyte lineages, focusing on NK cells and their phenotypes, in two independent breast cancer patient cohorts (n = 26 and n = 30). Owing to the clinical evidence supporting a significant role for NK cells in HER2+ breast cancer in mediating responses to Trastuzumab, we further evaluated HER2- and HER2+ specimens separately. Consistent with literature, we found that CD3+ T cells were the dominant leukocyte subset across breast cancer specimens. In comparison, NK cells, identified by CD56 or NKp46 expression, were scarce in all specimens with low granzyme B expression indicating reduced cytotoxic functionality. Whereas NK cell density and phenotype did not appear to be influenced by HER2 status, spatial analysis revealed distinct NK cells phenotypes regarding their proximity to neoplastic tumor cells that associated with HER2 status. Spatial cellular neighborhood analysis revealed multiple unique neighborhood compositions surrounding NK cells, where NK cells from HER2- tumors were more frequently found proximal to neoplastic tumor cells, whereas NK cells from HER2+ tumors were instead more frequently found proximal to CD3+ T cells. This study establishes the utility of quantitative mIHC to evaluate NK cells at the single-cell spatial proteomics level and illustrates how spatial characteristics of NK cell neighborhoods vary within the context of HER2- and HER2+ breast cancers.

DOP030 Mucosal deficiency of mitochondria-driven humoral immunity is linked to Crohn’s Disease

Abstract Background Secretory IgA (sIgA) is critical for the maintenance of the mucosal barrier function, preventing bacterial translocation and reducing the risk of chronic inflammation. Several decades of research have revealed a dysregulation of the B-cell compartment in inflammatory bowel disease (IBD) of yet unknown origin1,2. While increased serum levels of (auto)antibodies against S. cerevisiae or the intestinal M-cell expressed FimH receptor glycoprotein 2 (GP2) are observed in patients with Crohn’s disease (CD)3, some studies indicated an impaired generation of intestinal immunoglobulin-secreting plasma cells (PCs)4. To unravel the role of mucosal humoral immunity in CD pathogenesis, we aimed to provide an in-depth characterisation of colonic PC differentiation in patients with CD. Methods In-depth phenotyping of patients with CD in remission and non-IBD controls (hospitalised normals) was performed using multi-omics analyses (spatial single-cell proteomics, proteomics, metabolomics, and transcriptomics) of plasma or colon biopsy samples. Colonic switched memory B cells (sMBCs) were sorted from lamina propria mononuclear cells (LPMNCs) by magnetic-activated cell sorting and ex vivo differentiated into PCs. The impact of mitochondrial function on colonic PC maturation was studied in mitochondrial respiratory chain complex V-deficient ATP8 mutant mice and mice that underwent nutritional intervention. Results Despite a hyperactive colonic B-cell compartment, patients with CD displayed significantly diminished mucosal dimeric IgA (dIgA) and fecal IgA compared to non-IBD controls. Polymeric immunoglobulin receptor (PIGR) expression did not differ, excluding a defective epithelial transcytosis of dIgA. Spatial single-cell proteomics uncovered that colonic plasmablasts and immature CD19+CD45+ PCs were increased at the expense of the fully mature CD19-CD45- phenotype. Consistently, PCs generated ex vivo from CD-derived sMBCs revealed impaired capacity to differentiate into IgA-secreting PCs. Metabolic phenotyping indicated a colonic mitochondrial dysfunction in patients with CD that was also reflected in colonic PCs by increased expression of the cell-surface NADase CD38 in concert with decreased expression of mitochondrially encoded transcripts. Of note, splenic PCs isolated from ATP8 mutant mice displayed a hyperproliferative and low-differentiated phenotype, while colonic IgA-secreting PCs were reduced. Conversely, feeding mice an isocaloric glucose-free, high-protein diet, which promotes mitochondrial activity, increased colonic IgA levels. Conclusion In summary, this study links mitochondrial dysfunction to impaired PC differentiation in patients with CD, resulting in reduced levels of sIgA and thereby compromised mucosal barrier integrity.

