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Measurable residual disease (MRD) surveillance in acute myeloid leukemia (AML) may identify patients destined for relapse and thus provide the option of pre-emptive therapy to improve their outcome. Whilst flow cytometric MRD (Flow-MRD) can be applied to high-risk AML/ myelodysplasia patients, its diagnostic performance for detecting impending relapse is unknown. We evaluated this in a cohort comprising 136 true positives (bone marrows preceding relapse by a median of 2.45 months) and 155 true negatives (bone marrows during sustained remission). At an optimal Flow-MRD threshold of 0.040%, clinical sensitivity and specificity for relapse was 74% and 87% respectively (51% and 98% for Flow-MRD ≥ 0.1%) by 'different-from-normal' analysis. Median relapse kinetics were 0.78 log 10 /month but significantly higher at 0.92 log 10 /month for FLT3-mutated AML. Computational (unsupervised) Flow-MRD (C-Flow-MRD) generated optimal MRD thresholds of 0.036% and 0.082% with equivalent clinical sensitivity to standard analysis. C-Flow-MRD-identified aberrancies in HLADRlow or CD34+CD38low (LSC-type) subpopulations contributed the greatest clinical accuracy (56% sensitivity, 90% specificity) and notably, by longitudinal profiling expanded rapidly within blasts in > 40% of 86 paired MRD and relapse samples. In conclusion, flow MRD surveillance can detect MRD relapse in high risk AML and its evaluation may be enhanced by computational analysis., (© 2024. The Author(s).)

Multiparameter flow cytometry is widely used for acute myeloid leukemia minimal residual disease testing (AML MRD) but is time consuming and demands substantial expertise. Machine learning offers potential advancements in accuracy and efficiency, but has yet to be widely adopted for this application. To explore this, we trained single cell XGBoost classifiers from 98 diagnostic AML cell populations and 30 MRD negative samples. Performance was assessed by cross-validation. Predictions were integrated with UMAP as a heatmap parameter for an augmented/interactive AML MRD analysis framework, which was benchmarked against traditional MRD analysis for 25 test cases. The results showed that XGBoost achieved a median AUC of 0.97, effectively distinguishing diverse AML cell populations from normal cells. When integrated with UMAP, the classifiers highlighted MRD populations against the background of normal events. Our pipeline, MAGIC-DR, incorporated classifier predictions and UMAP into flow cytometry standard (FCS) files. This enabled a human-in-the-loop machine learning guided MRD workflow. Validation against conventional analysis for 25 MRD samples showed 100% concordance in myeloid blast detection, with MAGIC-DR also identifying several immature monocytic populations not readily found by conventional analysis. In conclusion, Integrating a supervised classifier with unsupervised dimension reduction offers a robust method for AML MRD analysis that can be seamlessly integrated into conventional workflows. Our approach can support and augment human analysis by highlighting abnormal populations that can be gated on for quantification and further assessment. This has the potential to speed up MRD analysis, and potentially improve detection sensitivity for certain AML immunophenotypes., (© 2024 International Clinical Cytometry Society.)

Acute myeloid leukemia (AML) is a heterogenous blood cancer with a dismal prognosis. It emanates from leukemic stem cells (LSCs) arising from the genetic transformation of hematopoietic stem cells (HSCs). LSCs hold prognostic value, but their molecular and immunophenotypic heterogeneity poses challenges: there is no single marker for identifying all LSCs across AML samples. We hypothesized that imaging flow cytometry (IFC) paired with artificial intelligence-driven image analysis could visually distinguish LSCs from HSCs based solely on morphology. Initially, a seven-color IFC panel was employed to immunophenotypically identify LSCs and HSCs in bone marrow samples from five AML patients and ten healthy donors, respectively. Next, we developed convolutional neural network (CNN) models for HSC-LSC discrimination using brightfield (BF), side scatter (SSC), and DNA images. Classification using only BF images achieved 86.96% accuracy, indicating significant morphological differences. Accuracy increased to 93.42% when combining BF with DNA images, highlighting differences in nuclear morphology, although DNA images alone were inadequate for accurate HSC-LSC discrimination. Model development using SSC images revealed minor granularity differences. Performance metrics varied substantially between AML patients, indicating considerable morphologic variations among LSCs. Overall, we demonstrate proof-of-concept results for accurate CNN-based HSC-LSC differentiation, instigating the development of a novel technique within AML monitoring.

Rationale: The abnormal expression of B-cell lymphoma-2 (Bcl-2) family members is often associated with the progression of the disease. Bcl-2 inhibitors (eg, venetoclax) were first reported to inhibit the proliferation of malignant lymphocytes and have a significant effect on patients with chronic lymphoblastic leukemia, but research on myeloid tumors is relatively delayed. Venetoclax was approved in 2018 for the treatment of acute myeloid leukemia (AML) patients who were not suitable for high-dose chemotherapy. The approval of venetoclax is an advance in the treatment of hematological tumors., Patient Concerns: Here we report a 64-year-old male with an increased white blood cell (WBC) count (39.0 × 109/L) and lymphocyte count (30.6 × 109/L) on physical examination in July 2014. The patients were diagnosed with chronic lymphocytic leukemia (CLL) through bone marrow (BM) smears and immunophenotyping without any cytogenetic or molecular abnormalities. Chlorambucil was prescribed, WBC was stable between 15 × 109/L and 25 × 109/L in the past 6 years. He came to the hospital again in May 2020 and complained of fatigue for 2 weeks. WBC (16.7 × 109/L) and lymphocyte (14.76 × 109/L) counts were increased, hemoglobin (HGB) and platelet (PLT) were decreased in peripheral blood, which indicated the progression of the disease., Diagnoses: The patient was diagnosed as secondary AML after CLL based on the clinical and laboratory findings., Interventions: He achieved a morphological complete remission in both AML and CLL without any adverse reactions after one course of venetoclax monotherapy., Outcomes: He received standard daunorubicin and cytarabine combined with venetoclax as consolidation therapy and is now ready for allogeneic-hematopoietic stem cell transplantation., Lessons: Our case presents a challenge to traditional treatment. New drugs such as venetoclax have shown outstanding effects in this respect. High expression of Bcl-2 can identify the responders of venetoclax. These findings should be validated in future clinical trials. We fully believe that in the near future, the comprehensive use of targeted drugs with different mechanisms will not only improve the quality of life of patients, but also completely change the prognosis of patients with recurrent and refractory hematological malignancies., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r 2  > 0.95 for all cell types in PB and r 2  > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Chromosomal aberrations play an important role in the incidence of myelodysplastic syndromes (MDS) and development to acute myeloid leukemia (AML). We report a case of a 62-year-old male patient diagnosed with MDS with excess blasts. The karyotype was 45, XY,+1,+1,-7,-10,-22,t(1;14) (q21;q32),t(1;17)(q21;p13),t(1;19)(q21;p13). The patient and his family refused treatment for financial reasons. After 2 months, the patient's MDS transformed into acute myeloid monocytic leukemia (AML-M5). This case of MDS with poor prognosis shows that patients with chromosomal numerical abnormality and balanced translocations should be treated early to prevent transition to AML. Further study of this case will reveal the molecular mechanism of MDS-to-AML transformation and identify new leukemic fusion genes., (© 2020 by the Association of Clinical Scientists, Inc.)

