Juvenile myelomonocytic leukemia presenting in an infant with a subdural hematoma.

BACKGROUND: Juvenile myelomonocytic leukemia (JMML) is a rare childhood hematopoietic disorder typically presenting with hepatosplenomegaly, lymphadenopathy, pallor, fever, and cutaneous findings. The authors report the first case, to our knowledge, of JMML presenting in a pediatric patient with a subdural hematoma. CASE DESCRIPTION: A 7-month old male with recurrent respiratory infections and a low-grade fever presented with a full fontanelle and an increasing head circumference and was found to have chronic bilateral subdural collections. Abusive head trauma, infectious, and coagulopathy workups were unremarkable, and the patient underwent bilateral burr holes for evacuation of the subdural collections. The postoperative course was complicated by the development of thrombocytopenia, anemia, and an acute subdural hemorrhage which required evacuation. Cytologic analysis of the subdural fluid demonstrated atypical cells, which prompted flow cytometric analysis, a bone marrow biopsy, and ultimately a diagnosis of JMML. Following chemotherapy, the patient's counts improved, and he subsequently underwent a hematopoietic stem cell transplant. CONCLUSION: Subdural collections may rarely represent the first presenting sign of hematologic malignancies. In patients with a history of recurrent infections and a negative workup for abusive head trauma, clinicians should include neoplastic etiologies in the differential for chronic subdural collections and have a low threshold for fluid analysis.

CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia and is distinctly low in KMT2A-rearranged cases.

BACKGROUND: CD49f (integrin α6) is a useful marker for minimal residual disease (MRD) detection in B lymphoblastic leukemia and has recently been suggested to mediate infiltration of the central nervous system by leukemic B lymphoblasts. However, data regarding expression of CD49f protein in B lymphoblastic leukemia are limited, and whether CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia is unknown. METHODS: CD49f protein expression was characterized by flow cytometry in a series of 40 cases of B lymphoblastic leukemia, which included the genetic subgroups: KMT2A-rerranged, BCR-ABL1+, ETV6-RUNX1+, hypodiploidy, and hyperdiploidy. RESULTS: Expression of CD49f differed significantly among the five genetic subgroups studied, whether assessed by percentage of blasts positive for the antigen (p = .0001, Kruskal-Wallis) or median fluorescence intensity (MFI) (p = .0001, Kruskal-Wallis). Moreover, the percentage of CD49f+ blasts and MFI of CD49f were significantly lower in KMT2A-rearranged cases than in cases without KMT2A rearrangement (p = .0002 for both, Mann-Whitney). CONCLUSIONS: CD49f protein expression varies among genetic subgroups of B lymphoblastic leukemia, and is distinctly low in KMT2A-rearranged cases.

Immunophenotyping of Acute Lymphoblastic Leukemia.

Immunophenotyping by flow cytometry is an important component in the diagnostic evaluation of patients with acute lymphoblastic leukemia. This technique further permits the detection of minimal residual disease after therapy, a robust prognostic factor that may guide individualized treatment. We describe here laboratory methods for both the initial characterization of lymphoblasts at diagnosis, and the detection of rare leukemic lymphoblasts after treatment. In addition to antibody combinations suitable for diagnosis and detection of minimal residual disease, we describe procedures for peripheral blood and bone marrow sample preparation, procedures for labeling of cell-surface and intracellular proteins with fluorochrome-conjugated antibodies, and approaches to analysis of immunophenotypic data.

Intracytoplasmic crystalline inclusions in chronic lymphocytic leukemia.

Neoplastic B cells may have cytoplasmic inclusions that are visible in routine peripheral blood smears by light microscopy.

Applications of Flow Cytometric Immunophenotyping in the Diagnosis and Posttreatment Monitoring of B and T Lymphoblastic Leukemia/Lymphoma.