Bispecific Aptamer-Drug Conjugates Selectively Eliminate Malignant Hematologic Cells for Treating Acute Myeloid Leukemia.

Surface antigen-directed immunotherapy is a curative treatment modality for acute myeloid leukemia (AML) that is characterized by the abundance and stability expression of surface antigens. However, current surface antigen-directed immunotherapies have shown poor outcomes and undesirable mortality rates in treating AML patients, primarily due to acquired resistance that arises from using single-target therapies to address the heterogeneous expression of surface antigens. Hence, in order to improve the efficacy of antigen-specific therapies for treating AML, we designed a bispecific aptamer-drug conjugate. In particular, cell-SELEX incorporating cell lysate-SELEX for aptamers with HEL cells yielded AptCD117, which specifically binds to CD117 (a highly expressed marker on both hematopoietic stem cells and primary AML cells) and has excellent performance in targeting human AML cells. Combined with CD71-binding aptamer LXD-11b (another broadly expressed surface antigen on leukemia cells), bispecific aptamers were designed to couple with monomethyl auristatin F (MMAF) for fabricating aptamer-drug conjugates. Results demonstrated that bispecific aptamer-MMAF conjugates efficiently kill different CD117 and CD71 expression levels of target AML cell lines in vitro. Importantly, the exposure of AML marrow specimens to bispecific aptamer-MMAF conjugates resulted in the selective elimination of primary AML cells in vitro and had no effect on healthy lymphocytes within the same specimens. Thus, these results provide a proof of concept for the generation of bispecific aptamer-drug conjugates directed against human AML cells, which hold the promise of advancing treatment strategies and improving AML patient outcomes.

DOP126 Circulating neutrophils exhibit distinct transcriptional and maturity changes in CD and UC that relate to pathogenic Th1/17 CD4+ memory T cell responses

Abstract Background Neutrophils are key players in intestinal damage in Crohn’s disease (CD) and ulcerative colitis (UC) but their immunological role in disease heterogeneity is largely overlooked. In both diseases, mature circulating neutrophils enter the intestinal tissue to clear invading bacteria but cause irreversible tissue damage. However, neutrophil function can be altered by pathogenic Th1/17 CD4+ memory T cell (Tm) responses even before the cells enter the tissue. We hypothesize that altered neutrophil function differs between, and within, CD and UC and that interpatient differences in pathogenic Tm responses change transcriptional profiles in circulating neutrophils, impacting their function in the inflamed mucosa. Methods We analyzed clinically well-characterized therapy-naive pediatric CD (n=33) and UC (n=12) patients and IBD-negative controls (IBDneg; n=11) from the PIBD-SETQ cohort and performed RNAseq on purified circulating neutrophils, neutrophil flow cytometry and plasma proteomics using Proximity Extension Assay technology. Results Comparison of gene expression profiles at diagnosis revealed 263 differentially expressed genes (DEG) in neutrophils from CD vs IBDneg, and 421 DEG in UC vs IBDneg, with 137 genes common to both comparisons and 137 DEG in CD vs UC. CD and UC neutrophil transcriptomes were enriched for granule biology pathways (MMP8, S100A8/9), while IFN-related pathways were only present in CD neutrophils. As granule proteins are expressed in neutrophils during maturation in bone marrow but not in circulation, we assessed the maturity of circulating neutrophils by flow cytometry. Neutrophils from UC, but not CD patients had phenotypic features of immaturity (lower surface expression of CD10, CD16 and CXCR2). As predicted, neutrophil gene expression profiles showed high interpatient heterogeneity within CD and UC. Within both CD and UC, hierarchical clustering on the basis of neutrophil DEG identified two patient clusters. In UC1 patients, neutrophils had higher expression of genes for granule proteins which related to higher plasma concentrations of IL-17A, CCL20 and MMP10 compared to UC2. In CD2 patients, neutrophils had increased gene expression for IFN-signaling and extracellular matrix re-organization relating to higher plasma IFNg, CDCP1 and CXCL9 compared to CD1. Conclusion We demonstrate that at time of diagnosis circulating neutrophils have acquired changes in transcriptional and maturity profiles that differ between and within CD and UC. In individual patients, transcriptomic changes correlate to pathogenic Th1/17 CD4+ Tm profiles. These changes may functionally alter neutrophil-mediated bacterial clearance and tissue damage in the inflamed mucosa and help decipher disease pathogenesis in individual patients.

