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Purpose: There is compelling evidence that CD4 + and CD8 + T cells are dysfunctional in multiple myeloma, compromising their ability to control disease progression. Pre-clinical models suggest that exercise represents a non-pharmacologic means to reduce immune exhaustion, but no studies to date have examined the relationship between an exercise intervention and biomarkers of immune exhaustion in multiple myeloma patients., Patients and Methods: The current study includes 24 multiple myeloma patients who participated in a six-month physical activity intervention, consisting of supervised strength training (n = 12) and unsupervised home-based walking arms (n = 12). Comprehensive flow cytometry was utilized to assess the frequency of CD4 + and CD8 + T cells and subpopulations expressing the markers of exhaustion PD-1, TIGIT, TIM3 and/or LAG3. Ratios of exhausted to non-exhausted cell populations, and percentages of exhausted to total populations of the same lineage, were calculated for the baseline and final timepoints., Results: Eighteen of 20 exhaustion measures were lower at the end of the intervention than at baseline, and several were significantly or borderline significantly reduced in the entire sample or in one of the arms. The entire sample saw improvements in the ratios of CD4 + TIGIT + to non-exhausted CD4 + (0.7 [0.6] to 0.6 [0.4], P = .04) and CD8 + PD1 + to non-exhausted CD8 + (1.8 [2.6] to 1.5 [2.0], P = .06), and in total exhausted CD8 + as a percent of total CD8 + (72.9 [21.9] to 68.3 [19.6], P < .01)., Conclusions: This pilot study suggests that physical activity induces changes in MM patients' immune systems, potentially rendering a less exhausted T cell state., Competing Interests: Disclosure JH serves on the advisory boards of Adaptive, Amgen, Angitia, Axxess Networks, Bristol Myers Squibb, GlaxoSmithKline, Janssen Biotech, Oncopeptides, ONCOtracker, Prothena, Sanofi, and Skyline; has given talks at Amgen, BeiGene, Beijing Medical Award Foundation, Curio Science, and Janssen Biotech; and serves on the data and safety monitoring board of Janssen Biotech. JMJ, JDT, AG, KBM, MH, HJ, BW, HM, PKW, KA, RC, and SA declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Clinical and Laboratory Standards Institute (CLSI) H62-Validation of Assays Performed by Flow Cytometry guideline, released in 2021, provides recommendations for platform workflow and quality system essentials, instrument setup and standardization, assay development and optimization and fit-for-purpose analytical method validation. In addition, CLSI H62 includes some recommendations for the validation strategies after a validated flow cytometric method has been modified. This manuscript builds on those recommendations and discusses the impact of different types of assay modifications on assay performance. Recommendations regarding which validation parameters to evaluate depending on the type of modification are provided. The impact of assay modification on the assay's intended use is discussed. When recommending minor deviations from the CLSI H62 process for a laboratory-initiated assay revision (e.g., specimen numbers for sensitivity, specificity, or precision studies), a rationale based on expert opinion is provided with the understanding that not every laboratory, assay type, and circumstance can be comprehensively addressed in this paper. These recommendations are meant as a practical recommendation and are not intended to be restrictive, prescriptive, or understood as necessarily sufficient to meet every specific requirement from regulatory bodies (e.g., FDA or New York State Department of Health)., (© 2024 International Clinical Cytometry Society.)

Purpose: Prognostic Immunophenotyping in Myeloma Response (PRIMeR) is an ancillary study of minimal residual disease (MRD) assessment for multiple myeloma by next-generation multiparameter flow cytometry (MFC). Patients were enrolled on a three-arm randomized control trial (Blood and Marrow Transplants Clinical Trials Network 0702 Stem Cell Transplant for Myeloma in Combination of Novel Agents [STaMINA]; ClinicalTrials.gov identifier: NCT01109004)., Methods: Four hundred and thirty-five patients consented to the MRD panel, which included 10 monoclonal antibodies measured via six-color MFC. MRD was measured at baseline/preautologous hematopoietic cell transplant (BL/preAutoHCT), premaintenance (PM), and 1 year (Y1) after AutoHCT with a sensitivity of 10 -5 to 10 -6 . The primary objective was to assess MRD-negative (MRD neg ) at 1 year after AutoHCT and progression-free survival and overall survival (PFS/OS)., Results: Similar to the STaMINA results, at a median follow-up of 70 months, there was no significant difference in PFS/OS by treatment arm in the PRIMeR patients. MRD neg at all three time points was associated with significantly improved PFS, and MRD neg at Y1 had significantly longer OS. Multivariate analysis of PFS, adjusting for disease risk and treatment arm, demonstrated hazard ratios (HRs) in MRD-positive patients compared with MRD neg patients at BL, PM, and Y1 of 1.55 ( P = .0074), 1.83 ( P = .0007), and 3.61 ( P < .0001), respectively. Corresponding HRs for OS were 1.19 ( P = .48), 0.88 ( P = .68), and 3.36 ( P < .001). Patients with sustained MRD neg or who converted to MRD neg by Y1 had similar PFS/OS., Conclusion: To our knowledge, this first, prospective US cooperative group, multicenter study demonstrates that MRD neg at Y1 after AutoHCT with lenalidomide maintenance is prognostic for improved 6-year PFS and OS. Serial MRD measurements may direct trials to test how further therapy may improve long-term PFS and OS.

