Multiparameter Flow Cytometry Analysis Research Articles

128: DECIPHERING THE IMMUNOLOGICAL DETERMINANTS OF RESPONSE TO NEOADJUVANT CHEMO-RADIATION IN ESOPHAGEAL ADENOCARCINOMA

Abstract Background and aim The standard therapy for esophageal adenocarcinoma (EAC) is pre-operative neoadjuvant chemo-radiation (NACR) and surgery. Less that 30% of patients achieve a pathological complete response (pCR) and increased 5-year survival after NACR. Understanding the mechanisms of response to NACR is pivotal to better stratify patients and inform the design of more efficacious therapies. Cancer progression partly depends on defective immune surveillance, and the presence of tumor infiltrating T lymphocytes (TILs) is associated with better prognosis of several malignancies. We hypothesize that NACR induces immunogenic cell death that stimulates immune responses against tumor neoantigens (TNAs), derived from cancer somatic mutations, in the responders. We thus compared responders and non-responders for tumor immune contexture, immune molecular pathways, circulating immune markers and TNAs-specific T cells responses. Methods Pre-NACR tumor biopsies prospectively collected from consecutive EAC patients were subjected to DNA whole exome and RNA sequencing, high dimensional (HD) multiparametric flow cytometry and immune contexture analysis by histology and by proteome digital spatial profiling (n = 53 targets), while their peripheral blood mononuclear cells and plasma collected pre- and post- NACR were analyzed by HD flow cytometry and cytokine multiplexing assay respectively. Results Treatment-naïve EACs of NACR responders showed higher TNA load and enriched immune signatures compared to non-responders. Moreover, pre-treatment EACs of responders demonstrate augmented effector T cell infiltration and reduced Treg cells and M2-like pro-tumoral macrophages. High M2-like macrophages score associated with lower overall survival. Increased frequencies of circulating myeloid suppressor populations were detected both at baseline and after NACR in non-responder patients. These observations were associated with circulating levels of n = 30 cytokines/chemokines and with spatial expression of immune markers in tumor biopsies. Conclusion Collectively, these results support pre-existing immune-mediated mechanisms of anti-tumor response stimulated by NACR in the fraction of responder EAC patients, suggesting possible ways to stratify patients and direct them to more tailored neoadjuvant and/or adjuvant treatments, possibly in combination with personalized immunotherapy approaches.

Multiparametric Flow Cytometry versus Conventional Cytology in the Study of Leptomeningeal Involvement in Malignant Hematological Diseases

CNS involvement is a complication in hematologic malignant neoplasms. The advantage of multiparametric flow cytometry (MFC) over conventional cytology (CC) in detecting occult leptomeningeal disease in CSF has been proven previously, as reported in the literature. In this study, we reviewed the experience of our laboratory in evaluating CSF specimens by MFC and CC after refinement of technical procedures. MFC analysis was performed in 159 specimens. In 91 specimens, simultaneous CC and MFC analysis was requested and results compared. Neoplastic cells were identified in 27 (17.0%) of the total samples and in 17 (18.7%) of the paired specimens group by MFC, compared with 2 (2.2%) specimens with positive results as determined by CC. MFC enabled identification of malignant cells in low-cellularity specimens (<5 cells/μL) and all neoplasm categories. MFC allowed the detection of minimal numbers of tumor cells in CSF specimens from individuals with leukemia and lymphoma in whom CC had not been able to identify those tumor cells.

P009 Integrin-b7+ cytotoxic MAIT cells are expanded in the colon of Ulcerative Colitis patients, correlate with disease severity and are targeted by vedolizumab therapy

Abstract Background Mucosal-associated invariant T (MAIT) cells are major histocompatibility complex (MHC) class Ib-restricted innate-like lymphocytes with anti-bacterial functions that are enriched at mucosal sites. Their role in ulcerative colitis (UC) is unknown. Methods Colonic biopsies and peripheral blood were collected from patients with active UC (n=34) and healthy control subjects (n=14). Peripheral blood mononuclear cells (PBMC) and Lamina propria mononuclear cells (LPMC) were isolated and activated in vitro with PMA/ionomycin followed by surface and intracellular staining and multiparametric flow cytometry analysis. T cell populations, including gamma delta T cells (TCR gamma delta+), iNKT cells (TCR Vα24-Jα18+), MAIT cells (TCR Vα7.2+ CD161+), CD4, CD8 T cells and their intracellular expression of IL17A, TNF, IFN-g, IL22, IL13 and granzyme B (GZMB) were determined. MAIT cells were further characterised as CD8+, CD4+, CD8 CD4 double positive (DP), CD8 CD4 double negative (DN) cells and expression of the gut homing integrin-b7. Results DN cells were the most common MAIT subset in the colon and were significantly increased in both UC patients and HC subjects compared to peripheral blood. In UC, DN MAIT cells exhibited a cytotoxic phenotype, with significantly higher production of Granzyme B (median 45.0% of cells) compared to DN MAIT cells in HC (median 17%, P&amp;lt;0.005). DN MAIT cells in UC gut also co-produced significantly higher proportion of IL17A (median 22% in UC vs 7% in HC, P&amp;lt;0.006) and TNF (median 61% in UC vs 10% in HC, P&amp;lt;0.02) compared to DN MAIT cells from HC. In UC patients, the proportional abundance of cytotoxic DN MAIT cells accumulating in the colon significantly correlated with disease activity (UCEIS) score (r2=0.34, P&amp;lt;0.0007). The number of DN MAIT cells expressing integrin-b7 was also significantly higher in UC patients compared to HC (median 66% in UC vs 32% in HC, p&amp;lt;0.001). In UC patients initiated on vedolizumab therapy, the proportional abundance of cytotoxic, DN MAIT cells accumulating in the colon significantly fell at week 14 in comparison with their pre-treatment abundance (median 25% vs 51%, P&amp;lt;0.001). Following vedolizumab induction, the reduction in the abundance of colonic cytotoxic DN MAIT cells correlated with endoscopic improvement (r2=0.45, p=0.0085). Conclusion DN MAIT cells with cytolytic activity and augmented expression of multiple cytokines are enriched in the colon of UC patients in numbers that correlate with the magnitude of mucosal injury. Vedolizumab treatment reduces DN MAIT cell accumulation in the colon and the magnitude of reduction correlates with treatment response.

Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.

Autoimmune Cytopenias and Dysregulated Immunophenotype Act as Warning Signs of Inborn Errors of Immunity: Results From a Prospective Study

Inborn errors of immunity (IEI) are genetic disorders characterized by a wide spectrum of clinical manifestations, ranging from increased susceptibility to infections to significant immune dysregulation. Among these, primary immune regulatory disorders (PIRDs) are mainly presenting with autoimmune manifestations, and autoimmune cytopenias (AICs) can be the first clinical sign. Significantly, AICs in patients with IEI often fail to respond to first-line therapy. In pediatric patients, autoimmune cytopenias can be red flags for IEI. However, for these cases precise indicators or parameters useful to suspect and screen for a hidden congenital immune defect are lacking. Therefore, we focused on chronic/refractory AIC patients to perform an extensive clinical evaluation and multiparametric flow cytometry analysis to select patients in whom PIRD was strongly suspected as candidates for genetic analysis. Key IEI-associated alterations causative of STAT3 GOF disease, IKAROS haploinsufficiency, activated PI3Kδ syndrome (APDS), Kabuki syndrome and autoimmune lymphoproliferative syndrome (ALPS) were identified. In this scenario, a dysregulated immunophenotype acted as a potential screening tool for an early IEI diagnosis, pivotal for appropriate clinical management and for the identification of new therapeutic targets.

Impact of Sex on Circulating Leukocytes Composition in COPD Patients.

PurposeChronic obstructive pulmonary disease is characterized by chronic inflammatory response both at the lung site and at the systemic level. Abnormalities in circulating leukocytes have been reported to occur in COPD patients and have been often shown to correlate with the decline in lung function. COPD affects men and women at a virtually comparable rate, even though distinct sex specific symptoms, progression and therapeutic implications have been described. Nonetheless, these sex-associated differences have not been analyzed in terms of circulating leukocytes. To assess the impact of sex on the changes of circulating immune cells in COPD patients.Patients and MethodsBlood samples were collected from 50 COPD patients (31 males, 19 females) and 63 age and sex-matched controls (35 males, 28 females) enrolled in this pilot study. Complete blood cell count and multi-parametric flow cytometry analysis were performed to characterize the leukocyte populations and subsets.ResultsMale COPD patients are distinguished from controls by a significant increase in white blood cell counts, neutrophil total and differential counts, and neutrophil-to-lymphocyte ratio. Conversely, a generalized leukocyte decrease discriminated female COPD patients from the related controls. The impact of sex is further remarked by a decrease in adaptive immune cell subpopulations in males as opposed to a consistent increase of innate immune cell types in females correlating with disease severity.ConclusionThese data indicate that the definition of specific changes of circulating leukocytes to be used as reliable biomarkers of the disease severity cannot be accomplished irrespectively of sex.

Vascular repair and regeneration in cardiometabolic diseases.

Chronic cardiometabolic assaults during type 2 diabetes (T2D) and obesity induce a progenitor cell imbalance in the circulation characterized by overproduction and release of pro-inflammatory monocytes and granulocytes from the bone marrow alongside aberrant differentiation and mobilization of pro-vascular progenitor cells that generate downstream progeny for the coordination of blood vessel repair. This imbalance can be detected in the peripheral blood of individuals with established T2D and severe obesity using multiparametric flow cytometry analyses to discern pro-inflammatory vs. pro-angiogenic progenitor cell subsets identified by high aldehyde dehydrogenase activity, a conserved progenitor cell protective function, combined with lineage-restricted cell surface marker analyses. Recent evidence suggests that progenitor cell imbalance can be reversed by treatment with pharmacological agents or surgical interventions that reduce hyperglycaemia or excess adiposity. In this state-of-the-art review, we present current strategies to assess the progression of pro-vascular regenerative cell depletion in peripheral blood samples of individuals with T2D and obesity and we summarize novel clinical data that intervention using sodium-glucose co-transporter 2 inhibition or gastric bypass surgery can efficiently restore cell-mediated vascular repair mechanisms associated with profound cardiovascular benefits in recent outcome trials. Collectively, this thesis generates a compelling argument for early intervention using current pharmacological agents to prevent or restore imbalanced circulating progenitor content and maintain vascular regenerative cell trafficking to sites of ischaemic damage. This conceptual advancement may lead to the design of novel therapeutic approaches to prevent or reverse the devastating cardiovascular comorbidities currently associated with T2D and obesity.