P1293 Novel mutations in SASH3 cause very early onset IBD by impairing the NF-kB and mTORC1 pathways

Abstract Background Human SAM and SH3 domain-containing 3 (SASH3) variants globally affect TCR signaling including its downstream mTORC1 and NF-kB signaling. These 3 pathys play great roles in the pathogenesis of very early-onset inflammatory bowel diseases (VEO-IBD). Here, we report a now 4-year-old patient (P1) who was ultimately diagnosed with SASH3 deficiency in the nuclear localization signal 1(NLS1) of the protein presenting with VEO-IBD along with his adult uncle clinically asymptomatic (P2) who carries the identical variant. Methods a 3.2-year-old boy suspected VEO-IBD was evaluated in our clinic. Tissues were obtained by endoscopy. Genomic DNA, extracted from the peripheral blood mononuclear cells of the child and parents, were subjected to whole-exome sequencing(WES) and sanger sequencing. After mutations in the SASH3 were identified, sanger sequencing were performed for their relatives,the immunological features of our patients were analyzed including the cell proliferation assay by flow cytometry. Western blot(WB) was performed to study the protein level of SASH3 and the NF-kB and mTORC1 pathways in patients. Results P1 was diagnosed with Crohn’s disease according the images of endoscopy and biopsies (Figure A). WES and sanger sequencing identical a variant at exon1 in SASH3 (c.10 C&amp;gt;T/p.R4C ) in P1, his mother was a heterozygous carrier (Figure B). Sanger sequencing confirmed that P2 who was clinically asymptomatic to date, carries the identical variant at exon1 in SASH3 (c.10 C&amp;gt;T/p.R4C ) which inherited from P1’ grandmother(Figure C,D). The SASH3 variants identified in them is located in the NLS1 of the protein(Figure E), it caused the protein is exclusively located in the cytoplasm and no protein was found inside the nucleus which were proved by WB( Figure F). The immune phenotype were not seriously damaged in our 2 patients at homeostatic conditions, but upon stimulation with anti-CD3 and anti-CD28, both CD4+ and CD8+ T cells from P1, demonstrated impaired expression of CD71 and GLUT1 (FigureG, H), indicating defective cell proliferation and activation. The WB demonstrated that both the level of p-NFkB-p65 and p-S6 was markedly reduced in P1 (Figure I). We used Infliximab to treat P1, one year later the endoscopy showed mucosal healing and the pathology showed improved in inflammation and absent in granulation tissue. (Figure J) Conclusion We identified a novel mutations in SASH3 located in the NLS1 of the protein which cause VEO-IBD by impairing the NF-kB and mTORC1 pathways.

Clinical Translation of a Dual-Integrin αvβ3- and CD13-Targeting PET Tracer.

