Carol J. Holman

Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.

A Prospective Clinical Study of Epstein‐Barr Virus and Host Interactions during Acute Infectious Mononucleosis

BACKGROUND Characterizing virus-host interactions during self-limited infectious mononucleosis could explain how Epstein-Barr virus (EBV) replication is normally controlled and provide insight into why certain immunocompromised patients fail to contain it. METHODS University students had an average of 7 clinical and virologic evaluations during acute infectious mononucleosis. EBV was quantified in 697 samples of oral wash fluid, whole blood, peripheral blood mononuclear cells (PBMCs), and plasma by a real-time (TaqMan) polymerase chain reaction (qEBV) assay developed in our laboratory. RESULTS Twenty of 25 subjects had serologically confirmed primary EBV infection. EBV was cleared from whole blood by a first-order process with a median half-life of 3 days, and its quantity was associated with severity of illness (r2=0.82). Oral shedding persisted at a median of >or=1x104 copies/mL for 32 weeks and was unrelated to severity of illness. Subjects with nonprimary EBV infection shed virus intermittently, and median quantities for all samples became undetectable within 4 weeks. CONCLUSIONS Using a novel qEBV assay, we demonstrated that young adults with primary EBV infection rapidly cleared virus from blood but not from the oropharynx. High oral concentrations of EBV in asymptomatic persons who have resumed normal activities support the concept that infectious mononucleosis is most likely acquired by kissing.

Ganciclovir-resistant cytomegalovirus encephalitis in a bone marrow transplant recipient.

Abstract: A 20‐year‐old patient, who received a bone marrow transplant in order to treat metachromatic leukodystrophy (MLD), succumbed to cytomegalovirus (CMV) encephalitis. After CMV viremia developed, the patient received ganciclovir, but he was switched to foscarnet when ganciclovir resistance was suspected. Foscarnet was discontinued because of concern about its potential central nervous system toxicity. Autopsy samples of brain and cerebrospinal fluid contained CMV DNA with a UL97 mutation (M460V) known to confer ganciclovir resistance. No foscarnet resistance mutations were found.

Quantitative Epstein–Barr virus shedding and its correlation with the risk of post-transplant lymphoproliferative disorder

We postulated that quantitative monitoring of Epstein–Barr virus (EBV) shedding after transplantation could distinguish EBV‐associated illnesses and predict clinical outcome. EBV DNA was measured in solid organ (SOT) and hematopoietic cell transplants (HCT) using our own real‐time TaqMan EBV PCR. The proportion of patients who had EBV DNAemia post‐transplant was significantly lower in HCT vs. SOT (p < 0.001). Over a 7.5‐yr period, post‐transplant lymphoproliferative disorder (PTLD) occurred in 66 (5.8%) of 1131 patients who met adequate monitoring criteria. SOT recipients developed PTLD significantly later than HCT recipients (median, 2.8 yr vs. 121 d; p < 0.001). PTLD was documented in 53 (14%) of 376 patients who had EBV in ≥1 whole blood sample vs. 13 (2%) of 755 patients who had at least three EBV‐negative blood samples and were never positive. PTLD risk in viremic patients increased with the peak quantity of EBV DNAemia (p < 0.001). PTLD occurred in 37/333 (11%) of patients with peak blood levels 103–105 copies/mL vs. 16/43 (37%) of patients with levels >105 (p < 0.001). EBV PCR was predictive in 29 (78%) of 37 patients tested within three wk prior to tissue diagnosis of PTLD, and thus, we conclude that EBV PCR with careful attention paid to changes in EBV DNAemia could lead to earlier diagnosis and treatment of PTLD.

Post-transplant lymphoproliferative disorders in lung transplant recipients: 20-yr experience at the University of Minnesota.

Wudhikarn K, Holman CJ, Linan M, Blaes AH, Dunitz JM, Hertz ME, Peterson BA. Post‐transplant lymphoproliferative disorders in lung transplant recipients: 20‐yr experience at the University of Minnesota.
Clin Transplant 2011: 25: 705–713.

Preclinical validation of interleukin 6 as a therapeutic target in multiple myeloma

AbstractStudies on the biologic and molecular genetic underpinnings of multiple myeloma (MM) have identified the pleiotropic, pro-inflammatory cytokine, interleukin-6 (IL-6), as a factor crucial to the growth, proliferation and survival of myeloma cells. IL-6 is also a potent stimulator of osteoclastogenesis and a sculptor of the tumor microenvironment in the bone marrow of patients with myeloma. This knowledge has engendered considerable interest in targeting IL-6 for therapeutic purposes, using a variety of antibody- and small-molecule-based therapies. However, despite the early recognition of the importance of IL-6 for myeloma and the steady progress in our knowledge of IL-6 in normal and malignant development of plasma cells, additional efforts will be required to translate the promise of IL-6 as a target for new myeloma therapies into significant clinical benefits for patients with myeloma. This review summarizes published research on the role of IL-6 in myeloma development and describes ongoing efforts by the University of Iowa Myeloma Multidisciplinary Oncology Group to develop new approaches to the design and testing of IL-6-targeted therapies and preventions of MM.

