Parul Bhargava

Detection of JC Virus DNA and Proteins in the Bone Marrow of HIV-Positive and HIV-Negative Patients: Implications for Viral Latency and Neurotropic Transformation

BACKGROUND We sought to determine the prevalence of JC virus (JCV) in bone marrow samples from human immunodeficiency virus (HIV)-positive and HIV-negative patients and to determine whether bone marrow is a site of latency and neurotropic transformation of JCV, the agent of progressive multifocal leukoencephalopathy (PML). METHODS We collected bone marrow aspirates, archival bone marrow samples, and blood and urine samples from 75 HIV-negative and 47 HIV-positive patients without PML as well as bone marrow and urine or kidney samples from 8 HIV-negative and 15 HIV-positive patients with PML. Samples were tested for JCV DNA by quantitative polymerase chain reaction and for JCV protein expression by immunohistochemical analysis. JCV regulatory regions (RRs) were characterized by sequencing. RESULTS JCV DNA was detected in bone marrow samples from 10 (13%) of 75 and 22 (47%) of 47 of the HIV-negative and HIV-positive patients without PML, respectively, compared with 3 (38%) of 8 and 4 (27%) of 15 of the HIV-negative and HIV-positive patients with PML. JCV DNA (range, 2-1081 copies/microg of cellular DNA) was detected in multiple leukocyte subpopulations of blood and bone marrow samples. JCV large T antigen, but not VP1 capsid protein, was expressed in bone marrow plasma cells. Bone marrow JCV RR sequences were similar to those usually found in the brains of patients with PML. CONCLUSIONS Bone marrow is an important reservoir and a possible site of neurotropic transformation for JCV.

Rearrangement of the JC virus regulatory region sequence in the bone marrow of a patient with rheumatoid arthritis and progressive multifocal leukoencephalopathy

The polyomavirus JC (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML). JCV remains quiescent in kidneys, where it displays a stable archetypal regulatory region (RR). Conversely, rearranged JCV RR, including tandem repeat patterns found in the central nervous system (CNS) of PML patients, have been associated with neurovirulence. The precise site and mechanism of JCV RR transformation is unknown. We present herein a patient with rheumatoid arthritis treated with methotrexate, who developed PML and had a rapid fatal outcome. JCV DNA polymerase chain reaction (PCR) was positive in cerebrospinal fluid (CSF), bone marrow, blood, and urine. Double-immunohistochemical staining demonstrated that 9% of bone marrow CD138+ plasma cells sustained productive infection by JCV, accounting for 94% of JCV-infected cells. JCV RR analysis revealed archetype and rearranged RR forms in bone marrow, whereas RR with tandem repeat was predominant in blood. These results suggest that the bone marrow may be a potential site of JCV pathogenic transformation. Further studies will be needed to determine the prevalence of JCV in bone marrow of immunosuppressed individuals at risk of PML and characterize the RR and phenotype of these JCV isolates.

Qualitative mapping of Barrett’s metaplasia: a prerequisite for intervention trials

BACKGROUND Barretts esophagus may present as a cellular mosaic with irregular longitudinal extensions of intestinal epithelium, spotty areas of dysplasia and other intermediate markers for cancer risk. It may not be possible to detect and reproducibly localize these findings with routine endoscopic biopsies. A more systematic biopsy protocol is necessary for chemopreventive studies to be feasible. METHODS Utilizing an adapted upper endoscope that allows accurate evaluation of distance from the incisors and rotatory position, chromoendoscopy with toluidine blue and systematic mapping (4 quadrant jumbo biopsies at 1 cm intervals) were performed twice on 18 patients with Barretts esophagus (second procedure 1 to 3 months after baseline study). All biopsy specimens were subjected to routine and immunohistochemical staining and flow cytometry to create baseline and follow-up maps for each patient. Eight of the 18 patients also underwent standard surveillance biopsies within 6 months of the systematic mapping procedures. RESULTS Epithelium type was reproducibly identified with 94% accuracy on second endoscopic maps. Ploidy, p53, and Ki-67 status were also reproducibly identified on second endoscopic maps (97%, 89%, and 85%, respectively). Dysplasia was found in 7 of 18 patients at similar sites at each mapping procedure (3 patients with high-grade dysplasia, 4 with low-grade dysplasia). Five of the patients who had dysplasia on mapping had also undergone standard surveillance. Low-grade dysplasia was missed in 2 of 3 patients and 1 patient with high-grade dysplasia had only low-grade dysplasia detected with standard biopsies. CONCLUSIONS Utilizing a modified gastroscope and this methodology, we reliably located sites of dysplasia and other biomarkers within a field of Barretts esophagus. Patients had variable areas of dysplasia that were missed on standard endoscopic surveillance.