Haploidentical (Haplo) allogeneic HCTs (alloHCT) have been used more frequently over the last decade as survival is similar to HLA-matched related donor (MRD) alloHCTs. We aimed to identify donor and recipient immune signatures before alloHCT that are associated with clinically meaningful outcomes in MRD vs Haplo alloHCT recipients. This retrospective cohort study of 165 MRD (n = 132) and Haplo (n = 33) alloHCT recipients and their related donors between 2007-2019 with paired peripheral blood samples immunophenotyped for T-cell, B-cell, NK cell and dendritic cell (DC) subsets. Immune cells were quantified before alloHCT in donors and recipients; calculations of immune cell ratios were classified as high, intermediate, and low and analyzed with alloHCT outcomes. Haplo donors were younger than MRD donors (median: 35 vs 51 years), whereas Haplo recipients were older than MRD recipients (median: 68 vs 54 years), were more likely to have a Karnofsky Performance Score ≤ 70 (76% vs 57%), 3+ comorbidities (54% vs 47%), and were in complete remission prior to alloHCT (58% vs 42%). In MRD alloHCT, a lower ratio of CD4+ to CD8+ effector memory cells in the donor was associated with lower 4-yr overall survival (OS; 25% vs 61%; P = .009), lower 4-yr progression free survival (PFS; 25% vs 58%; P = .014) and higher incidence of 1-yr transplant-related mortality (TRM; 39% vs 7%; P = .009) in recipients. A higher ratio of CD8+ effector memory to total NK cells measured in MRD recipients was associated with a higher incidence of grade II-IV aGvHD (63% vs 37%; P = .004) but was not statistically significant for III-IV aGvHD (23% vs 12%). In Haplo alloHCT, a lower ratio of total T-regulatory to CD4+ central memory cells in the donor was associated with lower 4-yr PFS (22% vs 60%; P = .0091). A higher ratio of CD4+ effector memory to CD8+ effector memory cells measured in Haplo recipients pre-alloHCT was associated with lower 4-yr OS (25% vs 88%; P = .0039). In both MRD and Haplo recipients, a higher ratio of CD4+ naïve to CD4+ central memory cells was associated with a higher incidence of grade II-IV aGvHD (64% vs 38%; P = .04). Evaluation of pre-alloHCT immune signatures of the donor and recipient may influence clinically meaningful patient outcomes in both MRD and Haplo transplants., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Identifying plasma biomarkers early after allo-HCT may become crucial to prevent and treat severe aGvHD. We utilized samples from 203 allo-HCT patients selected from the Blood & Marrow Transplant Clinical Trials Network (BMT CTN) to identify new biomarker models to predict aGvHD and overall mortality. Two new biomarkers (Gal-3 and LAG-3), and previously identified biomarkers (ST2/IL33R, IL6, Reg3A, PD-1, TIM-3, TNFR1) were screened. Increased Gal-3 levels measured at Day +7 post-transplant predicted the development of aGvHD (grade 2-4) in the total population [AUC: 0.602; P = 0.045] while higher Day +14 levels predicted overall mortality due to toxicity among patients receiving reduced intensity conditioning [P = 0.028] but not myeloablative conditioning. Elevated LAG-3 levels (Day +21) were associated with less severe aGvHD [159.1 ng/mL vs 222.0 ng/mL; P = 0.046]. We developed a model utilizing Gal-3, LAG-3, and PD-1 levels at Days +14 and +21 with an improved performance to predict aGvHD and overall non-relapse mortality. We confirmed four informative biomarkers (Reg3A, ST2, TIM-3, and TNFR1) predict severe aGvHD at day +14 and day +21 (grade 3-4). In conclusion, the combination of Gal-3 alone or in combination with LAG-3, and PD-1 is a new informative model to predict aGvHD development and overall non-relapse mortality after allo-HCT., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue ® Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the "know your dye" and "know your cytometer" principles described herein., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Flow cytometry (FC) is routinely used for hematological disease diagnosis and monitoring. Advancement in this technology allows us to measure an increasing number of markers simultaneously, generating complex high-dimensional datasets. However, current analytic software and methods rely on experienced analysts to perform labor-intensive manual inspection and interpretation on a series of 2-dimensional plots via a complex, sequential gating process. With an aggravating shortage of professionals and growing demands, it is very challenging to provide the FC analysis results in a fast, accurate, and reproducible way. Artificial intelligence has been widely used in many sectors to develop automated detection or classification tools. Here we describe a type of machine learning method for developing automated disease classification and residual disease monitoring on clinical flow datasets., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Presence of cytotoxic T lymphocytes (CTL) in the tumor microenvironment (TME) predicts the effectiveness of cancer immunotherapies. The ability of toll-like receptor 3 (TLR3) ligands, interferons (IFNs) and COX2 inhibitors to synergistically induce CTL-attracting chemokines (but not regulatory T cell (Treg)-attractants) in the TME, but not in healthy tissues, observed in our preclinical studies, suggested that their systemic application can reprogram local TMEs., Methods: Six evaluable patients (33-69 years) with metastatic triple-negative breast cancer received six doses of systemic chemokine-modulating (CKM) regimen composed of TLR3 ligand (rintatolimod; 200 mg; intravenous), IFN-α2b (20 MU/m 2 ; intravenous) and COX2 inhibitor (celecoxib; 2×200 mg; oral) over 2 weeks. The predetermined primary endpoint was the intratumoral change in the expression of CTL marker, CD8α, in the post-CKM versus pre-CKM tumor biopsies. Patients received follow-up pembrolizumab (200 mg, intravenously, every 3 weeks), starting 3-8 days after completion of CKM., Results: Post-CKM biopsies showed selectively increased CTL markers CD8α (average 10.2-fold, median 5.5-fold, p=0.034) and granzyme B (GZMB; 6.1-fold, median 5.8-fold, p=0.02), but not FOXP3 (Treg marker) relative to HPRT1 expression, resulting in the increases in average CD8α/FOXP3 ratio and GZMB/FOXP3 ratio. CKM increased intratumoral CTL-attractants CCL5 and CXCL10, but not Treg-attractants CCL22 or CXCL12. In contrast, CD8 + T cells and their CXCR3 + subset showed transient decreases in blood. One clinical response (breast tumor autoamputation) and three stable diseases were observed. The patient with clinical response remains disease free, with a follow-up of 46 months as of data cut-off., Conclusions: Short-term systemic CKM selectively increases CTL numbers and CTL/Treg ratios in the TME, while transiently decreasing CTL numbers in the blood. Transient effects of CKM suggest that its simultaneous application with checkpoint blockade and other forms of immunotherapy may be needed for optimal outcomes., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Studies evaluating peripheral patient samples show radiation can modulate immune responses, yet the biological changes in human tumors particularly at the cellular level remain largely unknown. Here, we address how radiation treatment shapes the immune compartment and interactions with cancer cells within renal cell carcinoma (RCC) patient tumors., Methods: To identify how radiation shaped the immune compartment and potential immune interactions with tumor cells we evaluated RCC tumors from patients treated only with nephrectomy or with radiation followed by nephrectomy. Spectral flow cytometry using a 35-marker panel was performed on cell suspensions to evaluate protein expression within immune subsets. To reveal how radiation alters programming of immune populations and interactions with tumor cells, we examined transcriptional changes by single-cell RNA sequencing (scRNAseq)., Results: Spectral flow cytometry analysis revealed increased levels of early-activated as well as effector programmed cell death protein-1 (PD-1) + CD8 T-cell subsets within irradiated tumors. Following quality control, scRNAseq of tumor samples from nephrectomy-only or radiation followed by nephrectomy-treated patients generated an atlas containing 34,626 total cells. Transcriptional analysis revealed increased transition from stem-like T-cell populations to effector T cells in irradiated tumors. Interferon (IFN) pathways, that are central to radiation-induced immunogenicity, were enriched in irradiated lymphoid, myeloid, and cancer cell populations. Focused cancer cell analysis showed enhanced antigen presentation and increased predicted TRAIL-mediated and IFN-mediated interactions between tumor cells and the same effector T-cell subsets increased by radiation. TRAIL and IFN pathways enriched in irradiated tumors were associated with survival in patients treated with immunotherapy., Conclusions: These findings identify the source of IFN enrichment within irradiated RCC and reveal heightened levels of PD-1 + CD8 + T-cell subsets and increased probability of interactions with tumor cells following standalone radiation treatment. This study provides a window into the irradiated tumor-immune microenvironment of patients and rationale for treatment combinations., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Spatial heterogeneity is a common phenomenon in metastatic solid tumors and an evolving concept in multiple myeloma (MM). The interplay between malignant plasma cells (PCs) and the microenvironment has not yet been analyzed in MM. For this purpose, we performed bone marrow aspirates and imaging-guided biopsies of corresponding lesions in newly diagnosed MM (NDMM) and relapsed/refractory MM (RRMM) patients. PCs were isolated and subjected to whole-exome sequencing (WES). Non-PCs were studied with next-generation flow (NGF) and T-cell receptor sequencing (TCRseq) to analyze the connection between malignant and nonmalignant cells in the bone marrow and in lesions. Although we observed a strong overlap from WES, NGF, and TCRseq in patients with intramedullary disease, WES revealed significant spatial heterogeneity in patients with extramedullary disease. NGF showed significant immunosuppression in RRMM compared with NDMM as indicated by fewer myeloid dendritic cells, unswitched memory B cells, Th9 cells, and CD8 effector memory T cells but more natural killer and regulatory T cells. Additionally, fewer T-cell receptor (TCR) sequences were detected in RRMM compared with NDMM and healthy individuals. After induction therapy, TCR repertoire richness increased to levels of healthy individuals, and NGF showed more regulatory T cells and myeloid-derived suppressor cells, regardless of depth of response. Clinical significance of imaging-guided biopsies of lesions was demonstrated by detection of monoclonal PCs in patients without measurable residual disease (MRD) in aspirates from the iliac crest as well as identification of secondary primary malignancies in MRD- patients. Furthermore, site-specific clones with different drug susceptibilities and genetically defined high-risk features were detected by our workflow., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Acute myeloid leukemia (AML) measurable residual disease (MRD) evaluated by multiparametric flow cytometry (MFC) is a surrogate for progression-free and overall survival in clinical trials and patient management. Due to the limited number of detection channels available in conventional flow cytometers, panels used for assessing AML MRD are typically split into multiple tubes. This cripples the simultaneous and correlated assessment of all myeloblast measurements. In response, we prototyped a single-tube 27-color MFC assay for the evaluation of AML MRD, incorporating all recommended markers. Marrow aspirates from 22 patients were processed for analysis using full spectrum flow cytometry (FSFC). The signal resolution of each marker was compared between samples stained with single antibody vs. the fully stained panel. The analytical accuracy for quantifying hematopoietic cells between our established 8-color assay and the new 27-color method were compared. Variations within an operator and between separate operators were assessed to evaluate the assays reproducibility. The limited of blank (LOB), limit of detection (LOD), and lower limit of quantification (LLOQ) of the 27-color method were empirically determined using limiting dilution experiments. The stability of antibody cocktails over a period of 120 h was also studied using cryopreserved marrow cells. The stain indices for all antibodies were lower in the fully stained panel compared to cells stained with one antibody but clear separations between negative and positive signals were achieved for all antibodies. Our results demonstrated a high concordance between the established 8-color method and the new 27-color assay for enumerating myeloblasts and MRD interpretation within and between operators. The data further showed that the single-tube 27-color assay easily achieved the minimum required detection sensitivity of 0.1%. When antibodies were combined, however, expression intensity of some antigens deteriorated significantly when stored. Our single-tube 27-color panel is a suitable, high sensitivity flow cytometric approach that can be used for AML MRD testing, which improves the correlation of aberrant antigens and detection of asynchronous differentiation patterns. Based on the stability study, we recommend the full panel be made prior to staining., (© 2022 International Society for Advancement of Cytometry.)