Background: The assessment of minimal residual disease (MRD) by flow cytometry (FC) has a prognostic impact in acute myeloid leukemia (AML), despite the low sensitivity in predicting relapse. Nonetheless, the role of leukemic-associated immunophenotypes (LAIPs)-related specificity on the sensitivity of MRD has not been clarified yet. In this respect, we accomplished this study., Methods: LAIP-frequencies of bone marrow samples from healthy donors and patients after treatment were quantified and subdivided in "categories of specificity" named as: "strong," "good," and "weak." At the following, the diagnostic performance of MRD was investigated in terms of sensitivity, specificity, predictive values, likelihood ratio (LR)., Results: "Strong" LAIPs were identified by CD7, CD2, CD4, and CD56 markers while "weak" LAIPs, independently of coexpressed markers, were mainly observed in CD33+ cells. MRD identified patients with significantly low DFS and OS but showed a low sensitivity in predicting relapse. Interestingly, majority of recurrences was noticed in patients with two LAIPs and lacking of "strong" LAIPs or only with one "good" LAIP. Thus, only patients showing one "strong" or two "good" LAIPs were considered suitable for MRD monitoring and selected to be further investigated. In this subset, positive MRD predicted a poor prognosis. Moreover, a higher sensitivity, negative predictive value (NPV) and LR- were observed after comparison with the previous series., Conclusions: These data highlight the relevant role of LAIP classification in "categories of specificity" in improving the sensitivity of MRD as assessed by FC., (© 2019 International Clinical Cytometry Society.)

Background: Leukemia stem cells (LSCs) in play an important role in the initiation, relapse, and progression of acute myeloid leukemia (AML), and in the development of chemotherapeutic drug resistance in AML. Studies regarding the detection of LSCs and the development of novel therapies for targeting them are extensive. The identification of LSCs and targeting therapies for them has been continuously under investigation., Methods: We examined the levels of CD45 dim CD34 + CD38 - CD133 + cells in bone marrow samples from patients with hematological malignancies and healthy controls, using four-color flow cytometry., Results: Interestingly, the CD45 dim CD34 + CD38 - CD133 + cells were highly expressed in the bone marrow of patients with AML compared to that in healthy controls (HC). Moreover, the proportions of CD45 dim CD34 + CD38 - CD133 + cells were also examined in diverse hematological malignancies, including AML, CML, DLBCL, MM, MDS, HL, ALL, and CLL. LSCs were prominently detected in the BMCs isolated from patients with AML and CML, but rarely in BMCs isolated from patients with DLBCL, MM, MDS, ALL, CLL, and HL. Additionally, the high CD45 dim CD34 + CD38 - CD133 + cell counts in AML patients served as a significantly poor risk factor for overall and event free survival., Conclusions: Therefore, our results suggest that CD45 dim CD34 + CD38 - CD133 + cells in AML might potentially serve as LSCs. In addition, this cell population might represent a novel therapeutic target in AML.

Purpose: This study aims to provide comprehensive insights into longitudinal immune landscape in acute myeloid leukemia (AML) development and treatment, which may contribute to predict prognosis and guide clinical decisions., Experimental Design: Periphery blood samples from 79 patients with AML (at diagnosis or/and after chemotherapy or at relapse) and 24 healthy controls were prospectively collected. We performed phenotypic and functional analysis of various lymphocytes through multiparametric flow cytometry and investigated prognostic immune-related risk factors., Results: Immune defects in AML were reflected in T and natural killer (NK) cells, whereas B-cell function remained unaffected. Both CD8 + T and CD4 + T cells exhibited features of senescence and exhaustion at diagnosis. NK dysfunction was supported by excessive maturation and downregulation of NKG2D and NKP30. Diseased γδ T cells demonstrated a highly activated or even exhausted state through PD-1 upregulation and NKG2D downregulation. Effective therapeutic response following chemotherapy correlated with T and NK function restoration. Refractory and relapsed patients demonstrated even worse immune impairments, and selective immune signatures apparently correlated clinical outcomes and survival. PD-1 expression in CD8 + T cells was independently predictive of poor overall survival and event-free survival., Conclusions: T-cell senescence and exhaustion, together with impaired NK and γδ T-cell function, are dominant aspects involved in immune dysfunction in AML. Noninvasive immune testing of blood samples could be applied to predict therapeutic reactivity, high risk for relapse, and unfavorable prognosis., (©2020 American Association for Cancer Research.)

