Multiparameter Flow Cytometry Research Articles

Circulating immune landscape in melanoma patients undergoing anti-PD1 therapy reveals key immune features according to clinical response to treatment

IntroductionImmune checkpoint blockers (ICB) bring unprecedented clinical success, yet many patients endure immune mediated adverse effects and/or fail to respond. Predictive signatures of response to ICB and mechanisms of clinical efficacy or failure remain understudied. DC subsets, in network with conventional αβ T (Tconv), NK, γδ T and iNKT cells, harbor pivotal roles in tumor control, yet their involvement in response to ICB remained underexplored.MethodsWe performed an extensive longitudinal monitoring of circulating immune cells from melanoma patients treated with first-line anti-PD1, before (T0) and during treatment. We assessed the phenotypic and functional features of DC and effector cells’ subsets by multi-parametric flow cytometry and ProcartaPlex® dosages.ResultsWe revealed differences according to response to treatment and modulations of patterns during treatment, highlighting a strong link between the immune landscape and the outcome of anti-PD1 therapy. Responders exhibited higher frequencies of circulating cDC1s, CD8+ T cells, and γδ2+ T cells in central memory (CM) stage. Notably, we observed a distinct remodeling of ICP expression profile, activation status and natural cytotoxicity receptor patterns of immune subsets during treatment. Anti-PD1 modulated DCs’ functionality and triggered deep changes in the functional orientation of Tconv and γδT cells.DiscussionOverall, our work provides new insights into the immunological landscape sustaining favorable clinical responses or resistance to first-line anti-PD1 therapy in melanoma patients. Such exploration participates in uncovering the mechanism of action of anti-PD1, discovering innovative predictive signatures of response, and paves the way to design pertinent combination strategies to improve patient clinical benefits in the future.

Natural Killer Cell Phenotypes and Clinical Outcomes in Pediatric Kidney Transplantation.

Natural killer (NK) cells have gained recognition for playing an integral role in both alloimmunity and protective immunity, particularly viral infection control, in solid organ transplantation. Using data from the Clinical Trials in Organ Transplantation in Children (CTOTC) study entitled, "Immune Development in Pediatric Transplantation," (NCT00951353), we aimed to identify NK cell phenotypes that were associated with viral infection versus alloreactive events during the first year after transplantation. We also examined the relationship between NK cells with 7-year patient and allograft survival using the Scientific Registry for Transplant Recipients (SRTR) database. A secondary analysis of peripheral blood mononuclear cells from 98 children aged 1-20 years old with kidney transplants was conducted using multiparameter flow cytometry for the following NK cell phenotypes: CD56bright, CD56dim, and CD56negative. We associated these phenotypes with either viral infection or alloimmunity (de novo donor-specific antibody (dnDSA) development or acute rejection), using Fine-Gray subdistribution hazard models for competing risks. Secondary outcomes included allograft and patient survival. We demonstrated that specific baseline NK cell phenotypes obtained prior to transplantation may be associated with either viral infection or alloimmunity. An elevation in CD56dim frequency was associated with an increased risk of infection, while an increase in CD56negative absolute count was associated with an increased risk of an alloimmune event. NK cells were not associated with graft survival. NK cell phenotyping may be a useful tool to help differentiate infectious from alloimmune risk.

The Functional Role and Prognostic Significance of TIM-3 Expression on NK Cells in the Diagnostic Bone Marrows in Acute Myeloid Leukemia