Objectives: The Scleroderma: Cyclophosphamide or Transplantation (SCOT) trial compared hematopoietic stem cell transplant to CYC treatment in patients with early SSc with progressive skin and lung or kidney involvement. Here we describe lymphocyte phenotype abnormalities at study entry and the relation to prior DMARD therapy., Methods: Lymphocyte subsets (n = 26) measured by flow cytometry were compared in 123 heathy controls and 71 SCOT participants, including those given (n = 57) or not given (n = 14) DMARDs within 12 months of randomization., Results: Compared with healthy controls, individuals with SSc showed significant reductions in central memory CD8 T cells, activated total and CD4 T cells, γ/δ T cells, memory B cells, myeloid and plasmacytoid dendritic cells and FOXP3+CD25+ Treg cells and increases in naïve CD4 T cells, effector memory CD4 T cells and effector CD8 T cells. A greater bias towards a IL-4+ Th2/T cytotoxic 2 (Tc2) phenotype based on the Th2:Th1 CD4 ratio and Tc2:Tc1 CD8 T cells was also found. Notably, no difference in any lymphocyte subset was observed between those given or not given prior DMARDs., Conclusions: In patients with early, severe SSc, significant lymphocyte subset abnormalities were observed. Prior treatment with immunosuppressive therapy did not impact the immunophenotype, suggesting that lymphocyte disturbances in scleroderma appeared to be due to the disease itself., Trial Registration: ClinicalTrials.gov (https://clinicaltrials.gov), NCT00114530., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: CIMAvax-EGF is an epidermal growth factor (EGF)-depleting immunotherapy which has shown survival benefit as a switch maintenance treatment after platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC). The primary objective of this trial is to establish the safety and recommended phase II dose (RP2D) of CIMAvax-EGF in combination with nivolumab as second-line therapy for NSCLC., Methods: Patients with immune checkpoint inhibitor-naive metastatic NSCLC were enrolled using a "3+3" dose-escalation design. Toxicities were graded according to CTCAE V4.03. Thirteen patients (one unevaluable), the majority with PD-L1 0%, were enrolled into two dose levels of CIMAvax-EGF., Findings: The combination was determined to be safe and tolerable. The recommended phase 2 dose of CIMAvax-EGF was 2.4 mg. Humoral response to CIMAvax-EGF was achieved earlier and in a greater number of patients with the combination compared to historical control. Four out of 12 evaluable patients had an objective response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Evans, Lee, Wallace, Reid, Muhitch, Dozier, Mesa, Luaces, Santos-Morales, Groman, Cedeno, Cinquino, Fisher, Puzanov, Opyrchal, Fountzilas, Dai, Ernstoff, Attwood, Hutson, Johnson, Mazorra, Saavedra, Leon, Lage, Crombet and Dy.)

