Subject(s)
Anemia , Mental Disorders , Aged , Anemia/complications , Anemia/diagnosis , Female , HumansABSTRACT
Myeloid neoplasms occasionally occur in patients with sickle cell disease, and the underlying connection between the two diseases is unclear. Herein, we retrospectively analyzed four cases of sickle cell disease patients who developed myeloid neoplasm. Age at time of diagnosis ranged from 27 to 59 years with a median of 35.5 years. Two patients were treated with hydroxyurea and the other two with supportive care alone, with one out of the four patients receiving additional treatment with hematopoietic stem cell transplant. Three patients presented with leukocytosis, and the remaining patient presented with pancytopenia. Two patients were diagnosed with myelodysplastic syndrome/myeloproliferative neoplasm, one with myelodysplastic syndrome, and the other with acute myeloid leukemia. All four cases demonstrated certain degrees of myelodysplasia and complex cytogenetic abnormalities with - 7/7q- and/or - 5/5q- or with 11q23 (KMT2A) rearrangement. This cytogenetic profile resembles that seen in therapy-related myeloid neoplasm, suggesting that myeloid neoplasm in the setting of sickle cell disease may represent a subcategory of the disease distinct from de novo myeloid neoplasm in general. Extensive literature review further demonstrates this similarity in cytogenetic profile, as well as in other associated pathologic features. Potential etiology includes therapy for sickle cell disease, disease-related immunomodulation, or disease-related chronic inflammation. We hypothesize that constant hematopoietic hyperplasia, stimulated by a hemolysis-induced cytokine storm, may increase the chance of somatic mutations/cytogenetic aberrations, resulting in transformation of myeloid precursors. This group of myeloid neoplasms seems to herald a dismal clinical outcome, with median survival <1 year, although the exact pathogenesis and biology of the disease remain to be investigated by large cohorts in future studies.
Subject(s)
Anemia, Sickle Cell/complications , Leukemia, Myeloid, Acute/complications , Myelodysplastic Syndromes/complications , Adult , Chromosome Aberrations , Female , Humans , Leukemia, Myeloid, Acute/genetics , Male , Middle Aged , Myelodysplastic Syndromes/genetics , Retrospective StudiesSubject(s)
Antineoplastic Agents, Immunological/therapeutic use , Bone Neoplasms/drug therapy , Histiocytic Sarcoma/drug therapy , Nivolumab/therapeutic use , Adolescent , Bone Neoplasms/immunology , Bone Neoplasms/secondary , Female , Histiocytic Sarcoma/immunology , Histiocytic Sarcoma/pathology , Humans , Prognosis , Remission InductionABSTRACT
OBJECTIVES: Lineage switch occurs in rare leukemias, and the mechanism is unclear. We report two cases of B-lymphoblastic leukemia (B-ALL) relapsed as acute myeloid leukemia (AML). METHODS: Retrospective review of clinical and laboratory data. RESULTS: Complex cytogenetic abnormalities were detected in B-ALL for both cases with subclone heterogeneity. Postchemotherapy marrow biopsies showed trilineage hematopoiesis without detectable B-ALL. Cytogenetics in both showed stemline abnormalities. The cases were considered "occult" myelodysplastic syndrome (MDS) preceding B-ALL. The patients relapsed 6.5 and 9 months following induction, respectively. Case 1 relapsed as AML-M5 initially, was treated as such, and then relapsed again as B-ALL. Case 2 relapsed as AML-M6. Cytogenetics demonstrated persistent abnormalities. Both patients died soon after relapse. CONCLUSIONS: Lineage switch between B-ALL and AML could be intermediated by occult MDS. A pluripotent progenitor likely undergoes neoplastic transformation, resulting in a genomically unstable clone. This leads to a repertoire of heterogeneous subclones that may be selected by chemotherapy. Lineage switch heralds a dismal clinical outcome.