In this review, we discuss applications of flow cytometric immunophenotyping (FCI) in the diagnostic evaluation and posttreatment monitoring of B and T lymphoblastic leukemia/lymphoma. We describe practical approaches to FCI at the time of diagnosis, with an emphasis on blast identification, lineage assignment, and distinction of B and T lymphoblastic leukemia/lymphoma from their morphologic and immunophenotypic mimics. We further review flow cytometric assays for the detection of minimal or measurable residual disease (MRD) after treatment, and illustrate both standard approaches, and newer strategies for improving sensitivity and circumventing the loss of immunophenotypic targets after immunotherapy. © 2019 International Clinical Cytometry Society.

Breast implant-associated anaplastic large-cell lymphoma.

In patients with suspected breast implant-associated anaplastic large-cell lymphoma, cytologic evaluation of fine-needle aspirate specimens from the peri-implant seroma, together with flow cytometric immunophenotyping and immunohistochemistry, represents a suitable preoperative diagnostic approach when planning for surgical management.

A QA Program for MRD Testing Demonstrates That Systematic Education Can Reduce Discordance Among Experienced Interpreters.

BACKGROUND: Minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) by flow cytometry is an established prognostic factor used to adjust treatment in most pediatric therapeutic protocols. MRD in B-ALL has been standardized by the Children's Oncology Group (COG) in North America, but not routine clinical labs. The Foundation for National Institutes of Health sought to harmonize MRD measurement among COG, oncology groups, academic, community and government, laboratories. METHODS: Listmode data from post-induction marrows were distributed from a reference lab to seven different clinical FCM labs with variable experience in B-ALL MRD. Labs were provided with the COG protocol. Files from 15 cases were distributed to the seven labs. Educational sessions were implemented, and 10 more listmode file cases analyzed. RESULTS: Among 105 initial challenges, the overall discordance rate was 26%. In the final round, performance improved considerably; out of 70 challenges, there were five false positives and one false negative (9% discordance), and no quantitative discordance. Four of six deviations occurred in a single lab. Three samples with hematogones were still misclassified as MRD. CONCLUSIONS: Despite the provision of the COG standardized analysis protocol, even experienced laboratories require an educational component for B-ALL MRD analysis by FCM. Recognition of hematogones remains challenging for some labs when using the COG protocol. The results from this study suggest that dissemination of MRD testing to other North American laboratories as part of routine clinical management of B-ALL is possible but requires additional educational components to complement standardized methodology. © 2017 International Clinical Cytometry Society.

PhenoGraph and viSNE facilitate the identification of abnormal T-cell populations in routine clinical flow cytometric data.

BACKGROUND: Flow cytometric identification of neoplastic T-cell populations is complicated by the wide range of phenotypic abnormalities in T-cell neoplasia, and the diverse repertoire of reactive T-cell phenotypes. We evaluated whether a recently described clustering algorithm, PhenoGraph, and dimensionality-reduction algorithm, viSNE, might facilitate the identification of abnormal T-cell populations in routine clinical flow cytometric data. METHODS: We applied PhenoGraph and viSNE to peripheral blood mononuclear cells labeled with a single 8-color T/NK-cell antibody combination. Individual peripheral blood samples containing either a T-cell neoplasm or reactive lymphocytosis were analyzed together with a cohort of 10 normal samples, which established the location and identity of normal mononuclear-cell subsets in viSNE displays. RESULTS: PhenoGraph-derived subpopulations from the normal samples formed regions of phenotypic similarity in the viSNE display describing normal mononuclear-cell subsets, which correlated with those obtained by manual gating (r2 = 0.99, P < 0.0001). In 24 of 24 cases of T-cell neoplasia with an aberrant phenotype, compared with 4 of 17 cases of reactive lymphocytosis (P = 1.4 × 10-7 , Fisher Exact test), PhenoGraph-derived subpopulations originating exclusively from the abnormal sample formed one or more distinct phenotypic regions in the viSNE display, which represented the neoplastic T cells, and reactive T-cell subpopulations not present in the normal cohort, respectively. The numbers of neoplastic T cells identified using PhenoGraph/viSNE correlated with those obtained by manual gating (r2 = 0.99; P < 0.0001). CONCLUSIONS: PhenoGraph and viSNE may facilitate the identification of abnormal T-cell populations in routine clinical flow cytometric data. © 2017 Clinical Cytometry Society.

