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Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is an uncommon mature T-cell neoplasm occurring in patients with textured breast implants, typically after 7-10 years of exposure. Although cytopathologic or histopathologic assessment is considered the gold standard diagnostic method for BIA-ALCL, flow cytometry (FC)-based immunophenotyping is recommended as an adjunct test. However, the diagnostic efficacy of FC is not well reported. We reviewed 290 FC tests from breast implant pericapsular fluid and capsule tissue from 182 patients, including 16 patients with BIA-ALCL over a 6-year period, calculating diagnostic rates and test efficacy. FC showed an overall sensitivity of 75.9%, specificity of 100%, and negative and positive predictive values of 95.4% and 100%, respectively. Blinded expert review of false-negative cases identified diagnostic pitfalls, improving sensitivity to 96.6%. Fluid samples had better rates of adequate samples for FC testing compared with tissue samples. Paired with FC testing of operating room (OR)-acquired fluid samples, capsulectomy FC specimens added no diagnostic value in patients with concurrent fluid samples; no cases had positive capsule FC with negative fluid FC. Fluid samples are adequate for FC testing more often than tissue. Capsule tissue FC specimens do not improve FC efficacy when paired with OR-acquired fluid FC samples and are often inadequate samples. FC is 100% specific for BIA-ALCL and can serve as a confirmatory test but should not be the sole diagnostic method. Awareness of sample-specific diagnostic pitfalls greatly improves the sensitivity of BIA-ALCL testing by FC., (© 2024 International Clinical Cytometry Society.)

Background: Measurement of minimal/measurable residual disease (MRD) in B-lymphoblastic leukemia/lymphoma (B-ALL) has become a routine clinical evaluation tool and remains the strongest predictor of treatment outcome. In recent years, new targeted anti-CD19 and anti-CD22 antibody-based and cellular therapies have revolutionized the treatment of the high-risk B-ALL. The new treatments raise challenges for diagnostic flow cytometry, which relies on the presence of specific surface antigens to identify the population of interest. So far, reported flow cytometry-based assays are developed to either achieve a deeper MRD level or to accommodate the loss of surface antigens post-target therapies, but not both., Methods: We developed a single tube flow cytometry assay (14-color-16-parameters). The method was validated using 94 clinical samples as well as spike-in and replicate experiments., Results: The assay was well suited for monitoring response to targeted therapies and reached a sensitivity below 10 -5 with acceptable precision (coefficient of variation < 20%), accuracy, and interobserver variability (κ = 1)., Conclusions: The assay allows for sensitive disease detection of B-ALL MRD independent of CD19 and CD22 expression and allows uniform analysis of samples regardless of anti-CD19 and CD22 therapy., (© 2023 International Clinical Cytometry Society.)

Minimal/measurable residual disease (MRD) is the most important independent prognostic factor for patients with B-lymphoblastic leukemia (B-LL). MRD post therapy has been incorporated into risk stratification and clinical management, resulting in substantially improved outcomes in pediatric and adult patients. Currently, MRD in B-ALL is most commonly assessed by multiparametric flow cytometry and molecular (polymerase chain reaction or high-throughput sequencing based) methods. The detection of MRD by flow cytometry in B-ALL often begins with B cell antigen-based gating strategies. Over the past several years, targeted immunotherapy directed against B cell markers has been introduced in patients with relapsed or refractory B-ALL and has demonstrated encouraging results. However, targeted therapies have significant impact on the immunophenotype of leukemic blasts, in particular, downregulation or loss of targeted antigens on blasts and normal B cell precursors, posing challenges for MRD detection using standard gating strategies. Novel flow cytometric approaches, using alternative strategies for population identification, sometimes including alternative gating reagents, have been developed and implemented to monitor MRD in the setting of post targeted therapy., (© 2023 International Clinical Cytometry Society.)

