Multiparameter Flow Cytometry Panel Research Articles

Abstract 3863: Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies

Abstract Flow cytometry assays using fresh whole blood which are interrogating either an extremely rare cell subset or a depleting immunotherapy, may require samples to be processed from global clinical sites over a wide geographical area, which can pose a major challenge for clinical operations. Precision for Medicine (PfM) has addressed these operational challenges using commercial stabilizers which are added to the blood allowing samples to be frozen at -80°C and subsequently batch processed for sample testing. We have performed a fit for purpose assay qualification of a multiparameter flow cytometry panel allowing the immunophenotypic analysis of monocyte, T cell, B cell and NK immune populations using Cytodelics™ fixed/stabilized healthy donor whole blood. Additionally, the assay was verified in whole blood samples from multiple solid tumor disease states including melanoma, colorectal cancer, head & neck, non-small cell lung cancer to demonstrate clinical application. In these oncology samples, we were able to assess both immune cell frequencies and their activation status in both fresh whole blood as well as fixed whole blood over a short-term sample stability study, where the samples had been stored at -80°C for several months. This data provides a representative example of the use of commercial stabilizing fixatives for clinical studies to extend the stability window of whole blood for subsequent assessment by flow cytometry. Fixed whole blood can be stored at -80°C, shipped on dry ice allowing multiple timepoints in a cohort to be assessed together, thus minimizing assay variability as compared to fresh whole blood analysis, and mitigating the impact of shipping delays and temperature excursions on the samples. With sufficient time for assay development there are several commercial stabilizers that can be tested to determine if the markers in the flow assay are compatible with these reagents, providing a valuable option to assess the impact of immunotherapies on the immune cells of interest and reducing operational challenges in global studies. Citation Format: Jackie Benko, Samantha Splitt, Angelina Bisconte, Laura Lozza, Rachel Owen, Deborah Phippard. Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3863.

Abstract 298: Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal prostate cancer

Abstract Patients with multifocal, locally advanced prostate cancer are at the highest risk of recurrence and death. Neoadjuvant therapies have limited response rates in this patient cohort and there remains a critical need to develop curative treatments. Prostate cancer (PC) shows marked heterogeneity, which is not limited to tumor cells but extends across immune and stromal compartments and has been linked to metastatic disease and therapeutic resistance. Cancer associated fibroblasts (CAFs) have been proposed to play a critical role in PC progression and invasion, but it is still unclear how CAFs interact with tumor cells. We evaluated CAFs from prostatectomy specimens as drivers of resistance to neoadjuvant androgen receptor signaling inhibitor (ARSi) and chemotherapy and assessed treatment response in co-culture systems in the context of CAF presence. We have utilized a novel radio-pathology tool that integrates PSMA PET/MRI scans to identify regions of interest that associate with heterogenous treatment response. MRI scans were utilized to print patient-specific 3D molds to micro dissect live tissue for subsequent cell sorting and molecular analyses. We have established a multi-parameter flow cytometry panel to characterize primary prostate fibroblasts from tumor foci and adjacent normal prostate tissue. In addition, co-culture assays were performed including immortalized CAF (hPrCSC-44) and MSC-derived fibroblast. To examine the tumor-promoting role of CAFs, fibroblasts were co-cultured with androgen-sensitive 3D tumor PC spheroids (LNCaP, C42B and LAPC4) in the integrated microfluidic STACKs platform that allows for the assessment of spatio-temporal interaction in a variety of culture microenvironments to model complex interactions. Cytotoxicity in 3D PC spheroids was assessed after Apalutamide, Darolutamide, or Docetaxel treatment by confocal microscopy. Integrated DNA, RNA, and radiology analysis of tumor biopsies have identified gene signatures associated with early biochemical recurrence. Most biopsies had low tumor purity, evidence of the cytotoxic impact of chemohormonal therapy while high tumor purity predicted high tumor expression. Poor clinical outcome was significantly associated with elevated stromal scores while epithelial and stromal enrichment were inversely correlated (rho= -0.80, p< 5 × 10−16). The presence of CAFs significantly decreased Docetaxel (C42B only vs C42B+CAF, 86.07% vs. 44.57%, respectively, p<0.001; LAPC4 only vs LAPC4+CAF, 70.98% vs 26.64%, respectively, p<0.001) and Darolutamide-induced (C42B only vs C42B+CAF, 52.59% vs 37.87%, respectively, p=0.0407). In conclusion, recurrent PC is linked to increased stromal content and the presence of CAFs supported tumor survival in response to ARSi and Docetaxel treatment. The molecular drivers of this phenomenon are currently being investigated. Citation Format: Nikolett Lupsa, Erika Heninger, Adeline Ding, Shannon R. Reese, Xavier T. Hazelberg, Aaron M. LeBeau, Brian P. Johnson, Peter P. Geiger, David J. Beebe, David A. Quigley, Joshua M. Lang. Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 298.