Detection of small abnormal B-Lymphoblast populations at diagnosis of chronic myelogenous leukemia, BCR-ABL1+: Incidence, phenotypic features, and clinical implications.

BACKGROUND: According to the 2008 World Health Organization (WHO) Classification of Tumors of the Haematopoietic and Lymphoid Tissues, the finding of B lymphoblasts in the blood or bone marrow of a patient with chronic myelogenous leukemia, BCR-ABL1+ (CML) should raise a concern for progression of the disease to B-lymphoblastic blast phase. Data addressing the incidence and phenotypic features of abnormal B lymphoblasts in CML, and whether the detection of B lymphoblasts inexorably heralds blast phase in CML, though, are limited. METHODS: We reviewed a consecutive series of patients with newly diagnosed CML who had undergone bone marrow examination with flow cytometric immunophenotyping. Polychromatic immunophenotyping data were reviewed, and clinical follow-up data were obtained. RESULTS: A precursor B-cell population with an abnormal composite immunophenotype was detected in 4 of 36 (11.1%) diagnostic bone marrow samples, at levels ranging from 0.01% to 0.30% of viable single cells acquired. The most common phenotypic aberrations were abnormally bright expression of CD10 and CD19 (seen in four and three cases, respectively), and abnormally dim expression of CD38 (seen in four cases). All three patients with adequate clinical follow-up have achieved and maintained a deep or major molecular response with a tyrosine kinase inhibitor, and none has progressed to B-lymphoblastic blast phase (follow-up duration: 17-46 months). CONCLUSIONS: In chronic-phase CML, a small (<0.5%) abnormal B-lymphoblast population is present in a significant minority of diagnostic bone marrow samples, but does not inevitably herald progression to B-lymphoblastic blast phase. © 2015 International Clinical Cytometry Society.

Risk stratification of chromosomal abnormalities in chronic myelogenous leukemia in the era of tyrosine kinase inhibitor therapy.

Clonal cytogenetic evolution with additional chromosomal abnormalities (ACAs) in chronic myelogenous leukemia (CML) is generally associated with decreased response to tyrosine kinase inhibitor (TKI) therapy and adverse survival. Although ACAs are considered as a sign of disease progression and have been used as one of the criteria for accelerated phase, the differential prognostic impact of individual ACAs in CML is unknown, and a classification system to reflect such prognostic impact is lacking. In this study, we aimed to address these questions using a large cohort of CML patients treated in the era of TKIs. We focused on cases with single chromosomal changes at the time of ACA emergence and stratified the 6 most common ACAs into 2 groups: group 1 with a relatively good prognosis including trisomy 8, -Y, and an extra copy of Philadelphia chromosome; and group 2 with a relatively poor prognosis including i(17)(q10), -7/del7q, and 3q26.2 rearrangements. Patients in group 1 showed much better treatment response and survival than patients in group 2. When compared with cases with no ACAs, ACAs in group 2 conferred a worse survival irrelevant to the emergence phase and time. In contrast, ACAs in group 1 had no adverse impact on survival when they emerged from chronic phase or at the time of CML diagnosis. The concurrent presence of 2 or more ACAs conferred an inferior survival and can be categorized into the poor prognostic group.

Detection of minimal residual disease in B lymphoblastic leukemia using viSNE.