Background: Compared to other immune checkpoint molecules, T cell immunoglobulin domain and mucin domain-3 (TIM-3) is highly expressed on natural killer (NK) cells, but its functional role and prognostic significance in acute myeloid leukemia (AML) remains unclear. This study aims to evaluate the role of TIM-3 expression on the cytotoxic and killing capacity of NK cells and its prognostic significance in AML. Methods: AML public single-cell RNA sequencing (scRNAseq) data were used to analyze the correlation of transcript levels between HAVCR2 (encoding TIM-3) and cytotoxic molecules in NK cells. NK cells from the bone marrows of seven newly diagnosed AML patients and five healthy donors (HDs) were stimulated in vitro and cell-killing activity was evaluated. A total of one hundred and five newly diagnosed adult AML patients and seven HDs were tested the expression of TIM-3 and cytotoxic molecules on the bone marrow NK cells by multi-parameter flow cytometry (MFC). Results: Both scRNAseq and MFC analysis demonstrated that TIM-3 expression on NK cells was positively related to the levels of perforin (PFP) and granzyme B (GZMB) (all p < 0.05) in AML. It was PFP and GZMB but not the TIM-3 level that was related to NK-cell-killing activity against K562 cells (p = 0.027, 0.042 and 0.55). A high frequency of TIM-3+ NK cells predicted poorer relapse-free survival (RFS) and event-free survival (EFS) (p = 0.013 and 0.0074), but was not an independent prognostic factor, whereas low GZMB levels in TIM-3+ NK cells independently predicted poorer RFS (p = 0.0032). Conclusions: TIM-3 expression on NK cells is positively related to PFP and GZMB levels but has no relation to cell-killing activity in AML, and low GZMB levels in TIM-3+ NK cells in the diagnostic bone marrows predicts poor outcomes. This study lays a theoretical foundation for the clinical application of immune checkpoint inhibitor treatment.

Measurable residual disease testing and allogeneic hematopoietic cell transplantation for AML: adapting Pre-MEASURE to clinical practice.

Measurable residual disease (MRD) testing in patients with acute myelogenous leukemia (AML) represents a heterogenous assessment process designed to quantify leukemia-specific biomarkers that are not ascertainable by routine pathologic evaluation. The most common tools used to assess MRD are multiparameter flow cytometry (MPFC), and polymerase chain reaction (PCR) based tools, including quantitative or digital droplet PCR (qPCR, ddPCR), or next-generation sequencing (NGS) technologies. Collectively, MRD assessments have become an important clinical tool in the management of patients with AML. Despite progress, significant questions remain with respect to the appropriate timing, frequency, and methodology of MRD assessment, and whether or how to adapt therapy based on MRD results. Recent data from the Pre-MEASURE study, a retrospective cohort analysis of error corrected NGS based MRD assessment prior to allogeneic hematopoietic cell transplantation (alloHCT) in patients with AML, provides additional key information with respect to the emerging role of NGS-based technology in MRD assessment. In the context of this review, we evaluate the Pre-MEASURE study as well as other recent, high-quality assessments of MRD in AML. Our focus is to provide a practical assessment of the use of emerging MRD technologies in patients with AML with an emphasis on the role of peri-transplant MRD for the practicing clinician.

Differential Effector Function of Tissue-Specific Natural Killer Cells Against Lung Tumors.

Natural killer (NK) cells are innate lymphoid cells capable of directly killing target cells while modulating immune effector responses. Despite their multifunctional capacities, a limited understanding of their plasticity and heterogeneity has impeded progress in developing effective NK cell-based cancer therapies. In this study, we investigated NK cell tissue heterogeneity in relation to their phenotype and effector functions against lung tumors. Using hanging-drop tumor spheroid and subcutaneously established LL/2 lung tumor models, we examined NK cell receptor diversity and its correlation with tissue-specific cytotoxicity through multiparametric flow cytometry, fluorescence imaging, and cytotoxicity assays. We identified distinct patterns of cell surface receptor expression on tissue-specific NK cells that are crucial for antitumor activity. Linear regression mathematical analyses further revealed significant positive correlations between activation-associated cell surface receptors and cytotoxic capacity in NK cells from tissues such as the liver and bone marrow. These findings underscore the differential effector capacities of NK cells from distinct tissues, even prior to exposure to LL/2 tumor cells. This highlights the significance of tissue-specific NK cell heterogeneity and its impact on their antitumor cytotoxicity. Recognizing these distinct tissue-specific receptor expression patterns will be instrumental in developing more efficacious NK cell-based cancer treatments.