Subject(s)
Leukemia, B-Cell/genetics , Leukemia, B-Cell/pathology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Neoplasms, Second Primary/genetics , Neoplasms, Second Primary/pathology , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Chromosome Aberrations , Genomic Instability , Humans , Leukemia, Myeloid, Acute/drug therapy , Male , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Young AdultABSTRACT
Plasmacytosis is a common finding in lymph node biopsies and can be seen in diverse circumstances ranging from reactive lymphadenopathy to malignant lymphoma. Familiarity with various histopathologic features of the different entities and awareness of their typical clinical and ancillary study findings are essential for an accurate diagnosis. In this review, we present common and representative nonneoplastic entities and lymphomas that have plasmacytic differentiation or associated plasmacytosis. We focus on the histological classification with an emphasis on the diagnostic approach and areas of diagnostic challenge.
Subject(s)
Lymph Nodes/pathology , Plasma Cells/cytology , Waldenstrom Macroglobulinemia/diagnosis , Waldenstrom Macroglobulinemia/pathology , Biomarkers, Tumor/analysis , Histocytochemistry , Humans , Immunohistochemistry , MicroscopyABSTRACT
BACKGROUND: Flow cytometric immunophenotyping (FCI) is recognized as a rapid, sensitive, and accurate method for diagnosis of B-cell lymphomas. We observed that FCI failed to identify the clonal B-cell population in several cases of large B-cell lymphoma (DLBCL) when tissue samples were prepared by a commercially available mechanical tissue disaggregation method. We tested a manual tissue disaggregation method and compared it with the mechanical method. METHODS: FCI findings from 51 cases of DLBCL processed with the mechanical tissue disaggregation method, 27 cases processed using the manual method, and 15 cases processed using a combination of both methods were compared. The histological and immunohistochemical findings in each case were reviewed. RESULTS: FCI detected a clonal B-cell population in 88.9% of cases processed by the manual tissue disaggregation method, 66.7% of cases processed by a combination of the manual and mechanical disaggregation methods, and in 62.7% of cases processed solely by the mechanical tissue disaggregation method (P < 0.01 Fisher exact). Manual processing yielded positive FCI results in 81.8% of the nodal tissue samples and 93.8% of the extra-nodal tissue samples, whereas mechanical disaggregation was particularly inefficient in preserving large lymphoma cells from extra-nodal tissue: 71.4% of the nodal and 56.8% of the extra-nodal tissue samples processed by the mechanical method showed clonal B-cells by flow cytometry (P < 0.006, Fisher exact). CONCLUSIONS: The diagnostic yield of FCI in DLBCL can be significantly improved by utilizing a manual disaggregation method, particularly in extra-nodal tissue samples. © 2015 International Clinical Cytometry Society.
Subject(s)
B-Lymphocytes/immunology , Flow Cytometry , Immunophenotyping , Lymphoma, Large B-Cell, Diffuse/pathology , Adult , Cell Aggregation/physiology , Female , Flow Cytometry/methods , Humans , Lymphoma, Large B-Cell, Diffuse/diagnosis , Male , Middle Aged , Tissue and Organ Harvesting/instrumentation , Tissue and Organ Harvesting/methodsABSTRACT
We present a case report of an 80-year-old woman with volume overload thought initially to be secondary to heart failure, but determined to be amiodarone-induced acute and chronic liver injury leading to submassive necrosis and bridging fibrosis consistent with early cirrhosis. Her histopathology was uniquely absent of steatosis and phospholipidosis, which are commonly seen in AIC.
ABSTRACT
OBJECTIVES: To compare the features of the blast phase of chronic myelogenous leukemia (CML) in patients treated with tyrosine kinase inhibitors (TKIs) with those in the pre-TKI era. METHODS: Sixty-seven patients with blast phase CML were identified in the Duke Pathology database from 1991 to 2011. The morphology and immunophenotype of blasts were evaluated, along with cytogenetic studies and associated findings in the peripheral blood and bone marrow. RESULTS: In the TKI era, the blasts were more frequently of a type other than the usual myeloid or lymphoid types when compared with the pre-TKI era. Blast phase in TKI-treated patients was associated with a higher peripheral WBC count and a lower blast percentage in the bone marrow. Of the 23 patients with cytogenetic studies during blast phase, additional cytogenetic changes more frequently occurred in patients with an unusual blast type, and some patients showed these changes months before the onset of blast phase. CONCLUSIONS: Blast phase CML in TKI- and non-TKI-treated patients differs in the morphology and immunophenotype of blasts, cytogenetic findings, and associated findings in the peripheral blood and bone marrow.
