Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.

Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples.

A limited plasma cell flow cytometry panel with reflex CD138 immunohistochemistry is an optimal workflow process for evaluating plasma cell neoplasms in bone marrow specimens.

Objectives: To determine the optimal workflow combination of flow cytometry (FC) and immunohistochemistry tests for efficient and cost-effective evaluation of plasma cell (PC) neoplasms (PCNs) in bone marrow (BM) specimens.

Methods: Various workflow combinations of immunohistochemistry and FC for 4,031 BM specimens worked up for PCNs were compared. Turnaround time (TAT), immunohistochemistry usage, technical charges, and addendum/amendment rates were compared between periods to determine the optimal workflow combination.

Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in the Western hemisphere. Tumor-specific chromosomal translocations, characteristic findings in several human malignancies that directly lead to malignant transformation, have not been identified in CLL. Using paired-end transcriptome sequencing, we identified recurrent and reciprocal RNA chimeras involving yippee like 5 (YPEL5) and serine/threonine-protein phosphatase PP1-beta-catalytic subunit (PPP1CB) in CLL.

A quantitative allele-specific PCR test for the BRAF V600E mutation using a single heterozygous control plasmid for quantitation: a model for qPCR testing without standard curves.

We describe a novel method for mutant allele quantitation using allele-specific PCR. The method uses a heterozygous plasmid containing one wild-type and one mutant sequence as a calibrator that is run at a single concentration, with each quantitative allele-specific PCR run. PCR data from both calibrator alleles, together with predetermined PCR efficiencies, are used to quantitate the mutant allele burden in unknown specimens.

Detection of Merkel cell polyomavirus in chronic lymphocytic leukemia T-cells.

Chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL) is the most common B-cell leukemia/lymphoma, effecting >15,000 patients/year. There has been a proposed limited antigenic etiology, at least in some cases, of CLL/SLL based upon immunoglobulin heavy chain stereotypy found across unrelated cases, suggesting viral source may provide such antigenic stimulation. With an established epidemiological link between CLL/SLL and Merkel cell carcinoma (MCC), there has been some interest in investigating a possible leukemogenic role of Merkel cell polyomavirus (MCPyV), which is found in 80% of MCC cases.

Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL), the most common primary lymphoma of spleen, is poorly understood at the genetic level. In this study, using whole-genome DNA sequencing (WGS) and confirmation by Sanger sequencing, we observed mutations identified in several genes not previously known to be recurrently altered in SMZL. In particular, we identified recurrent somatic gain-of-function mutations in NOTCH2, a gene encoding a protein required for marginal zone B cell development, in 25 of 99 (∼25%) cases of SMZL and in 1 of 19 (∼5%) cases of nonsplenic MZLs.

Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic control antibodies.

Introduction: Expression of ZAP-70 by chronic lymphocytic leukemia (CLL) is associated with more aggressive disease and can help differentiate CLL using mutated immunoglobulin heavy chain variable genes (VH) from cases expressing unmutated VH genes. However, flow cytometric detection of ZAP-70 in CLL shows considerable variability and may be of questionable significance because most laboratories cannot correlate their results to clinical outcome or VH mutational data.

Methods: Seventy cases of CLL were evaluated for ZAP-70 using a previously optimized staining procedure and two different methods to eliminate nonspecific background staining.

A pyrosequencing-based test for detection and relative quantification of the BCR-ABL1 T315I point mutation.

Aims: The BCR-ABL1 T315I mutation imparts resistance to tyrosine kinase inhibitors currently available for treatment of chronic myelogenous leukaemia. Thus, quantitative monitoring of the emergence and expansion of T315I-positive subclones may be clinically useful. The goals of this study were to retrospectively review the authors' experience with Sanger sequencing-based BCR-ABL1 kinase domain mutation testing, paying particular attention to the T315I mutation, and to develop an alternative test for relative quantification of T315I using pyrosequencing.

Combined testing for CCAAT/enhancer-binding protein alpha (CEBPA) mutations and promoter methylation in acute myeloid leukemia demonstrates shared phenotypic features.

Loss of function mutations in CCAAT/enhancer binding protein alpha (CEBPA) have been identified in acute myeloid leukemia (AML) and bi-allelic (double) CEBPA mutations are associated with improved prognosis in cases of cytogenetically normal-AML. In a subset of AML patients lacking CEBPA mutations, core promotor methylation of CEBPA has been described and is associated with a gene expression profile similar to the mutated cases including the expression of T cell associated genes such as CD7. However, the overall incidence and pattern of CEBPA mutations and core promoter methylation has not been thoroughly explored in a larger subset of AML with expression of CD7.

Clinical laboratory analysis of immunoglobulin heavy chain variable region genes for chronic lymphocytic leukemia prognosis.

Chronic lymphocytic leukemia (CLL) is the most common leukemia affecting adults in the western world. The clinical course of CLL is highly variable: cases that express mutated immunoglobulin heavy chain variable regions (IgV(H)) typically have a more indolent clinical course compared with those with unmutated IgV(H). The use of the V(H)3-21 variable region has also been found to confer a poor prognosis, independent of mutation status.

Immunomagnetic bead separation of mononuclear cells from contaminating granulocytes in cryopreserved blood samples.

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads.

Flow cytometric detection and serotyping of enterovirus for the clinical laboratory.

Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA).

Use of similar immunoglobulin VH gene segments by MALT lymphomas of the ocular adnexa.

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue type (MALT lymphomas) develop from acquired reactive infiltrates directed against external or autoantigens. Although some European cases of ocular adnexal MALT lymphoma have been associated with Chlamydia psittaci infections, C. psittaci has not been detected in large studies of US-based cases.