Initial Clinical Results and Immune Correlates of a Phase 2 Study of Daratumumab, Bortezomib, Dexamethasone Followed By a Proteasome Inhibitor in-Class Transition ( iCT) to Daratumumab, Ixazomib, Dexamethasone in Relapsed Refractory Multiple Myeloma

Background: The regimen of daratumumab, bortezomib, and dexamethasone (DVd) was approved in relapsed refractory multiple myeloma (RRMM) patients (pts) with >1 line of therapy based on the pivotal phase 3 CASTOR study. Notably in CASTOR, bortezomib was administered for a fixed duration of 8 cycles, and both the parenteral administration route and treatment-related peripheral neuropathy (PN) may limit continuous bortezomib exposure in clinical practice. As an alternative, we conducted a phase 2 study of DVd, followed by daratumumab, ixazomib, dexamethasone (DId) utilizing a proteasome inhibitor (PI) in-class transition ( iCT) with the hypothesis that oral ixazomib may provide a more tolerable and convenient way to deliver continuous PI- and anti-CD38-based therapy to improve efficacy (NCT03763162). Methods: This phase 2 single-center study enrolled RRMM pts with 1-3 lines of prior therapy with disease progression on or following the last line of therapy. Key exclusion criteria included prior ixazomib exposure, refractoriness to anti-CD38 therapy, and refractoriness to bortezomib or carfilzomib therapy at last exposure. Enrolled pts received DVd for 3 cycles on a 21-day (D) cycle with daratumumab on D1, D8, and D15 and subcutaneous (SC) bortezomib 1.3 mg/m 2 on D1, D4, D8, and D11. Starting cycle (C) 4, pts transitioned to DId on a 28-day cycle, with daratumumab on D1 and D15 for C3-C7 and on D1 starting C8 and oral ixazomib 4 mg on D1, D8 and D15. Daratumumab was initially administered intravenously (16 mg/kg) but later changed to SC injection (1800 mg) with a protocol amendment. Bone marrow (BM) aspirates were performed pre-treatment, post-C3, post-C6 (optional), and at progression to evaluate immune correlates of therapeutic response and resistance using 4 16-color multiparameter flow cytometry panels (2 T-cell, 1 NK/myeloid, and 1 B-cell). The primary endpoint of the study was progression free survival (PFS) with planned enrollment of 40 pts. Results: As of 15 May 2023, 36 pts have enrolled with a median of 1 prior line of therapy. Median age was 66, 67% were male, 28% were Black, 97% were lenalidomide-refractory, and 42% had high-risk cytogenetics (Cg; del 17p, t(4;14), t(14;16), and/or +1q21 gain/amplification). Overall response rate was 83%, ≥very good partial response (VGPR) rate was 53%, and ≥complete response rate was 19%. At a median follow-up of 24.8 months, median PFS was 21.1 months and median response duration was 26.2 months. Median PFS among pts with high-risk Cg, 1 line of prior therapy, and 2-3 lines of prior therapy was 21.1 months, 22.8 months, and 14.3 months, respectively. The most common treatment emergent adverse events (TEAEs) of any grade were diarrhea (69%), thrombocytopenia (69%), fatigue (67%), and lymphopenia (67%). The most common ≥grade 3 TEAEs were lymphopenia (42%), thrombocytopenia (42%), and hypertension (31%). Treatment emergent PN occurred in 61% pts (11% ≥ grade 3). The most common reason for treatment discontinuation was disease progression (42%), and 16 (44%) pts remain on study with ongoing responses. Efficacy and safety stopping rules for the study have not been met. Multiparameter flow cytometry immune correlates from BM aspirates demonstrated significantly lower Ki67 + cell frequency and significantly higher PD-1 + cell frequency within the CD8 + T-cell population at baseline in pts with no response versus those with optimal responses (≥VGPR). Among pts with short PFS (<12 months), CD4 +/CD8 + ratio and CD4 + non-T-reg naïve T-cells were significantly higher post-C3 and post-C6 compared to pts with longer PFS (>12 months). Among initial responders who later progressed on therapy, T-regulatory cells were significant increased at progression compared to pre-C4, and NK cell CD16 expression was significantly reduced at progression compared to baseline. Finally, distinct patterns of CD4 + T effector memory (EM) cell subsets were observed with significantly decreased EM1 and increased EM3 subsets at progression compared to baseline. Conclusion: Treatment with DVd followed by DId with a PI iCT from bortezomib to ixazomib is a feasible approach for continuous PI and anti-CD38 therapy, leading to durable responses in RRMM pts, among whom nearly all (97%) were lenalidomide refractory. Immune correlates revealed distinct patterns of T-cell and NK-cell phenotypes when profiled serially in responding and non-responding pts. Additional correlative analyses are ongoing.

The Laip-Based-Dfn Approach Is Superior in Terms of Useful MRD Results As Compared to the Laip Approach after Cycle II in Acute Myeloid Leukemia