FOXM1 is a therapeutic target for high-risk multiple myeloma

The transcription factor forkhead box M1 (FOXM1) is a validated oncoprotein in solid cancers, but its role in malignant plasma cell tumors such as multiple myeloma (MM) is unknown. We analyzed publicly available MM data sets and found that overexpression of FOXM1 prognosticates inferior outcome in a subset (~15%) of newly diagnosed cases, particularly patients with high-risk disease based on global gene expression changes. Follow-up studies using human myeloma cell lines (HMCLs) as the principal experimental model system demonstrated that enforced expression of FOXM1 increased growth, survival and clonogenicity of myeloma cells, whereas knockdown of FOXM1 abolished these features. In agreement with that, constitutive upregulation of FOXM1 promoted HMCL xenografts in laboratory mice, whereas inducible knockdown of FOXM1 led to growth inhibition. Expression of cyclin-dependent kinase 6 (CDK6) and NIMA-related kinase 2 (NEK2) was coregulated with FOXM1 in both HMCLs and myeloma patient samples, suggesting interaction of these three genes in a genetic network that may lend itself to targeting with small-drug inhibitors for new approaches to myeloma therapy and prevention. These results establish FOXM1 as high-risk myeloma gene and provide support for the design and testing of FOXM1-targeted therapies specifically for the FOXM1High subset of myeloma.

Adrenal extranodal NK/T-cell lymphoma diagnosed by fine-needle aspiration and cerebrospinal fluid cytology and immunophenotyping: A case report

The cytologic findings of an extranodal NK/T‐cell lymphoma (NKTCL) presenting as a large adrenal mass with leptomeningeal involvement diagnosed by CT‐guided fine‐needle aspiration and cerebrospinal fluid (CSF) cytology are described. The 65‐year‐old Caucasian patient presented with progressive headache and multiple cranial nerve neuropathies. Magnetic resonance imaging showed leptomeningeal enhancement surrounding the conus medullaris and cauda equine, and a subsequent PET/CT demonstrated a large right adrenal gland mass. Fine‐needle aspiration of the adrenal mass showed occasional large pleomorphic cells with prominent nucleoli, moderate amounts of cytoplasm, and rare large cells with sparse cytoplasmic granules admixed with numerous small lymphocytes. Initial flow cytometry from this sample showed no clonal B‐cell population. Immunoperoxidase stains performed on the cell block/core specimen showed that the large atypical cells were positive for CD2, CD30, CD43 and CD56, TIA‐1, granzyme, and perforin, but for none of the other T‐cell markers used (CD3, CD4, CD5, CD8, CD45RO), which stained the abundant background lymphocytes. A CSF specimen showed similar neoplastic cells and flow cytometry showed an NK‐cell population with aberrant immunophenotype. The cytologic findings of the neoplastic cells and the extensive panel of immunoperoxidase stains allowed the diagnosis of NKTCL, which was confirmed by the subsequent flow‐cytometric immunophenotyping performed on the CSF. This is, to the best of our knowledge, the first case of NKTCL diagnosed by FNA of the adrenal gland and by CSF cytology. Diagn. Cytopathol. 2009.

Specific detection of human BK polyomavirus in urine samples of immunocompromised patients

ABSTRACT A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were postive when specimens were submitted because of clinical suspicion of BK virus infection.

Follicular lymphoma transformed to "double-hit" B lymphoblastic lymphoma presenting in the peritoneal fluid

Lymphomas showing both MYC/8q24 rearrangement and IGH@BCL2/t(14;18)(q32;q21), also referred to as “double‐hit” or “dual‐hit” lymphomas (DHL) are rare B‐cell malignancies with a germinal center B‐cell immunophenotype and heterogeneous cytologic and histologic features. Such lymphomas may arise de novo or through transformation of follicular lymphomas and are classified either as “B‐cell lymphoma, unclassifiable, with features intermediate between diffuse large B‐cell lymphoma (DLBCL) and Burkitt lymphoma (BL)” (most commonly), DLBCL, or, rarely, as B‐lymphoblastic lymphoma. We report a case of B‐lymphobastic lymphoma arising through transformation of follicular lymphoma diagnosed on peritoneal fluid cytology, flow cytometry, and cytogenetic studies in a 53‐year‐old man who presented with abdominal pain, shortness of breath, night sweats, extensive lymphadenopathy, pleural effusion, and ascites. Cytologic examination of the ascitic fluid showed two distinct populations of neoplastic lymphoid cells, a predominant population of larger cells with fine powdery (“blastic”) chromatin, visible to prominent nucleoli and occasional small cytoplasmic vacuoles and a less numerous population of smaller cells with centrocytic morphology. Flow cytometry also showed two distinct monotypic B‐cell populations, both expressing CD10, and TdT‐positivity was demonstrated immunohistochemically. Fluorescence in situ hybridization (FISH) demonstrated both MYC rearrangement and IGH/BCL2 gene fusion and cytogenetic analysis showed a complex karyotype including both t(14;18)(q32;q21) and t(8;22)(q24.1;q11.2). Since DHL pursue an aggressive clinical course, respond poorly to therapy, and have a poor outcome, it is important to suspect the diagnosis when encountering neoplastic lymphoid cells that are difficult to classify in effusion cytology specimens and to order the appropriate immunophenotyping and cytogenetic studies. Diagn. Cytopathol. 2013;41:986–990.