BACKGROUND: Minimal residual disease (MRD) following treatment is a robust prognostic marker in B lymphoblastic leukemia. However, the detection of MRD by flow cytometric immunophenotyping is technically challenging, and an automated method to detect MRD is therefore desirable. viSNE, a recently developed computational tool based on the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm, has been shown to be capable of detecting synthetic "MRD-like" populations of leukemic cells created in vitro, but whether viSNE can facilitate the immunophenotypic detection of MRD in clinical samples has not been evaluated. METHODS: We applied viSNE retrospectively to 8-color flow cytometric immunophenotyping data from normal bone marrow samples, and samples from B lymphoblastic leukemia patients with or without suspected MRD on the basis of conventional manual gating. RESULTS: In each of 14 bone marrow specimens containing MRD or suspected MRD, viSNE identified a putative MRD population; an abnormal composite immunophenotype was confirmed for the putative MRD in each case. MRD populations were not identified by viSNE in control bone marrow samples from patients with increased normal B-cell precursors, or in post-treatment samples from B lymphoblastic leukemia patients who did not have detectable MRD by manual gating. CONCLUSION: viSNE shows promise as an automated method to facilitate immunophenotypic MRD detection in patients treated for B lymphoblastic leukemia.

Detection of B-cell populations with monotypic light chain expression in cerebrospinal fluid specimens from patients with multiple sclerosis by polychromatic flow cytometry.

BACKGROUND: Flow cytometric immunophenotyping is an established method for the detection of occult leptomeningeal disease in patients with aggressive B-cell non-Hodgkin lymphoma, and is increasingly being used in the evaluation of patients without an established diagnosis of lymphoma who present with signs and/or symptoms referable to the central nervous system. However, the specificity of flow cytometric immunophenotyping in the identification of monoclonal B-cell populations in cerebrospinal fluid (CSF) has not been systematically evaluated. METHODS: We searched a consecutive series of CSF specimens submitted to our laboratory for polychromatic (8-color) flow cytometric immunophenotyping between June, 2010 and December, 2012 for cases in which a B-cell population with monotypic immunoglobulin light chain expression was detected in patients without clinical or radiographic evidence of lymphoma. RESULTS: A B-cell population with monotypic light chain expression was identified in CSF specimens from three patients whose subsequent clinical and radiologic evaluation provided no evidence for lymphoma. In all three patients, a diagnosis of multiple sclerosis was ultimately rendered upon completion of the clinical evaluation. CONCLUSIONS: We conclude that the detection of a B-cell population with monotypic light chain expression in CSF by polychromatic flow cytometry is not diagnostic of occult leptomeningeal involvement by a B-cell non-Hodgkin lymphoma. Moreover, these findings suggest that monotypic B-cell populations detectable by polychromatic flow cytometry may be prevalent in patients with multiple sclerosis, and highlight the importance of clinicopathologic correlation in this application of polychromatic flow cytometry.

Improved compensation of the fluorochrome AmCyan using cellular controls.

BACKGROUND: Implementation of polychromatic flow cytometry in the clinical laboratory often requires the use of newer fluorochromes, with which experience may be comparatively limited. In the course of implementing polychromatic flow cytometry in our laboratory, we have observed significant differences in compensation values derived for the violet-excited dye, AmCyan, when cells rather than a commercially available set of polystyrene microparticles (BD CompBeads) are used as compensation controls. METHODS: Compensation values were calculated for AmCyan and several other fluorochromes using the BD CompBeads Set according to the manufacturer's protocol, and using unstained and singly stained lymphocytes as compensation controls. RESULTS: When the BD CompBeads Set was used to determine compensation values, spillover from AmCyan into V450 was overcompensated, while spillover from AmCyan into FITC was undercompensated. In contrast, when compensation values were calculated using unstained and singly stained lymphocytes, spillover into V450 and FITC from cells stained brightly with AmCyan-conjugates was compensated appropriately. Although significant differences were observed in the compensation of spillover from AmCyan into V450 and FITC using cells rather than the BD CompBeads Set as compensation controls (P < 0.0001, two-tailed Wilcoxon signed-rank test), such differences were not observed in control experiments using fluorochromes excited by the blue (FITC and PE) or red (APC) lasers. CONCLUSION: Improved compensation of the violet-excited dye, AmCyan, is obtained when cells rather than the BD CompBeads Set are used as compensation controls.