Clinical decision-making based on measurable residual disease (MRD) in acute myeloid leukemia (AML) is steadily increasing; therefore, accurate MRD results are imperative. Multi-parameter flow cytometry (MFC) is often used due to wide availability, particularly when no suitable mutation is present. Two approaches are most common in MFC-MRD: 1. Leukemia-associated immunophenotype (LAIP) approach, in which the aberrant phenotype is identified at diagnosis and tracked during follow-up. 2. Different-from-Normal (DfN) approach, in which aberrant surface antigen expression patterns not present in normal bone marrow (empty spaces in 2D dot plots) are identified at follow-up. The LAIP approach is sensitive, but does not consider immunophenotypic shifts and cannot be used when no LAIP is identified at diagnosis or when a diagnosis sample is unavailable. The DfN approach can detect these shifts, but ample expertise is needed to determine a malignant population. In the ELN-DAVID AML-MRD guidelines 2022, it is recommended to use the combination of the LAIP approach and the DfN approach to use the advantages of both approaches. In the HOVON-SAKK132 (HO132), the MRD was based on the combination of MFC-MRD and NPM1 RQ-PCR. Initially, the MFC-MRD in this study was based on the LAIP approach. However, the complete MFC panel (four 8-color tubes) published by Cloos et al. (J.Vis.Exp, 2018) was used for measuring MRD during follow-up. Therefore in this study, we had the unique opportunity to retrospectively analyze the samples after induction cycle 2 (C2) of patients in complete remission (CR/CRi) and determine additional MRD results with the LAIP-based-DfN approach. The original LAIP approach encompasses determining the percentages of the LAIPs in the follow-up sample, which were also identified at diagnosis. For the LAIP-based-DfN approach, we followed the same approach, but determined the percentages of all possible LAIPs in the MFC-MRD panel at diagnosis and after C2. An emerging LAIP is defined as a LAIP that is present in the follow-up but was not found at diagnosis. MRD positivity was defined as ≥ 0.1% LAIP cells of white blood cells. Patients with an emerging LAIP < 0.1% or no LAIPs in follow-up would result in MRD negativity for LAIP-based-DfN, but was only possible when at least 500,000 cells were acquired in all four tubes. At diagnosis, we received 884 samples of 905 patients (98%), in which we found a LAIP ≥ 10% of CD45dim blasts in 83% of the samples (Figure 1A). After C2, we received 615 samples, for which we had MFC-MRD results for 79% (487/615) by the LAIP approach. When we applied the LAIP-based-DfN approach, we scored 69 of 615 extra MRD results: an emerging LAIP ≥ 0.1% (MRDpos, N = 14), no LAIPs (MRDneg, N = 25) or an emerging LAIP < 0.1% (MRDneg, N = 30). When including these results in the data of the LAIP approach, the percentage of patients with an MRD result increased from 79% (487/615) to 90% (556/615), and the MRD positive percentage increased from 13% (81/615) to 19% (118/615)(Figure 1B). The LAIPs of emerging clones most often included CD56, CD15, and CD22. To assess the prognostic value in the HO132, we combined the reanalyzed LAIP-based-DfN MFC-MRD with the NPM1-MRD (N = 567). In these analyses, overall survival (OS) hazard ratio (HR) 95% confidence interval (CI) 1.92 (1.44-2.56) and relapse-free survival (RFS) HR 95% CI 1.7 (1.31-2.19) were similar to the original LAIP based/ NPM1 approach: OS HR: 95%CI 2.02 (1.48-2.76) and RFS HR: 95%CI 1.78 (1.34-2.35), respectively. In conclusion, the LAIP-based-DfN approach adds additional MRD results while retaining the prognostic value. Since HOVON-SAKK centers are highly compliant with the protocol and 98% of diagnosis samples were available, the LAIP-based-DfN approach can offer additional value for studies with limited access to diagnosis samples.

One High Efficient Detection Panel of Multiparamer Flow Cytometry for Non-Hematopoietic Tumors

Background: In the past half century, flow cytometry has been successfully applied to detection of hematopoietic tumors. In fact, the incidence rate of non-hematopoietic tumors is higher than hematologic malignancies. However, the application of flow cytometry has been limited for a long time due to samples, lack of specific markers and other reasons. With the development of immunological technique, the detection of solid tumors by flow cytometry has attracted renewed attention. We designed a highly sensitive and specific assay panel for multiparameter flow cytometry(MFC) screening, diagnosis and follow-up of non-hematopoietic tumors. Aims:find a highly efficient panel of MFC for screening, diagnosis and follow-up of non-hematopoietic tumors. methods: From Aug. 1st, 2008 to Mar. 31st, 2022, 858 samples from patients with non-hematopoietic tumor were detected in the flow cytometry lab of Hebei Yanda Lu Daopei Hospital, of which 617 samples were BM, 3 PB, 196 cerebrospinal fluid(CSF), 9 Pleural effusion or ascites, 3 lymph node, and 30 fine needle aspiration(FNA) from various tissue. In these 850 person, 476 were males, 374 females, the median age was 4 years old (0-80 years), and the median percentage of tumor cells in the sample was 0.16% (0.01%-96.23%).After rounds of optimization, CD9 FITC/GD2 PE/CD3 percp/CD4 pecy7/CD56 APC/CD36 Apccy7/CD81 BV421/CD45 V500, cytokeratin(CK) or CD326 FITC/HLA-ABC PE/CD38 percp/CD19 pecy7/CD56 APC/CD36 Apccy7/CD7 BV421/CD45V500 were selected as the final detection panel. The analysis was performed using 3 laser 8-10 color canto flow cytometers, and diva/flowjo software. The results were compared with pathological and clinical diagnosis to evaluate the sensitivity and specificity. Results: Firstly, Sensitivity and specificity analysis. Of 858 samples, 567 were positive, of which 442 were BM, 2 PB, 84 CSF, 9 fluid, 2 lymph nodes, and 28 FNA. The coincidence rate was more than 90% with pathology, other laboratory examinations and clinical diagnosis. It covers most of tumors nominated by WHO diagnostic criteria. The coverage and specificity were more than 99%, and the sensitivity was 0.01%. Both false positive rate and false negative rate were less than 1%. Secondly, the selection and verification of immunological markers. The positive rate and specificity of each immunological markers were analyzed in 567 positive samples, as shown in Figure 1. The specificity of CK combined with GD2 in the diagnosis of non-hematopoietic tumors was almost 100%, and the positive rate of CD56 was significantly different between epithelial and non-epithelial tumors. CD3/CD19/CD4/CD38/HLA-ABC/CD36/CD7 is almost not expressed in non-hematopoietic tumors but expressed at least one by almost all hematopoietic cells, which can exclude hematological malignancies. CD9/CD81, as a sensitive but non-specific marker, can assist in judgment, and combined with CD45-/CD36-, exclude non-specific staining to prevent missed diagnosis. Thirdly, tumor sources can be roughly distinguished. PCA (Principal component analysis) can accurately classify epithelial and non-epithelial tumors by analyzing 38 cases of neuroblastoma and 5 cases of epithelial-derived tumors. Flowsom cluster analysis of some common solid tumors in children showed that the expression intensity of different tumor markers was different. As shown in Figure 2. Conclusion: Although the high heterogeneity of non-hematopoietic tumors and the lack of specific immune markers, we found one high sensitive and specific MFC panel for detection of solid tumors after rounds of trials. The panel includes some non-lineage specific markers but commonly expressed in hematologic neoplasms and rarely in non-hematopoietic tumors. Different from the precise classification of euroflow protocol, our panel focuses on the identification of tumor cells, and exclusion of hematological malignancies and non-specific staining. It is an important detection and analysis panel with high coverage, strong practicability, high sensitivity and specificity, which may reduce the rate of missed diagnosis.