Background: Lineage infidelity is characteristic of mixed phenotype acute leukemia and is also seen in blast phase of chronic myeloid leukemia (CML), myeloid/lymphoid neoplasia with eosinophilia and gene rearrangements, and subtypes of acute myeloid leukemia. Driver genetic events often occur in multipotent progenitor cells in myeloid neoplasms, suggesting that multilineage output may be more common than appreciated. This phenomenon is not well studied in myelodysplastic syndrome (MDS) and non-CML myeloproliferative neoplasms (MPN)., Methods: We systematically evaluated phenotypic lineage infidelity by reviewing bone marrow pathology and flow cytometry (FC) studies of 1262 consecutive patients with a diagnosis of MDS and/or non-CML MPN. We assessed B- and T-cells in these patients by FC. When abnormal B-lymphoblast (ABLB) populations were detected, we additionally evaluated immature B-cells using a high sensitivity FC assay for B-lymphoblastic leukemia/lymphoma (B-ALL)., Results: We identified 9 patients (7 MDS, 7/713, 1%; 2 non-CML MPN, 2/312, 0.6%; 0 in MDS/MPN) with low-level ABLB populations (0.012%-3.6% of WBCs in marrow) with abnormal immunophenotypes. Genetic studies on flow sorted cell populations confirmed that some ABLB populations were clonally related to myeloid blasts (4/6, 67%). On follow-up, ABLB populations in 8/9 patients remained stable or disappeared. Only 1 case progressed to B-ALL., Conclusions: These findings demonstrate that phenotypically detectable abnormal immature B lineage output occurs in MDS and non-CML MPN, albeit rarely. While presence of ABLB does not necessarily reflect blast crisis, the underlying disease biology of our findings may ultimately be relevant to patient management and warrants further investigation., (© 2021 International Clinical Cytometry Society.)

Flow cytometry has been indispensable in diagnosing B cell lymphoma and plasma cell neoplasms. The advances in novel multicolor flow cytometry have also made this technology a robust tool for monitoring minimal/measurable residual disease in chronic lymphocytic leukemia and multiple myeloma. However, challenges using conventional gating strategies to isolate neoplastic B or plasma cells are emerging due to the rapidly increasing number of antibody therapeutics targeting single or multiple classic B/plasma cell-lineage markers, such as CD19, CD20, and CD22 in B cells and CD38 in plasma cells. This review is the first of a two-part series that summarizes the most current targeted therapies used in B and plasma cell neoplasms and proposes detailed alternative approaches to overcome post-targeted therapy analysis challenges by flow cytometry. The second review in this series (Chen et al.) focuses on challenges encountered in the use of targeted therapy in precursor B cell neoplasms., (© 2022 International Clinical Cytometry Society.)

Background: Accurate Sezary cell detection in peripheral blood of mycosis fungoides/Sezary syndrome (MF/SS) patients by flow cytometry can be difficult due to overlapping immunophenotypes with normal T cells using standard markers. We assessed the utility of programmed death-1 (PD-1/CD279), a transmembrane protein expressed in some hematopoietic cells, for identification and quantitation of circulating Sezary cells among established markers using flow cytometry., Methods: 50 MF/SS and 20 control blood samples were immunophenotyped by flow cytometry. Principal component analysis (PCA) assessed contributions of antigens to separation of abnormal from normal T cell populations. PD-1 was assessed over time in blood and bone marrow of available MF/SS cases., Results: Normal CD4+ T cells showed dim/intermediate to absent PD-1 expression. PD-1 in Sezary cells was informatively brighter (≥1/3 log) than internal normal CD4+ T cells in 39/50 (78%) cases. By PCA, PD-1 ranked 3rd behind CD7 and CD26 in population separation as a whole; it ranked in the top 3 markers in 32/50 (64%) cases and 1st in 4/50 (8%) cases when individual abnormal populations were compared to total normal CD4+ T cells. PD-1 clearly separated Sezary from normal CD4+ T cells in 15/26 (58%, 30% of total) cases with few and subtle alterations of pan-T cell antigens/CD26 and was critical in 6 (12% of total), without which identification and quantification were significantly affected or nearly impossible. PD-1 remained informative in blood/bone marrow over time in most patients., Conclusions: PD-1 significantly contributes to accurate flow cytometric Sezary cell assessment in a routine Sezary panel., (© 2022 International Clinical Cytometry Society.)