Small B-cell neoplasms with typical mantle cell lymphoma immunophenotypes often include chronic lymphocytic leukemias.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are CD5+ small B-cell neoplasms (SBCNs) with overlapping features. Flow cytometric immunophenotyping is often used to help differentiate CLL from MCL, and a characteristic CLL phenotype is considered essentially diagnostic. However, previous studies have not specifically examined how well a typical MCL immunophenotype distinguishes MCL from CLL.

Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene.

We used BAC array-based CGH to detect genomic imbalances in 187 CLL cases. Submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most common genomic aberration in CLL. Oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.

Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays.

Expression of T-plastin, FoxP3 and other tumor-associated markers by leukemic T-cells of cutaneous T-cell lymphoma.

Peripheral blood cells from 28 patients with leukemic cutaneous T-cell lymphoma including 25 patients with Sezary syndrome were evaluated for expression of regulatory T-cell-associated markers (FoxP3, CD25, CTLA-4, neurophilin-1), T-cell activation markers (CD28 and its ligands B7.1 and B7.2) and NK cell-associated markers (NKG2D and its ligands Mic-A and Mic-B) using real-time quantitative polymerase chain reaction.

Fatal non-transplant-related epstein-barr virus-associated atypical lymphoid proliferations in infants and children: a clinicopathologic study.

Most Epstein-Barr virus (EBV)-related infections in infants and children are asymptomatic or self-limited mild viral illnesses, but rare cases of a rapidly fatal disorder have been described. Failure of the cellular response to control EBV-related lymphoid proliferation leads to severe disease with multiple complications, including a fatal outcome or development of an EBV-driven, clonal lymphoid neoplasm. In this report we characterize 3 cases of fatal, nontransplant, or immunodeficiency-related EBV infection in very young children with immunophenotypic and molecular evidence of B/natural killer (NK)-T cell clonal expansion.

A new DNA-based test for detection of nucleophosmin exon 12 mutations by capillary electrophoresis.

Mutations in nucleophosmin (NPM1) exon 12 are thought to be the most common genetic event in acute myelogenous leukemia (AML) and to confer favorable clinical prognoses. In this report, we describe a simple molecular test for the detection of NPM1 exon 12 mutations in patients with AML using polymerase chain reaction amplification of genomic DNA followed by the analysis of amplification products by capillary electrophoresis. Mutations were reproducibly detected when present in at least 5% of cells, and all NPM1 exon 12 mutations reported to date in AML could be identified using this method.

CD158k/KIR3DL2 is a useful marker for identifying neoplastic T-cells in Sézary syndrome by flow cytometry.

Enumeration of neoplastic T-cells in peripheral blood specimens is necessary for the diagnosis of Sézary syndrome (SS) and monitoring treatment responses. Because neoplastic T-cells in SS can be difficult to identify by morphology alone, flow cytometry immunophenotyping is often utilized. However, the reported immunophenotypic criteria for identifying neoplastic T-cells in SS are variable, not present in all cases, or sometimes found in reactive T-cell populations.

Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia.

Background: The goal of this study was to optimize a cell staining procedure for flow cytometric detection of zeta-chain associated protein-70 (ZAP-70). Our specific objectives were to improve antibody selection criteria, identify a cell permeabilization procedure better tailored to ZAP-70 analysis, as well as to establish objective criteria to control antigen stability.

Methods: Sequentially titrated 2F3.

Quantitation of plasma cells in bone marrow aspirates by flow cytometric analysis compared with morphologic assessment.

Context: Accurate quantitation of bone marrow plasma cells is an important component in the diagnosis and posttreatment assessment of plasma cell dyscrasias. Although flow cytometry is sometimes used for this purpose and can rapidly evaluate many cells, the accuracy of flow-based plasma cell quantitation compared with morphologic assessment (currently the gold standard) is uncertain as direct comparison studies have not been previously reported.

Objective: To determine how percentages of plasma cells in diagnostic aspirate smears quantitated by morphologic assessment relate to percentages of plasma cells quantified by flow cytometry.

Analysis of immunoglobulin V genes suggests cutaneous marginal zone B-cell lymphomas recognise similar antigens.

Extranodal marginal zone B-cell lymphomas (EMZL) are thought to develop from reactive infiltrates that represent immune responses to external or auto-antigens. Except for gastric EMZL, the antigenic triggers of EMZL development are mostly unknown, although a subset of cutaneous EMZL have been associated with Borrelia burgdorferi infections. To further evaluate whether a common antigen may be promoting the development of cutaneous EMZL, the immunoglobulin heavy chain variable (V(H)) genes from eight USA cases were sequenced and analysed.

T-cell large granular lymphocyte leukemias have multiple phenotypic abnormalities involving pan-T-cell antigens and receptors for MHC molecules.

T-cell large granular lymphocyte (T-LGL) leukemias represent monoclonal T-cell expansions that express CD16, CD56, or CD57 and cause cytopenias. The identification of T-LGL leukemias can be difficult because reactive T-LGL cells also can express CD16, CD56, and CD57, and many leukemia cases show only mild lymphocytoses. In this study, 23 T-LGL leukemia cases were analyzed by 3- and 4-color flow cytometry to identify markers that could aid in discriminating leukemic from normal T-LGL.

Flow cytometric analysis of different CD14 epitopes can help identify immature monocytic populations.

Accurate diagnosis of monocytic neoplasms often is dependent on quantitating monoblasts, promonocytes, and monocytes. However, distinguishing these populations by morphologic assessment alone can be difficult and subject to significant intraobserver variability. We evaluated a 4-color flow cytometry technique using different anti-CD14 antibodies that recognize the MO2 and MY4 epitopes to identify monoblasts, promonocytes, and monocytes.