Kristy L. Wolniak

Leukemic IDH1 and IDH2 Mutations Result in a Hypermethylation Phenotype, Disrupt TET2 Function, and Impair Hematopoietic Differentiation

Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore, expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort, IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2, and TET2 loss-of-function mutations were associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data, expression of IDH mutants impaired TET2 catalytic function in cells. Finally, either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression, suggesting a shared proleukemogenic effect.

T regulatory cells participate in the control of germinal centre reactions.

Germinal centre (GC) reactions are central features of T‐cell‐driven B‐cell responses, and the site where antibody‐producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity antibodies. These processes require exquisite regulation not only to ensure the production of desired antibodies, but to minimize unwanted autoreactive or low affinity antibodies. To assess whether T regulatory (Treg) cells participate in the control of GC responses, immunized mice were treated with an anti‐glucocorticoid‐induced tumour necrosis factor receptor‐related protein (GITR) monoclonal antibody (mAb) to disrupt Treg‐cell activity. In anti‐GITR‐treated mice, the GC B‐cell pool was significantly larger compared with control‐treated animals, with switched GC B cells composing an abnormally high proportion of the response. Dysregulated GCs were also observed regardless of strain, T helper type 1 or 2 polarizing antigens, and were also seen after anti‐CD25 mAb treatment. Within the spleens of immunized mice, CXCR5+ and CCR7− Treg cells were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Final studies demonstrated administration of either anti‐transforming growth factor‐β or anti‐interleukin‐10 receptor blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Treg cells is important in controlling the GC response. Taken together, these findings indicate that Treg cells contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs.

Characterization of (4-Hydroxy-3-Nitrophenyl)Acetyl (NP)-Specific Germinal Center B Cells and Antigen-Binding B220− Cells after Primary NP Challenge in Mice

Previous studies examining the primary germinal center (GC) response to SRBC in mice demonstrated a steady ratio of IgM+ to isotype-switched GC B cells and a persistent population of GC B cells with a founder phenotype. These characteristics held true at the inductive, plateau, and dissociative phases of the GC response, suggesting a steady-state environment. To test whether these characteristics apply to the primary response of other T cell-dependent Ags, the present study examined the GC response after challenge with (4-hydroxy-3-nitrophenyl)acetyl (NP) in C57BL/6 mice. Multiparameter flow cytometric analysis was used to assess the phenotype of splenic NP-reactive cells at multiple time points after immunization. Results of these studies demonstrated the characteristics of the SRBC-induced GC reaction to be fully maintained in the NP response. In particular, there was a steady ratio of nonswitched to switched B cells, with the majority of NP-reactive GC B cells displaying IgM. In addition, a substantial frequency of B220− NP-binding cells was observed in the spleen at later time points after NP challenge. Although these cells were IgE+, they were found to express both κ and λ L chains and display the high-affinity IgE Fc (FcεRI) receptor, suggesting that this population is not of B cell origin. Adoptive transfer studies further demonstrated the B220− NP-binding subset to be derived from the myeloid lineage.

Expansion of a clonal CD8+CD57+ large granular lymphocyte population after autologous stem cell transplant in multiple myeloma.

Clonal expansions of large granular lymphocytes (LGLs) have been identified in patients following stem cell transplants and may represent posttransplant LGL leukemias or reactive immune responses. To differentiate between these 2 possibilities, we assessed peripheral blood and bone marrow of patients with myeloma after autologous stem cell transplant. All patients examined shortly after autologous stem cell transplant had significant increases in the LGLs in the peripheral blood and bone marrow (71% of lymphocytes) as compared with controls (39%). This increase was detectable years after transplant. The LGLs had a reproducible immunophenotype of CD8+CD57+ T cells without phenotypic abnormalities in 19 of 20 patients. Sixty-five percent of the post-autologous stem cell transplant patients had clonal T-cell receptor gene rearrangements in the bone marrow, yet no patients had neutropenia or splenomegaly. Although the LGL expansions were clonal and persistent, the lack of clinical sequelae suggests the clonal LGL expansion is a reactive, potentially beneficial, immune response to autologous stem cell transplant.

