High-resolution melting assay for rapid, simultaneous detection of JAK2, MPL and CALR variants.

AIMS: Identification of recurrent genetic alterations in JAK2, MPL and CALR remains crucial in the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPNs). Current laboratory testing algorithms may entail batching and/or sequential testing, involving multiple testing modalities and sometimes send-out testing that increase the technical and economic demands on laboratories while delaying patient diagnoses. To address this gap, an assay based on PCR and high-resolution melting (HRM) analysis was developed for simultaneous evaluation of JAK2 exons 12-14, MPL exon 10 and CALR exon 9, embodied in the HemeScreen® (hereafter 'HemeScreen') MPN assay. METHODS: The HemeScreen MPN assay was validated with blood and bone marrow samples from 982 patients with clinical suspicion for MPN. The HRM assay and Sanger sequencing were performed in independent Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories with Sanger sequencing (supported by droplet digital PCR) serving as the gold standard. RESULTS: HRM and Sanger sequencing had an overall concordance of 99.4% with HRM detecting 133/139 (96%) variants confirmed by sequencing (9/10 MPL, 25/25 CALR, 99/104 JAK2), including 114 single nucleotide variants and 25 indels (3-52 bp). Variants consisted of disease-associated (DA) variants (89%), variants of unclear significance (2%) and non-DA variants (9%) with a positive predictive value of 92.3% and negative predictive value of 99.5%. CONCLUSIONS: These studies demonstrate the exquisite accuracy, sensitivity and specificity of the HRM-based HemeScreen MPN assay, which serves as a powerful, clinically applicable platform for rapid, simultaneous detection of clinically relevant, somatic disease variants.

Aggressive B cell lymphomas in the 2017 revised WHO classification of tumors of hematopoietic and lymphoid tissues.

The recent 2017 update of the World Health Organization classification of lymphomas has significant changes from the previous edition. Subtypes of large B cell lymphoma and related aggressive B cell lymphomas are addressed. Clinicopathological features of entities as related to morphology, immunophenotype, cell of origin, and molecular/genetic findings are reviewed with emphasis on changes or updates in findings. Specific subtypes addressed include: T cell/histiocyte-rich large B cell lymphoma, primary diffuse large B cell lymphoma (DLBCL) of the CNS, primary cutaneous DLBCL leg-type, EBV-positive DLBCL, NOS, DLBCL associated with chronic inflammation, primary mediastinal large B cell lymphoma, intravascular large B cell lymphoma, ALK-positive large B cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, HHV8-positive diffuse large B-cell lymphoma, NOS, Burkitt lymphoma, Burkitt-like lymphoma with 11q aberration, high-grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, high grade B cell lymphoma, NOS, B cell lymphoma, unclassifiable, with features intermediate between DLBCL and classic Hodgkin lymphoma and large B cell lymphoma with IRF4 translocation. In addition, EBV positive mucocutaneous ulcer is addressed.

Evidence of BRAF V600E in indeterminate cell tumor and interdigitating dendritic cell sarcoma.

BRAF V600E mutations have been reported in several histiocytic and dendritic cell neoplasms. In this case series, we report BRAF V600E-positive histiocytic and dendritic cell neoplasms in association with lymphomas and lymphoid proliferations. This is a review of cases with immunohistochemistry for BRAF V600E, with additional immunohistochemistry to categorize tumors. We report the first case of BRAF V600E-positive indeterminate cell tumor in association with angioimmunoblastic T-cell lymphoma. We also report a case of BRAF V600E-positive interdigitating dendritic cell sarcoma in a patient with positive B-cell polymerase chain reaction. It is unclear if these neoplasms developed as transdifferentiation of lymphoid neoplasms or if they developed independently. These cases illustrate the expanding spectrum of BRAF V600E-positive histiocytic and dendritic cell tumors and suggest that attention should be paid to lymphomas for possible coincident presentation of these disorders.

Utility of BCL2, PD1, and CD25 immunohistochemical expression in the diagnosis of T-cell lymphomas.