Angiogenesis is essential in the development and progression of tumors. This study aimed to investigate the clinical application of 68Ga-labeled heterodimeric peptide (68Ga-HX01) targeting integrin αvβ3 and CD13 in tumor neovascularization. Six healthy volunteers were recruited to study the biodistribution, pharmacokinetics, and radiation of 68Ga-HX01. Twelve patients with various malignancies were enrolled to seek the preliminary clinical value of 68Ga-HX01. In healthy volunteers, SUVs of each major organ on 68Ga-HX01 PET were measured. The clinical data, lesion numbers, and uptake were recorded in patients. The integrin αvβ3 and CD13 expression of the resected tumors was checked via immunohistochemistry staining. With a mean injected dose of 167.98 ± 26.32 MBq, 68Ga-HX01 was well tolerated and safe without side effects in 6 healthy volunteers. The radiation absorbed effective dose of 68Ga-HX01 was 1.94 × 10-2 mSv/MBq, and the urinary bladder wall held the highest absorbed effective dose (0.15 ± 5.87 × 10-2 mSv/MBq). In 12 patients with various malignancies, 68Ga-HX01 PET could clearly visualize the lesions from the surrounding tissues. The SUVmax values in tumors were significantly higher than those in the surrounding tissues (P < 0.05). A positive correlation trend between tumor SUVmax and semiquantitative integrin αvβ3 and CD13 expression was determined (P < 0.05). For clinical use, 68Ga-HX01 is safe with low radiation absorbed effective dose. It also indicates the efficiency of dual integrin αvβ3 and CD13-targeting PET radiotracer in tumor diagnosis, which may assist in patient prognosis and selecting eligible patients for antiangiogenic therapy.

Vδ2 T-cells response in people with Mpox infection: a three-month longitudinal assessment

The first evidence that Orthopoxvirus induced the expansion in vivo and the recall of effector innate Vδ2 T-cells was described in a macaque model. Although, an engagement of αβ T-cells specific response in patients infected with human monkeypox (Mpox) was demonstrated, little is known about the role of γδ T-cells during Mpox infection. IFN-γ-producing γδ T-cells in the resistance to poxviruses may a key role in inducing a protective type 1 memory immunity. We analyzed the kinetics of Vδ2 T-cell response from the acute phase up to three months after Mpox infection. Fourteen MSM subjects (5 PWH, 35.7%) were enrolled in a longitudinal study from May to July 2022. Blood samples were collected in the early phase of infection (T1, T2) and at 3 months (T3M) post-symptom onset. Vδ2 T-cell profiles (CD45RA/CCR7), activation/exhaustion markers (CD38/HLA-DR/CD57/PD-1/TIM-3), cytokine production (IFN-γ/TNF-α) and CD107a expression were assessed by multiparametric flow cytometry. Ten healthy donors (HD) were used as a control group. At T1, Vδ2 T-cell frequency of patients decreased, and effector memory Vδ2 T-cells increased with respect to HD. Activation/exhaustion markers were higher than HD. Vδ2 functionality decreased at T1 related to HD, and it was associated with CD38 and HLA-DR higher expression as well as TIM-3. Vδ2 T-cells restored their profile at T3M. The presence of effector/activated Vδ2 T-cells in the early stages of Mpox infection and their capability to activate quickly, producing pro-inflammatory cytokines, may be useful to enhance the early adaptive response to human Mpox, maintaining a protective memory/effector T-cell response.

A case series of ultrasound and pathological assessment of follicular thyroid tumors: Addressing indeterminate malignancy.

Follicular tumors with uncertain malignant potential of the thyroid are a new classification of thyroid tumors in the World Health Organization (2017). Only a few cases of evaluating the value of ultrasound combined with pathological examination in diagnosing follicular tumors with uncertain malignant potential of the thyroid have been reported in recent decades. A retrospective analysis was performed on 18 patients with follicular tumors with uncertain thyroid malignant potential who underwent preoperative ultrasonography and were confirmed by operation and pathology in our hospital from January 2019 to November 2024. Ultrasonic characteristics, histopathology, and immunohistology were recorded. The patient was pathologically diagnosed as follicular tumor of uncertain malignant potential. All the 18 patients underwent ultrasound, surgical treatment and immunohistochemical staining in our hospital. The patients underwent surgery. There was no complication or recurrence. This case series indicates that the ultrasonographic manifestations of follicular tumors with uncertain thyroid malignant potential were regular and well-defined hypoechoic thyroid masses with an elastic grade of 2. The pathological characteristics were positive expression of P53, TG, CD56, Ki67, TTF-1, and Gal-3 mutations in most lesions.