Strong expression of EZH2 and accumulation of trimethylated H3K27 in diffuse large B-cell lymphoma independent of cell of origin and EZH2 codon 641 mutation

Gain-of-function EZH2 mutation promotes H3K27 trimethylation (H3K27me3) and lymphoid transformation of germinal center (GC) derived B-cell lymphoma, such as GCB diffuse large B-cell lymphoma (DLBCL), but not activated B-cell (ABC) DLBCL. It is unclear whether expression levels of EZH2 and consequential H3K27me3 vary by EZH2 mutation and/or cell-of-origin in DLBCL. Ninety lymphoma samples including 40 DLBCLs were studied by immunohistochemistry. EZH2 Y641 mutations were detected in three of 20 (15%) GCB and none of 20 ABC types. All 40 DLBCLs showed strong EZH2, expression with high-level H3K27me3 in 90% GCBs and 95% ABCs. In 50 other B-cell lymphomas except for follicular lymphoma, strong EZH2 expression correlated with high-grade features. Immunoblot of DLBCL cell lines and microarray gene expression study of EZH2 in B-cell lymphomas were consistent with the immunohistochemistry findings. High-level EZH2 and H3K27me3 were common in DLBCL independent of cell-of-origin and EZH2 mutation. High-level EZH2 in lymphoma of aggressive features suggests additional therapeutic targets.

Early Posttransplant Lymphoproliferative Disease

Early posttransplant lymphoproliferative disorders (EPTLDs) represent the first changes in posttransplant lymphoproliferative disorders (PTLDs) morphologic spectrum. EPTLD data are available mostly from case reports and series that include other types of PTLD. Fifteen EPTLDs were reviewed retrospectively. Clinical data, histopathology, clonality, and Epstein- Barr virus (EBV) status were correlated with staining intensity to an antibody for phosphorylated S6 (pS6) ribosomal protein, a downstream effector of mammalian target of rapamycin (mTOR). Median time from transplantation to EPTLD was 50 months (range, 7-135 mo). EPTLDs involved tonsil and/or adenoids (n = 11) and lymph nodes (n = 4), all of which were nonclonal and EBV-encoded RNA–positive. Most (n = 11) were plasmacytic hyperplasia and florid follicular hyperplasia (n = 4). All regressed with reduced immunosuppression, and had increased pS6 staining compared with normal tonsil ( P = .002, F test). EPTLDs developed later than previously reported, involved mostly tonsils/adenoids, were EBV-encoded RNA (EBER) positive, showed increased pS6, and had excellent clinical outcome with reduction of immunosuppression.

EBV-negative aggressive NK-cell leukemia/ lymphoma: A clinical and pathological study from a single institution