Background: Ofatumumab is a humanized type 1 anti-CD20 monoclonal antibody. Preclinical studies show improved complement-mediated cytotoxicity (CMC) compared to rituximab in mantle cell lymphoma (MCL). This study evaluates the safety and efficacy of combining ofatumumab with HyperCVAD/MA (O-HyperCVAD) in newly diagnosed MCL., Methods: In this single-arm phase 2 study, 37 patients were treated with the combination of O-HyperCVAD for 4 or 6 cycles, followed by high dose chemotherapy and autologous stem cell transplant. Primary objectives were overall response rate (ORR) and complete response (CR) rate at the end of therapy. Secondary objectives included minimal residual disease (MRD) negativity, progression-free survival (PFS), and overall survival (OS)., Results: Median age was 60 years; ORR was 86% and 73% achieved a CR by modified Cheson criteria. The MRD negativity rate was 78% after 2 cycles of therapy, increasing to 96% at the end of induction; median PFS and OS were 45.5 months and 56 months, respectively. Achieving a post-induction CR by both imaging and flow cytometry was associated with improved PFS and OS. Early MRD negativity (post-2 cycles) was also associated with an improved PFS but not OS. There were 3 deaths while on therapy, and grades 3 and 4 adverse events (AEs) were observed in 22% and 68% of the patients., Conclusion: The addition of ofatumumab to HyperCVAD/HD-MA led to high rates of MRD negativity by flow cytometry in patients with newly diagnosed MCL. Achieving a CR post-induction by both imaging and flow cytometry is associated with improved overall survival., (© 2022 American Cancer Society.)

Background: Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression-free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter-laboratory variation in MM MRD reporting., Methods: Seventeen participants from 13 countries performed blinded analysis of the same eight de-identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re-analyzed for MRD and other bone marrow cells (Stage 2). Inter-laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3)., Results: In Stage 1, participants agreed on MRD+/MRD- status 89% and 68% of the time respectively. Inter-observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD- status improved to 74%. Better consistency enumerating all parameters among operators resulted in near-unanimous agreement on sample adequacy., Conclusion: Uniform flow cytometry data analysis substantially reduced inter-laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint., (© 2022 International Clinical Cytometry Society.)

Osteolytic lesions (OL) characterize symptomatic multiple myeloma. The mechanisms of how malignant plasma cells (PC) cause OL in one region while others show no signs of bone destruction despite subtotal infiltration remain unknown. We report on a single-cell RNA sequencing (scRNA-seq) study of PC obtained prospectively from random bone marrow aspirates (BM) and paired imaging-guided biopsies of OL. We analyze 148,630 PC from 24 different locations in 10 patients and observe vast inter- and intra-patient heterogeneity based on scRNA-seq analyses. Beyond the limited evidence for spatial heterogeneity from whole-exome sequencing, we find an additional layer of complexity by integrated analysis of anchored scRNA-seq datasets from the BM and OL. PC from OL are characterized by differentially expressed genes compared to PC from BM, including upregulation of genes associated with myeloma bone disease like DKK1, HGF and TIMP-1 as well as recurrent downregulation of JUN/FOS, DUSP1 and HBB. Assessment of PC from longitudinally collected samples reveals transcriptional changes after induction therapy. Our study contributes to the understanding of destructive myeloma bone disease., (© 2022. The Author(s).)

Measuring response among patients with multiple myeloma is essential for the care of patients. Deeper responses are associated with better progression free survival (PFS) and overall survival (OS). To test the hypothesis that Mass-Fix, a mass spectrometry-based means to detect monoclonal proteins, is superior to existing methodologies to predict for survival outcomes, samples from the STAMINA trial (NCT01109004), a trial comparing three transplant approaches, were employed. Samples from 575 patients from as many as three time points (post-induction [post-I; pre-maintenance [pre-M]; 1 year post enrollment [1YR]) were tested when available. Four response parameters were assessed: Mass-Fix, serum immunofixation, complete response, and measurable residual disease (MRD) by next generation flow cytometry. Of the four response measures, only MRD and Mass-Fix predicted for PFS and OS at multiple testing points on multivariate analyses. Although MRD drove Mass-Fix from the model for PFS at post-I and pre-M, 1YR Mass-Fix was independent of 1YR MRD. For OS, the only prognostic pre-I measure was Mass-Fix, and the only 1YR measures that were prognostic on multivariate analysis were 1YR MRD and 1YR Mass-Fix. SIFE and CR were not. Mass-Fix is a powerful means to track response., (© 2022. The Author(s).)

Background: Daratumumab is an anti-CD38 immunotherapeutic drug that has increasingly been used to treat patients with heavily pre-treated and relapsed/refractory multiple myeloma. In so doing, the detection of CD38 antigen on plasma cells by flow cytometry is impeded. We hypothesized that alternative markers can be used in place or in addition to CD38 when detecting plasma cells post-treated with daratumumab., Methods: A total of 16 alternative markers were tested using 22 bone marrow aspirates from patients with plasma cell neoplasm. The ability of selected markers to discern plasma cells from other hematopoietic cells were evaluated. The stability of tested markers when stored at 4 or 25°C after T = 0, 24, 48, and 72 h was also established. Finally, selected markers were incorporated into a panel used for monitoring multiple myeloma measurable residual disease to test their utility to identify plasma cells in the presence of daratumumab and/or elotuzumab (anti-CD319) drugs., Results: Out of the 16 tested markers, CD319, CD54, CD229, CD317, and p63 were expressed by >90% of the plasma cells. Only CD319, CD54, and CD229 achieved 100% detection sensitivity. Further analysis showed that CD319 was better than CD229 and CD54 at resolving plasma cells from background hematopoietic cells, with CD54 being the worst (resolution metric, mean ± SD: CD319 [2.04 ± 0.86]; CD229 [1.47 ± 0.45]; and CD54 [1.22 ± 0.60]). CD229 was expressed by >90% of T lymphocytes, whereas CD319 was expressed preferentially by the CD8+ T cells and less frequently in CD4+ T cells. Additionally, CD229 was found on >60% of B and NK cells, as well as minor subsets of monocytes and granulocytes. CD319 was expressed on most NK cells and a minor subset of B cells, granulocytes, and monocytes. Even though CD229 and CD319 were expressed by different leukocyte subsets, their expression levels were highest on plasma cells. The expression of CD138 on plasma cells was significantly lower after storage at 4°C, while the expression levels of CD38, CD229, and CD319 remained stable at 4 or 25°C. Using limiting dilution experiments, the treatment of cells with daratumumab severely impeded the detection of CD38 antigen on plasma cells, whereas elotuzumab treatment did not block detection of CD319 on plasma cells., Conclusions: CD319 is a suitable alternative to CD38 for identifying plasma cells. Our results showed that a panel used for monitoring multiple myeloma measurable residual disease could be modified by using CD319 alone or in combination with CD38 to detect PCs in daratumumab or elotuzumab treated patients., (© 2020 International Clinical Cytometry Society.)