Mucin 1 is a potential therapeutic target in cutaneous T-cell lymphoma

Cutaneous T-cell lymphoma (CTCL) is an aggressive neoplasm with limited treatments for patients with advanced disease. The mucin 1 C-terminal subunit (MUC1-C) oncoprotein plays a critical role in regulating cell proliferation, apoptosis, and protection from cytotoxic injury mediated by reactive oxygen species (ROS). Although CTCL cells exhibit resistance to ROS-induced apoptosis, the expression and functional significance of MUC1 in CTCL have not been previously investigated. Present studies demonstrate that MUC1-C is overexpressed in CTCL cell lines and primary CTCL cells but is absent in resting T cells from healthy donors and B-cell lymphoma cells. We have developed a cell-penetrating peptide that disrupts homodimerization of the MUC1-C subunit necessary for its nuclear translocation and downstream signaling. We show that treatment of CTCL cells with the MUC1-C inhibitor is associated with downregulation of the p53-inducible regulator of glycolysis and apoptosis and decreases in reduced NAD phosphate and glutathione levels. In concert with these results, targeting MUC1-C in CTCL cells increased ROS and, in turn, induced ROS-mediated late apoptosis/necrosis. Targeting MUC1-C in CTCL tumor xenograft models demonstrated significant decreases in disease burden. These findings indicate that MUC1-C maintains redox balance in CTCL cells and is thereby a novel target for the treatment of patients with CTCL.

CD79a Is Heterogeneously Expressed in Neoplastic and Normal Myeloid Precursors and Megakaryocytes in an Antibody Clone–Dependent Manner

CD79a, a component of the B-cell antigen receptor complex, can also be expressed in certain non-B-cell malignancies. The reported frequency of CD79a expression in acute myeloid leukemias (AML) ranges from 0% to 90%. We evaluated 39 bone marrow biopsy specimens (29 AML and 10 normal cases) using 5 different commercially available anti-CD79a monoclonal antibody (MoAb) clones. Of 7 acute promyelocytic leukemia (APL) cases, 6 (86%) stained for CD79a with clones HM47/A9 (Novocastra, Newcastle Upon Tyne, England) and HM57 (DAKO, Carpinteria, CA) but were negative with clones 11E3 (Novocastra), and JCB117 (DAKO). Half of 6 acute megakaryoblastic leukemia (AMKL) cases and normal megakaryocytes in 14 (67%) of 21 cases were immunoreactive using clone 11D10 (Novocastra). Approximately one third of non-APL/non-AMKL AML and myeloid precursors in normal marrow specimens stained with clones HM57 and 11D10. This heterogeneity of CD79a expression in AML, megakaryocytes, and myeloid precursors is MoAb clone-dependent, likely owing to different epitope detection, and may be of diagnostic usefulness.

Pitfalls of Diagnosis Based on Abnormal Flow Cytometry and [18F]Fluorodeoxyglucose Positron Emission Tomography