Co-Expression of GPRC5D, FcRH5 and BCMA Suggests That Targeting More Than One Cell Surface Marker May be a Viable Strategy in Relapsed/Refractory Multiple Myeloma (RRMM): Biomarker Results from the Phase I Study of Forimtamig, a GPRC5DxCD3 Bispecific Antibody

Background: Forimtamig is a GPRC5DxCD3 T-cell engaging bispecific antibody that induces T-cell mediated multiple myeloma (MM) cell killing and has shown promising clinical activity in a Phase I study (NCT04557150) in patients with RRMM (Carlo-Stella et al. ASH 2022). GPRC5D is known to be overexpressed in MM plasma cells (MMPCs), but little is known about its prevalence in relation to risk status and other identified MM targets like FcRH5 and BCMA. We developed a unique exploratory multiparameter flow cytometry panel to characterize GPRC5D, FcRH5 and BCMA expression on MMPCs (Lelios et al. Cyto 2023). We present data on the prevalence and co-expression of these targets, and their association with baseline patient characteristics and response to forimtamig. Methods: Baseline bone marrow aspirate samples were collected from patients who received intravenous or subcutaneous forimtamig during dose-escalation, and were analyzed with flow cytometry. Target expression was depicted as frequency of positive MMPCs and as receptor density (molecules of equivalent soluble fluorochrome). Patients were grouped by number of prior lines of therapy, ISS score, cytogenetic risk (high risk defined as presence of one or more of the following abnormalities: del(17p), t(4;14), t(14;16), and response to forimtamig (responder [≥PR] or non-responder [<PR]). All patients provided informed consent. Results: At data cut-off (June 15, 2023), 48/163 patients were biomarker evaluable (samples passing pre-defined quality standards and containing ≥100 acquired MMPCs). GPRC5D was detected at baseline in 46/48 (96%) patients, with most MMPCs expressing the target (median 95.6%; min-max: 0-100%). GPRC5D expression was more prevalent on MMPCs than FcRH5 (median 78.6%; min-max: 3.7-100%) and BCMA (median 40.3%; min-max: 0-100%; Figure). Interestingly, GPRC5D receptor density (median 20,612; min-max: 6495-208,072) was 47-100-fold higher and more homogenous than FcRH5 (median 436; min-max: 8-3921) and BCMA (median 206; min-max: 14-6657). Clinical response to forimtamig was not correlated with GPRC5D+ MMPC frequency or GPRC5D receptor density measured by flow cytometry ( Table). Percentages of GPRC5D+, FcRH5+ and BCMA+ MMPCs and receptor densities did not significantly differ between patient groups defined by ISS score, cytogenetic risk and number of prior lines of therapy. Patients previously exposed to BCMA-targeted agents (n=7) tended to have lower BCMA expression levels. GPRC5D and FcRH5 were co-expressed on 75.4% of MMPCs (min-max: 0-100%) followed by MMPCs co-expressing GPRC5D and BCMA (median 31.9%; min-max: 0-100%) or BCMA and FcRH5 (median 25.3%; min- max: 0-94.3%). In the majority of patients, double and triple negative MMPCs were rarely observed (0.5-6% of MMPCs expressed only one or none of the three targets across all analyzed patients). Conclusions: GPRC5D, FcRH5 and BCMA are highly prevalent across MM patient subgroups, including high-risk patients who may warrant targeted therapy approaches. Based on our data and previously published work for FcRH5- and BCMA-targeted T-cell bispecifics (Sumiyoshi et al. EHA 2021; Cortes-Selva et al. ASH 2022), target expression on MMPCs, as measured by flow cytometry, has not been associated with clinical response to the corresponding CD3 bispecifics, likely due to the high efficacy of T-cell mediated tumor cell killing. Co-expression of MM targets suggests that targeting more than one MM surface marker may result in double/triple hits on individual tumor cells. Our data support further testing of dual/triple MMPC targeting as a promising concept to promote fast and deep tumor clearance, and to achieve durable responses, while potentially avoiding clonal selection, in patients with RRMM. Further investigation is needed to understand the relevance of target expression differences measured by flow cytometry compared to other methods like immunohistochemistry, as well as to understand T-cell bispecific resistance mechanisms beyond target expression. Updated data will be presented.