Prediction of Clinical Response to Venetoclax Plus Decitabine in AML Using a 7-Day Ex Vivo Assay

Prediction of Clinical Response to Venetoclax Plus Decitabine in AML Using a 7-Day Ex Vivo Assay

Early Immune Profile Assessment By Flow Cytometry Predicts Severe COVID-19 Pneumonia

▪Background and ObjectiveThe rapid spread of the COVID -19 pandemic and the high variability of the course of the disease make it essential to search for early predictors of outcome. The objective of our study is to predict severe SARS COV2 pneumonia using early cytometric profilesMaterial and MethodsProspective and observational study of adults with confirmed COVID-19 infection admitted on Emergency Department (ED). We collected epidemiological, clinical and laboratory data of every patient until they were discharged or died. Multiparametric flow cytometry (FC) analysis of T-lymphocytes (CD4, CD8, CD4 activated, CD8 activated, naïve (Tn), central-memory (Tcm), effector-memory (Tem), effector (Te) and Th17 subsets), B-lymphocytes (naïve, memory, transitional subsets, and assessment of clonality), NK cells, plasmablasts, p-DCs (plasmacytoid dendritic cells), m-DCs (myeloid dendritic cells), basophils, and monocytes (MO1, MO2, MO3, slan+ MO3) was performed on whole peripheral blood collected on EDTA, before immunosuppressive therapy was started. We designed a 7-tube 8-color experimental panel. Cell surface staining of 2 × 106 cells was performed and at least 500 000 total events were acquired for the assessment of plasmablasts, p-DCs, m-DCs, basophils, and the monocyte subsets; for the study of B, T and NK-lymphocyte populations we acquired at least 100 000 total events (FACS Canto II; BD Biosciences). Severity was assessed on the basis of World Health Organization´s (WHO) international 10 level ordinal scale (WHOs) and also according to 4 respiratory status, based on SpO2(peripheral blood oxygen saturation)/FIO2 (fraction of inspired oxygen) ratio (SpFi). SpFi group 1 >452, SpFi2 2: 315-452; SpFi 3 236-315, SpFi 4: <236 (respiratory distress)Results53 patients were included: epidemiological and clinical data available on table 1. 23 patients (43.39%) arrived to SpFi4 status. WHOs >6 (WHO 6:oxygen by non invasive ventilation or high flow) was achieved by 20 patients (37.7%)Good prognosis (meaning SpFi1 as the worse respiratory status in the follow -up) was associated to cytometric profiles: there was a significant increase in CD3, p-DCs, m-DCs, basophils, monocytes, Tcm, % of lymphocytes and CD3/CD19 ratio whereas there was a significant reduction in CD19, % of neutrophils and % Neutrophils/% lymphocyte ratio. In the SpFi4 group, there was a significant reduction of CD3, p-DCs, m-DCs, plasmablasts and CD3/NK ratio. In patients starting in SpFi group 1-2 in the ED but progressing to SpFi 3-4 during the follow up (27 patients), there was a statistically significant relation with Tn, Te and Tn/Te ratio (Tn/Te ratio <0.717: OR 13.5 (p 0.002, [95% CI 2.552-71.403). Initial SpFi1 patients that evolved to SpFi 3-4 during follow up (10 patients), presented a Tn/Te ratio < 0.717 with an OR 11.556 (p =0.005, [95% CI 2.059-64.853]).Plasmablasts < 0.075 and CD3/NK <5.71, were identified as independent risk factors for SpFI4 during follow up. After multivariable analysis, both variables kept their significance: CD3/NK (OR 11,247, p=0.005) and plasmablasts (OR 12,524 , p=0.004). About prediction of WHOs >6, multivariable analysis showed CD3/NK <5.71 (OR 22,240 [95%CI 2,340-211,342] p= 0.007) and plasmablasts<0.075 (OR 28.635 [95% CI 3,187-257,301] p=0.003).A score (0,1,2) comprising both risk factors, was significantly predictive of SpFI4, regardless of the initial respiratory status, age or days from symptoms onset. In our cohort, only 1/15 (6.7%) patients with 0 points (neither plasmablasts nor CD3/NK score), arrived to SpFi4. However, 10/11 (90.9%) patients with 2 points, reached to SpFi4 respiratory status (C-index = 0.837) Same score was applied to predict WHOs > 6: 90.9% with 2 points progressed to WHO>6 and 0/15 patients with 0 points reached the same goal (C-index = 0.872)An incidental finding of 4 indolent B-lymphoproliferative disorders (2CLL-like MBLs and 2non -CLL-like MBL), was found, and they were associated with older age and progression to death.ConclusionsFlow cytometry on whole peripheral blood samples of SARS-COV2 pneumonia patients, collected before corticosteroid or immunosuppressive therapy, could identify cytometric patterns associated to prognosis. Plasmablasts, mDCs and pDCs levels as well as CD3/NK ratio, are associated to a worse respiratory status, while Tn/Te ratio could detect non-severe patients who will require high-flow oxygen devices during follow up. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multiple TET2 Mutations As a New Biological Clue for Differentiating Oligomonocytic Chronic Myelomonocytic Leukemia from Myelodysplastic Syndromes