T-cell lymphomas (TCLs) are a heterogenous group of diseases that show histologic and immunophenotypic features overlapping with reactive lymphoid proliferations and often require the use of ancillary testing for accurate diagnosis. The oncoprotein, bcl-2, is expressed in various types of lymphoma. At present, expression of this protein is useful for distinguishing several B-cell lymphomas. Although there are some anecdotal reports that the lack of bcl-2 expression by T cells might also be a useful marker for the diagnosis of TCL, there are no focused studies to address this hypothesis. Another antigen with value in TCL diagnosis is programmed death-1 (PD-1), a marker of follicular helper T cells, which has been reported to be sensitive in the detection of angioimmunoblastic TCL and peripheral T-cell lymphoma, unclassified. However, several reports have also shown that PD-1-positive cells may be increased in a number of settings other than TCL, including reactive and atypical lymphadenopathies. Finally, lymphoma cells express a variety of cytokine receptors and signaling molecules that are current or potential targets for immunomodulatory therapy. One such target is the interleukin (IL)-2 receptor (CD25), which is acted on by denileukin diftitox/ONTAK, a recombinant diphtheria toxin-IL-2 fusion protein. Selection of suitable patients for therapy often includes pretreatment assessment of CD25 expression in tumor cells. In order to further assess the diagnostic and therapeutic utility of these antigens, we compared the expression of the CD25, PD-1, and bcl-2 in 119 cases of T-cell non-Hodgkin lymphoma using immunohistochemical techniques applied to routinely processed and paraffin-embedded tissues. We show that lack of expression of bcl-2 was observed in 52% cases of TCL and may aid in identification of neoplastic T-cell populations. In combination, bcl-2, CD25, and PD-1 provide diagnostic utility and may aid in selecting appropriate patients for immunomodulatory therapy.

Abnormal expression of CD20 on IgG4 plasma cells associated with IgG4-related lymphadenopathy.

CONTEXT: Immunoglobulin G4 (IgG4)-related disease is a recently described entity that presents as mass-forming lesions in soft tissue, exocrine glands, and in lymph nodes as IgG4-related lymphadenopathy. The underlying pathologic mechanism of IgG4-related disease is unclear; however, rituximab (an anti-CD20 monoclonal antibody) has been shown to have clinical efficacy. OBJECTIVE: To look for the presence or absence of CD20 on the IgG4-expressing plasma cells in IgG4-related lymphadenopathy. DESIGN: Twelve flow cytometry cases were identified through a retrospective review from the authors' institutions files. Cases were selected by the presence of a lymph node biopsy specimen with increased IgG4 plasma cells by immunohistochemistry and a histologic diagnosis compatible with IgG4-related lymphadenopathy. RESULTS: We report dim CD20 expression on plasma cells in all cases for which a plasma cell population was clearly identified by flow cytometry. These cases were from patients with lymph node biopsy specimens that met published criteria for IgG4-related lymphadenopathy. CONCLUSIONS: This finding may be one potential explanation for the clinical efficacy of rituximab in IgG4-related disease.

Reactive lymphadenopathies that mimic lymphoma: entities of unknown etiology.

Kikuchi-Fujimoto disease, Kimura disease, Rosai-Dorfman disease and IgG4 related lymphadenopathy may present with enlarging masses clinically mimicking lymphoma. A combination of clinical and histologic findings is necessary to diagnose these important rare entities, which may occasionally have aggressive clinical behavior. The recognition of these disorders is important in order to avoid misdiagnosis of malignancy, other systemic diseases such as systemic lupus, and to institute correct management and therapy, such as steroid treatment for IgG4 related lymphadenopathy. The underlying etiologies of these diseases are not completely clear at present, however, their recognition has become more common as diagnostic techniques improve. Their diagnosis and recognition may help to elucidate their underlying pathobiology.

Histopathological findings in 29 lymph node biopsies with increased IgG4 plasma cells.