Subject(s)
Blast Crisis/drug therapy , Blast Crisis/pathology , Protein Kinase Inhibitors/therapeutic use , Adult , Aged , Cytogenetic Analysis/methods , Female , Humans , Immunophenotyping , Male , Middle Aged , Treatment OutcomeABSTRACT
Castleman disease is a rare lymphoproliferative disorder of unknown etiology that most commonly presents as a mediastinal nodal mass or, in the extranodal form of the disease, a mass located in the mediastinum or retroperitoneum. It is exceptionally uncommon for Castleman disease to present in the extremities. We report a rare case of extranodal Castleman disease presenting as a muscular forearm mass. We compare our case with the seven other reported cases in which Castleman disease presented as an isolated soft tissue mass in the extremities.
Subject(s)
Castleman Disease/diagnosis , Muscular Diseases/diagnosis , Aged , Diagnosis, Differential , Female , Forearm/diagnostic imaging , Forearm/pathology , Humans , Lymph Nodes/diagnostic imaging , Lymph Nodes/pathology , Magnetic Resonance Imaging , Rare Diseases/diagnosis , Tomography, X-Ray ComputedABSTRACT
CONTEXT: Plasma cell myeloma and chronic lymphocytic leukemia are both common hematologic malignancies, sharing many epidemiologic features. Concomitant detection of the 2 conditions poses special diagnostic challenges for the pathologist. OBJECTIVE: To describe the pathologic findings in cases of concomitant bone marrow involvement by myeloma and CD5(+) monoclonal B cells and to outline the differential diagnostic possibilities, suggest a workup for correct diagnosis, and examine clinical outcome. DESIGN: Fifteen cases that met the diagnostic criteria were identified from pathology databases at 4 participating institutions. Morphologic findings were reviewed, additional immunohistochemical stains performed, and flow cytometric, cytogenetic, and relevant laboratory and clinical information was summarized. Previously published cases were searched from electronic databases and cross-references. RESULTS: Most patients (13 of 15) were older males. Often (11 of 15) they presented clinically with myeloma, yet had both monotypic plasma cells and B cells in the diagnostic marrow. In 4 patients, myeloma developed 24 months or later after chronic lymphocytic leukemia. In 7 patients, myeloma and CD5(+) B cells showed identical immunoglobulin light-chain restriction. Primary differential diagnoses include lymphoplasmacytic lymphoma, marginal zone lymphoma, and chronic lymphocytic leukemia with plasmacytoid differentiation. CD56 and/or cyclin D1 expression by plasma cells was helpful for correct diagnosis. Most patients in our cohort and published reports were treated for plasma cell myeloma. CONCLUSIONS: Concomitant detection of myeloma and chronic lymphocytic leukemia in the bone marrow is a rare event, which must be carefully differentiated from lymphomas with lymphoplasmacytic differentiation for correct treatment.
Subject(s)
B-Lymphocytes/pathology , Bone Marrow/pathology , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Lymphocytosis/diagnosis , Multiple Myeloma/diagnosis , Plasma Cells/pathology , Aged , Aged, 80 and over , B-Lymphocytes/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , CD5 Antigens/metabolism , Clone Cells , Diagnosis, Differential , Female , Gene Rearrangement, B-Lymphocyte, Light Chain , Humans , Immunoglobulin Light Chains/analysis , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lymphocytosis/genetics , Lymphocytosis/metabolism , Lymphoma, B-Cell, Marginal Zone/diagnosis , Male , Middle Aged , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Neoplasms, Multiple Primary/diagnosis , Neoplasms, Multiple Primary/genetics , Neoplasms, Multiple Primary/metabolism , Neoplasms, Second Primary/diagnosis , Neoplasms, Second Primary/genetics , Neoplasms, Second Primary/metabolism , Plasma Cells/metabolism , Waldenstrom Macroglobulinemia/diagnosisABSTRACT
An elderly patient with watery diarrhea for 3 months received extensive laboratory, radiographic and upper and lower gastrointestinal (GI) endoscopic work up including colonic biopsies, but a diagnosis was not established before death. At autopsy enteropathy associated T-cell lymphoma-type II (EATL-II) with multifocal mucosal involvement of the jejunum was identified. The colon was completely uninvolved grossly and microscopically. The stomach showed only subtle lesions grossly but microscopic examination revealed involvement by lymphoma in the stomach as well as other organs in abdomen and chest. The relationship between celiac disease and enteropathy associated T-cell lymphoma, the practical difficulties in establishing the diagnosis and the pathology of T-cell lymphomas affecting the gastrointestinal tract are discussed.
ABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.
Subject(s)
Genetic Heterogeneity , Lymphoma, Large B-Cell, Diffuse/genetics , Adult , Base Sequence , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases , DNA Mutational Analysis , DNA, Neoplasm/genetics , Exome , Gene Expression , Genetic Variation , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Models, Molecular , Molecular Sequence Data , Molecular Targeted Therapy , Mutation , Oncogenes , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/genetics , Protein Conformation , Proto-Oncogene Proteins c-kit/genetics , Receptor, Platelet-Derived Growth Factor alpha/genetics , Sequence Homology, Nucleic Acid , Signal Transduction/geneticsABSTRACT
AIMS: To determine the roles of the presence of malignancy, tumour proliferation fraction, vascular compromise and therapeutic and diagnostic manipulations in lymph node infarction (LNI). METHODS AND RESULTS: Thirty-five cases of LNI were identified over a 20-year period. Of the 35 patients, 31 (89%) had an underlying malignancy: 27 of the 31 (87%) were haematologic malignancies, the rest being metastatic carcinoma (two), melanoma, and seminoma. Of the four patients without evidence of malignancy, two were diagnosed with viral infection, one had LNI adjacent to a thrombosed pancreas graft, and one was lost to follow-up. Ki67 immunostaining in viable tumour demonstrated a range (5-60%) of proliferation fractions. A history of fine needle aspiration alone was present in seven of the 35 patients (20%), a history of chemotherapy alone in 11 (31%), and a history of both in two (5.7%). Factor VIII immunostaining highlighted thrombosed and recanalized vessels next to the infarction. CONCLUSIONS: Infarction of lymph nodes is associated with previous, concurrent or subsequent diagnosis of malignancy in the vast majority of cases. Chemotherapy or previous fine needle aspiration can precipitate infarction in some cases, but infarction may occur without such intervention, possibly because of an underlying subacute or chronic vascular compromise produced by vascular thrombosis.
Subject(s)
Infarction/pathology , Lymph Nodes/pathology , Lymphatic Diseases/pathology , Lymphoma/pathology , Thrombosis/pathology , Adolescent , Adult , Aged , Biomarkers, Tumor/metabolism , Biopsy, Fine-Needle , Databases, Factual , Factor VIII/metabolism , Female , Humans , Infarction/etiology , Lymph Nodes/blood supply , Lymph Nodes/injuries , Lymph Nodes/metabolism , Lymphatic Diseases/etiology , Lymphoma/complications , Lymphoma/metabolism , Male , Middle Aged , Neck , Thrombosis/complications , Young AdultABSTRACT
PURPOSE: The decision to re-induce patients with acute myeloid leukemia (AML) based on results of the day 14 bone marrow (BM) biopsy is variable and lacks evidence based data. The aim of our review was to evaluate the accuracy of a day 14 BM biopsy in determining the need for re-induction chemotherapy. METHODS: Seventy-four patients with newly diagnosed de novo AML treated with induction chemotherapy were retrospectively reviewed for the purpose of evaluating treatment decisions and outcomes based on their day 14 BM biopsy. Response to therapy in this analysis was based on morphology alone. RESULTS: Of the 74 patients undergoing standard induction, 45 patients (61%) had no evidence of leukemia on their day 14 BM biopsy. Eighteen patients (24%) had definitive residual disease (RD), and 11 patient's (15%) were classified as indeterminate response (IR). Fifteen patients with RD and one with IR underwent re-induction chemotherapy. However, thirteen patients (3 RD and 10 IR) were observed until count recovery without any re-induction therapy. Eleven of these 13 patients who were observed eventually attained a morphologic complete remission (CR), including two patients with RD. CONCLUSIONS: A day 14 BM biopsy may have suboptimal sensitivity for the detection of residual leukemia. Some patients with an IR on day 14 may not require re-induction chemotherapy, but instead, may benefit from careful observation until count recovery to avoid the mortality and morbidity associated with re-induction chemotherapy.