Detection of minimal/measurable residual disease (MRD) in acute myeloid leukemia (AML) is important for guiding patient-specific clinical management. Natural killer (NK) cells can express various markers not typically associated with NK lineage, potentially confounding the detection of MRD by flow cytometry. We have observed CD33 expression on NK cells when evaluating for AML MRD in routine clinical practice in multiple patient samples. To characterize CD33 expression on NK cells, 40 peripheral blood or bone marrow samples with NK cells present at >5% of lymphocytes were selected for further assessment of NK cell phenotype and CD33 expression. Seven of the 40 samples (17.5%) were found to have CD33 expression on at least 5% of the NK cells. The CD33-positive NK cell population accounted for an average of 11.4% of NK cells (median 11.9%, range 8.0-15.3%) and 2.2% of total white cells (median 1.1%, range 0.1-10.1%). This NK cell subset expressed bright CD2, bright CD56, and dim CD16. On average, CD33 expression on NK cells was dimmer than on monocytes (mean median fluorescence intensity ratio 0.4; range 0.1-1.0). This study characterizes expression of CD33 on NK cells. Recognition of this pattern of antigen expression is critical in evaluating samples for MRD in patients with myeloid neoplasms, particularly AML., (© 2019 International Clinical Cytometry Society.)

Although genetics is a relevant risk factor in acute myeloid leukemia (AML), it can be minimally informative and/or not readily available for the early identification of patients at risk for treatment failure. In a randomized trial comparing standard vs high-dose induction (ClinicalTrials.gov #NCT00495287), we studied early peripheral blast cell clearance (PBC) as a rapid predictive assay of chemotherapy response to determine whether it correlates with the achievement of complete remission (CR), as well as postremission outcome, according to induction intensity. Individual leukemia-associated immunophenotypes (LAIPs) identified pretherapy by flow cytometry were validated and quantified centrally after 3 days of treatment, expressing PBC on a logarithmic scale as the ratio of absolute LAIP+ cells on day 1 and day 4. Of 178 patients, 151 (84.8%) were evaluable. Patients in CR exhibited significantly higher median PBC (2.3 log) compared with chemoresistant patients (1.0 log; P < .0001). PBC < 1.0 predicted the worst outcome (CR, 28%). With 1.5 log established as the most accurate cutoff predicting CR, 87.5% of patients with PBC >1.5 (PBChigh, n = 96) and 43.6% of patients with PBC ≤1.5 (PBClow, n = 55) achieved CR after single-course induction (P < .0001). CR and PBChigh rates were increased in patients randomized to the high-dose induction arm (P = .04) and correlated strongly with genetic/cytogenetic risk. In multivariate analysis, PBC retained significant predictive power for CR, relapse risk, and survival. Thus, PBC analysis can provide a very early prediction of outcome, correlates with treatment intensity and disease subset, and may support studies of customized AML therapy., (© 2020 by The American Society of Hematology.)

Background: Neoplasms derived from plasmacytoid dendritic cells (PDCs) are currently divided into two broad categories: mature PDC proliferations associated with myeloid neoplasms (MPDMN) and blastic plasmacytoid dendritic cell neoplasm (BPDCN); only BPDCN is recognized in the WHO 2016 classification of hematopoietic neoplasms. We present seven patients with high grade myeloid neoplasms (MNs), mostly acute leukemias, having a spectrum of PDC differentiation and not fitting with MPDMN or BPDCN., Methods: We analyzed seven MN cases having increased myeloblasts and prominent CD56-negative PDC proliferations comprising 5-26% of bone marrow or blood cellularity as measured by flow cytometry. The cases included five acute myeloid leukemia (three FAB M4 subtype, two unclassified), one mixed phenotype acute leukemia, and one case of unclassified MN., Results: Six cases demonstrated immunophenotypic evidence of PDC differentiation from leukemic blasts, based on variable expression of CD34, CD45, CD123, and CD304 by the leukemic cells. Four cases had circulating PDC populations in blood. None of the cases met clinical or pathologic criteria for BPDCN. Morphologic review was available for four acute leukemia cases and demonstrated either nodular or interstitial infiltrates of PDCs. All cases had an aggressive clinical course, and three cases had FLT3 ITD mutation., Conclusions: These cases demonstrate that high grade MNs, in particular AML, can exhibit PDC differentiation, with or without monocytic differentiation, in a manner distinct from MPDMN or BPDCN. The existence of MNs with immature PDC proliferations suggests that there is a broader spectrum of PDC-associated neoplasms than currently recognized. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: In patients with acute myeloid leukemia (AML), CD56 expression has been associated with adverse clinical outcome. We reported on a phenotype associated with very poor prognosis (RAM) in children enrolled in the Children's Oncology Group trial AAML0531 (Brodersen et al. Leukemia 30 (2016) 2077-2080). RAM is also characterized in part by high-intensity expression of the CD56 antigen. Herein, we investigate underlying biological and clinical differences among CD56-positive AMLs for patients in AAML0531., Methods: For 769 newly diagnosed pediatric patients with de novo AML enrolled in AAML0531, bone marrow specimens were submitted for flow cytometric analysis. For each patient, an immunophenotypic expression profile (IEP) was defined by mean fluorescent intensities of assayed surface antigens. Unsupervised hierarchical clustering analysis (HCA) was completed to group patients with similar immunophenotypes. Clusters were then evaluated for CD56 expression. Principal component analysis (PCA) was subsequently applied to determine whether CD56-positive patient groups were nonoverlapping., Results: HCA of IEPs revealed three unique phenotypic clusters of patients with CD56-positive AML, and PCA showed that these three cohorts are distinct. Cohort 1 (N = 77) showed a prevalence of t(8;21) patients (72%), Cohort 2 (N = 52) a prevalence of 11q23 patients (69%), and Cohort 3 (RAM) (N = 16) a prevalence of patients with co-occurrence of the CBFA2T3-GLIS2 fusion transcript (63%). The 5-year event-free survival (EFS) for Cohorts 1, 2, and 3 were 69, 39, and 19%, respectively., Conclusions: When leukemia is considered by its multidimensional immunophenotype and not by the expression of a single antigen, correlations are seen between genotype and there are significant differences in patient outcomes. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background: Aberrant phenotypes in acute leukemia have been reported with varying frequencies in independent studies and their association with prognostic factors is still a matter of debate., Aim: This study aims to identify the frequency of aberrant immunophenotypes in de novo acute myeloid leukemia (AML) and to evaluate their association with initial clinical and hematological features., Materials and Methods: A total of 181 patients of de novo AML were included during the time (July 2010-June 2012). The immunophenotype of all cases of AML was studied by using flow cytometry., Results: Aberrant lymphoid antigen expression was seen in 43.1% cases. Most frequent aberrant lymphoid antigen was CD7, seen in 26.5% cases. All French-American-British (FAB) subtypes except AML-M3 expressed aberrant lymphoid antigens. The expression was most common in AML-M4 in the current study. CD34 expression in AMLs was significantly associated with the expression of aberrant lymphoid antigens. Lymphoid antigen expression in adult AML was significantly associated with higher white blood cell (WBC) count (>50,000/mm 3 ) and higher number of peripheral blasts (>70%)., Conclusion: In summary, CD7 is the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is usually not associated with aberrant lymphoid antigen expression. AML cases with CD34 positivity are more likely to express aberrant lymphoid markers. The current study also supports that aberrant lymphoid antigen expression in adult AML is associated with adverse presenting hematological features (WBC count >50,000/mm 3 , peripheral blasts >70%). Pediatric Ly + AML cases are not associated with adverse presenting clinical and biological features., Competing Interests: None