IgG4-related sclerosing disease encompasses a family of disorders associated with increased numbers of IgG4 plasma cells and mass forming lesions in various tissues. Lymphadenopathy is a common finding, seen in up to 80% of cases. In the largest series of cases to date, we describe histologic, immunohistochemical, special stain and flow cytometric findings in 29 cases of enlarged lymph nodes with increased IgG4 plasma cells. Lymph node biopsies showed all resection specimens; no needle core biopsies of tissue were evaluated. Cases were considered to have increased numbers of IgG4 plasma cells using the histological criteria outlined by Cheuk and Chan (2010): IgG4 plasma cells >50 cells in a high-power field and >40% of IgG-positive plasma cells positive for IgG4. Additionally, increased intrafollicular plasma cells were a common finding. The lymph nodes showed a variety of reactive histological features including follicular hyperplasia, progressive transformation of germinal centers, interfollicular expansions, variable degrees of fibrosis, increased histiocytes and occasionally an appearance similar to that of plasma cell Castleman disease.

Necessity of bilateral bone marrow biopsies for ancillary cytogenetic studies in the pediatric population.

The need for bilateral pediatric bone marrow biopsies for cytogenetic and fluorescent in situ hybridization (FISH) studies has not been clearly delineated. We retrospectively identified 166 pediatric bilateral bone marrow biopsy specimens obtained from patients with a variety of clinical diagnoses, including solid tumors, lymphoma, leukemia, and other hematologic conditions. The cases included all pediatric bilateral bone marrow biopsies performed at our hospital spanning the years of 1992 to 2008. Agreement of FISH and classical cytogenetic results between the 2 sides was assessed. Of a total of 166 bilateral cases, 2 cases showed disagreement (1.2%), both from patients with solid tumors. One case was a rhabdomyosarcoma, in which FISH only was performed; the second was a neuroblastoma in which FISH and cytogenetics were performed (both FISH and classical cytogenetic results disagreed). The remainder of the cases showed complete agreement between the 2 sides (total 98.8%). We conclude that it is usually not necessary to perform bilateral bone marrow biopsies for FISH and cytogenetics in the pediatric population outside of the setting of solid tumor staging.

Bone acquisition in healthy young females is reciprocally related to marrow adiposity.

CONTEXT: Considerable evidence indicates that osteoblasts and adipocytes share a common progenitor cell in the bone marrow that is capable of mutually exclusive differentiation into the cell lineages responsible for bone and fat formation. OBJECTIVE: The purpose of this study was to examine the relation between bone acquisition and changes in marrow adiposity. DESIGN: This was a longitudinal study. OUTCOME MEASURES AND SUBJECTS: Computed tomography measurements of femoral cortical bone area (CBA), cross-sectional area (CSA), and marrow density, and dual-energy x-ray absorptiometry (DXA) measurements of total body fat and lean mass (LM) were obtained in 39 healthy females (15-20 yr of age) at baseline and 18-24 months later. RESULTS: Marrow adiposity was inversely related to CBA at baseline and follow-up (r = 0.39 and 0.33; P = 0.015 and 0.039, respectively) but was not associated to CSA (r = 0.19 and 0.17; P = 0.24 and 0.32, respectively). The association between marrow fat and CBA persisted, even after controlling for body mass and DXA values of LM and femoral CSA. Gains in CBA during the course of the study were related to decreases in marrow fat (r = 0.41; P = 0.009), a relation that persisted, even after accounting for changes in bone size. Marrow fat was not associated to anthropometric measures or DXA values of body fat and LM (all r's between -0.15 and 0.19; P > 0.05). CONCLUSIONS: Bone acquisition in the appendicular skeleton of healthy young females is inversely related to changes in marrow adiposity. These results provide support for the growing body of evidence indicating an inversely coupled relationship between osteogenesis and adipogenesis in the skeleton.

Transoral Surgical Approach for Retropharyngeal Node Involvement in I-131-Negative 18-fluoro-2-deoxyglucose Positron Emission Tomography-Positive Recurrent Thyroid Cancer.

Transoral pharyngotomy is a viable minimally invasive approach for resection of metastatic thyroid cancer in retropharyngeal lymph nodes in highly select patients. A few authors have already reported on its safe application with excellent outcomes. We herein describe a case where the technique is assisted with the Omniguide CO(2) laser system to safely access and remove a metastatic node in the retropharyngeal space of a 24 year-old Caucasian woman. Furthermore, her disease was I-131-negative and positron emission tomography-positive, demonstrating the technique is still feasible in this dedifferentiated cancer state.