Introduction: The diagnosis of CLL/SLL relies on flow cytometric immunophenotyping. Increasing emphasis is being placed on precise detection of the minimal residual disease. Following antigen recommendations of ERIC and ESCCA's Harmonization Project, we validated a 14-color assay for the characterization CD5+ lymphoproliferative neoplasms and CLL MRD with a sensitivity of at least 10 -4 ., Methods: The assay was designed based on ERIC/ESCCA recommended antigens with the addition of CD40 for alternate gating when CD19 expression is reduced. Lower limit of quantitation/lower limit of detection, assay procedural precision, linearity, and limit of blank were established. Then, 52 CD5+ B-cell lymphoproliferative neoplasms (41 CLL/11 non-CLL) and 29 normal samples were used for parallel evaluation. Automated cluster identification and quantitation of CLL clones in MRD setting was performed using Barned-Hutt SNE. Separation analysis between CLL and non-CLL phenotypes was performed by PCA and bh-SNE., Results: Separation ratios for each antigen exceeded ERIC/ESCCA guidelines. Precision was <20% at LLOQ (0.01%). The limit of blank was <10/500,000 cells. Concordance between the 14-color and legacy assay (Deming regression y = 1.01x, r 2  = .99) was seen. All 20 samples with MRD levels 0.5%-0.006% (median 0.04%) showed an abnormal cell cluster by bh-SNE, with concordant results between manual and automated quantitation (y = x, r 2  = 1). CLL cases clustered together and away from mantle cell lymphoma by bh-SNE and PCA with outlier atypical phenotype CLL cases posing diagnostic challenges by both manual and automated analysis., Conclusion: The 14-color CD5+ LPD assay provides a robust standardization platform for MRD and disease characterization using both manual and automated analysis., (© 2020 International Clinical Cytometry Society.)

Chromosome translocations involving the RUNX1 gene at 21q22 are recurring abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), that is, t(8;21) and t(3;21) and in B-cell acute lymphoblastic leukemia with t(12;21). These translocations result in the fusion of RUNX1 with RUNX1T1, MECOM, and ETV6, respectively, and are implicated in leukemogenesis. Here we describe 10 rare RUNX1 fusion gene partners, including six novel fusions, in myeloid neoplasia. Comprehensive molecular testing revealed the partner genes and features of these fusions in all the tested patients, and detected various recurring myeloid related gene mutations in eight patients. In two patients, RUNX1 mutations were identified. Most of these fusions were detected in patients with high-grade MDS and AML with a relatively short survival. Integration of conventional chromosome analysis, FISH testing and molecular genetic studies allow a comprehensive characterization of these rare RUNX1 fusions. Our study may help define myeloid neoplasms with rare and novel RUNX1 translocations and support appropriate patient management., (© 2020 Wiley Periodicals LLC.)

Background: Induction chemotherapy for acute leukemia often leads to antigenic shifts in residual abnormal blast populations. Studies in precursor B cell ALL (B-ALL) have demonstrated that chemotherapy commonly results in the loss of antigens associated with immaturity, limiting their utility for minimal residual disease (MRD) detection. Little information is available about the stability of these antigens in precursor T cell ALL (T-ALL) though it is presumed that CD99 and terminal deoxynucleotidyl transferase (TdT) are highly informative based on limited studies., Methods: In a longitudinal investigation, we explored patterns of lineage specific and immaturity-associated antigens in T-ALL in a large cohort of patients treated under the multicenter Children's Oncology Group (COG) protocol. All samples were analyzed using multicolor flow cytometry in a standardized fashion at a single institution., Results: We report that markers of immaturity particularly, TdT and CD99, dramatically decline on leukemic blasts during therapy. CD34 and CD10 expression is confined to a minority of pretreatment samples and is also not stable. In contrast, lineage-associated markers including CD2, CD3, CD4, CD5, CD7, and CD8 failed to show significant trends., Conclusions: Our study strongly argues for expansion of immunophenotyping panels for T-ALL MRD to decrease reliance on immature antigens. This study represents the first demonstration of consistent immunophenotypic shifts in T-ALL., (2010 Clinical Cytometry Society.)