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post-treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors. Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoring Basic Protocol 2: Immunophenotypic detection of CD34+CD38- leukemic stem cells., (© 2019 The Authors.)

Acute myeloid leukemia with biallelic mutation of CEBPA (CEBPA-dm AML) is a distinct good prognosis entity recognized by WHO 2016 classification. However, testing for CEBPA mutation is challenging, due to the intrinsic characteristics of the mutation itself. Indeed, molecular analysis cannot be performed with NGS technique and requires Sanger sequencing. The association of recurrent mutations or translocations with specific immunophenotypic patterns has been already reported in other AML subtypes. The aim of this study was the development of a specific cytofluorimetric score (CEBPA-dm score), in order to distinguish patients who are unlikely to harbor the mutation. To this end, the correlation of CEBPA-dm score with the presence of the mutation was analyzed in 50 consecutive AML patients with normal karyotype and without NPM1 mutation (that is mutually exclusive with CEBPA mutation). One point each was assigned for expression of HLA DR, CD7, CD13, CD15, CD33, CD34 and one point for lack of expression of CD14. OS was not influenced by sex, age and CEBPA-dm score. Multivariate OS analysis showed that CEBPA-dm (p < 0.02) and FLT3-ITD (p < 0.01) were the strongest independent predictors of OS. With a high negative predictive value (100%), CEBPA-dm score < 6 was able to identify patients who are unlikely to have the mutation. Therefore, the application of this simple score might optimize the use of expensive and time-consuming diagnostic and prognostic assessment in the baseline work up of AML patients., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Increasing evidence supports the prognostic significance of measurable residual disease (MRD) in acute myeloid leukemia (AML). Dynamic MRD assessment for patients with AML complements baseline patient risk assessment factors in determining patient prognosis. MRD status may also be helpful in informing therapeutic decisions. The European Leukemia Net MRD working party recently issued consensus recommendations for the use of MRD in AML. The Food and Drug Administration also issued advice for using MRD in trials of hematologic malignancies. This article discusses MRD testing, highlights the challenges in adopting MRD testing in clinical practice, and provides insights into the future of the field., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: Acute leukemia (AL) is a highly heterogeneous malignant disease caused by hematopoietic cell abnormalities. Our study investigated the potential for immunophenotyping of leukemic cells via flow cytometry and the clinical usefulness of this approach in treatment of AL., Methods: Bone marrow (BM) specimens were collected to detect antigen expression on hematopoietic cells in pre-treatment samples from patients with AL. In addition, fraction survival curves were calculated using the Kaplan-Meier method to explore the effect of markers on prognosis in AL., Results: Expression levels of immunophenotypic markers in patients with acute lymphoblastic leukemia (ALL) were significantly different from those in patients with acute myeloid leukemia (AML). In addition, there was a potential association between the surface marker, cluster of differentiation 2 (CD2), and fraction survival in AML. However, no similar result was found in ALL. Moreover, genetic tests showed greater positive variation of the break point cluster-Abelson tyrosine kinase ( BCR-ABL) fusion gene in samples from patients with ALL than in samples from patients with AML., Conclusions: We have shown a rapid and effective flow cytometry method that enables the identification of immunophenotype in AL. Moreover, CD2 may constitute a predictive marker for prognosis in patients with AML.

Flow-cytometric detection of now termed measurable residual disease (MRD) in acute myeloid leukemia (AML) has proven to have an independent prognostic impact. In a previous multicenter study we developed protocols to accurately define leukemia-associated immunophenotypes (LAIPs) at diagnosis. It has, however, not been demonstrated whether the use of the defined LAIPs in the same multicenter setting results in a high concordance between centers in MRD assessment. In the present paper we evaluated whether interpretation of list-mode data (LMD) files, obtained from MRD assessment of previously determined LAIPs during and after treatment, could reliably be performed in a multicenter setting. The percentage of MRD positive cells was simultaneously determined in totally 173 LMD files from 77 AML patients by six participating centers. The quantitative concordance between the six participating centers was meanly 84%, with slight variation of 75%-89%. In addition our data showed that the type and number of LAIPs were of influence on the performance outcome. The highest concordance was observed for LAIPs with cross-lineage expression, followed by LAIPs with an asynchronous antigen expression. Our results imply that immunophenotypic MRD assessment in AML will only be feasible when fully standardized methods are used for reliable multicenter assessment., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Recent data have demonstrated the high sensitivity and specificity of peripheral blood flow cytometry (PBFC) for the diagnosis of pediatric leukemia; however, diagnostically significant immunophenotypic discrepancies between PBFC and bone marrow (BM) evaluation, which result in different lineage assignment and treatment protocols, can rarely occur. Here, we sought to further characterize the performance of PBFC for pediatric leukemia and highlight the exceptions when PBFC can result in misdiagnosis., Methods: An institutional database was searched between 2012 and 2016 for cases of acute leukemia with concurrent PBFC and BM evaluation. Immunophenotyping results from the peripheral blood and BM using four or eight color flow cytometry, as well as BM cytochemical staining and immunohistochemistry, were compared., Results: Two hundred ninety PBFC samples with concurrent BM evaluation were identified. Based on the final immunophenotypic classification, the cases were distributed as follows: 108 B-lymphoblastic leukemia (B-ALL), 57 T-lymphoblastic leukemia (T-ALL), 116 acute myeloid leukemia (AML), and 9 mixed-phenotype acute leukemia (MPAL). Among all cases, five had a diagnostically significant discrepancy between PBFC and BM evaluation. In three cases, the immunophenotype by PBFC was consistent with early T-cell precursor ALL (ETP-ALL), whereas BM evaluation demonstrated MPAL. Two cases were suspicious for acute megakaryoblastic leukemia (AMKL) and MPAL, T/myeloid by PBFC but were diagnosed as B-ALL and T-ALL in the BM., Conclusion: Immunophenotypic classification by PBFC is accurate (>98%) in almost all cases of pediatric leukemia with the rare exceptions of suspected ETP-ALL, MPAL, and AMKL. These PBFC diagnoses should be confirmed with BM immunophenotyping., (© 2018 Wiley Periodicals, Inc.)