Regulation of PHD2 by HIF-1α in Erythroid Cells: Insights into Erythropoiesis Under Hypoxia.

The hypoxia-inducible factor (HIF) pathway has been demonstrated to play a pivotal role in the process of high-altitude adaptation. PHD2, a key regulator of the HIF pathway, has been found to be associated with erythropoiesis. However, the relationship between changes in Phd2 abundance and erythroid differentiation under hypoxic conditions remains to be elucidated. A hemin-induced K562 erythroid differentiation model was used to explore the effects of PHD2 knockdown under hypoxia. Erythroid differentiation was assessed by flow cytometry and immunofluorescence. HIF-1α's regulation of PHD2 was examined using luciferase assays and ChIP-seq. CRISPR/Cas9 was applied to knock out EGLN1 and HIF1A, and a fluorescent reporter system was developed to track PHD2 expression. PHD2 knockdown enhanced erythroid differentiation, evident by increased CD71 and CD235a expression. Reporter assays and ChIP-seq identified an HIF-1α binding site in the EGLN1 5' UTR, confirming HIF-1α as a regulator of PHD2 expression. The fluorescent reporter system provided real-time monitoring of endogenous PHD2 expression, showing that HIF-1α significantly modulates PHD2 levels under hypoxic conditions. PHD2 influences erythropoiesis under hypoxia, with HIF-1α regulating its expression. This feedback loop between HIF-1α and PHD2 sheds light on mechanisms driving erythroid differentiation under low-oxygen conditions.

Prognostic impact of expression of CD2, CD25, and/or CD30 in/on mast cells in systemic mastocytosis: a registry study of the European Competence Network on Mastocytosis.

Expression of CD2, CD25 and/or CD30 in extracutaneous mast cells (MC) is a minor diagnostic criterion for systemic mastocytosis (SM) in the classification of the World Health Organization and International Consensus Classification. So far, it remains unknown whether expression of these antigens on MC is of prognostic significance in SM. We performed a retrospective multi-center study of patients with SM using the data set of the registry of the European Competence Network on Mastocytosis, including 5034 patients with various MC disorders. The percentage of CD2-, CD25+ and/or CD30+ MC was considerably lower in patients with indolent SM compared to patients with advanced SM, including aggressive SM and MC leukemia. Whereas CD25 and CD30 expression in MC could not be associated with prognosis, we found that lack of CD2 expression in MC is associated with a significantly reduced overall survival (OS) in patients with SM (p < 0.0001). Lack of CD2 was also associated with the presence of extramedullary involvement affecting the spleen, liver, and/or lymph nodes (odds ratio 2.63 compared to SM with CD2+ MC). Together, lack of CD2 expression in MC is a prognostic marker and indicator of reduced OS and extramedullary disease expansion in patients with SM.

Imaging Flow Cytometric Identification of Chromosomal Defects in Paediatric Acute Lymphoblastic Leukaemia.

Acute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) ETV6::RUNX1. We aimed to assess the applicability of a new imaging flow cytometry methodology that incorporates cell morphology, immunophenotype, and fluorescence in situ hybridisation (FISH) to identify aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. We evaluated this new "immuno-flowFISH" platform on 39 cases of paediatric ALL of B-lineage known to have aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. After identifying the leukaemic population based on immunophenotype (i.e., expression of CD34, CD10, and CD19 antigens), we assessed for copy numbers of loci for the centromeres of chromosomes 4 and 21 and the ETV6 and RUNX1 regions using fluorophore-labelled DNA probes in more than 1000 cells per sample. Trisomy 4 and 21, tetrasomy 21, and translocations of ETV6::RUNX1, as well as gains and losses of ETV6 and RUNX1, could all be identified based on FISH spot counts and digital imagery. There was variability in clonal makeup in individual cases, suggesting the presence of sub-clones. Copy number alterations and translocations could be detected even when the cell population comprised less than 1% of cells and included cells with a mature B-cell phenotype, i.e., CD19-positive, lacking CD34 and CD10. In this proof-of-principle study of 39 cases, this sensitive and specific semi-automated high-throughput imaging flow cytometric immuno-flowFISH method has been able to show that alterations in ploidy and ETV6::RUNX1 could be detected in the 39 cases of paediatric ALL. This imaging flow cytometric FISH method has potential applications for diagnosis and monitoring disease and marrow regeneration (i.e., distinguishing residual ALL from regenerating haematogones) following chemotherapy.