Abstract C046: PARP inhibitors as immune modulators in metastatic ovarian cancer treatment

Abstract Epithelial ovarian carcinoma (EOC) is one of the top five causes of cancer-related death in women. Most patients with EOC achieve initial remission after primary or interval cytoreductive surgery combined with platinum-taxane chemotherapy. However, mutations in BRCA1 or BRCA2 genes, which lead to homologous recombination (HR) defects, play a crucial role in platinum sensitivity and justify the use of poly (ADP-ribose) polymerase (PARP) inhibitors as maintenance therapy, commonly linked to extended progression-free survival (PFS). Besides their direct cytotoxic and cytostatic effects, PARP inhibitors (PARPi) have shown significant immunostimulatory properties by disrupting DNA repair in cancer cells, and opening possibilities for synergy with immune checkpoint inhibitors (ICIs). In this study, we investigate the immunomodulatory effects of PARPi using multiparametric flow cytometry, multiplexed immunolabeling, single-cell transcriptomics, and functional assays in an experimental BR5Brca1-/- syngeneic mouse model and human EOC tumor samples. We examine the molecular and cellular mechanisms that can be exploited to develop more effective combination therapies. PARPi may increase the mutational burden in EOCs due to unresolved DNA damage and the release of damage-associated molecular patterns (DAMPs), thereby increasing T-cell infiltration. In addition, PARPi appear to promote potent type I interferon (IFN) secretion through the activation of cGAMP synthase and the stimulator of the interferon genes (STING) pathway. In combination with ICIs, PARPi showed a beneficial effect on the balance between adaptive anti-tumor immunity and innate myeloid components, leading to an improved cytotoxic T-cell response, as observed in mouse models and HGSOC tumor samples. These observations emphasize the role of strategically designed combinations of PARPi and immunotherapeutic agents, which could be the key to overcoming immunosuppression in the EOC microenvironment, thereby improving clinical outcomes. Citation Format: Sarka Vosahlikova, Peter Holicek, Irena Moserova, Michal Hensler, Romana Mikyskova, Lenka Kasikova, Josef Pasuvka, Jana Drozenova, Katerina Mojzisova, Marek Kovar, Iain McNiesh, Michael Halaska, Lukas Rob, Sandra Orsulic, Milan Reinis, Lorenzo Galluzzi, Radek Spisek, Jitka Palich Fucikova. PARP inhibitors as immune modulators in metastatic ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C046.

Hemin promotes platelet activation and plasma membrane disintegration regulated by the subtilisin-like proprotein convertase furin.

Platelet activation plays a critical role in thrombosis and hemostasis. Several pathophysiological situations lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. Hemin has been shown to activate platelets and induce thrombo-inflammation. Classical antiplatelet therapy failed to prevent hemin-induced platelet activation. Thus, the aim of the present study was to characterize the mechanism of hemin-induced platelet death (ferroptosis). We evaluated the invitro effect of hemin on platelet activation, signaling, oxylipins, and plasma membrane destruction using light transmission aggregometry, exvivo thrombus formation, multiparametric flow cytometry, micro-UHPLC mass spectrometry for oxylipin profiling, and scanning ion conductance microscopy (SICM). We found that hemin induces platelet cell death indicated by increased ROS levels, phosphatidyl serine (PS) exposure, and loss of mitochondrial membrane potential (ΔΨm). Further, hemin causes lipid peroxidation and generation of distinct oxylipins, which strongly affects plasma membrane integrity leading to generation of platelet-derived microvesicles. Interestingly, hemin-dependent platelet death (ferroptosis) is specifically regulated by the subtilisin-like proprotein convertase furin. In summary, platelet undergo a non-apoptotic cell death mediated by furin. Inhibition of furin may offer a therapeutic strategy to control hemin-induced thrombosis and thrombo-inflammation at a site of hemolysis.