Abstract 898: Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of cells with greater persistence, there is a need in multiple phases of the CAR-T development pipeline for highly specific detection reagents that can be leveraged to monitor the expression of CARs on the cell surface. Many commercially available CAR detection reagents, however, either lack specificity or are not versatile in their ability to detect CARs of differing antigen specificity. Here, we report on the generation and validation of rabbit monoclonal antibodies raised against two linker sequences that are commonly integrated into single-chain variable fragment (scFv)-based CARs. These antibodies serve as versatile detection reagents that can be used to interrogate the surface expression of CARs. Methods: The monoclonal antibodies, E7O2V and E3U7Q, were generated by rabbits immunized with peptide sequences most commonly used to construct the linker region of scFv based CARs, Gly4Ser and Whitlow, respectively. E7O2V and E3U7Q were validated for specificity and versatility using flow cytometric analysis of non-transduced versus CAR-transduced cell lines and primary human T cells. Results: Flow cytometric analysis of live Jurkat cells and primary human T cells transduced with CAR constructs revealed that E7O2V and E3U7Q could detect surface expressed CARs containing the appropriate linker sequence, independently of scFv specificity. No specific staining was observed on non-transduced cells. Conclusions: In a flow cytometry assay, E7O2V and E3U7Q specifically detect surface expressed scFv-based CARs containing either a Gly4Ser linker or a Whitlow linker, respectively. Furthermore, these monoclonal antibodies are versatile in that they can also detect their respective linker sequence independently of scFv specificity. The potential exists to leverage these antibodies for CAR-T cell enrichment and for incorporation into multiparametric flow cytometry panels used to phenotype CAR-T cells during the discovery, manufacturing, and clinical phases of the development pipeline. Citation Format: Amrik Singh, Sarah L'Heureux, Ryan Sinapius, Michael Kvorjak, Emma Wade, Jason Lohmueller, Jeremy Fisher, Reginaldo Prioli. Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 898.

Application of Multiparametric Flow Cytometry Panels to Study Lymphocyte Subpopulations in Tuberculin-Positive Cattle.

Flow cytometry (FC) is widely used in microbiology, immunology, hematology, and oncology. In the veterinary field, FC enabled the study of the immune response in cattle infected with different pathogens, as well as vaccine testing. However, few fluorochrome-conjugated antibodies recognize bovine antigens, limiting the possible benefits of FC and the implementation of multiparametric analysis for more complex studies. Two cytometry panels with five colors each were designed and implemented for the study and identification of populations and subpopulations of T cells derived from the peripheral blood mononuclear cells of dairy heifers. Both panels detected differences in T cell subpopulations between heifers positively and negatively tested for tuberculin; they detected overexpression of CD25+ and CD45RO+ in tuberculin-positive heifers after stimulation with a culture filtrate protein extract (CFPE) from Mycobacterium bovis (M. bovis). We identified subpopulations of T cells from peripheral blood mononuclear cells using two multicolor panels. These panels could be used to analyze total bovine blood in immunopathogenic studies and vaccine development. The same strategy could be implemented in other species of veterinary interest.

A comprehensive multiparameter flow cytometry panel for immune profiling and functional studies of frozen tissue, bone marrow, and spleen

Flow cytometry (FC) is a highly informative technology that can provide valuable information about immune phenotype monitoring and immune cell states. However, there is a paucity of comprehensive panels developed and validated for use on frozen samples. Here, we developed a 17-plex flow cytometry panel to detect subtypes, frequencies, and functions of different immune cells that can be leveraged to study the different cellular characteristics in different disease models, physiological, and pathological conditions. This panel identifies surface markers to characterize T cells (CD8+, CD4+), natural killer (NK) cells and their subtypes (immature, cytotoxic, exhausted, activated),natural killer T (NKT) cells, neutrophils, macrophages (M1 (pro-inflammatory) and M2 (anti-inflammatory)), monocytes and their subtypes (classical and non-classical), dendritic cells (DC) and their subtypes (DC1, DC2), and eosinophils. The panel was designed to include only surface markers to avoid the necessity for fixation and permeabilization steps. This panel was optimized using cryopreserved cells.Immunophenotyping of spleen and bone marrow using the proposed panel was efficient in correctly differentiating the immune cell subtypes in inflammatory model of ligature-induced periodontitis, in which we found increased percentage of NKT cells, activated and mature/cytotoxic NK cells in the bone marrow of affected mice. This panel enables in-depth immunophenotyping of murine immune cells in bone marrow, spleen, tumors, and other non-immune tissues of mice. It could be a tool for systematic analysis of immune cell profiling in inflammatory conditions, systemic diseases, and tumor microenvironments.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Unsupervised cluster analysis and subset characterization of abnormal erythropoiesis using the bioinformatic Flow-Self Organizing Maps algorithm.

BackgroundThe Flow‐Self Organizing Maps (FlowSOM) artificial intelligence (AI) program, available within the Bioconductor open‐source R‐project, allows for an unsupervised visualization and interpretation of multiparameter flow cytometry (MFC) data.MethodsApplied to a reference merged file from 11 normal bone marrows (BM) analyzed with an MFC panel targeting erythropoiesis, FlowSOM allowed to identify six subpopulations of erythropoietic precursors (EPs). In order to find out how this program would help in the characterization of abnormalities in erythropoiesis, MFC data from list‐mode files of 16 patients (5 with non‐clonal anemia and 11 with myelodysplastic syndrome [MDS] at diagnosis) were analyzed.ResultsUnsupervised FlowSOM analysis identified 18 additional subsets of EPs not present in the merged normal BM samples. Most of them involved subtle unexpected and previously unreported modifications in CD36 and/or CD71 antigen expression and in side scatter characteristics. Three patterns were observed in MDS patient samples: i) EPs with decreased proliferation and abnormal proliferating precursors, ii) EPs with a normal proliferating fraction and maturation defects in late precursors, and iii) EPs with a reduced erythropoietic fraction but mostly normal patterns suggesting that erythropoiesis was less affected. Additionally, analysis of sequential samples from an MDS patient under treatment showed a decrease of abnormal subsets after azacytidine treatment and near normalization after allogeneic hematopoietic stem‐cell transplantation.ConclusionUnsupervised clustering analysis of MFC data discloses subtle alterations in erythropoiesis not detectable by cytology nor FCM supervised analysis. This novel AI analytical approach sheds some new light on the pathophysiology of these conditions.