Immunophenotyping by multiparameter flow cytometry is a rapid and efficient technique to simultaneously assess and correlate multiple individual cell properties like size and internal complexity along with antigen expression in a population of cells. This method is utilized for rapid characterization of the blasts and classification of acute myeloid leukemia (AML), in both the peripheral blood (PB) and bone marrow (BM). This technique is not only useful in the initial diagnosis but also in monitoring and determining prognosis of the disease through minimal residual disease (MRD) testing. This chapter provides an overview of procedures for specimen processing, staining, and immunophenotyping of AML and describes the principles of data analysis for AML classification and MRD testing.

Introduction: Flow cytometric quantification of minimal residual disease (MRD) in acute leukemia (AL) represents an indispensable tool to guide modern therapeutic protocols toward a precision medicine approach, being a powerful predictor of the overall response to treatment. This review covers the most challenging aspects and developments of this method, aiming at supporting further its implementation in clinical practices. Area covered: Flow cytometric MRD is based on the discrimination of leukemia cells from their physiological counterparts by the recognition of the leukemia-associated immunophenotypes. Technical and standardization advances along the last decades have been implemented allowing flow cytometric MRD to consolidate its role in modern therapeutic protocols for ALs. However, gaps in sensitivity and data interpretation are still present together with the need for further optimization of MRD-based clinical protocols. In this review, we critically analyze and discuss the most relevant and representative contributions in the field by accurate selection of the literature available in PubMed. Expert commentary: Further research in flow cytometric MRD can bring this technology toward wider and consistent applications in multiple acute leukemia settings rendering this tool a future golden standard and providing clinicians with more reliable and accurate tools for clinical decisions.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Background: In acute myeloid leukemia controversy exists about the role of immunophenotyping of the blasts at diagnosis as a potential prognostic factor., Methods: We retrospectively analyzed immunophenotypic marker expression on blasts in relation to genetic aberrancies and survival data of 684 patients. All patients were included in different studies from the HOVON/SAKK Consortium., Results: Markers CD2, CD7, CD11b, CD19, CD22, and CD56 all appeared to be associated with one or more established prognostic genetic aberrancies. In the overall population, differences in univariate survival analyses were observed for CD2, CD11b, and CD22. After correcting these survival differences for currently used risk profile data, only CD11b expression remained of prognostic value for poor overall survival (OS) and event-free survival (EFS). CD11b expression turned out to be an independent factor for poor OS and EFS in the subgroup of patients who lacked cytogenetic and molecular aberrancies., Conclusions: In this study, we demonstrate that associations between immunophenotypic markers and genetic aberrancies interfere with the prognostic properties of immunophenotypic markers. Such may account for most of the previously reported prognostic impact of these markers. Only CD11b expression remained as an independent prognostic marker. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia., Methods: MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics., Results: The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa., Conclusions: MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Diagnostic biomarkers can be used to determine relapse risk in acute myeloid leukemia, and certain genetic aberrancies have prognostic relevance. A diagnostic immunophenotypic expression profile, which quantifies the amounts of distinct gene products, not just their presence or absence, was established in order to improve outcome prediction for patients with acute myeloid leukemia. The immunophenotypic expression profile, which defines each patient's leukemia as a location in 15-dimensional space, was generated for 769 patients enrolled in the Children's Oncology Group AAML0531 protocol. Unsupervised hierarchical clustering grouped patients with similar immunophenotypic expression profiles into eleven patient cohorts, demonstrating high associations among phenotype, genotype, morphology, and outcome. Of 95 patients with inv(16), 79% segregated in Cluster A. Of 109 patients with t(8;21), 92% segregated in Clusters A and B. Of 152 patients with 11q23 alterations, 78% segregated in Clusters D, E, F, G, or H. For both inv(16) and 11q23 abnormalities, differential phenotypic expression identified patient groups with different survival characteristics ( P <0.05). Clinical outcome analysis revealed that Cluster B (predominantly t(8;21)) was associated with favorable outcome ( P <0.001) and Clusters E, G, H, and K were associated with adverse outcomes ( P <0.05). Multivariable regression analysis revealed that Clusters E, G, H, and K were independently associated with worse survival ( P range <0.001 to 0.008). The Children's Oncology Group AAML0531 trial: clinicaltrials.gov Identifier: 00372593 ., (Copyright© 2017 Ferrata Storti Foundation.)