SURG-48. LASER INTERSTITIAL THERMAL THERAPY REMODELS THE TUMOR IMMUNE LANDSCAPE IN A MOUSE GLIOMA MODEL

Abstract BACKGROUND Laser interstitial thermal therapy (LITT), a minimally invasive surgical technique for tumor ablation, is increasingly being used to treat primary and recurrent glioma. While the primary goal of using LITT has been to destroy tumor tissue, little is known about the impact of LITT on the overall immune response. To investigate changes in the tumor immune landscape, we utilized a mouse model of LITT and applied it to a poorly immunogenic mouse model of glioma. METHODS SB28 glioma cells were implanted in the right frontal lobe of C57BL/6 mice, and on day 7, an optical fiber is inserted into the tumor, paired with a thermocouple for real-time temperature monitoring. A 30W laser was activated in pulses to maintain a peri-lesional temperature of 44C for 150-180 seconds. The tumor and LITT-associated immune response was analyzed by multiparameter flow cytometry, single cell RNA-seq, and multiplex immunofluorescence. RESULTS LITT (versus sham control) triggered an increase in neutrophils in the tumor during the acute phase at day 3 following treatment. Total immune cell numbers increased over time in both sham- and LITT-treated mice, but there was a significant increase at post-LITT day 7 in select adaptive immune cell populations including type 1 conventional dendritic cells, NK cells, and gamma delta T cells. Monocytes/macrophages comprised the majority of the immune infiltrate, and multiplex immunofluorescence revealed these populations localized to the perimeter of the LITT lesion. scRNA-seq also revealed enrichment of a Fabp5- and Gpnmb-expressing macrophage subset following LITT. CONCLUSIONS LITT modifies the tumor immune infiltrate with increases in neutrophils as well as select adaptive cell and macrophage subsets. Future studies will explore adjunct therapies to further augment the immune response in the setting of LITT.

TMIC-28. TITLE: AN IMMUNE-RELATED, SEX-SPECIFIC T-CELL SIGNATURE IN GLIOBLASTOMA MICROENVIRONMENT WITH PROGNOSTIC IMPACT

Abstract BACKGROUND Glioblastoma, IDH-wildtype (GBM), is the most prevalent and aggressive primary brain cancer with a poor prognosis. Immune checkpoint inhibitors (ICIs) have been explored in GBM with limited success, highlighting the need for a better understanding of the tumor microenvironment (TME). This study investigated whether the expression of specific immune checkpoints on tissue-resident memory T cells (Trm) predicts patient outcomes. METHODS In a single cohort observational study, tumor samples from 45 histologically confirmed GBM patients were processed to obtain single-cell suspensions. Multiparametric flow cytometry was used to analyze the correlation of Trm phenotypes with overall survival (OS) and progression-free survival (PFS). Univariate and multivariate analyses were conducted to assess the prognostic significance of Trm subsets. RESULTS Low frequency of exausted Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) was associated with better clinical outcomes. Multivariate analysis identified low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 as the most significant independent prognostic factors for longer OS (hazard ratios—HR [95%CI]: 0.14 [0.04–0.52] p < 0.001, 0.39 [0.16–0.96] p = 0.04, respectively). Additionally, younger patients (<63 years) and males had distinct immune profiles affecting prognosis. Notably, the proportion of exausted CD8+CD103+PD1+ Trms had a prognostic correlate only in male patients, consistent with findings by other groups on the existance of gender-specific immune TME in GBM. CONCLUSIONS Further exploration of Trm-based immune profiling has the potential to shed light on GBM resistance to ICIs and to help in personalizing treatment strategies.

IMMU-28. LOW-INTENSITY PULSED ULTRASOUND COMBINED WITH POLY-ICLC AND INTERLEUKIN-2 UNLOCKS THE IMMUNE PRIVILEGE OF THE BRAIN BY PROMOTING EPITOPE SPREADING AND ACTIVATION OF CNS-SPECIFIC NAÏVE T CELLS