A 30-year-old, HIV-positive man with a previous history of an atypical nasopharyngeal Burkitt lymphoma developed fluorodeoxyglucose (FDG) avidity on a routine FDG-positron emission tomography (PET)/computed tomography scan performed 10 months after the completion of all treatment. This new FDG-avid disease was in the area of his initial disease. Flow cytometric assessment of a fine needle aspiration showed a CD10-expressing B-cell population with kappa predominance. The corresponding cytology smears had large atypical lymphoid cells along with smaller lymphocytes and macrophages. Because of the patients previous history of a CD10(+), high-grade B-cell lymphoma, the cytologic and flow cytometric findings were considered highly suspicious for a B-cell lymphoma. Because the differential diagnosis included a relapsed Burkitt lymphoma versus a second, unrelated lymphoma (the former with a dismal prognosis) it was deemed prudent to obtain more tissue via an open biopsy for confirmation of diagnosis and exact subclassification. An open biopsy, however, revealed a reactive lymph node with enlarged geographic follicles; no lymphoma was demonstrable and c-Myc studies were negative. The patient remains without evidence of disease. Retrospectively, the original flow cytometric assessment was believed to likely represent sampling of hyperplastic germinal centers with significantly expanded CD10(+) B cells. The FDG uptake and the kappa predominance further confounded the interpretation. This case illustrates the pitfalls of standard diagnostic techniques, including PET scanning, cytology, and flow cytometry, particularly in the setting of HIV. It further underscores the importance of adequate clinical correlation and a low threshold for performing open biopsies in such patients.

Recurrent histiocytic necrotizing lymphadenitis with a long latency in a patient with autoimmunity: a case report and review of literature.

Kikuchi–Fujimoto disease (KFD), a histiocytic necrotizing lymphadenitis (HNL), characteristically presents as cervical lymphadenopathy in young Asian women. Most resolve spontaneously with rare recurrences described. We report a patient with biopsy-proven recurrence of KFD-like HNL after almost 8 years and analyze 65 additional published cases with recurrences. While those with recurrences similarly affect young (average age = 27 years), Asian (80%) women (76%), 73% had multiple sites of involvement and 32% of those tested had underlying autoimmune conditions. Our case is unusual with respect to the following: (a) Age: 50 years, the oldest among the reported patients with recurrences. (b) Race: African descent, with only 3 others reported with recurrent HNL. Of these 4 cases, 2 had underlying autoimmunity. (c) Underlying condition: Her clinical and laboratory features were best felt to represent Sjögren’s syndrome (SjS). Only 2 other cases of SjS-associated HNL have been reported; in 2 recently reported cases SjS developed subsequently.

Plasmacytoid dendritic cells in lymph nodes of patients with human immunodeficiency virus.

Circulating plasmacytoid dendritic cells (PDC) decrease in human immunodeficiency virus (HIV) infection, either from loss or redistribution to lymph nodes (LN). Limited animal and human studies variably showed increased or decreased nodal PDC. CD123 immunostaining was performed on 28 archived LN biopsies (20 reactive) from 25 HIV patients. PDC clustering was graded (1: none; 2: rare small; 3: medium-sized, loose; and 4: large tight clusters) and correlated with HIV-lymphadenitis stage, blood CD4 counts, time since HIV diagnosis, and treatment duration. Increased PDC clustering was seen with decreasing CD4 counts (P=0.001), shorter treatment duration (P=0.0268), and advancing HIV-lymphadenitis stage (P=0.06). No correlation with time since HIV diagnosis was noted. To our knowledge, this is the first human study assessing relationship of nodal PDC in HIV to CD4 counts, treatment duration, and lymphadenitis pattern. Our findings suggest that PDC redistribute to LN with advancing immunodeficiency and stage of HIV infection.