Identifying Leukemia-associated Immunophenotypes in Acute Myeloid Leukemia Patients Using Multiparameter Flow Cytometry.

ObjectivesWe sought to identify leukemia-associated immunophenotypes (LAIPs) in 50 acute myeloid leukemia (AML) patients at diagnosis using an eight-color multiparameter flow cytometry (MFC) panel and to detect if they showed any alteration in relapsed/refractory cases.MethodsWe used the eight-color MFC panel with CD45/side scatter log gating strategy to analyze LAIPs in 50 AML patients presenting to Alexandria University Hospitals, Egypt at diagnosis and relapse and refractory cases. Twenty age and sex matched bone marrow samples from patients performing bone marrow aspirate for non-malignant hematological indications were included as controls.ResultsLAIPs were observed in 43 (86.0%) cases. Only one aberrant immunophenotype was identified in four cases (9.3%), while two to 12 aberrant immunophenotypes were found in the other 39 (90.7%) cases. Strong LAIPs were obtained by combining CD2, CD4, CD56, with either CD34 or CD117, in contrast to CD19, which has to be combined with CD117. Refractory cases showed the presence of the same LAIPs at both initial diagnosis and persistent disease. One case showed the acquisition of new LAIPs after relapse.ConclusionsThe good choice of LAIPs depends on their specificity rather than their frequency. The results of this study can help in increasing the sensitivity of LAIPs strategy in minimal residual disease using MFC in AML patients, which is considered an important post-diagnosis parameter associated with prognosis and clinical outcome.

Multiparameter flow cytometry in plasma cell disorders: when in doubt, go with the flow.

Multiparameter flow cytometry in plasma cell disorders: when in doubt, go with the flow.

Ten Color Multiparameter Flow Cytometry in Bone Marrow and Apheresis Products for Assessment and Outcome Prediction in Multiple Myeloma Patients.

ObjectiveIn clinical trials (CTs), the assessment of minimal residual disease (MRD) has proven to have prognostic value for multiple myeloma (MM) patients. Multiparameter flow cytometry (MFC) and next-generation sequencing are currently used in CTs as effective tools for outcome prediction. We have previously described 6- and 8-color MFC panels with and without kappa/lambda, which were equally reliable in detecting aberrant plasma cells (aPC) in myeloma bone marrow (BM) specimens. This follow-up study a) established a highly sensitive single-tube 10-color MFC panel for MRD detection in myeloma samples carrying different disease burden (monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma (SMM), MM), b) evaluated additional, rarely used markers included in this panel, and c) assessed MRD levels and the predictive value in apheresis vs. BM samples of MM patients undergoing autologous stem cell transplantation (ASCT).Methods + ResultsThe 10-color MFC was performed in BM and apheresis samples of 128 MM and pre-MM (MGUS/SMM) patients. The markers CD28, CD200, CD19, and CD117 underwent closer examination. The analysis revealed distinct differences in these antigens between MM, MGUS/SMM, and patients under treatment. In apheresis samples, the 10-color panel determined MRD negativity in 44% of patients. Absence of aPC in apheresis corresponded with disease burden, cytogenetics, and response to induction. It also determined MRD negativity in BM samples after ASCT and was associated with improved progression-free survival.ConclusionThese results highlight the significance of the evaluation of both BM and apheresis samples with a novel highly sensitive 10-color MFC panel.

Distinctive Platelet Features and Lymphocyte Sets Distribution in Immune Thrombocytopaenia Patients Responders to Thrombopoietin Receptor Agonists

Background: The heterogeneous pathophysiology of immune thrombocytopaenia (ITP) may explain the wide variation in the presentation of the disease, the clinical course and the response to therapeutic treatments. Among them, agonists of thrombopoietin receptor (TPO-RA) were incorporated as a second line agent for the management of ITP patients who do not respond to other treatments. We hypothesized that platelet and white cells from these patients might present some characteristics different from those observed in ITP patients who don't need to be treated and those responders to a first line therapeutic agent. In order to test this hypothesis we designed this study to evaluate platelet function and distribution of lymphocyte´s compartments in three ITP patient's groups: 1) untreated (UT) and treated with 2) TPO-RA or 3) corticoids.Materials and Methods: We recruited 33 UT ITP patients (30% male), 35 treated with TPO-RA (45% male), 15 with corticoids (25 % male) and 96 healthy controls (42% male).The La Paz Universitary Hospital Ethics Committee approved the experimental protocol.Platelet-activation, granule's content release, surface exposure of phosphatidylserine (PS) and activities of caspase-3, -8 or -9 were assessed by flow cytometry (respectively performed with PAC1, anti P-selectin and anti-CD63 antibodies, from BD; Annexin V from BD Pharmingen and Millipore kit).The frequency of T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes, natural killer (NK) CD56bright and CD56dim and regulatory T (Treg) [CD4+CD25+ forkhead box protein 3(FoxP3)+] were determined by multi-parametric flow cytometry panels (BD Biosciences).The statistical analysis of the experimental data was using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: Platelet count were similar in all ITP groups but lower than controls (p<0.001). Platelet activation, evaluated by binding of PAC1 (antibody that only recognizes the active form of fibrinogen receptor) after the stimulation with TRAP (an agonist for PAR-1 receptors), were equally reduced in all ITP groups. Platelet activation with ADP was only reduced in the TPO-RA ITP patients. A diminished exposure of P-selectin and CD63 was also observed in UT and TPO-RA ITP patients after platelet stimulation with TRAP (Table 1). No differences were observed in surface expression of CD41, CD61, CD42a and CD42b between ITP patients and healthy controls. Platelet apoptosis evaluation showed that ITP patients tended to have an increased activity of caspases -3,7,-8 and -9 activities, but this observation was significant only in the ITP group of treated with TPO-RA (Table 1).Regarding lymphocytes, no differences were observed in LT subsets among all groups. Intermediate (IM, CD14++CD16+), but not non-classical (NCM, CD14+CD16++) and classical (CM, CD14++CD16-) monocytes were increased in UT and TPO-RA ITP patients. CD56brightCD16- NKand CD56brightCD16+NK, abundant cytokine producers with a weak cytotoxic activity, were increased only in TPO-RA ITP patients. In this group a diminution was observed in memory LB cells. Regarding LTreg population, their count was reduced in UT and corticoid-treated patients, whereas their values reach normality in the TPO-RA treated group (Table 2).Conclusions: All ITP patients, untreated or responders to treatment with TPO-RA and corticoids recovered platelet count but not its function. TPO-RA-responders patients showed an increased apoptosis compared to the other ITP patients recruited. Nevertheless, TPO-RA seems to have a beneficial regulatory effect on immune system that might counteract platelet destruction.Work supported by grant from FIS-FEDER PI15/01457. NVB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresÁlvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