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective., (© 2021 John Wiley & Sons Ltd.)

Problem: Previous studies identified circulating CD14 + HLA-DR lo/- monocytic cells as an immune suppressive subset in solid malignancies, such as prostate, renal cell carcinoma, and pancreatic cancer. Such monocytic cells have been implicated not only in tumour progression but also as a potential barrier for immunotherapy. This study examined the relationship between the frequency of circulating monocytic cells and epithelial ovarian cancer (EOC) progression pre- and post-frontline chemotherapy, defined by disease stage, which is a leading prognostic factor for this malignancy., Method of Study: Incident cases of 236 women with EOC were recruited and comprehensive flow cytometry was utilized to assess the frequency of peripheral blood CD33 + CD11b + HLA-DR -/low CD14 + CD15 - monocytic cells, henceforth termed CD14 + HLA-DR lo/- monocytic cells, prior to and after completion of frontline chemotherapy. Multivariable odds ratios (OR) were used to estimate the association between CD14 + HLA-DR lo/- monocytic cell percentages and disease stage. Wilcoxon signed-rank tests evaluated changes in these monocytic cell levels pre- and post-chemotherapy in a patient subset (n = 70)., Results: Patients with elevated frequencies of circulating CD14 + HLA-DR lo/- monocytic cells at diagnosis were at 3.33-fold greater odds of having advanced stage (III/IV) EOC (CI: 1.04-10.64), with a significant trend in increasing CD14 + HLA-DR lo/- monocytic cell levels (P = .04). There was a 2.02% median decrease of these monocytic cells post-chemotherapy among a subset of patients with advanced stage disease (P < .0001)., Conclusion: These findings support the potential clinical relevance of CD14 + HLA-DR lo/- monocytic cells in EOC for prognosis and may indicate a non-invasive biomarker to measure disease progression., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Increased β-adrenergic receptor (β-AR) signaling has been shown to promote the creation of an immunosuppressive tumor microenvironment (TME). Preclinical studies have shown that abrogation of this signaling pathway, particularly β2-AR, provides a more favorable TME that enhances the activity of anti-PD-1 checkpoint inhibitors. We hypothesize that blocking stress-related immunosuppressive pathways would improve tumor response to immune checkpoint inhibitors in patients. Here, we report the results of dose escalation of a nonselective β-blocker (propranolol) with pembrolizumab in patients with metastatic melanoma., Patients and Methods: A 3 + 3 dose escalation study for propranolol twice a day with pembrolizumab (200 mg every 3 weeks) was completed. The primary objective was to determine the recommended phase II dose (RP2D). Additional objectives included safety, antitumor activity, and biomarker analyses. Responders were defined as patients with complete or partial response per immune-modified RECIST at 6 months., Results: Nine patients with metastatic melanoma received increasing doses of propranolol in cohorts of 10, 20, and 30 mg twice a day. No dose-limiting toxicities were observed. Most common treatment-related adverse events (TRAEs) were rash, fatigue, and vitiligo, observed in 44% patients. One patient developed two grade ≥3 TRAEs. Objective response rate was 78%. While no significant changes in treatment-associated biomarkers were observed, an increase in IFNγ and a decrease in IL6 was noted in responders., Conclusions: Combination of propranolol with pembrolizumab in treatment-naïve metastatic melanoma is safe and shows very promising activity. Propranolol 30 mg twice a day was selected as RP2D in addition to pembrolizumab based on safety, tolerability, and preliminary antitumor activity., (©2020 American Association for Cancer Research.)

Previous studies have reported a beneficial effect from cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation (alloHCT) on immune reconstitution. We determined the CMV antigenemia level associated with increased CMV antigen-specific T cells (CASTs) at day +100 and decreased CMV reactivation after day +100. CMV reactivation and CASTs were measured with CMV antigenemia and CMV-specific major histocompatibility complex multimers. The analysis consisted of 775 CAST measurements obtained before and 30, 100, and 365 days post-alloHCT from 327 consecutive patients treated between 2008 and 2016. Detectable CASTs correlated with recipient (P < .0001) and donor (P < .0001) CMV seropositivity pre-alloHCT. CMV reactivation before day +100 was associated with a higher proportion of patients who achieved ≥3 CASTs/µL by day +100 (61% with versus 39% without reactivation, P < .001). In alloHCT recipients at high risk for CMV reactivation (R + D ± ) with a maximum of grade II acute graft-versus-host-disease, reactivating CMV before day +100 and achieving ≥3 versus <3 CASTs/µL at day +100 was associated with reduced CMV reactivation from day +100 to +365 (27% versus 62%, P = .04). This protective effect was observed with low-level but not high-level CMV reactivation (<5 versus ≥5/50,000 polymorphonuclear leukocytes + pp65, respectively). These findings suggest low-level CMV reactivation may be beneficial and that treatment may be delayed until progression. These findings will need validation in prospective clinical trials using CMV PCR and antigenemia assays., (Copyright © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post-treatment. To achieve the high sensitivity (one abnormal cell in 10 5 -10 6 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, "Pre-lysis" method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the "Pooled-tube" and "Dextran Sedimentation" staining procedures and correlated them with the "Pre-lysis" method as potential alternative approaches., Methods: A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the "Pre-lysis," "Pooled-tube," and "Dextran Sedimentation" techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments., Results: The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the "Pooled-tube" method when compared to "Pre-lysis." "Pre-lysis" was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by "Pooled-tube" when alternative CD56 PE clones were used. "Dextran sedimentation" yielded similar staining results when compared to "Pre-lysis" for all the markers analyzed. The "Pooled-tube" method, when normalized to "Pre-lysis," recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between "Pre-lysis" versus "Dextran Sedimentation." All three flow cytometric assays achieved a minimum sensitivity of 10 -5 and approached that of 10 -6 for detecting rare events., Conclusion: Both "Pooled-tube" and "Dextran Sedimentation" staining procedures were comparable to the "Pre-lysis" method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing., (© 2019 International Clinical Cytometry Society.)

Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer-review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry., (© 2019 International Society for Advancement of Cytometry.)

This is the first longitudinal study of immune profiles and autologous hematopoietic cell transplant (AHCT) survival in B-cell non-Hodgkin lymphoma (B-NHL) patients and the effect of plerixafor mobilization on immune reconstitution in this population. A comprehensive immunophenotyping panel was performed in 104 consecutive adult B-NHL patients (58% diffuse large B cell and 42% mantle cell) who received AHCT (1/2008-11/2014), at a median of 28 days pre-AHCT (N = 104) and Day +100 (N = 83) post-AHCT. Median follow-up post-AHCT was 61 months (range: 8-120 months). Compared to patients mobilized with filgrastim and plerixafor, patients mobilized with filgrastim alone had a higher proportion of CD4+ naïve (p = 0.006) and CD8+ central memory T-cells (p = 0.006) pre-AHCT. For patients transplanted in complete remission (CR), a higher proportion of CD8+ effector memory T-cells pre-AHCT was associated with worse progression-free survival (PFS; p < 0.01) and overall survival (OS; p < 0.01). A higher ratio of CD8:CD4+ central memory T-cells pre-AHCT was associated with worse PFS (p < 0.0001) and OS (p = 0.0034). This same ratio measured post-AHCT among patients in CR on Day +100 was associated with worse and OS (p = 0.008) but not PFS (p = not significant). These immune subsets are complementary biomarkers which identify patients transplanted in CR who have poor survival prognoses and may warrant further clinical interventions.

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol., (© 2019 John Wiley & Sons, Inc.)

Background: An interlaboratory study was performed to compare the performance of the 3 mL TransFix®/EDTA Vacuum Blood Collection Tube (TVT), with the 5 mL Cyto-Chex® BCT tube (BCT). Both devices are intended for collection and storage of whole blood specimens for immunophenotyping of leukocytes by flow cytometry for up to 14 days., Methods: One site in the United States and two in the United Kingdom tested samples from 10 HIV positive patients and four healthy subjects for a total of 42 samples. From each subject, three blood samples were collected: a BD 4 mL K 3 EDTA Vacutainer (Vacutainer), a TVT, and a BCT. At all sites, samples were analyzed on a BD FACS Canto II flow cytometer for a full lymphocyte subset count within 6 h of collection (all devices) and on Day 11 and Day 15 (TVT and BCT only). Data obtained from the Vacutainer were used as the control data set with which TVT and BCT data were statistically compared., Results and Conclusions: Statistical concordance was demonstrated for both devices in relation to cell absolute count recovery. For cell marker signal, both devices exhibited a significant decrease in mean fluorescence intensity (MFI) for the detection of lymphocyte subsets and their target markers. There was a marked increase in autofluorescence observed for BCT stabilized lymphocytes whereas values for the TVTs were comparable to the control. There were eight instances of statistical equivalence between the level of antibody autofluorescence observed in the control tube and the TVT across both patient cohorts, versus two for the BCT. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Objective: In the randomised scleroderma: Cyclophosphamide Or Transplantation (SCOT trial) (NCT00114530), myeloablation, followed by haematopoietic stem cell transplantation (HSCT), led to improved clinical outcomes compared with monthly cyclophosphamide (CYC) treatment in systemic sclerosis (SSc). Herein, the study aimed to determine global molecular changes at the whole blood transcript and serum protein levels ensuing from HSCT in comparison to intravenous monthly CYC in 62 participants enrolled in the SCOT study., Methods: Global transcript studies were performed at pretreatment baseline, 8 months and 26 months postrandomisation using Illumina HT-12 arrays. Levels of 102 proteins were measured in the concomitantly collected serum samples., Results: At the baseline visit, interferon (IFN) and neutrophil transcript modules were upregulated and the cytotoxic/NK module was downregulated in SSc compared with unaffected controls. A paired comparison of the 26 months to the baseline samples revealed a significant decrease of the IFN and neutrophil modules and an increase in the cytotoxic/NK module in the HSCT arm while there was no significant change in the CYC control arm. Also, a composite score of correlating serum proteins with IFN and neutrophil transcript modules, as well as a multilevel analysis showed significant changes in SSc molecular signatures after HSCT while similar changes were not observed in the CYC arm. Lastly, a decline in the IFN and neutrophil modules was associated with an improvement in pulmonary forced vital capacity and an increase in the cytotoxic/NK module correlated with improvement in skin score., Conclusion: HSCT contrary to conventional treatment leads to a significant 'correction' in disease-related molecular signatures., Competing Interests: Competing interests: SA reports grants from National Institute of Health, grants from Karen Brown Scleroderma Foundation, grants from Department of Defense, during the conduct of the study; grants and personal fees from Boehringer Ingelheim, personal fees from Integrity Continuing Education, personal fees from Medscape, outside the submitted work. MDM reports personal fees from Boehringer Ingelheim, during the conduct of the study; personal fees from Galapagos NV (Pharma), personal fees from Medtelligence, personal fees from Actelion Pharma, personal fees from Astellas, personal fees from Mitsubishi-Tanabe, grants from Bayer, grants from Reata, grants from Sanofi, grants from Corbus, grants from Eicos/ Sciences, outside the submitted work. KMS reports grants from National Institutes of Health, NIAID, during the conduct of the study; personal fees from GlaxoSmith/Kline, grants from Astra Zeneca, grants from Takeda Millennium, personal fees from Magenta, personal fees from Aerotek, personal fees from Kiadis Pharma, from Genentech Roche, outside the submitted work., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent interest in high sensitivity multiple myeloma (MM) measurable residual disease (MRD) testing is a direct consequence of the high-quality responses achieved using novel therapeutic agents and better treatment strategies. Traditional diagnostic measures such as immunohistochemistry and morphology have detection sensitivities of only 10 -2 - 10 -3 , which do not reliably predict progression free survival (PFS) or overall survival (OS) after these treatments. Contemporary monitoring of MM MRD has switched to more sensitive platforms such as quantitative allele-specific oligonucleotide polymerase chain reaction (ASO-qPCR), next-generation sequencing (NGS), and multiparametric flow cytometry (MFC). Though both ASO-qPCR and NGS have excellent detection sensitivities (10 -5 - 10 -6 ), both technologies have lower applicability when compared to MFC. Conventional MFC can easily reach a detection sensitivity of 10 -4 and when optimized can achieve a sensitivity of 10 -5 - 10 -6 . Current consensus guidelines require a minimum of 2 million and recommend 5 million events be acquired to reach a minimum sensitivity of 10 -5 . As conventional immunophenotyping protocols are unable to attain these numbers, alternative MFC staining procedures are required. This manuscript describes two high-sensitivity MFC approaches that can be used for MM MRD testing., Competing Interests: Disclosure Statement: The authors have no commercial or financial conflicts of interest to disclose.