Mixed-phenotype acute leukemia (MPAL) is a heterogeneous group of hematopoietic malignancies in which blasts show markers of multiple developmental lineages and cannot be clearly classified as acute myeloid or lymphoblastic leukemias. Historically, various names and classifications were used for this rare entity accounting for 2-5% of all acute leukemias depending on the diagnostic criterias used. The currently valid classification of myeloid neoplasms and acute leukemia published by the World Health Organization (WHO) in 2016 refers to this group of diseases as MPAL. Because adverse cytogenetic abnormalities are frequently present, MPAL is generally considered a disease with a poor prognosis. Knowledge of its treatment is limited to retrospective analyses of small patient cohorts. So far, no treatment recommendations verified by prospective studies have been published. The reported data suggest that induction therapy for acute lymphoblastic leukemia followed by allogeneic hematopoietic cell transplantation is more effective than induction therapy for acute myeloid leukemia or consolidation chemotherapy. The establishment of cooperative groups and international registries based on the recent WHO criterias are required to ensure further progress in understanding and treatment of MPAL. This review summarizes current knowledge on the diagnosis, classification, prognosis and treatment of MPAL patients.

Background: SNAP-tag, a self-labeling protein tag, is commonly used for in vitro and in vivo analysis of bound target proteins. We report the first evidence that SNAP-tag could be used for ex vivo detection of enriched biological markers., Methods: Proof of concept was established for target c-kit receptor, a pathological and diagnostic marker for a variety of cancers. SNAP-tag conjugates with stem-cell factor (SCF) fusion proteins were designed and their binding and specificity was validated in vitro using flow cytometry and immunostaining., Results: Ex vivo diagnostic application of the fusion protein was demonstrated in comparison with anti-c-kit antibody for peripheral blood samples from leukemia patients and colorectal tissue specimens.

Minimal residual disease (MRD) after therapy has unequivocal prognostic value in acute leukemia. Over the past 20years, a number of techniques have evolved into routine laboratory tools to detect MRD, most notably, multiparametric flow cytometry (MFC) and quantitative polymerase chain reaction (PCR)-based molecular methods. There is growing evidence that the presence of MRD detected by MFC or molecular methods provides independent prognostic information and is associated with an increased risk of relapse and shortened survival. However, the predictive value of MRD may be affected by a lack of consensus as to the timing for assessment, the methodology used, and interlaboratory variation in test performance. Herein, we review the methodological principles of MRD assays, discuss the clinical implications of monitoring MRD for risk stratification and directing further therapy, and suggest potential areas for future investigation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Leukemia cell lines have been widely used in the hematology field to unravel mechanistic insights and to test new therapeutic strategies. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases that are characterized by ineffective hematopoiesis and frequent progress to acute myeloid leukemia (AML). A few cell lines have been established from MDS patients after progression to AML but their characterization is incomplete. Here we provide a detailed description of the immunophenotypic profile of the MDS-derived cell lines SKK-1, SKM-1, F-36P; and MOLM-13. Specifically, we analyzed a comprehensive panel of markers that are currently applied in the diagnostic routine for myeloid disorders. To provide high-resolution genetic data comprising copy number alterations and losses of heterozygosity we performed whole genome single nucleotide polymorphism-based arrays and included the cell line OHN-GM that harbors the frequent chromosome arm 5q deletion. Furthermore, we assessed the mutational status of 83 disease-relevant genes. Our results provide a resource to the MDS and AML field that allows researchers to choose the best-matching cell line for their functional studies. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Opinion Statement: New technology and improved understanding of the pathogenesis of acute leukemias have allowed for sensitive detection of minimal residual disease (MRD). Despite many years of research demonstrating the prognostic value of MRD, there is no standard of care for measurement of MRD in acute myeloid leukemia. The techniques for assessment are continuing to improve at a rapid pace; however, the benefit of risk-adapted approaches for MRD positive disease remains a major question. This review focuses on recent methodological advances for MRD detection, the role of MRD in prognostication, and current application of the available evidence in guiding therapy decisions.

Multiparameter flow cytometry has become an indispensable tool for the diagnosis and classification of acute myeloid leukemia (AML). The basic method relies on the unique ability to detect immunophenotypic abnormalities on discrete subpopulations. The primary roles in the initial assessment of AML are to determine the immaturity of the leukemic population, define the lineage and the immunophenotypic aberrancies in blasts, and identify characteristic immunophenotypic features to predict important recurrent cytogenetic and genetic abnormalities and prognosis. The established immunophenotypic profile, a baseline "fingerprint," is used for follow-up assessment of residual disease. This chapter provides an overview of procedures for specimen processing, staining, and immunophenotyping of AML and describes our strategy for data analysis supplemented with illustrative case examples.

Historically, the diagnosis and classification of acute leukemia involved morphologic review of blasts in the peripheral blood and bone marrow smears and cytochemical staining. Cytochemical stains, which are enzymatic colorimetric reactions that occur in the cells of interest, were necessary to assign and confirm myeloid and lymphoid lineage. In the current WHO 2008 Classification of leukemia, immunophenotyping and cytogenetic analysis have largely replaced cytochemical staining in the characterization of acute leukemias. Nonetheless, cytochemical testing remains a useful adjunct assay for the proper classification of acute leukemia in a number of diagnostic settings. This chapter reviews the principles of the most common cytochemical stains, their procedures and guides to interpretation, and results in acute myeloid leukemia.