Assessment of chilling injury in hypothermic stored boar spermatozoa by multicolor flow cytometry.

Hypothermic storage of boar semen may allow antibiotic-free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA-containing events, Yo-Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA-Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700-DNA to identify DNA-containing events, JC-1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo-Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC-1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t-distributed stochastic neighbor embedding (t-SNE) maps and moving radar plots revealed similar subpopulations as identified by three-dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long-term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling-induced alterations of cell function in sperm subpopulations.

Importance of validating antibody panels: Anti-PD-L1 clone binds AF700 fluorophore

Researchers routinely use antibodies to assess the expression levels of proteins on the surface or intracellularly in a variety of different cell types. In this current study we highlight the importance of careful validation of antibodies for analysis of protein expression by flow cytometry and how failure to do so can significantly impact the interpretation of the data generated leading to false-positive results. There has been increasing awareness of the role the programmed death receptor 1 (PD-1) pathway plays in health and disease and a potential that programme death ligand 1 (PD-L1) may play a role in inflammatory disease. We aimed to investigate PD-L1 expression on human neutrophils isolated from healthy individuals and patients diagnosed with chronic obstructive pulmonary disease (COPD). We observed an increase in surface expression of PD-L1 by human neutrophils when incubated with AlexaFluor™700-conjugated anti-CD16. Through careful interrogation and antibody validation, we found a novel interaction between a commercially available anti-PD-L1 antibody and the AlexaFluor™700 fluorophore, resulting in this observed increase in PD-L1 signal. Surface expression of PD-L1 was not observed on neutrophils from healthy volunteers or patients with COPD when clone 29E.2A3 of anti-PD-L1 was not used with AlexaFluor™700-conjugated anti-CD16. This highlights the importance of robust antibody validation to ensure antibody compatibility in the context of multi-parametric flow cytometry panels. We also show that, without these validation experiments, novel neutrophil phenotypes could be falsely reported – an important consideration when there is increasing interest in neutrophil heterogeneity.

A Novel 10-Fluorochrome Multiparameter Flow Cytometry (MFC) Panel for Precise Plasma Cell (PC) and MRD Assessment in Clinical Routine of Multiple Myeloma (MM) Patients and throughout the Disease Course

Introduction: Since the introduction of novel agents into the treatment of Multiple Myeloma (MM), much deeper remissions can be achieved. This leads to a demand for disease evaluation tools with higher sensitivity and clinical practicability. In various clinical trials the assessment of the MRD status has been proven to have strongest prognostic value for MM patients (pts). The Euroflow Consortium has pioneered a highly standardized next generation flow (NGF) approach with the establishment of two 8-color tubes tests (Flores-Montero 2017). However, due to cost-intensive and time-consuming preconditions, this MRD approach might not be feasible in every center. Thus, other readily available, validated MRD tools may also guarantee highly sensitive MRD evaluation which allows for early relapse detection and to steer treatment, such as decision upon need for consolidation, duration of maintenance, therapy pauses or even earlier FDA/EMA approval of novel antimyeloma agents. Methods: Our 10 color single tube MFC panel consisting of the antigens CD38, CD138, CD19, CD45, CD27, CD56, CD28, CD81, CD117 and CD200, was systematically validated using Fluorescence-minus-one controls, consecutive spike-in-controls, consistency analyses and measurement of 9 MM cell lines. Thereafter, we assessed 131 Bone Marrow (BM), peripheral blood (PB), and leukapheresis (LA) samples from 97 different MM, MGUS and SMM pts and 13 healthy individuals (HI) as controls. Samples were processed within 6-20 hours of aspiration and a bulk lysis was performed. Subsequently, &gt;3x106 viable nucleated cells were acquired on a BD LRSFortessa Cytometer. Data analysis was performed using the BeckmanCoulter software Kaluza. BM was sampled at initial diagnosis (ID) or at progression (PD), after standard therapy (ST) or stem cell transplantation (SCT). The study was performed with written consent from all pts and approval from the ethics committee. Results: Our MFC approach confirmed the antigen expression in all MM cell lines (RPMI 8226; U266; IM-9; MM1.S; MM1.R; L363; Karpas620; NCI-H929; OPM-2) as previously reported. The spike-in-controls determined the limit of detection (LOD) at 10-5. This LOD was achieved in 89% of our MRD samples. An easy-to-adapt gating strategy to identify abnormal plasma cells (aPC) vs. normal plasma cells (nPC) was also established in our MM pt samples. In this cohort, we identified 10 distinct aPC and nPC subpopulations that differed in their expression pattern, suggesting clonal subtypes of MM. Moreover, in 19% of BM samples, two malignant subpopulations coexisting in the same pt, were identified. Since our BM cohort consisted of 46% samples at ID or PD vs. 54% posttreatment samples after ST and SCT, clonality variation in the former, differences of aPCs vs. nPCs ratio and to the latter group could be determined. Moreover, significant differences of much higher percentage of aPC in symptomatic MM vs SMM/MGUS pts were readily verified with our panel (p=0.0087). With a median time of 51 days from treatment to MRD evaluation, 24% of post treatment samples were MRD negative (MRD-) at the 10-5 level (&lt;0.001% aPC). In line, MRD negativity induced an improved PFS, with OS analyses needing a longer follow-up. Since we also assessed pre- and post-treatment samples, antibody expression levels on PC from ID to post treatment demonstrated a significant normalization in Median Fluorescence Intensity (MFI) of 5 antibodies (CD56, CD81, CD45, CD200, CD27). These may thus serve as informative markers for the MRD assessment of these samples. Conversely, with progression from remission to PD, CD81 and CD45 antibodies showed significant decreases (p=0.0007 and p=0.0026, respectively) in expression, which suggest that both may serve as early relapse markers. Of the assessed LA samples at harvest before ASCT, 44% were MRD-, warranting future investigations into possible implications for pts' prognosis, treatment and PFS/OS data. Conclusion: Here we present a readily available, cost-effective, quick and highly validated MFC panel with an easy to adapt gating strategy that allows for precise aPC vs. nPC assessment in- and outside clinical trials. Our 10 color MFC panel is applicable in BM samples of MM pts and precursor diseases, as well as in LA samples. With the additional MFC information, ideally at ID and repeatedly after treatment, readily available individual treatment decisions seem possible to obtain for every MM patient. Disclosures Wäsch: Amgen: Other: travel, Research Funding; Sanofi: Other: Travel, Research Funding; Jazz: Other: travel, Research Funding; Celgene: Other: travel, Research Funding; Gilead: Other: travel, Research Funding; Takeda: Consultancy; Gilead: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy.