Subject(s)
Bone Marrow/pathology , Induction Chemotherapy , Leukemia, Myeloid, Acute/drug therapy , Adolescent , Adult , Aged , Biopsy , Humans , Leukemia, Myeloid, Acute/pathology , Middle Aged , Retrospective StudiesABSTRACT
Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.
Subject(s)
Burkitt Lymphoma/genetics , Mutation , Ammonia-Lyases/genetics , Base Sequence , Cell Line, Tumor , Chaperonin Containing TCP-1/genetics , DNA Helicases/genetics , DNA-Binding Proteins , GTP-Binding Protein alpha Subunits, G12-G13/genetics , Genes, myc/genetics , Genome, Human , Glutamate Formimidoyltransferase/genetics , Homeodomain Proteins/genetics , Humans , Inhibitor of Differentiation Proteins/genetics , Intracellular Signaling Peptides and Proteins , Lymphoma, Large B-Cell, Diffuse/genetics , Membrane Proteins/genetics , Molecular Sequence Data , Multifunctional Enzymes , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Proto-Oncogene Proteins c-ret/genetics , Sequence Analysis, DNA , Transcription Factors/genetics , Translocation, GeneticABSTRACT
The gastrointestinal tract (GIT) is the most commonly involved site of extranodal lymphomas. The close association between chronic inflammation and specific GIT lymphomas not only provide interesting insights into the pathobiology of lymphomas but also poses unique diagnostic challenges. A clear understanding of marginal zone and mucosa associated lymphoid tissue (MALT) in health and disease is helpful to place GIT lymphomas in proper context. A wide variety of lymphomas besides MALT lymphomas occur in various parts of the GIT. The characteristic pathological, immunophenotypic, and genetic features of different GIT lymphomas categorized according to World Health Organization (WHO) classification are presented. The epidemiological, clinical, and pathological features of lymphomas occurring in each part of the GIT are summarized and the key points regarding lymphomas at each site are emphasized. A tabular summary of the important differential diagnostic considerations at each site is given and suggestions for a minimal diagnostic work up are provided.
Subject(s)
Gastrointestinal Neoplasms/diagnosis , Gastrointestinal Neoplasms/pathology , Lymphoma, B-Cell, Marginal Zone/diagnosis , Lymphoma, B-Cell, Marginal Zone/pathology , Diagnosis, Differential , Gastrointestinal Neoplasms/epidemiology , Gastrointestinal Neoplasms/physiopathology , Humans , Lymphoma, B-Cell, Marginal Zone/epidemiology , Lymphoma, B-Cell, Marginal Zone/physiopathologyABSTRACT
INTRODUCTION: High relapse rates and infections remain primary causes of failure in nonmyeloablative transplantation. Interleukin-2 (IL-2) may stimulate the immune system and improve outcomes. The primary objective of this pilot study was to evaluate the feasibility of administering IL-2 following a T-cell-depleted nonmyeloablative hematopoietic stem cell transplant. METHODS: Patients received T-cell-depleted nonmyeloablative transplant from a matched or mismatched related donor. Those with allogeneic engraftment, Subject(s)
Antineoplastic Agents/administration & dosage
, HLA Antigens/immunology
, Hematopoietic Stem Cell Transplantation
, Histocompatibility
, Interleukin-2/administration & dosage
, Myelodysplastic Syndromes/therapy
, Neoplasms/therapy
, Tissue Donors
, Adult
, Aged
, Antineoplastic Agents/adverse effects
, Chemotherapy, Adjuvant
, Drug Administration Schedule
, Family
, Fatigue/etiology
, Feasibility Studies
, Graft vs Host Disease/immunology
, Hematopoietic Stem Cell Transplantation/adverse effects
, Humans
, Immunosuppressive Agents/therapeutic use
, Interleukin-2/adverse effects
, Middle Aged
, Myelodysplastic Syndromes/immunology
, Neoplasms/immunology
, North Carolina
, Pilot Projects
, Time Factors
, Transplantation Conditioning
, Transplantation, Homologous
, Treatment Outcome
ABSTRACT
A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-ß pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.