Introduction. Oligomonocytic chronic myelomonocytic leukemia (OM-CMML) is defined as those myelodysplastic syndromes (MDS) or MDS/MPN unclassifiable cases with relative monocytosis (≥ 10% monocytes) and a total monocyte count of 0.5-<1x10 9/L. These patients show similar clinical, genomic and immunophenotypic features than overt CMML, although the majority of them are currently classified as MDS following WHO 2017 classification. TET2 is the most prevalent mutated gene in CMML and it is frequent to find multiple TET2 mutations in the same patient.Objective. We investigated TET2 mutations in 42 OM-CMML, their diagnostic value and their correlation with immunophenotypic pattern, and compared them with that observed in 54 CMML and 86 MDS.Patients and methods. Samples were collected from 182 patients: 42 OM-CMML, 54 overt CMML, and 86 MDS. Molecular characterization was performed by next-generation sequencing (NGS) using a custom panel (QIAseq Custom DNA Panels, Qiagen) including the whole codifying region of 25 myeloid-associated genes and sequenced using Illumina technology. Multiparametric flow cytometry (FC) analysis of monocyte subsets was performed on whole PB collected on EDTA. Cell surface staining of 2 × 10 6 cells was performed and at least 500 000 total events were acquired per tube (FACS Canto II; BD Biosciences). The FC strategy analysis, the 5-tube experimental panel, and the NGS custom panel are described in Calvo & Garcia-Gisbert, Blood Adv 2020.Results. 1.Patients with multiple TET2 (multi-TET2) mutations (2 or more) were identified with similar percentages in OM-CMML (17/42, 40.5%) and CMML (29/54, 53.7%) while they were infrequent in MDS (5/86, 5.8%) (P<0.001). In MDS patients, 4/5 with multi-TET2 showed a percentage of monocytes >10% though they were not considered OM-CMML since the monocyte count was below 500/microL. CMML and OM-CMML patients with multi-TET2 showed predominantly 2 TET2 mutations (CMML: 20/29, 69%; OM-CMML: 14/17, 82%;). The distribution of patients with >2 TET2 mutations was similar in CMML and OM-CMML. We did not observe differences among the frequency of nonsense, frameshift or missense TET2 mutations, nor between early or late-truncating (early: aa 1-1128, late: 1128-1936) TET2 mutations between OM-CMML and CMML.2.TET2 variant allele frequency (VAF) was significantly lower in MDS (median 11.83%) than in OM-CMML and CMML (P<0.001 in both comparisons). OM-CMML and CMML showed similar TET2 VAF (medians 41.4% and 40.3%). In those patients with TET2 VAF >55% or a sum of muti-TET2 mutations VAF above 55% (17 OM, 19 CMML, 2 MDS), we could infer a biallelic alteration in the same clone (Coltro, Leukemia 2020). We did not observe differences in the frequency of OM-CMML showing biallelic TET2 mutations when compared to CMML.3.The main clinical characteristics of CMML patients (overt and OM-CMML), grouped by TET2 mutational status (unmutated: TET2 WT; 1 TET mutation: TET2 single; ≥2 mutations: multi-TET2) are depicted in Table 1. As shown, we did not observed significant differences when comparing TET2 single to TET2 WT or multi-TET2 Of note, multi-TET2 patients showed a higher percentage of PB and BM monocytes, BM promonocytes and more dysgranulopoiesis than TET2 WT patients.4.Patients with TET2 WT presented a lower number of mutated genes than TET single and multi-TET2 patients (P=0.019 and P=0.029). In line with previous studies, IDH mutations and TET2 mutations where mutually exclusive (P=0.001). Co-mutation of SRSF2 and TET2, the well-accepted gene signature of CMML, was found in 25/96 patients (26.0%, 13 OM-CMML, 12 CMML) in 10 cases co-mutated with TET2 single and in 15 cases with multi-TET2.5.We assessed the proportion of OM-CMML patients with MO1>94% since this has been shown as very sensitive and specific feature of CMML. In TET2 mutated OM-CMML, we observed a higher proportion of patients with MO1>94% (90% mut. vs 40% WT; P=0.001) and CD56 expression on monocytes (70% mut. vs 30% WT; P=0.025). We observed a trend (non-significant) difference in the proportion of patients showing these features when comparing TET2 single vs multi-TET2 OM-CMML (single, CD56: 57.1%, MO1>94%: 85.7%; multi-TET2, CD56: 81.3%, MO1>94%: 93.8%).Conclusion. The high occurrence of multiple TET2 mutations in overt and OM-CMML and its infrequency in MDS is a new biological clue for supporting the consideration of OM-CMML in the continuum of CMML. [Display omitted] DisclosuresSalar: Celgene: Consultancy, Speakers Bureau; Gilead: Research Funding; Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Bellosillo: Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau; Thermofisher Scientific: Consultancy, Speakers Bureau.

Significant Plasma Cell Loss (up to 90%) Despite Preserved Overall Cell Viability - Time Dependent Changes Observed in Multiparametric Flow Cytometry Analysis of Bone Marrow Samples in Multiple Myeloma