MiR-223 and Chromogranin A Affect Inflammatory Immune Cell Activation in Liver Metastasis of Neuroendocrine Neoplasms.

Neuroendocrine neoplasms (NENs) are a diverse group originating from endocrine cells/their precursors in pancreas, small intestine, or lung. The key serum marker is chromogranin A (CgA). While commonly elevated in patients with NEN, its prognostic value is still under discussion. Secretion/posttranslational proteolytic cleavage of CgA results in multiple bioactive fragments, which are essential regulators of the cardiovascular and immune system. miR-223, regulator of Nrlp3 inflammasome and neutrophil activation, was recently found to have decreased in patients with NEN. We performed flow cytometry of circulating neutrophils in a patient cohort (n = 10) with NEN, microdissection and histology of tumor tissue. Subsequently, in vitro transfections using the well-established human pancreatic NEN cell line (BON), and co-culture experiments with primary macrophages and neutrophils were performed. Serum miR-223 in patients correlated with the expression of the neutrophil activation marker CD15 in circulating cells. Neutrophilic CD62L/CD63 showed good discrimination compared to healthy controls. Immune cell-derived miR-155, miR-193 and miR-223 colocalize with neutrophil in the extra-tumoral tissue alongside Nlrp3-associated caspase-1 activation. miR-223 knockdown in BON decreased the CgA intracellularly, increased in cellular granularity and caspase-1 activation. Plasmin inhibitor a2-aP reverted those effects. Western Blot showed fragmented CgA following miR-223 knockdown, which altered the inflammatory potential of neutrophils. Our data hence provide initial insights into an immunoregulatory mechanism via miR-223 and CgA in NEN cells, as regulation of miR-223 in NEN may affect tumor-associated inflammation.

Illuminating the impact of CD38-induced adenosine formation in B-cell lymphoma

The expression of CD38 by cancer cells may mediate an immune-suppressive effect by producing Extracellular Adenosine (ADO) acting through G-protein-coupled cell surface receptors on cellular components and tumor cells. This can increase PD-1 expression and interaction with PD-L1, suppressing CD8 + cytotoxic T cells. This study examines the impact of heightened CD38 expression and extracellular ADO on various hematological and clinical parameters in patients with mature B-cell lymphoma, alongside their correlation with the soluble counterparts of the PD-1/PD-L1 axis. Our study was conducted on 90 patients, CD38-positive and CD38-negative (measured by flow cytometry), with mature B-cell lymphoma divided into CLL and B-NHL subtypes. Their serum ADO, soluble PD-1, and PD-L1 levels were measured using a sandwich ELISA. Our study revealed a positive correlation between CD38 expression, sADO, sPD-1, and sPD-L1 in mature B-cell lymphoma patients. CD38-positive patients had higher sADO, sPD-1, and sPD-L1 levels. Higher CD38 expression and extracellular ADO negatively affected HB level and PLT count and positively correlated with the higher risk stratification in mature B-cell lymphoma patients. This study explored the potential impact of CD38 expression and elevated extracellular ADO on B-cell lymphoma alongside their link with the PD-1/PD-L1 axis. Our findings underscore the influence of extracellular ADO on the neoplastic process of mature B-cell lymphoma. We also propose targeting the CD38-induced-ADO formation pathway, which could serve as a promising therapeutic immune target with multifaceted effects within mature B-cell neoplasms.