Ectonucleotidase CD39 and Checkpoint Signalling Receptor Programmed Death 1 are Highly Elevated in Intratumoral Immune Cells in Non–small-cell Lung Cancer

Lung cancer is the leading cause of cancer death in both sexes worldwide and has a predicted 5-year survival rate of <20%. Immunotherapy targeting immune checkpoints such as the programmed death 1 (PD-1) signaling pathway has led to a shift of paradigm in the treatment of advanced non–small-cell lung cancer (NSCLC) but remains without effect in ∼80% of patients. Accumulating evidence suggests that several immunosuppressive mechanisms may work together in NSCLC. The contribution and cooperation between different immunosuppressive mechanisms in NSCLC remain unknown. Recently, the CD39-adenosine pathway has gained increasing attention as a crucial immunosuppressive mechanism and possible target for immunotherapy. Immune cells were extracted from lung and tumor tissue after lung resection in 12 patients by combined enzymatic and mechanical tissue disaggregation. A multiparameter flow cytometry panel was established to investigate the expression and coexpression of CD39 and PD-1 on key lymphocyte subtypes. Frequencies of CD39+, PD-1+, and CD39+/PD-1+cells were higher among both CD4+ and CD8+ T cells isolated from NSCLC tumor tissue than in T cells from normal lung tissue. Similarly, the frequency of FoxP3+ CD4+ T cells (Tregs) was highly significantly elevated in tumor tissue compared to adjacent lung tissue. The consistent upregulation of CD39 on immune cells in tumor microenvironment indicates that the CD39 signaling pathway may, in addition to the PD-1 pathway, represent another important mechanism for tumor-induced immunosuppression in NSCLC. In addition, the present study indicates that a comprehensive immune response profiling with flow cytometry may be both feasible and clinically relevant.

A Multiparameter Flow Cytometry Analysis Panel to Assess CD163 mRNA and Protein in Monocyte and Macrophage Populations in Hyperinflammatory Diseases.

CD163 facilitates regulation and resolution of inflammation and removal of free hemoglobin and is highly expressed in myeloid cells from patients with inflammatory disorders, such as systemic juvenile idiopathic arthritis (SJIA) and macrophage activation syndrome (MAS). Our recent studies indicate that regulation of CD163 mRNA expression is a key functional property of polarized monocytes and macrophages and is mediated at the transcriptional and posttranscriptional level, including via microRNAs. The goal of the current study is to develop a multiparameter flow cytometry panel incorporating detection of CD163 mRNA for polarized monocyte and macrophage populations in disorders such as SJIA and MAS. THP-1 cells and CD14+ human monocytes were stained using fluorochrome-conjugated Abs to myeloid surface markers, along with CD163 mRNA. Staining for mRNA could reliably detect CD163 expression while simultaneously detecting different macrophage populations using Abs targeting CD14, CD64, CD80, CD163, and CD209. This approach was found to be highly sensitive for increased mRNA expression when macrophages were polarized with IL-10 [M(IL-10)], with a strong signal over a broad range of IL-10 concentrations, and showed distinct kinetics of CD163 mRNA and protein induction upon IL-10 stimulation. Finally, this panel demonstrated clear changes in polarization markers in unstimulated monocytes from patients with SJIA and MAS, including upregulated CD163 mRNA and increased CD64 expression. This approach represents a robust and sensitive system for RNA flow cytometry, useful for studying CD163 expression as part of a multimarker panel for human monocytes and macrophages, with broad applicability to the pathogenesis of hyperinflammatory diseases.