European flow cytometry quality assurance guidelines for the diagnosis of primary immune deficiencies and assessment of immune reconstitution following B cell depletion therapies and transplantation.

Over the last 15 years activity of diagnostic flow cytometry services have evolved from monitoring of CD4 T cell subsets in HIV-1 infection to screening for primary and secondary immune deficiencies syndromes and assessment of immune constitution following B cell depleting therapy and transplantation. Changes in laboratory activity in high income countries have been driven by initiation of anti-retroviral therapy (ART) in HIV-1 regardless of CD4 T cell counts, increasing recognition of primary immune deficiency syndromes and the wider application of B cell depleting therapy and transplantation in clinical practice. Laboratories should use their experience in standardization and quality assurance of CD4 T cell counting in HIV-1 infection to provide immune monitoring services to patients with primary and secondary immune deficiencies. Assessment of immune reconstitution post B cell depleting agents and transplantation can also draw on the expertise acquired by flow cytometry laboratories for detection of CD34 stem cell and assessment of MRD in hematological malignancies. This guideline provides recommendations for clinical laboratories on providing flow cytometry services in screening for immune deficiencies and its emerging role immune reconstitution after B cell targeting therapies and transplantation.

Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet.

BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

The flow cytometry myeloid progenitor count: A reproducible parameter for diagnosis and prognosis of myelodysplastic syndromes.

BACKGROUND: The bone marrow blast count is central to the diagnosis and monitoring of myelodysplastic syndromes (MDS). It is an independent risk factor for worse prognosis whether based on the morphology blast count or the flow cytometry (FC) myeloid progenitor (MyP) count. It is a principal population in FC MDS analysis also because once defined; it provides significant contributions to the overall FC MDS score. METHODS: We elected to investigate inter-analyst agreement for the most fundamental parameter of the FC MDS diagnostic score: the MyP count. A common gating strategy was agreed and used by seven cytometrists for blind analysis of 34 routine bone marrows sent for MDS work-up. Additionally, we compared the results with a computational approach. RESULTS: Concordance was excellent: Intraclass correlation was 0.993 whether measuring %MyP of total cells or CD45+ cells, and no significant difference was observed between files from different centers or for samples with abnormal MyP phenotypes. Computational and manual results were similar. Applying the common strategy to individual laboratories' control cohorts produced similar MyP reference ranges across centers. CONCLUSION: The FC MyP count offers a reliable diagnostic and prognostic measurement in MDS. The use of manual and computational approaches side by side may allow for optimizing both strategies. Considering its known prognostic power, the MyP count could be considered a useful and reliable addition to existing prognostic scoring systems.

A series of case studies illustrating the role of flow cytometry in the diagnostic work-up of myelodysplastic syndromes.

Current guidelines recommend flow cytometric analysis as part of the diagnostic assessment of patients with cytopenia suspected for myelodysplastic syndrome. Herein we describe the complete work-up of six cases using multimodal integrated diagnostics. Flow cytometry assessments are illustrated by plots from conventional and more recent analysis tools. The cases demonstrate the added value of flow cytometry in case of hypocellular, poor quality, or ambiguous bone marrow cytomorphology. Moreover, they demonstrate how immunophenotyping results support clinical decision-making in inconclusive and clinically 'difficult' cases.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

BACKGROUND: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML). METHODS: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML). RESULTS: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%). CONCLUSION: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter.

Allogeneic Anti-BCMA CAR T Cells Are Superior to Multiple Myeloma-derived CAR T Cells in Preclinical Studies and May Be Combined with Gamma Secretase Inhibitors.

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

Clinical and prognostic significance of small paroxysmal nocturnal hemoglobinuria clones in myelodysplastic syndrome and aplastic anemia.