Background: Minimal/measurable residual disease (MRD) testing by flow cytometry (FC) has been proposed as a potential surrogate clinical endpoint in plasma cell myeloma (PCM) clinical trials. As a result, effort has gone into standardizing this approach on PCM patients., Aims: To assess inter-laboratory variation in FC MRD testing of PCM patients in an independent inter-laboratory study., Methods: A dilution series of five stabilized bone marrow samples manufactured to contain 0%, 0.1%, 0.01%, 0.001%, and 0.0001% neoplastic plasma cells (PCs) were tested blind, using standardized FC PCM MRD assays by 10 international laboratories., Results: Laboratories' assays broadly adhered to the consensus guidelines; however, some deviations were identified in panel design, fluorochrome conjugates, and lysis reagents. Despite this, all laboratories that returned results detected neoplastic PCs down to 0.001% of leucocytes. 6/8 laboratories detected neoplastic PCs at a level of 0.0001%. Quantitative data returned by laboratories showed good consensus and linearity with increasing variation at lower levels of MRD. However, examples of analytical and post analytical error were identified., Summary/conclusion: Broadly standardized PCM MRD FC assays can attain the lower limit of detection (LOD) required by current and future clinical trials, an important consideration in establishing PCM MRD testing as a surrogate clinical marker in PCM clinical trials. Laboratories' assays showed good linearity, encouraging the prediction of survival based on log reduction in neoplastic PC populations in future clinical trials. However, the deviations from consensus guidelines identified in this study would suggest that if PCM MRD assays are further standardized interlaboratory variation could be reduced. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Disease relapse and toxicity are the shortcomings of reduced-intensity conditioning (RIC) for allogeneic hematopoietic cell transplantation (alloHCT). We hypothesized that adding total body irradiation (TBI) to and decreasing melphalan (Mel) from a base RIC regimen of fludarabine (Flu) and Mel would increase cytoreduction and improve disease control while decreasing toxicity. We performed a phase II trial of Flu 160 mg/m 2 , Mel 50 mg/m 2 , and TBI 400 cGy (FluMelTBI-50, n = 61), followed by a second phase II trial of Flu 160 mg/m 2 , Mel 75 mg/m 2 , and TBI 400cGy (FluMelTBI-75, n = 94) as RIC for alloHCT. Outcomes were compared with a contemporaneous cohort of 162 patients who received Flu 125 mg/m 2 and Mel 140 mg/m 2 . Eligibility criteria were equivalent for all 3 regimens. All patients were ineligible for myeloablative/intensive conditioning. The median (range) follow-up for all patients was 51 (15 to 103) months. Day 100 donor lymphoid chimerism and transplant-related mortality, neutrophil and platelet engraftment, acute and chronic graft versus host disease incidence, overall survival (OS), and progression-free survival (PFS) were equivalent between FluMel, FluMelTBI-50, and FluMelTBI-75. Stomatitis wasdecreased for FluMelTBI versus FluMel (P < .01). PFS for patients not in complete remission on alloHCT was improved for FluMelTBI-75 versus FluMel (P = .03). On multivariate analysis, OS (P = .05) and PFS (P = .05) were significantly improved for FluMelTBI-75 versus FluMel. FluMelTBI-75 is better tolerated than FluMel, with improved survival and disease control., (Copyright © 2018 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Aim: To investigate infused hematopoietic cell doses and their interaction with conditioning regimen intensity +/- total body irradiation (TBI) on outcomes after peripheral blood hematopoietic cell transplant (PBHCT)., Methods: Our retrospective cohort included 247 patients receiving a first, T-replete, human leukocyte antigen-matched allogeneic PBHCT and treated between 2001 and 2012. Correlations were calculated using the Pearson product-moment correlation coefficient. Overall survival and progression free survival curves were generated using the Kaplan-Meier method and compared using the log-rank test., Results: Neutrophil engraftment was significantly faster after reduced intensity TBI based conditioning [reduced intensity conditioning (RIC) + TBI] and > 4 × 10 6 CD34+ cells/kg infused. A higher total nucleated cell dose led to a higher incidence of grade II-IV acute graft-versus-host disease in the myeloablative + TBI regimen group ( P = 0.03), but no significant difference in grade III-IV graft-versus-host disease. A higher total nucleated cell dose was also associated with increased incidence of moderate/severe chronic graft-versus-host disease, regardless of conditioning regimen. Overall and progression-free survival were significantly better in patients with a RIC + TBI regimen and total nucleated cell dose > 8 × 10 8 /kg (3 years, overall survival: 70% vs 38%, P = 0.02, 3 years, progression free survival: 64% vs 38%, P = 0.02)., Conclusion: TBI and conditioning intensity may alter the relationship between infused cell doses and outcomes after PBHCT. Immune cell subsets may predict improved survival after unmanipulated PBHCT., Competing Interests: Conflict-of-interest statement: All authors declare no conflicts-of-interest related to this article.