Multiparametric flow cytometry (MPFC) is a mainstream laboratory method used in the diagnosis of multiple myeloma. Minimal residual disease (MRD) assessment by EuroFlow next-generation flow cytometry allows assessment down to an assay sensitivity of 1x10 -5.Delayed sample processing remains a common challenge due to logistical limitations. Specialized tests performed in central pathology laboratories are frequently located a considerable distance from healthcare providers.Our study aims to evaluate the impact of delayed sample processing on plasma cell yield and bone marrow sample stability. There is little published data available.Plasma cell yield and bone marrow sample stability were investigated in patients with multiple myeloma who underwent bone marrow biopsy. Participants were included based on ³10% plasma cell burden by morphological quantification on the bone marrow aspirate smear. Bone marrow aspirates were collected in EDTA (with three samples also collected in lithium heparin) and stored at four degrees Celsius. Samples were analyzed by MPFC within four hours of collection, at 24 and at 48 hours after collection. CD138 and CD38 co-expression were used to identify plasma cells, and absence of 7-AAD to determine cell viability. Mean fluorescence intensity (MFI) of CD138 and CD38 was recorded. Statistical analyses were performed using two-tailed Wilcoxon signed-rank tests and repeated measures ANOVA with significance assigned at p<0.05.Bone marrow aspirate samples of nine participants were evaluated. Significant reduction in plasma cell yield was observed over time (p<0.001) while sample integrity remained unchanged (p>0.05). The most marked reduction in plasma cell detection was seen between initial processing and 24 hours (median absolute reduction 9%, range 0 to 23% and median relative reduction 37%, range -8 to 90%, p<0.01). Further significant reduction of plasma cells occurred after an additional 24 hours (p=0.025). At 48 hours, the median absolute reduction in plasma cell yield from initial testing was 12% (range 1 to 24%) and median relative reduction was 40% (range 18 to 90%).Sample integrity remained constant. The median viability at collection, 24 hours and 48 hours was 91%, 93% and 95% respectively. The most significant specimen deterioration observed was 13% viability reduction to 75% overall by 48 hours.Three of the participants had additional samples collected in lithium heparin anticoagulant media that were analyzed in parallel with their EDTA samples. Plasma cell yield remained similar across the two different anticoagulants with overall cell viability remaining high in lithium heparin (³90%). A trend of time-dependent reduction of CD138 MFI was observed with lithium heparin but not with EDTA.This study demonstrates the significance of time to processing as a pre-analytical variable in MPFC in multiple myeloma. The greatest loss of plasma cells occurs within the first 24 hours after collection but continues to fall significantly out to 48 hours. Reductions of up to 90% were observed in our small cohort and represent a potential 1 log reduction in yield. This decrease in plasma cell yield raises questions of reliability and validity of flow cytometry, whereby the sensitivity depth may be compromised if the sample cannot be processed on the same day of collection. It is a technical limitation of flow cytometry in comparison to polymerase chain reaction methods where sensitivity is unaffected by delays in processing.The overall viability of cells within the samples remained stable over time, despite the decline in plasma cells. A reduction in CD138 MFI is observed in lithium heparin storage medium that may impact on standardized gating techniques. Further validation studies are warranted to explore these phenomena.MRD monitoring in multiple myeloma is rapidly becoming an accepted standard of care in the evaluation of treatment response and represents an independent prognostic maker of progression free survival that can be used to guide further therapy. Our findings indicate the potential of false negative MRD results with delays in sample processing. This questions the current consensus guidelines that recommend samples can be processed up to 2 days after collection. These guidelines may need to be revised in the near future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Functionally Mature CD1c+ Dendritic Cells Preferentially Accumulate in the Inflammatory Arthritis Synovium.

ObjectiveTo examine the role of synovial CD1c+DCs in patients with Inflammatory Arthritis (IA) with a specific focus on the transcriptional and maturation signatures that govern their function.MethodsRNA sequencing was performed on healthy control (HC) peripheral blood (PB), IA PB, and IA synovial fluid (SF) CD1c+DCs. Multiparametric flow-cytometry and SPICE analysis were used to examine site [SF and Synovial Tissue (ST) CD1c+DCs] and disease specific characteristics of CD1c+DCs, while functional assays such as antigen processing, activation, and MMP production were also performed.ResultsIncreased frequency of CD1c+DCs (p<0.01) with a concomitant increase in CD80, CCR7 (p<0.01), and CXCR3 (p<0.05) expression was identified in IA PB compared to HC PB. Enrichment of CD1c+DCs was identified in IA synovial tissue (ST) (p<0.01) and IA SF (p<0.0001) compared to IA PB, while RNAseq revealed distinct transcriptional variation between PB and SF CD1c+DCs. Flow cytometry revealed increased expression of CD83, CD80, PD-L1, and BTLA (all p<0.05) in IA SF CD1c+DCs compared to PB, while SPICE identified synovial cells with unique co-expression patterns, expressing multiple DC maturation markers simultaneously. Functionally, synovial CD1c+DCs are hyper-responsive to TLR7/8 ligation (p<0.05), have decreased antigen processing capacity (p=0.07), and display dysregulated production of MMPs. Finally, examination of both synovial CD1c+DCs and synovial CD141+DCs revealed distinct maturation and transcriptomic profiles.ConclusionSynovial CD1c+DCs accumulate in the inflamed IA synovium in a variety of distinct poly-maturational states, distinguishing them transcriptionally and functionally from CD1c+DCs in the periphery and synovial CD141+DCs.