Utility of fascin and JunB in distinguishing nodular lymphocyte predominant from classical lymphocyte-rich Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and lymphocyte-rich classical Hodgkin lymphoma (LRCHL), although clinically and morphologically similar, differ biologically and in prognosis. Immunolabeling of Reed-Sternberg (RS) cells in LRCHL and lymphocytic and/or histiocytic variants (L&H cells) in NLPHL is often required to help distinguish between the 2 variants. Our aim was to evaluate fascin (a distinct 55-kd actin-bundling protein) and JunB (an activator protein-1 family transcription factor) to differentiate NLPHL from LRCHL. A total of 35 archival cases of NLPHL (n=24) and LRCHL (n=11) from adults and children were studied. Slides were reviewed for all cases and clinical, morphologic, and immunohistochemical features were evaluated. Each case was immunostained for fascin and JunB, and immunoreactivity of RS cells, L&H cells, and background lymphocytes were recorded. Whereas occasional L&H cells were weakly positive for fascin in 3 out of 24 (12.5%) cases of NLPHL, RS cells in LRCHL were positive for fascin in 11 out of 11 (100%) cases with a strong cytoplasmic staining pattern. JunB was positive in 10 out of 24 (41.7%) of NLPHL cases, and 11 out of 11 (100%) of LRCHL cases, showing a stippled and/or diffuse nuclear staining pattern. In addition to L & H Cells, JunB also stained small background lymphocytes, particularly in areas of progressively transformed germinal centers of NLPHL. Either stains when tested alone, if negative, or with rare L&H cell weak positivity for fascin, is indicative of NLPHL. The L&H cells of NLPHL cases were negative for concomitant staining in 24 out of 24 (100%) cases. Concomitant positive staining of classic RS cells for fascin and JunB was found in 11 out of 11 (100%) of LRCHL cases. Although fascin positivity alone supports the diagnosis of LRCHL, concomitant positivity offers stronger support and is less likely to lead to a false conclusion if aberrant fascin staining were to be encountered in a case of NLPHL. Staining for fascin and JunB provides a basis for distinguishing NLPHL from LRCHL and offers an alternative to other antibody profiles.

A rare finding of brown fat in bone marrow as a mimic for metastatic disease.

A 54-year-old woman presented with newly elevated CA 27.29, a tumor marker for breast cancer, which rose from 32 U/mL (9 months prior) to 61 U/mL (reference range 0–39 U/mL) and was verified on repeat testing. She had a significant history of bilateral breast carcinoma, both right-sided ductal carcinoma and left-sided lobular carcinoma, status post bilateral mastectomies five years prior. Her surgical history also included a remote resection for Stage I renal cell carcinoma, and resections for multiple parathyroid adenomas. Additionally, her medical history included hypertension, Hashimoto’s thyroiditis, osteopenia secondary to hyperparathyroidism, and prior lithium toxicity resulting in Stage III/IV chronic kidney disease. Persistent elevation in the tumor marker prompted a PET-CT scan, which revealed a sclerotic lesion within the right sacral ala (Image 1A). A CT-guided biopsy was performed to rule-out metastatic disease. Histopathologic examination unexpectedly revealed a focus of brown adipose tissue. No carcinoma was seen. One-third of the core length displayed trilineage hematopoietic marrow with an age-appropriate cellularity of 30–40% (Image 1B, left half). The remaining core length was hypocellular, and largely devoid of hematopoietic precursors, with an abrupt transition to interstitial brown fat cells present singly and with focal clustering (Image 1B, right half & inset). There appeared to be prominent background vascularity, focal bony remodeling, and some stromal damage. Sections of the marrow clot showed hematopoietic elements with occasional scattered brown fat cells. Immunohistochemical stains were performed to show that these cells were immunoreactive for S100 (Image 1C) and negative for macrophage marker CD68 (Image 1D), consistent with brown adipocytes. CD20 and CD3 stained a subset of background lymphocytes; no lymphoid aggregates were appreciated. Mast cell tryptase highlighted few scattered mast cells, with no clustering. CD117 also highlighted mast cells, as well as normal myeloid elements. Cytokeratin was negative. Brown fat is a form of fetal/neonatal adipose tissue involved in thermoregulation. It gradually disappears with age, but can persist in adults, primarily in the interscapular region, axilla, chest wall, mediastinum, and retroperitoneum. These are sites where hibernomas normally arise. Hibernomas are benign tumors of brown fat, equivalent to lipomas in white fat. While white fat is a normal component of bone marrow, the finding of Image 1. A: CT scan showing sclerotic lesion of the right sacral ala. B: Bone biopsy, H&E: Trilineage hematopoiesis with abrupt transition (marked by black arrows) to brown fat. Inset (top right) shows high power image of adipocytes with multiple small cytoplasmic vacuoles (brown adipocytes). C,D: Immunohistochemical stains show that these cells are immunoreactive for S100 (C) and negative for macrophage marker CD68 (D), consistent with brown adipocytes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]