In this large single-centre study, we report high prevalence (25%) of, small (<10%) and very small (<1%), paroxysmal nocturnal hemoglobinuria (PNH) clones by high-sensitive cytometry among 3085 patients tested. Given PNH association with bone marrow failures, we analyzed 869 myelodysplastic syndromes (MDS) and 531 aplastic anemia (AA) within the cohort. PNH clones were more frequent and larger in AA vs. MDS (p = 0.04). PNH clone, irrespective of size, was a good predictor of response to immunosuppressive therapy (IST) and to stem cell transplant (HSCT) (in MDS: 84% if PNH+ vs. 44.7% if PNH-, p = 0.01 for IST, and 71% if PNH+ vs. 56.6% if PNH- for HSCT; in AA: 78 vs. 50% for IST, p < 0.0001, and 97 vs. 77%, p = 0.01 for HSCT). PNH positivity had a favorable impact on disease progression (0.6% vs. 4.9% IPSS-progression in MDS, p < 0.005; and 2.1 vs. 6.9% progression to MDS in AA, p = 0.01), leukemic evolution (6.8 vs. 12.7%, p = 0.01 in MDS), and overall survival [73% (95% CI 68-77) vs. 51% (48-54), p < 0.0001], with a relative HR for mortality of 2.37 (95% CI 1.8-3.1; p < 0.0001) in PNH negative cases, both in univariate and multivariable analysis. Our data suggest systematic PNH testing in AA/MDS, as it might allow better prediction/prognostication and consequent clinical/laboratory follow-up timing.

Monitoring Allogeneic CAR-T Cells Using Flow Cytometry.

Chimeric antigen receptor T cell (CAR-T) therapies have now entered mainstream clinical practice with two approved autologous CAR-T products targeting CD19 and numerous other products in early and late phase clinical trials. This has led to a demand for highly sensitive, specific, and easily reproducible methods to monitor CAR-T cells in patients. Here we describe a flow cytometry based protocol for detection of allogeneic CAR-T cells and for monitoring their phenotype and numbers in blood and bone marrow of patients following CAR-T treatment.

Aminopeptidase inhibition as a targeted treatment strategy in myeloma.

Myeloma cells are highly dependent on the unfolded protein response to assemble folded immunoglobulins correctly. Therefore, targeting protein handling within a myeloma cell by inhibiting the aminopeptidase enzyme system, which catalyses the hydrolysis of amino acids from the proteins NH2 terminus, represents a therapeutic approach. CHR-2797, a novel aminopeptidase inhibitor, is able to inhibit proliferation and induce growth arrest and apoptosis in myeloma cells, including cells resistant to conventional chemotherapeutics. It causes minimal inhibition of bone marrow stromal cell (BMSC) proliferation but is able to overcome the microenvironmental protective effects, inhibiting the proliferation of myeloma cells bound to BMSCs and the increase in vascular endothelial growth factor levels seen when myeloma cells and BMSCs are bound together. Additive and synergistic effects are seen with bortezomib, melphalan, and dexamethasone. Apoptosis occurs via both caspase-dependent and non-caspase-dependent pathways with an increase in Noxa, cleavage of Mcl-1, and activation of the unfolded protein response. Autophagy is also seen. CHR-2797 causes an up-regulation of genes involved in the proteasome/ubiquitin pathway, as well as aminopeptidases, and amino acid deprivation response genes. In conclusion, inhibiting protein turnover using the aminopeptidase inhibitor CHR-2797 results in myeloma cell apoptosis and represents a novel therapeutic approach that warrants further investigation in the clinical setting.

Heat shock protein inhibition is associated with activation of the unfolded protein response pathway in myeloma plasma cells.

Plasma cells producing high levels of paraprotein are dependent on the unfolded protein response (UPR) and chaperone proteins to ensure correct protein folding and cell survival. We hypothesized that disrupting client-chaperone interactions using heat shock protein 90 (Hsp90) inhibitors would result in an inability to handle immunoglobulin production with the induction of the UPR and myeloma cell death. To study this, myeloma cells were treated with Hsp90 inhibitors as well as known endoplasmic reticulum stress inducers and proteasome inhibitors. Treatment with thapsigargin and tunicamycin led to the activation of all 3 branches of the UPR, with early splicing of XBP1 indicative of IRE1 activation, upregulation of CHOP consistent with ER resident kinase (PERK) activation, and activating transcription factor 6 (ATF6) splicing. 17-AAG and radicicol also induced splicing of XBP1, with the induction of CHOP and activation of ATF6, whereas bortezomib resulted in the induction of CHOP and activation of ATF6 with minimal effects on XBP1. After treatment with all drugs, expression levels of the molecular chaperones BiP and GRP94 were increased. All drugs inhibited proliferation and induced cell death with activation of JNK and caspase cleavage. In conclusion, Hsp90 inhibitors induce myeloma cell death at least in part via endoplasmic reticulum stress and the UPR death pathway.