Subject(s)
B-Lymphocytes , Gene Expression Profiling/methods , Lymphoma, Large B-Cell, Diffuse/genetics , MicroRNAs/genetics , Base Sequence , Chromatin Immunoprecipitation , Gene Library , High-Throughput Nucleotide Sequencing , Humans , MicroRNAs/analysis , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNAABSTRACT
We describe a simple and robust flow cytometry assay for ZAP-70 and CD38 expression. The steps required to validate this assay in a clinical flow cytometry laboratory are described. Two criteria were used to characterize ZAP-70 expression into positive, negative, and indeterminate categories and applied to 111 cases of chronic lymphocytic leukemia (CLL) resulting in 29.7% positive, 56.8% negative, and 13.5% indeterminate cases. A sensitivity-specificity crossover plot between ZAP-70 and CD38 suggested a cutoff of 12.5% for defining CD38 positivity. ZAP-70+ cases were significantly more likely to be at a higher clinical stage and, together with CD38+ cases, were more likely to have unmutated IgV(H). However, for individual patients, the concordance between these markers was not perfect. It may be necessary to evaluate several prognostic markers simultaneously in CLL, and availability of convenient assays for ZAP-70 and CD38 is desirable for optimal clinical decision making.
Subject(s)
ADP-ribosyl Cyclase 1/blood , Flow Cytometry/methods , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Membrane Glycoproteins/blood , ZAP-70 Protein-Tyrosine Kinase/blood , ADP-ribosyl Cyclase 1/genetics , Biomarkers, Tumor/blood , Chromosome Aberrations , DNA Mutational Analysis , DNA, Neoplasm/analysis , Gene Expression Regulation , Humans , Immunoglobulin Heavy Chains/blood , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Membrane Glycoproteins/genetics , Neoplasm Staging , Predictive Value of Tests , Prognosis , ZAP-70 Protein-Tyrosine Kinase/geneticsABSTRACT
Initial exposure of monocytes/macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Molecular mechanisms responsible for endotoxin tolerance are not well defined. We and others have shown that IL-1R-associated kinase (IRAK)-M and SHIP-1 proteins, negative regulators of TLR4 signaling, increase in tolerized cells. TGF-beta1, an anti-inflammatory cytokine, is upregulated following LPS stimulation, mediating its effect through SMAD family proteins. Using a monocytic cell line, THP1, we show that LPS activates endogenous SMAD4, inducing its migration into the nucleus and increasing its expression. Secondary challenge with high dose LPS following initial low-dose LPS exposure does not increase IRAK-M or SHIP1 protein expression in small hairpin (sh)SMAD4 THP-1 cells compared with control shLUC THP1 cells. TNF-alpha concentrations in culture supernatants after second LPS challenge are higher in shSMAD4 THP-1 cells than shLUC THP1 cells, indicating failure to induce maximal tolerance in absence of SMAD4 signaling. Identical results are seen in primary murine macrophages and mouse embryonic fibroblasts, demonstrating the biological significance of our findings. TGF-beta1 treatment does not increase IRAK-M or SHIP1 protein expression in shSMAD4 THP-1 cells, whereas it does so in shLUC THP1 cells, indicating that TGF-beta1 regulates IRAK-M and SHIP1 expression through a SMAD4-dependent pathway. Knockdown of endogenous SHIP1 by shSHIP1 RNA decreases native and inducible IRAK-M protein expression and prevents development of endotoxin tolerance in THP1 cells. We conclude that in THP-1 cells and primary murine cells, SMAD4 signaling is required for maximal induction of endotoxin tolerance via modulation of SHIP1 and IRAK-M.