Background: Circulating hematopoietic progenitors (HPCs) are involved in inflammatory diseases such as atherosclerosis, the immune response to cancer, and disorders of the hematopoietic system. HPC characterization by flow cytometry typically utilizes CD34 in combination with other cell surface markers to identify cell populations that give rise to specific hematopoietic lineages. CD133, also known as prominin-1, is a cell surface protein found in HPCs that has a similar but not interchangeable expression pattern with CD34 for characterization of HPC populations. Our goal was to determine the distribution of CD133 expression within HPC sub-types amongst circulating CD34+ cells., Methods: The quantity of different HPC populations within the CD34+ CD133+ and CD34+ CD133- fractions of venous blood obtained from healthy human subjects was measured using multicolor flow cytometry., Results: The majority of circulating CD34+ cells is CD133-. CD133+ CD34+ cells express low levels of CD38, contain cell populations bearing cell surface markers of hematopoietic stem cells, multipotent progenitors, and multilymphoid progenitors, and are largely devoid of CD38 expressing lineage specified progenitors. These findings clarify the composition of circulating CD133+ CD34+ cell types in adult human subjects. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

The Blood and Marrow Transplant Clinical Trials Network Myeloma Intergroup Workshop on Minimal Residual Disease and Immune Profiling was convened on December 1, 2016 at the American Society of Hematology meeting to discuss the emerging data and technologies for minimal residual disease assessment and immune profiling in myeloma. Particular emphasis was placed on developing strategies to incorporate these techniques into clinical trial design. This document reviews the literature, summarizes the topics discussed in the workshop, and provides recommendations for integration of these techniques into future clinical trial design., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Nano-sized membrane-encapsulated extracellular vesicles isolated from the ascites fluids of ovarian cancer patients are identified as exosomes based on their biophysical and compositional characteristics. We report here that T cells pulsed with these tumor-associated exosomes during TCR-dependent activation inhibit various activation endpoints including translocation of NFκB and NFAT into the nucleus, upregulation of CD69 and CD107a, production of cytokines, and cell proliferation. In addition, the activation of virus-specific CD8 + T cells that are stimulated with the cognate viral peptides presented in the context of class I MHC is also suppressed by the exosomes. The inhibition occurs without loss of cell viability and coincidentally with the binding and internalization of these exosomes. This exosome-mediated inhibition of T cells was transient and reversible: T cells exposed to exosomes can be reactivated once exosomes are removed. We conclude that tumor-associated exosomes are immunosuppressive and represent a therapeutic target, blockade of which would enhance the antitumor response of quiescent tumor-associated T cells and prevent the functional arrest of adoptively transferred tumor-specific T cells or chimeric antigen receptor T cells. Cancer Immunol Res; 6(2); 236-47. ©2018 AACR ., (©2018 American Association for Cancer Research.)

In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells.The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: (a) Assessment of the dye's spectral profile on the laboratory's flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems; (b) Evaluating the effect of labeling on cell growth rate;, ((c) Testing the fidelity with which dye dilution reports cell division; (d) Determining the maximum number of generations to be included when using dye dilution profiles to estimate fold population expansion or frequency of responder cells; and (e) Verifying that relevant cell functions (e.g., effector activity) remain unaltered by tracking dye labeling.)

Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.

Since publication in 2010 of the International Clinical Cytometry Society (ICCS) Consensus Guidelines for detection of Paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometery, a great deal of work has been performed to develop, optimize, and validate a number of high-sensitivity assays to detect PNH phenotypes in both red blood cells (RBCs) and white blood cells (WBCs, neutrophils, and monocytes). This section (Part 2) of the updated ICCS PNH Consensus Guidelines will focus on specific instrument setup for these PNH assays, the identification and proper testing of appropriate antibody conjugates and combinations therof, and basic assay design. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Individuals with Down syndrome (DS) frequently have hematopoietic abnormalities, including transient myeloproliferative disorder and acute megakaryoblastic leukemia which are often accompanied by acquired GATA1 mutations that produce a truncated protein, GATA1s. The mouse has been used for modeling DS based on the syntenic conservation between human chromosome 21 (Hsa21) and three regions in the mouse genome located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. To assess the impact of the dosage increase of Hsa21 gene orthologs on the hematopoietic system, we characterized the related phenotype in the Dp(10)1Yey/+;Dp(16)1Yey/+;Dp(17)1Yey/+ model which carries duplications spanning the entire Hsa21 orthologous regions on Mmu10, Mmu16 and Mmu17, and the Dp(10)1Yey /+; Dp(16)1Yey /+; Dp(17)1Yey/+ ; Gata1 Yeym2 model which carries a Gata1s mutation we engineered. Both models exhibited anemia, macrocytosis, and myeloproliferative disorder. Similar to human DS, the megakaryocyte-erythrocyte progenitors (MEPs) and granulocyte-monocyte progenitors (GMPs) were significantly increased and reduced, respectively, in both models. The subsequent identification of all the aforementioned phenotypes in the Dp(16)1Yey/ + model suggests that the causative dosage sensitive gene(s) are in the Hsa21 orthologous region on Mmu16. Therefore, we reveal here for the first time that the human trisomy 21-associated major segmental chromosomal alterations in mice can lead to expanded MEP and reduced GMP populations, mimicking the dynamics of these myeloid progenitors in DS. These models will provide the critical systems for unraveling the molecular and cellular mechanism of DS-associated myeloproliferative disorder, and particularly for determining how human trisomy 21 leads to expansion of MEPs as well as how such an alteration leads to myeloproliferative disorder., Competing Interests: CONFLICTS OF INTEREST None of the authors has any conflicts of interest.

Plasma cell dyscrasia (PCD) is a heterogeneous disease that has seen a tremendous change in outcomes due to improved therapies. Over the past few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high-sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting, sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Multiple therapeutic options exist for multiple myeloma (MM), including autologous hematopoietic stem cell transplantation (AHSCT). Measurement of minimal residual disease (MRD) and immune reconstitution is rapidly becoming an integral part of the care of MM patients. We investigated comprehensive immune profiling (IP) associated with progression-free survival (PFS) and overall survival (OS). From August 2007 to January 2014, 101 consecutive MM patients underwent peripheral blood IP and marrow MRD testing before and approximately 100 days after AHSCT. Higher pre-AHSCT CD19 + B-cell counts correlated with improved 2-year PFS (83% [highest quartile] vs 53% [lowest quartile]; P = .01) and OS (93% [highest quartile] vs 63% [lowest quartile]; P = .0003). This effect was seen primarily in patients with MRD-positive marrow tests. Higher γδ T-cell counts post-AHSCT correlated with improved 2-year PFS (65% [highest quartile] vs 45% [lowest quartile]; P = .02) and OS (89% [highest quartile] vs 65% [lowest quartile]; P = .01). Higher CD4 + central memory (CM) cell counts post-AHSCT were associated with improved 2-year OS (95% [upper quartile] vs 47% [lowest quartile]; P = .0003) but not PFS. The higher γδ T-cell and CD4 + CM-cell count associations were primarily observed in MRD-negative patients post-AHSCT and in patients not receiving maintenance therapy. This proof-of-concept study demonstrates that IP before and after AHSCT can be of complementary prognostic value for depth of response. Maintenance therapy seems to overcome negative IP. IP and MRD should be measured in clinical trials of maintenance therapy with novel agents post-AHSCT for MM to confirm their utility for prognosis and management., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.