ABSENT CD33 IN AML PATIENT MIMICS MRD POSITIVE: A SINGLE CASE REPORT

Diagnosis and treatment of acute myeloid leukemia (AML) remains difficult to this day, cure rates remain low and measurable residual disease (MRD) assessment is still challenging. Early detection of residual leukemic cells is essential to predict therapeutic success and Multiparametric Flow Cytometry (MFC) has been an effective tool for it. Here, we focus on the importance in caution when interpreting MDR in AML. Bone marrow (BM) sample was assessed to FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype (KT) and immunophenotyping. Using an 8-colour antibody panel (CD45, CD34, CD117, CD 33, HLA-DR, CD38, CD123, CD13, CD19, CD7, CD11b, CD4, CD56, CD64, CD11c, CD15, CD133, CD41a and NG2), we applied a gating strategy with fixed gates comparing normal to AML BM, settled to catch LAIPs (Leukemia Associated-Immunophenotypes), abnormal cell maturation pattern and Leukemic Stem Cell, that were detected on diagnosis and evaluated for MRD. Assays were performed at the Hematology Laboratory of the Medical School of Ribeirão Preto, University of São Paulo, in accordance to Local Ethical Boards. A 21-year-old male patient with AML without maturation, diagnosed after 60 days of symptoms, presented anemia (7.9 g/dL), thrombocytopenia (26 000/μL) and white blood cell count of 5710/μL with 73% blasts in peripheral blood and 84% in BM. MFC revealed CD45dim and low side scatter; positive staining for CD117, CD34, HLA-DR, CD38, CD123, CD11c, CD64, CD15 and CD133; negative staining for CD33, CD14, CD7, CD11b, CD4, CD56, CD19, CD41a and NG2. We found no metaphases (KT), no molecular abnormality and he failed risk stratification. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day). BM was obtained at diagnosis, 30 and 33 days after 1 st and 2 nd induction, and 3, 9, 12 and 15 months after consolidation of chemotherapy. The positivity of a specific phenotype (CD117+/CD34+/CD33wk/CD13+ ≥ 0.1%) was preserved at diagnosis and follow-ups: 0,506%, 0,681%, 0,402%, 0,101%, 0,158% and 0,122%. Complete remission was achieved by day 30 after 1st induction and no change in the outcome was reported. Absent CD33 was observed in diagnosis and follow-ups, during and after treatment. Post-analytical phase of MFC is the most vulnerable to wrong interpretations. CD33 is a common myeloid antigen expressed on malignant blasts in AML and had been reported as a potential target therapy. CD33 low blasts are associated to a more mature AML and its high expression is related to adverse landscapes, highlighting the importance of CD33 evaluation. Standard care for AML enrolls MRD monitoring and failure to achieve an MRD-negative, CR or detected MRD during or after therapy is associated with relapse or poor outcomes. Still, MRD analysis must embrace a set of standards in order to prevent post-analytic errors. Here we present a case report of AML with absent CD33 maintained from diagnosis to MRD follow-ups, mimicking MRD positivity. This data emphasizes the importance of caution in MFC analysis and correlation of diagnostic and follow-ups results interpretation, as well as constant training of the team.

Unbiased immune profiling reveals a natural killer cell-peripheral nerve axis in fibromyalgia.

The pathophysiology of fibromyalgia syndrome (FMS) remains elusive, leading to a lack of objective diagnostic criteria and targeted treatment. We globally evaluated immune system changes in FMS by conducting multiparametric flow cytometry analyses of peripheral blood mononuclear cells and identified a natural killer (NK) cell decrease in patients with FMS. Circulating NK cells in FMS were exhausted yet activated, evidenced by lower surface expression of CD16, CD96, and CD226 and more CD107a and TIGIT. These NK cells were hyperresponsive, with increased CCL4 production and expression of CD107a when co-cultured with human leukocyte antigen null target cells. Genetic and transcriptomic pathway analyses identified significant enrichment of cell activation pathways in FMS driven by NK cells. Skin biopsies showed increased expression of NK activation ligand, unique long 16-binding protein, on subepidermal nerves of patients FMS and the presence of NK cells near peripheral nerves. Collectively, our results suggest that chronic activation and redistribution of circulating NK cells to the peripheral nerves contribute to the immunopathology associated with FMS.

African Trypanosomiasis: Extracellular Vesicles Shed by Trypanosoma brucei brucei Manipulate Host Mononuclear Cells.

African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by Glossina spp. (tsetse fly). Parasite introduction into mammal hosts triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defence since they are antigen-presenting cells and have a microbicidal function essential for trypanosome clearance. Adaptive immune defence is carried out by lymphocytes, especially by T cells that promote an integrated immune response. Like mammal cells, T. b. brucei parasites release extracellular vesicles (TbEVs), which carry macromolecules that can be transferred to host cells, transmitting biological information able to manipulate cell immune response. However, the exact role of TbEVs in host immune response remains poorly understood. Thus, the current study examined the effect elicited by TbEVs on MΦ and T lymphocytes. A combined approach of microscopy, nanoparticle tracking analysis, multiparametric flow cytometry, colourimetric assays and detailed statistical analyses were used to evaluate the influence of TbEVs in mouse mononuclear cells. It was shown that TbEVs can establish direct communication with cells of innate and adaptative immunity. TbEVs induce the differentiation of both M1- and M2-MΦ and elicit the expansion of MHCI+, MHCII+ and MHCI+MHCII+ MΦ subpopulations. In T lymphocytes, TbEVs drive the overexpression of cell-surface CD3 and the nuclear factor FoxP3, which lead to the differentiation of regulatory CD4+ and CD8+ T cells. Moreover, this study indicates that T. b. brucei and TbEVs seem to display opposite but complementary effects in the host, establishing a balance between parasite growth and controlled immune response, at least during the early phase of infection.

Abstract 1501: Employing 3D-ACT platform to assess the ability of stromal-targeting agents to improve penetration and efficacy of cell therapy