Abstract Immunotherapies targeting glioma, such as chimeric antigen receptor (CAR) T cell therapies, offer new therapeutic avenues; however, their application has yielded limited success in clinical trials so far. Due to the antigenic heterogeneity of gliomas, targeting a few or several antigens still results in recurrence with “antigen-loss” tumors, indicating the need to engage the endogenous immune system to eliminate these therapy-resistant tumor cells. However, natural anatomical barriers and an immunosuppressive environment allow brain tumors to evade detection by the endogenous immune system. Low-intensity pulsed ultrasound combined with microbubbles (LIPU/MB) has recently been shown to transiently open the blood-brain barrier (BBB), enabling penetration of cells and drugs into the CNS. Using intravital two-photon imaging, two-photon microscopy, multiparametric spectral flow cytometry, and bone marrow chimera mice, alongside blood samples from glioblastoma patients treated with LIPU/MB, we observed that LIPU/MB promotes migration of CNS antigen-presenting cells from the perivascular space into the periphery. To assess the immunological relevance of LIPU/MB-mediated CNS antigen presentation to the peripheral immune system, we have developed new transgenic mice (GFAP-minigene mice) with an intact BBB expressing four immunogenic peptides under the CNS-specific GFAP promoter. In GFAP-minigene mice, LIPU/MB combined with toll-like receptor 3 agonist poly-ICLC and Interleukin-2 promoted priming, activation, and homing of naïve CNS-specific T cells into the brain parenchyma. Cytotoxic T cell activity in the brain was accompanied by MHC-I and MHC-II epitope spreading, mirrored by the occurrence of endogenous T cells in the brain, deep cervical lymph nodes, and spleen harboring other CNS-(minigene) antigen-specific T cell receptors. Collectively, our findings suggest that LIPU/MB combined with poly-ICLC can overcome inherent limitations of immunotherapy by enhancing CNS epitope spreading and activation of naïve CNS-antigen-specific T cells within the context of an intact blood-brain barrier.

CD38 and CD58: A Gallant Savior in Under-Resourced Laboratories.

Background B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common childhood malignancies and comprises almost the majority of acute leukemias in children. At the end of induction, minimal residual disease (MRD) is one of the most important prognostic indicators. MRD is commonly detected by multiparametric flow cytometry. Interpreting panels consisting of multiple tubes requires a lot of expertise and is subject to stringent standardization processes. Hence, there arises a need for an easier alternative that can be more universally applicable and reduce complexity in interpretation, resulting in uniform reporting and cost-effectiveness, especially in resource-deficient settings, without compromising clinical outcomes. Therefore, the aim is to find the usefulness of the MRD "lite" panel for post-induction MRD detection in pediatric B-ALL using CD38, CD10, CD34, CD19, and CD45 compared to the "standard tube" panel. Methods The study included 25 children diagnosed with B-ALL and undergoing treatment. MRD detection was performed on day 28 of post-induction chemotherapy using a standard tube panel consisting of three tubes (gold standard) and our single tube MRD "lite" panel. The results obtained by the two were compared. Results The sensitivity and specificity of the MRD "lite" panel were calculated as 100% and 84.2%, respectively, taking the standard tube panel as the gold standard. CD10, CD20, and CD200 were valuable in distinguishing MRD-positive cases from MRD-negative cases. Moreover, the MRD "lite" panel had 100% concordance between the two observers, suggesting simplicity in assessment and, hence, wider applicability.

Changes in immune cell populations during acclimatization to high altitude.

The immune response to acute hypoxemia may play a critical role in high-altitude acclimatization and adaptation. However, if not properly controlled, hypoxemia-induced inflammation may exacerbate high-altitude pathologies, such as acute mountain sickness (AMS), or other hypoxia-related clinical conditions. Several studies report changes in immune cell subsets at high altitude. However, the mechanisms underlying these changes, and if these alterations are beneficial or maladaptive, remains unknown. To address this, we performed multiparameter flow cytometry on peripheral blood mononuclear cells (PBMCs) collected throughout 3 days of high-altitude acclimatization in healthy sea-level residents (n = 20). Additionally, we conducted invitro stimulation assays to test if high-altitude hypoxia exposure influences responses of immune cells to subsequent inflammatory stimuli. We found several immune populations were altered at high altitude, including monocytes, T cells, and B cells. Some changes in immune cell populations are potentially correlated with AMS incidence and severity. Invitro high-altitude PBMC cultures stimulated with lipopolysaccharide (LPS) showed no changes in pro-inflammatory cytokine production after 1 day at high-altitude. However, by day three pro-inflammatory cytokine production in response to LPS decreased significantly. These results indicate that high-altitude exposure may initiate an inflammatory response that encompasses innate immune sensitization, with adaptive immune suppression following acclimatization.

Immunophenotyping myelodysplastic neoplasms: the role of flow cytometry in the molecular classification era.