To discuss the expression of T helper cell 17 (Th17) cells and CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in peripheral blood (PB) of patients with acute leukemia (AL), and to explore the relationship between them and disease prognosis. 40 patients diagnosed with acute leukemia in The First Affiliated Hospital of Zhengzhou University from July 2012 to August 2014 were selected as the observation group. Meanwhile, 40 healthy people were taken as the control group. Flow Cytometry Method (FCM) was used to detect the level of Th17 cells and CD4 + CD25 + Foxp3 + cells in peripheral blood of the two groups, and enzyme-linked immuno sorbent assay (ELISA) method was used to test the level of IL17 and TGF-β in peripheral blood of two groups; reverse transcription-polymerase chain reaction (RT-PCR) was adopted to analyze the mRNA levels of RORγT and Foxp3 in peripheral blood. In addition, we examined the levels of Th17 and CD4 + CD25 + Foxp3 + cells and associated factor levels in patients with remission after AL chemotherapy. the Th17 cells (CD3 + CD4 + IL-17 + ) in acute leukemia patients accounted for (1.51±0.27)%, which was significantly higher than that of control group (0.36±0.23)%, with statistical significance (t=20.51, P<0.001); the percentage of CD4 + CD25 + Foxp3 + cells in AL patients was (3.37±0.48)%, which was significantly higher than that of control group of (1.26±0.27)%, with statistical significance (t=24.23, P<0.001); the serum levels of IL-17 and TGF-β in AL patients were (28.12±6.33) pg/ml and (38.41±8.44) pg/ml respectively, which were all significantly higher than that of control group of (14.41±6.21) pg/ml and (24.49±7.42) pg/ml, with statistical significance (t=7.83, P<0.001; t=7.83, P<0.001); the RORγT mRNA and Foxp3 mRNA levels in AL patients were all significantly higher than that of control group, with statistical significance (t=12.27, P<0.001; t=7.89, P<0.001). In addition, compared with before chemotherapy, the levels of Th17 cells and CD4 + CD25 + Foxp3 + cells, and the serum levels of IL-17 and TGF-β in acute leukemia patients all decreased significantly after 6 months of chemotherapy, and the difference was statistically significant (P<0.001). Th17 cells, CD4+ CD25+ Foxp3 + cells and their secretory proteins IL-17, TGF-β and transcription factors were significantly increased in AL patients. Therefore, regular detection of peripheral blood Th17 and Treg cells, as well as their secretory proteins are useful for monitoring the immune status and prognosis of patients.

Background: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder presenting with accumulation of proliferating undifferentiated blasts. Xenograft transplantation studies have demonstrated a rare population of leukemia-initiating cells called leukemic stem cells (LSCs) capable of propagating leukemia that are enriched in the CD34+/CD38- fraction. LSCs are quiescent, resistant to chemotherapy and likely responsible for relapse and therefore represent an ideal target for effective therapy. LSCs are reported to overexpress the alpha subunit of the IL-3 receptor (CD123) compared to normal CD34+/CD38- hematopoietic stem cells. It has not been demonstrated whether CD123-positive (CD34+/CD38-) subpopulation is enriched for any clonal markers of AML or any LSC properties. The aims of this study were to investigate whether FMS-like tyrosine kinase (FLT3)/internal tandem duplication (ITD) mutations are present at LSC level and whether FLT3/ITD mutation is confined to LSC as defined by CD34+/CD38-/CD123+ and not CD34+/CD38-/CD123-., Methods: Thirty-four AML cases were analyzed by five-color flow cytometry and sequential gating strategy to characterize of CD34+/CD38-/CD123+ cells. These cells were sorted, analyzed by PCR, and sequenced for FLT3/ITD., Results: In this study, we confirm significant expression of CD123 in 32/34 cases in the total blast population (median expression = 86 %). CD123 was also expressed in the CD34+/CD38- cells (96 ± 2 % positive) from 28/32 for CD123+ AML. CD123 was not expressed/low in normal bone marrow CD34+/CD38- cells (median expression = 0 %, range (0-.004 %). AML samples were tested for FLT3/ITD (10 positive/25). FLT3/ITD+ AML cases were sorted into two putative LSC populations according to the expression of CD123 and analyzed for FLT3/ITD again in the stem cell fractions CD34+/CD38-/CD123+ and CD34+/CD38-/CD123-. Interestingly, FLT3/ITD was only detected in CD34+/CD38-/CD123+ (7/7) and not in CD34+/CD38-/CD123- subpopulation (6/7)., Conclusions: This finding shows that FLT3/ITD are present at LSC level and may be a primary and not secondary event in leukemogenesis, and the oncogenic events of FLT3/ITD happen at a cell stage possessing CD123. It shows that CD123 immunoprofiling provides further delineation of FLT3+ LSC clone. This novel finding provides a rationale for treatment involving CD123-targeting antibodies with intracellular FLT3 inhibitors directed against CD34+/CD38-/CD123+. This may result in more effective anti-LSC eradication.

Objectives: To examine the value of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) in core binding factor (CBF) acute myeloid leukemia (AML)., Methods: We studied 42 patients with t(8;21)(q22;q22)/RUNX1-RUNX1T1 and 51 with inv(16)(p13.1q22)/CBFB-MYH11 Tandem MRD analyses by MFC and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed in 281 bone marrow (BM) samples., Results: Grouping qRT-PCR levels as ≤0.01, 0.01 to 0.1, 0.1 to 1, 1 to 10, and >10%, and reporting MFC (sensitivity, 0.1%-0.01%) as positive or negative, κ coefficient test showed no agreement between qRT-PCR and MFC in BM samples obtained postinduction (n = 44, κ = 0.041), and only weak agreement during consolidation (n = 108, κ = 0.083), maintenance/follow-up (n = 107, κ = 0.164), and salvage chemotherapy (n = 24, 0.376). In the post induction BM samples, while qRT-PCR <0.1% was associated with lower and ≥10% with higher AML relapse risk (P = .035), qRT-PCR between 0.1% to 1% and 1% to 10% failed to predict relapse. In the latter group with intermediate qRT-PCR results, MFC provided prognostic value for relapse (P = 0.006)., Conclusions: MFC and qRT-PCR are complementary tests in monitoring CBF AML MRD., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background & Objectives: Flow cytometry is an important tool to diagnose acute leukaemia. Attempts are being made to find the minimal number of antibodies for correctly diagnosing acute leukaemia subtypes. The present study was designed to evaluate the analysis of side scatter (SSC) versus CD45 flow dot plot to distinguish acute myeloid leukaemia (AML) from acute lymphoblastic leukaemia (ALL), with minimal immunological markers., Methods: One hundred consecutive cases of acute leukaemia were evaluated for blast cluster on SSC versus CD45 plots. The parameters studied included visual shape, CD45 and side scatter expression, continuity with residual granulocytes/lymphocytes/monocytes and ratio of maximum width to maximum height (w/h). The final diagnosis of ALL and AML and their subtypes was made by morphology, cytochemistry and immunophenotyping. Two sample Wilcoxon rank-sum (Mann Whitney) test and Kruskal-Wallis equality-of-populations rank tests were applied to elucidate the significance of the above ratios of blast cluster for diagnosis of ALL, AML and their subtypes. Receiver operating characteristic (ROC) curves were generated and the optimal cut-offs of the w/h ratio to distinguish between ALL and AML determined., Results: Of the 100 cases, 57 of ALL and 43 cases of AML were diagnosed. The median w/h ratio of blast population was 3.8 for ALL and 1 for AML (P<0.001). ROC had area under curve of 0.9772.The optimal cut-off of the w/h ratio for distinction of ALL from AML was found to be 1.6., Interpretation & Conclusions: Our findings suggest that if w/h ratio on SSC versus CD45 plot is less than 1.6, AML may be considered, and if it is more than 1.6, ALL may be diagnosed. Using morphometric analysis of the blast cluster on SSC versus CD45, it was possible to distinguish between ALL and AML, and their subtypes., Competing Interests: None.