Aggressive natural killer (NK)-cell leukemia/lymphoma is a systemic NK-cell neoplasm that preferentially affects Asians with a fulminant clinical course and is almost always associated with Epstein-Barr virus (EBV). The data on EBV-negative aggressive NK-cell leukemia/lymphoma are limited. Here we report a series of three patients (two Caucasians, one African-American) with EBV-negative aggressive NK-cell leukemia/lymphoma from a single institution, including a case diagnosed on post-mortem examination. Similar to EBV-positive aggressive NK-cell leukemia/lymphoma, our patients presented with constitutional symptoms and hepatosplenomegaly, and followed a highly aggressive clinical course. The disease involved peripheral blood, bone marrow, liver, spleen, and lymph node, and the neoplastic cells were pleomorphic with prominent azurophilic granules and demonstrated an atypical NK-cell phenotype. Lack of blood lymphocytosis (3 of 3), bone marrow interstitial infiltration (2 of 3), EBER negativity (3 of 3), and atypical phenotype including CD3 negativity by immunohistochemistry make an early recognition of the disease difficult. Ancillary studies revealed a complex karyotype (1 of 2), overexpression (3 of 3), and amplification (1 of 1) of c-MYC. The polycomb repressive complex 2 pathway-associated proteins EZH2 and H3K27me3 and immune checkpoint protein PD-L1 were overexpressed in three of three and two of three cases, respectively. Our findings indicate that the EBV-negative aggressive NK-cell leukemia/lymphoma shares similar clinicopathological features to the EBV-positive counterpart except for the high prevalence of Asian seen in EBV-positive cases. Overexpression of polycomb repressive complex 2 pathway-associated proteins and PD-L1 suggest potential therapeutic targets for this aggressive disease. Next-generation sequencing on two of three cases identified multiple genetic alterations but were negative for JAK–STAT pathway-associated gene mutations previously reported in EBV-positive NK/T-cell lymphoma, suggesting alternative molecular pathogenic mechanisms for EBV-negative aggressive NK-cell leukemia/lymphoma.

Report of the results of the international clinical cytometry society and american society for clinical pathology workload survey of clinical flow cytometry laboratories

Thorough review of current workload, staffing, and testing practices in clinical laboratories allows for optimization of laboratory efficiency and quality. This information is largely missing with regard to clinical flow cytometry laboratories. The purpose of this survey is to provide comprehensive, current, and accurate data on testing practices and laboratory staffing in clinical laboratories performing flow cytometric studies.

Role of Flow Cytometry in Plasma Cell Neoplasms

Plasma cell neoplasms (PCN) are a heterogeneous group of disorders with a spectrum of clinical presentations from asymptomatic monoclonal gammopathy of undetermined significance (MGUS) to smoldering multiple myeloma to symptomatic multiple myeloma (MM). Other less common categories of PCN include AL amyloidosis, POEMS syndrome, and plasma cell leukemia. Typically, the gold standard for diagnosis has been to correlate morphologic assessment with clinical information (hypercalcemia, renal failure, anemia, and lytic bone lesions) and serologic data, including protein and urine electrophoresis with immunofixation, along with serum-free light chain assay data. As technology has improved, especially monoclonal antibody production and instrumentation, multicolor flow cytometry has become an important tool to demonstrate that the plasma cells present in a bone marrow/tissue/fluid specimen are clonal or immunophenotypically abnormal.

A Small Case Series of Intravascular Large B-Cell Lymphoma with Unexpected Findings: Subset of Cases with Concomitant Extravascular Central Nervous System (CNS) Involvement Mimicking Primary CNS Lymphoma

Background Intravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal lymphoma with growth mainly in the lumina of vessels. We studied a small series of IVLBCL and focused on its central nervous system (CNS) involvement. Methods Searching the medical records of Northwestern Memorial Hospital, we identified five cases of IVLBCL from January 2007 to January 2015. Clinical information, hematoxylin and eosin stained histologic slides and immunohistochemistry studies were reviewed for all cases. Polymerase chain reaction (PCR) analysis for the immunoglobulin (Ig) heavy and light chain gene rearrangement was performed on all five cases. Results Three of the five cases of IVLBCL were autopsies. Patients’ age ranged from 56 to 84. CNS involvement was present in two cases—in both patients, the CNS involvement showed an extravascular pattern with confluent sheet-like formation. PCR analysis confirmed that in one case the systemic intravascular and CNS extravascular components were clonally identical. Conclusions In a small case series of IVLBCL, we observed that CNS involvement by IVLBCL often has an extravascular morphology, but is clonally identical to the intravascular counterpart by PCR analysis. As IVLBCL can have a rapidly progressing poor outcome, it should be kept in the differential diagnoses for patients presenting with lymphoma of the CNS. The presence of extravascular growth patterns in the CNS should not exclude IVLBCL as a diagnosis.