The unique heterogenous landscape of myelodysplastic syndromes/neoplasms (MDS) has resulted in continuous redefinition of disease sub-entities, in view of the novel translational research data that have clarified several areas of the pathogenesis and the progression of the disease. The new international classifications (WHO 2022, ICC 2022) have incorporated genomic data defining phenotypical alterations, that guide clinical management of specific patient subgroups. On the other hand, for over a decade, multiparameter flow cytometry (MFC) has proven its value as a complementary diagnostic tool for these diseases and although it has never been established as a mandatory test for the baseline evaluation of MDS patients in international guidelines, it is almost universally adopted in everyday clinical practice for the assessment of suspected cytopenias through simplified scoring systems or elaborate analytical strategies for the detection of immunophenotypical dysplastic features in every hematopoietic cell lineage in the bone marrow (BM). In this review, we explore the clinically meaningful interplay of MFC data and genetic profiles of MDS patients, to reveal the currently existing and the potential future role of each methodology for routine clinical practice, and the benefit of the patients. We reviewed the existing knowledge and recent advances in the field and discuss how an integrated approach could lead to patient re-stratification and guide personalized management.

A phase 2, multicenter, clinical trial of CPX-351 in older patients with secondary or high-risk acute myeloid leukemia: PETHEMA-LAMVYX.

LAMVYX was a multicenter, single-arm, phase 2 trial designed to validate the safety and efficacy of CPX-351 in patients aged 60-75 years with newly diagnosed, secondary acute myeloid leukemia and to generate evidence on key issues not addressed in the preceding regulatory pivotal trial. The primary end point of the study was the complete remission (CR)/CR with incomplete hematologic recovery (CRi) rate after induction. Eligible patients were recommended to undergo allogeneic hematopoietic stem cell transplantation after the first consolidation cycle. Alternatively, patients could undergo up to six maintenance cycles with CPX-351. Twenty-nine patients (49%; 95% exact confidence interval [CI], 37%-62%) patients achieved a CR/CRi after one or two cycles of induction, with a measurable residual disease negativity rate of 67% as assessed by centralized, multiparameter flow cytometry. Among patients who had serial next-generation sequencing analyses available, clearance of somatic mutations that were present at diagnosis was achieved in 7 (35%). The median follow-up among survivors was 16.8 months (range, 8.7-24.3 months). The median event-free survival was 3.0 months (95% CI, 1.4-7.3 months), and the median overall survival was 7.4 months (95% CI, 3.7-12.7 months). In landmark analyses at day +100 from diagnosis, the 1-year overall and event-free survival rate among patients who underwent allogeneic hematopoietic stem cell transplantation was 70% (95% CI, 47%-100%) and 70% (95% CI, 47%-100%), respectively. The corresponding values were 89% (95% CI, 71%-100%) and 44% (95% CI, 21%-92%), respectively, for patients who entered the maintenance phase. No significant longitudinal changes were observed in severity index or quality-of-life visual analog scale scores. The current data provide novel insights that might inform the clinical positioning and optimal use of CPX-351, complementing previous results (ClinicalTrials.gov identifier NCT04230239).

CD56briCD38+ as a novel neutrophil-specific marker in chronic myeloid leukemia

BackgroundChronic myeloid leukemia (CML) is classified as a subtype of myeloproliferative neoplasm, and bone marrow flow cytometry does not reveal any specific immunophenotype. Interestingly, aberrant expression of cluster of differentiation (CD) markers such as CD56 and CD38 has been observed on neutrophils in CML patients. Therefore, we investigated the abnormal expression of CD56 and CD38 in CML neutrophils to explore their diagnostic value in identifying CML through flow cytometry. MethodsWe have developed a multi-parameter flow cytometry assay to identify aberrant immunophenotypes in CML neutrophils among bone marrow nucleated cells. ResultsCompared to healthy donors and patients with a reactive neutrophilia or other hematological malignancies, the percentage of CD56briCD38+ neutrophil subsets in CML patients exhibits a distinctive increase (cut-off value, 2.0 %). The specificity and sensitivity associated with the learning cohort (168 samples) were 90.8 % and 84.9 %, respectively, while in the validation cohort (194 samples), they were 90.7 % and 84.7 %. The accumulation of CD56briCD38+ neutrophil subsets, which demonstrate are abnormal characteristics, is independent of the neutrophil count, BCR/ABL1 fusions and risk stratification but associated with blast cells immunophenotype. Moreover, this increase disappears in CML patients after treatment with tyrosine kinase inhibitors when the curative effect was satisfactory. ConclusionsWe conclude that an increase in the proportion of CD56briCD38+ neutrophil subsets exceeding 2.0 % of total neutrophils serves as a highly sensitive and specific flow cytometry marker, enabling rapid and accurate identification of CML.