Abstract Background: Adoptive T cell therapy (ACT) has demonstrated great clinical success for the treatment of hematological malignancies. However, the unique challenges presented by solid tumors has significantly limited their utility. The primary characteristic of an effective ACT is the ability to selective identify tumor cells. In case of solid tumors, the ACT faces the additional hurdle requiring penetration into the tumor microenvironment (TME) while retaining function. Tumors develop highly immunosuppressive stroma which serves as a mechanical hinderance for the penetration and function of activated effector cells. In this study we used our 3D-ACT platform to investigate the ability of focal adhesion kinase (FAK) inhibitors to enhance the penetration of engineered cells into the intact patient tumor stroma and their efficacy within the tumor immune microenvironment. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids with intact TME measuring 150 µm in size were prepared from fresh tumor samples of renal cell carcinoma (RCC), and colorectal carcinoma (CRC) using proprietary technology developed at Nilogen Oncosystems. Engineered T cells were labeled with different cell tracker fluorescent dyes to monitor cell movements and locations within tumoroids by confocal analysis. 3D tumoroids were treated with vehicle only or FAK inhibitors and exposed to CAR-T cells at 10:1 E:T ratio. Cell penetration within tumoroids and tumor cell killing was measured by high-content confocal imaging combined with 3D-image analysis. ACT cell activation was monitored using multiparameter flow cytometry analysis and multiplex cytokine release assays to assess changes in the TME. Results: Our studies demonstrated that the confocal-based high-content real time imaging platform, combined with custom image analysis algorithms, allowed for monitoring of treatment-mediated tumor cell killing with structural analysis of engineered T-cells in the intact 3D tumoroids. Our data revealed that CAR-T cell penetration into tumoroids greatly varied between different tumor types and was heavily influenced by the tumor's stromal components. Treatment with FAK-inhibitors led to alterations within the TME, which subsequently impacted penetration and efficacy of CAR-T cells as assessed by flow cytometry and confocal imaging. Conclusion: The ex vivo model described here (3D-ACT) retains the TME and replicate the heterogeneity of tumor stroma to provide provides a clinically relevant platform to evaluate the influence of the tumor stromal components on engineered T cell functions. We believe the 3D-ACT platform can effectively be used to screen for drugs that can promote ACT penetration into the tumor stroma and survival within the tumor microenvironment. Citation Format: Zhisong Tong, Stephen Iwanowycz, Jared Ehrhart, Mibel M. Pabón, Tina Pastoor, Soner Altiok. Employing 3D-ACT platform to assess the ability of stromal-targeting agents to improve penetration and efficacy of cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1501.

Hyper-Activation of the Adenosinergic Axis in the Eμ-TCL1 Chronic Lymphocytic Leukemia Mouse Model Offers Therapeutic Opportunities

Background. Tumor immunosuppression is a major cause for treatment failure and disease relapse, both in solid tumors and leukemia. Several indications suggest that increased extracellular adenosine levels, which potently suppress T cell responses and skew macrophages towards an M2 phenotype, characterize the tumor microenvironment. Hence, there is intense investigation to identify drugs that target this axis.We previously showed that CLL cells, particularly those endowed in the proliferation centers, express CD39 and CD73, the enzymes that lead to extracellular adenosine production. We also showed that the A2A adenosine receptor is expressed at high levels in CLL lymph nodes, likely due to the effect of hypoxia in positively modulating adenosine production.Aim of the work. The main aim of this work is to study the expression and the role of the adenosinergic axis in vivo, determining the impact of this signaling pathway in CLL progression and modulation of immune responses. To this purpose, we exploited the Eμ-TCL1 (TCL1) mouse model based on the adoptive transfer of spleen-purified leukemic cells in naïve C57BL/6 immunocompetent recipients (WT).Results. Firstly, expression of the adenosinergic axis (CD39, CD73, CD26 and A2A) both on the leukemic side and on bystander immune cells was assessed by comparing cells obtained from the spleen, the bone marrow (BM) and peripheral blood (PB) of TCL1 and WT mice. Flow cytometry analysis of B220+/CD5+ leukemic cells showed that CD39 was constantly expressed at high levels, in contrast to normal circulating B cells from naïve mice. CD73 expression was more heterogeneous, with both CD73+ and CD73- leukemic clones. These data recapitulate the phenotype observed in CLL patients and sustain the hypothesis of variable levels of adenosine in tissues colonized by leukemic cells. In contrast, CD26, the adenosine deaminase-anchoring protein was poorly detected, as opposed to WT B cells. Real-time PCR data obtained from purified cell populations confirmed this expression pattern and also indicated dramatic up-regulation in the expression of the A2A receptor, suggesting that leukemia development is accompanied by increased ability to respond to extracellular adenosine. The development of CLL is usually associated with specific T cell and myeloid subset changes. Multiparametric flow cytometry analyses, with selective markers, were adopted to identify naïve, memory, central memory, effector memory and effector T cells, as well as inflammatory, intermediate or patrolling monocytes. Data obtained confirm that in TCL1 mice the naïve T cell subset was dramatically reduced compared to WT mice, with the simultaneous increase in memory, exhausted and regulatory T cell sub-populations. Both CD4+ and CD8+ lymphocyte subsets in TCL1 mice were highly CD39+ compared to the normal counterpart, as opposed to CD73 that was expressed in distinct subpopulation of cells, including regulatory T cells. No significant differences were highlighted for CD26, in all the tissues considered, while the A2A receptor became expressed at very high levels in all cellular components of the leukemic niche. In the monocyte compartment, we observed a reduction of the inflammatory subset, with a parallel increase of patrolling population, functionally similar to M2 tumor-supportive macrophages.Given the role of low oxygen tension as a critical modulator of the adenosinergic system, hypoxia levels were determined, comparing tumor-bearing and WT mice. Mice were treated with pimonidazole hydrochloride, a hypoxia marker, 2 hours prior euthanasia. Flow cytometry and immuno-histochemistry analyses indicated that leukemic engraftment is paralleled by increased hypoxia levels in the lymphoid tissues, such as spleen or BM.Conclusions.Taken together, these results show that the adenosinergic axis is over-expressed in the TCL1 mouse model and confirm that the presence of leukemic cells polarizes the host immune cells, favoring the establishment of a tumor supportive environment. This could be due, at least partly, to the presence of a highly hypoxic leukemic niche. These observations confirm what described for CLL patients, indicating that this animal model can be used to test the impact of adenosine-targeting therapies, in combination with drugs that directly target the clone. DisclosuresNo relevant conflicts of interest to declare.