Interphase heterogenous chromosomes spatially close to each other are predominantly located near the center of nuclei and are prone to incur translocations. We screened a t(8;21) (q22;q22) acute myeloid leukemia-M2 patient during three phases (post-chemotherapy, remittent stage, and relapse) and a donor of normal karyotype as control by two-(2D) and three-dimensional (3D)-fluorescence in situ hybridization (FISH). Our classification of nuclei (normal, transitional, and malignant nuclei) by 3D-FISH analyses may provide a more precise prognosis than 2D-FISH results, especially for remittent stage sample in our study, in which 2D-FISH findings showed normal results, whereas 3D-FISH results showed extreme abnormalities (normal nuclei 27%, transitional nuclei 36%, malignant nuclei 37%). The relative radial positions (d/R) of chromosomes 8 were similar to d/R of chromosomes 21 for the relapse sample. We classified heterogenous chromosome pairs into close pairs and normal pairs based on their relative distances (d'/(2R)). The centers of close pairs were more internal than normal pairs in nuclei in all samples, and the d/R values of a given-type pairwise heterogenous chromosomes were similar among four samples. Our data demonstrate that the classification of nuclei based on spatial organization of chromosomes by 3D-FISH is reasonable and essential for evaluating acute myeloid leukemia prognosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Several factors are known to affect prognosis of acute leukemia such as age, high leukocyte count, cytogenetic abnormality, performance status and recurrent leukemia. We aimed to investigate the association between cell surface markers and prognostic determinants such as recurrence at 6 and 12 months and survival at 6, 12 and 18 months in acute leukemia patients., Patients and Methods: A total of 142 patients, 101 with acute myeloid leukemia (AML) and 41 with B-cell acute lymphoblastic leukemia (B-ALL) were included. The effects of surface markers on survival and recurrence rates were evaluated retrospectively., Results: In AML patients, CD5+ and CD34+ immunophenotypes and in ALL patients cCD22+, CD34+ and CD49f + CD19+ immunophenotypes were positive prognostic indicators. In AML patients CD7 expression, and in ALL patients CD5+, CD7+ and CD117+ immunophenotypes and >90% CD45 expression were negative prognostic indicators., Conclusions: This study demonstrates that flow cytometry, a common diagnostic tool in acute leukemia, may also have prognostic value in acute leukemia in the future.

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.

Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2)., (© 2015 International Clinical Cytometry Society.)

Purpose of Review: Patients with acute myeloid leukemia typically are monitored with bone marrow biopsy 7-10 days after completion of induction therapy. However, this test has relatively poor sensitivity and specificity for classifying patients likely to have long-term cure or even patients who will need additional therapy to achieve complete remission. Several new methods of assessing response are being studied and are summarized in this review., Recent Findings: Many new methods are being used to evaluate response assessment. Emerging data suggest that various types of clinical, chemical, and imaging biomarkers, as well as more sensitive techniques to measure minimal residual disease, can all help provide more accurate response assessment and prognostic information., Summary: New techniques will improve the way we assess response to acute myeloid leukemia therapy. Larger confirmatory studies utilizing these novel markers of response to change treatment and to improve clinical outcomes are urgently needed.

Researchers have recognized that aberrant methylation is an important initiating event in the pathogenesis of hematological malignancies. DNMT3A is a DNA methyltransferase that plays a vital role in de novo methylation of DNA. Somatic mutation of DNMT3A, especially at the Arg882 (R882) site of the DNMT3A coding sequence, has been identified in pre-leukemic stem cell clones as one of the driver mutations of acute myeloid leukemia (AML). Statistical analysis has indicated that patients with AML with DNMT3A mutation tend to have the M4/M5 subtype of AML according to the French-American-British classification. In this study we aimed to investigate the association between the typical immunophenotype of leukemic blasts and mutation of DNMT3A R882. In addition, we further determined the relationship between DNMT3A R882 mutation and the expression of monocytic differentiation genes, and its clinical significance.

Current chemotherapeutic regimens achieve CR in a large percentage of patients with AML. However, relapse after CR remains a significant problem. The presence of leukemic cells at levels too low to be detected by conventional microscopy, termed minimal residual disease (MRD), has been associated with an increased risk of relapse and shortened survival. Detection of MRD requires the use of highly sensitive ancillary techniques. Multi-color flow cytometric immunophenotyping is a sensitive method for quick and accurate detection of MRD. Use of this method in patient management may result in lower rates of relapse and improved survival, and is an effective means of assessing novel therapeutic agents. This method can be used in the vast majority of patients with AML, regardless of the immunophenotypic, cytogenetic and molecular genetic abnormalities present. Unfortunately, conflicting data regarding optimum methods of measurement and reporting, as well as the expertize required to interpret results have limited broad application of this technique. We provide a broad overview of this technique, including its advantages and limitations, and discuss the methods employed at our institution. We also review several possible areas of future investigation.

Acute myeloblastic leukemia accounts for approximately 20% of acute leukemias in children. The days the microscope represented the main tool in the diagnosis and classification of Acute Myeloblastic Leukemia seem to be very far. This review summarizes the current diagnosis of this malignancy, where the morphological, cytochemical, immunophenotyping, cytogenetic and molecular characterization represents the basement of a risk group related therapy.