Detection of circulating tumor cells in non-metastatic prostate cancer through integration of a microfluidic CTC enrichment system and multiparametric flow cytometry.

Prostate cancer (PCa) is the second most common cancer among men and the fifth leading cause of cancer death. Circulating tumor cell (CTC) enumeration and characterisation in PCa has been shown to provide valuable information on prognosis of disease, therapy management and detection of resistance. Here, Cellsway's microfluidic platform for high-throughput enrichment of intact CTC populations was used to isolate CTCs from the blood of 20 localised PCa patients and 10 healthy donor samples to evaluate the clinical performance of the technology. To enumerate and characterise CTCs, a multi-parameter flow cytometry analysis was performed on the enriched CTC suspension using CTC-specific biomarkers. CTCs were detected in 17 of 20 patient samples, which corresponds to 85% CTC positivity. The median CTC count per 7.5 ml blood was 2 (1-9). In 80% of patients (n = 16), the number of CTCs ranged from 1 to 5, and in 5% of patients (n = 1) the number of CTCs was above 5. No CTCs were observed in the blood samples of 10 healthy volunteers, demonstrating the high specificity and low risk of false positives of the technology.

Peripheral immune profiling of soft tissue sarcoma: perspectives for disease monitoring.

Studying the tumor microenvironment and surrounding lymph nodes is the main focus of current immunological research on soft tissue sarcomas (STS). However, due to the restricted opportunity to examine tumor samples, alternative approaches are required to evaluate immune responses in non-surgical patients. Therefore, the purpose of this study was to evaluate the peripheral immune profile of STS patients, characterize patients accordingly and explore the impact of peripheral immunotypes on patient survival. Blood samples were collected from 55 STS patients and age-matched healthy donors (HD) controls. Deep immunophenotyping and gene expression analysis of whole blood was analyzed using multiparametric flow cytometry and real-time RT-qPCR, respectively. Using xMAP technology, proteomic analysis was also carried out on plasma samples. Unsupervised clustering analysis was used to classify patients based on their immune profiles to further analyze the impact of peripheral immunotypes on patient survival. Significant differences were found between STS patients and HD controls. It was found a contraction of B cells and CD4 T cells compartment, along with decreased expression levels of ICOSLG and CD40LG; a major contribution of suppressor factors, as increased frequency of M-MDSC and memory Tregs, increased expression levels of ARG1, and increased plasma levels of IL-10, soluble VISTA and soluble TIMD-4; and a compromised cytotoxic potential associated with NK and CD8 T cells, namely decreased frequency of CD56dim NK cells, and decreased levels of PRF1, GZMB, and KLRK1. In addition, the patients were classified into three peripheral immunotype groups: "immune-high," "immune-intermediate," and "immune-low." Furthermore, it was found a correlation between these immunotypes and patient survival. Patients classified as "immune-high" exhibited higher levels of immune-related factors linked to cytotoxic/effector activity and longer survival times, whereas patients classified as "immune-low" displayed higher levels of immune factors associated with immunosuppression and shorter survival times. In conclusion, it can be suggested that STS patients have a compromised systemic immunity, and the correlation between immunotypes and survival emphasizes the importance of studying peripheral blood samples in STS. Assessing the peripheral immune response holds promise as a useful method for monitoring and forecasting outcomes in STS.

Flow Cytometry for Non-Hodgkin and Hodgkin Lymphomas.