Molecular Subtypes of Plasma Cell Neoplasms with Circulating Neoplastic Plasma Cells

Background:Circulating neoplastic plasma cells (CNPCs) are frequently seen in plasma cell neoplasms (PCN). Prior studies have shown that CNPCs are usually associated with a higher tumor burden and disease progression. Additionally the percentage of CNPCs in newly diagnosed PCN as quantified by flow cytometric analysis is found to be an independent predictor of survival.The UAMS myeloma group has established a molecular classification of PCNs based on unsupervised hierarchical clustering of gene expression profiling (GEP) data, and has identified 7 subgroups: Hyperdiploidy (HY), Cyclin D-1 (CD-1), CD-2, Low Bone (LB), Proliferation (PR), MMSET/FGFR3 (MS) and MAF (MF). This molecular subtyping in conjunction with the UAMS GEP 70 gene signature profile has been found to be clinically useful in identifying low risk group with superior event-free and overall survival from high-risk group.The purpose of this study was to determine if a particular PCN molecular subtype was more likely to be associated with CNPCs. To the best of our knowledge, the association between the different molecular subtypes and CNPCs has not been studied before.Design:A retrospective review was performed to determine the number of PCN patients with CNPCs during 2015-2016. Peripheral blood immunophenotyping was performed using 8-color multi-parameter flow cytometry analysis. PCN molecular subtypes and GEP risk stratification was performed on CD138-enriched plasma cells from bone marrow aspirate based on our previously established protocol. Additional data including clinical classification, bone marrow plasma cell tumor burden and karyotype analysis were also collected.Results:A total of 305 patient had peripheral blood flow cytometric analysis performed during the study period; 147 patients showed presence of CNPCs (48.19%). Only 120 of the 147 cases with CNPCs had all the additional data and were included for further analysis.Based on UAMS GEP 70 signature risk score, 86 patients (71.7%) showed low risk gene classification compared to 34 patients (28.3%) with high risk gene classification. Distribution of molecular subgroups defined by GEP data, identified that the HY group was the largest subgroup (n=29, 24.2%), followed by CD-2 (n= 24, 20%), PR (n=19, 15.8%), MF (n=15, 12.5%), MS (n=12, 10%), CD-1 (n=12, 10%) and LB (n=9, 7.5%). However, further analysis of low risk group patients identified equal distribution of HY (n=24) and CD-2 (n=23) subgroups. Further analysis of molecular subgroups based on bone marrow tumor burden is listed in Table 1.Based on the international myeloma working group diagnostic criteria, most patients had multiple myeloma (MM) (n=112, 93.3%); smoldering myeloma (n=5, 4.2%) and MGUS (n=3, 2.5%) was less common. Of the MM group, two patients met the criteria for plasma cell leukemia and showed extensive bone marrow involvement. Bone marrow involvement in a cases ranged from <5% to 95% plasma cells; with most patients (72.5%) showing <60% cellularity replacement by plasma cells. Cytogenetic analysis revealed abnormal/complex karyotype in the majority of the patients (n=89, 73%) compared to normal karyotype (n=31, 26%).Conclusions:Our study indicates that CNPCs occurs more frequently in patients with low risk multiple myeloma based on UAMS GEP 70 signature classification especially CD-2 and Hyperdipoidy (HY) subtypes. This correlation is best observed in cases with <60% bone marrow tumor burden. These patients are also more likely to have abnormal/complex karyotype. Further long-term studies should be performed to determine the prognostic significance of these findings. [Display omitted] DisclosuresMorgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria. Davies:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants

BackgroundHigh-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of...

Unsupervised Flow Cytometry Analysis Allows for an Accurate Identification of Minimal Residual Disease Assessment in Acute Myeloid Leukemia.

Simple SummaryIn acute myeloid leukemia (AML), minimal/measurable residual disease (MRD) can be assessed based on molecular markers or immunophenotypic features evaluated at diagnosis, through multiparameter flow cytometry (MFC) for the latter. New artificial intelligence tools allow to perform unsupervised analysis of MFC data. The Flow-Self-Organizing-Maps (FlowSOM) tool was used here to concomitantly compare MFC features of normal bone marrow together with diagnosis and follow-up bone marrow samples from 40 AML patients for the evaluation of MRD. MFC results were compared to molecular MRD, showing high concordance. This opens the road for a new easy and objective way of assessing MRD even in AML patients without molecular markers.The assessment of minimal residual disease (MRD) is increasingly considered to monitor response to therapy in hematological malignancies. In acute myeloblastic leukemia (AML), molecular MRD (mMRD) is possible for about half the patients while multiparameter flow cytometry (MFC) is more broadly available. However, MFC analysis strategies are highly operator-dependent. Recently, new tools have been designed for unsupervised MFC analysis, segregating cell-clusters with the same immunophenotypic characteristics. Here, the Flow-Self-Organizing-Maps (FlowSOM) tool was applied to assess MFC-MRD in 96 bone marrow (BM) follow-up (FU) time-points from 40 AML patients with available mMRD. A reference FlowSOM display was built from 19 healthy/normal BM samples (NBM), then simultaneously compared to the patient’s diagnosis and FU samples at each time-point. MRD clusters were characterized individually in terms of cell numbers and immunophenotype. This strategy disclosed subclones with varying immunophenotype within single diagnosis and FU samples including populations absent from NBM. Detectable MRD was as low as 0.09% in MFC and 0.051% for mMRD. The concordance between mMRD and MFC-MRD was 80.2%. MFC yielded 85% specificity and 69% sensitivity compared to mMRD. Unsupervised MFC is shown here to allow for an easy and robust assessment of MRD, applicable also to AML patients without molecular markers.