Multiparametric flow cytometry is a powerful diagnostic tool that permits rapid assessment of cellular antigen expression to quickly provide immunophenotypic information suitable for disease classification. This chapter describes a general approach for the identification of abnormal lymphoid populations by flow cytometry, including B, T, NK, and Hodgkin lymphoma cells suitable for the clinical and research environment. Knowledge of the common patterns of antigen expression of normal lymphoid cells is critical to permit identification of abnormal populations at disease presentation and for minimal residual disease assessment. We highlight an overview of procedures for processing and immunophenotyping non-Hodgkin B- and T-cell lymphomas and also describe our strategy for the sensitive and specific diagnosis of classic Hodgkin lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and T-cell/histiocyte-rich large B-cell lymphoma.

Novel Compound Heterozygous Variants in the FAS Gene Lead to Fetal Onset of Autoimmune Lymphoproliferative Syndrome (ALPS).

FAS gene defects lead to autoimmune lymphoproliferative syndrome (ALPS), which is often inherited in an autosomal dominant and rarely in an autosomal recessive manner. We report a case of a newborn girl with novel compound heterozygous variants in FAS and reveal the underlying mechanism. Whole-exome sequencing (WES) was used to identify pathogenic variants. Multiparametric flow cytometry analysis, phosflow analysis, and FAS-induced apoptosis assays were used to explore the effects of the variants on FAS expression, apoptosis, and immunophenotype. The HEK293T cells were used to assess the impact of the variants on protein expression and FAS-induced apoptosis. The patient was born with hepatosplenomegaly, anemia, and thrombocytopenia. She also experienced COVID-19, rotavirus infection, herpes simplex virus infection, and severe pneumonia. The proportion of double-negative T cells (DNTs) was significantly elevated. Novel FAS compound heterozygous variants c.310T > A (p.C104S) and c.702_704del (p.T235del) were identified. The apoptotic ability of T cells was defective, and FAS expression on the surface of T cells was deficient. The T235del variant decreased FAS expression, and the C104S protein remained in the endoplasmic reticulum (ER) and could not translocate to the cell surface. Both mutations resulted in loss-of-function in terms of FAS-induced apoptosis in HEK293T cells. The DNTs were mainly terminally differentiated T (TEMRA) and CD45RA+HLA-DR+, with high expression of CD85j, PD-1, and CD57. The percentage of Th1, Tfh, and autoreactive B cells were significantly increased in the patient. The abnormal immunophenotyping was partially attenuated by sirolimus treatment. We identified two variants that significantly affect FAS expression or localization, leading to early disease onset of in the fetus. Abnormalities in the mTOR pathway are associated with a favorable response to sirolimus.

Measurable residual disease as predictor of post–day +100 relapses after allografting in adult AML

AbstractMeasurable residual disease (MRD) by multiparametric flow cytometry (MFC) before allogeneic hematopoietic cell transplantation (HCT) identifies patients at high risk of acute myeloid leukemia (AML) relapse, often occurring early after allografting. To examine the role of MFC MRD testing to predict later relapses, we examined 935 adults with AML or myelodysplastic neoplasm/AML transplanted in first or second morphologic remission who underwent bone marrow restaging studies between day 70 and 100 after HCT and were alive and without relapse by day +100. Of 935 adults, 136 (15%) had MRD before HCT, whereas only 11 (1%) had MRD at day +70 to +100. In day +100 landmark analyses, pre-HCT and day +70 to +100 MFC MRD were both associated with relapse (both P < .001), relapse-free survival (RFS; both P < .001) overall survival (OS; both P < .001), and, for post-HCT MRD, nonrelapse mortality (P = .001) after multivariable adjustment. Importantly, although 126/136 patients (92%) with MRD before HCT tested negative for MRD at day +70 to +100, their outcomes were inferior to those without MRD before HCT and at day +70 to +100, with 3-year relapse risk of 40% vs 15% (P < .001), 3-year RFS of 50% vs 72% (P < .001), and 3-year OS of 56% vs 76% (P < .001), whereas 3-year nonrelapse mortality estimates were similar (P = .53). Thus, despite high MRD conversion rates, outcomes MRD positive/MRD negative (MRDneg) patients are inferior to those of MRDneg/MRDneg patients, suggesting all patients with pre-HCT MRD should be considered for preemptive therapies after allografting.