Kazuki Iwaki

Antifungal prophylaxis with micafungin in neutropenic patients with hematological malignancies.

The aim of the study was to assess the antifungal prophylactic efficacy, safety, and tolerability of micafungin, 150 mg daily, and to evaluate the usefulness of monitoring 1,3-β-d-glucan (BG) in neutropenic patients undergoing chemotherapy for hematological malignancies. This investigation was a retrospective, non-randomized study. A group of patients who did not receive systemic antifungal prophylaxis was compared to another group of patients who received micafungin 150 mg daily. All patients admitted with hematological malignancy and undergoing chemotherapy or stem cell transplant were included. The plasma BG level was measured once weekly. The clinical endpoint was the diagnosis of invasive fungal infection (IFI). Antifungal prophylaxis led to a significant decrease in the occurrence of IFI (from 12.3% to 1.5%, p = 0.001). Few severe adverse effects clearly attributable to micafungin were seen. Sensitivity, specificity, positive predictive value, negative predictive value, and efficiency of BG values >8.9 pg/mL for diagnosis of IFI were 0.90, 0.99, 0.82, 0.99, and 0.99, respectively. Micafungin, 150 mg daily, is an effective and safe drug for antifungal prophylaxis, and monitoring of BG antigenemia is a useful tool for diagnosis of IFI in neutropenic patients with hematological malignancies.

TNF-α expression in tumor cells as a novel prognostic marker for diffuse large B-cell lymphoma, not otherwise specified.

Several cytokines promote malignant cell growth and are therefore believed to contribute to disease aggressiveness. The cytokine tumor necrosis factor-&agr; (TNF-&agr;) acts as a tumor-promoting factor and has been linked to all tumorigenic stages in many cancers. Here, we evaluated 62 lymphoma tissue specimens from patients having diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) by immunostaining with anti–TNF-&agr; antibody. Cytoplasmic TNF-&agr; reactivity in ≥20% of the tumor cells was considered positive. Our results demonstrated that tumor specimens from DLBCL, NOS patients could be divided into 2 types—TNF-&agr; positive (38 cases, 61%) and TNF-&agr; negative (24 cases, 39%)—and that TNF-&agr; positivity in DLBCL, NOS was correlated with poorer overall survival (OS; P=0.0005, log rank test) and progression-free survival (PFS; P=0.0330, log rank test) compared with TNF-&agr; negativity. Cox regression analysis showed that TNF-&agr; expression was a significant prognostic factor for OS (P<0.0001) and PFS (P=0.0323). Regarding OS and PFS, multivariate analysis showed that TNF-&agr; expression in tumor cells was an independent prognostic factor for the International Prognostic Index (IPI). Therefore, TNF-&agr;-positive DLBCL, NOS may constitute a unique subtype of DLBCL, NOS with an aggressive clinical course. The addition of TNF-&agr; expression to the IPI may significantly improve the predictive prognostic value. The therapeutic strategy of DLBCL, NOS patients should be based on correct prognosis; therefore, patients with poor prognoses could be more accurately detected by evaluating both TNF-&agr; expression levels and the IPI.

Expression of tumour necrosis factor‐α and its receptors in Hodgkin lymphoma

mus-associated post-transplant autoimmune hemolytic anemia. Pediatric Transplantation, 10, 358–361. Zhan, P., Tey, S.K., Koyama, M., Kuns, R.D., Olver, S.D., Lineburg, K.E., Lor, M., Teal, B.E., Raffelt, N.C., Raju, J., Levegue, L., Markey, K.A., Varelias, A., Clouston, A.D., Lane, S.W., MacDOnald, K.P. & Hill, G.R. (2013) Induced regulatory T cells promote tolerance when stabilized by Rapamycin and IL-2 in vivo. Journal of Immunology, 191, 5291–5303.

Co‐infection of human herpesvirus‐6 and human herpesvirus‐8 in primary cutaneous diffuse large B‐cell lymphoma, leg type

Primary cutaneous diffuse large B-cell lymphoma (PCLBCL), leg type is a distinct clinicopathological entity found predominantly in elderly women. Compared with primary cutaneous lymphomas of other skin sites, PCLBCL, leg type exhibits a more aggressive behaviour and worse outcome. Thus, after several years of considerable debate, PCLBCL, leg type was determined to be a subtype of diffuse large B-cell lymphoma (DLBCL), and is now recognized as a separate entity by the World Health Organization and the European Organization for Research and Treatment of Cancer consensus classification for primary cutaneous lymphomas (Swerdlow et al, 2008). A 91-year-old woman presented with a rapidly growing, proliferative, cutaneous lesion on her left lower limb. One month later, a swelling developed in her left inguinal region and subsequently on the left side of her neck, for which she was admitted to our hospital. Physical examination revealed a slightly tender, purplish red, erythematous, reticular, focally indurated plaque with irregular borders on her left lower limb. The skin lesion continued to expand and multiple nodules appeared on the surface of the plaque (Fig 1). The results of blood analysis were as follows: white blood cell count, 9Æ58 · 10/l (82Æ8% neutrophils and 10Æ4% lymphocytes); red blood cell count, 3Æ66 · 10/l; haemoglobin, 97 g/l; platelet count, 481 · 10/l; lactate dehydrogenase, 774 iu/l and C-reactive protein, 135Æ3 mg/l. Serological tests for human immunodeficiency virus (HIV) and human T-cell leukaemia virus type 1 were negative. A biopsy specimen obtained from the leg lesion showed diffuse cellular infiltration throughout the dermis and subcutaneous tissue composed predominantly of abnormally large lymphoma cells. The lymphoma cells had oval to round vesicular nuclei with prominent nucleoli (Fig 2A). Immunohistochemical analysis of the tumour cells indicated that they were positive for CD20, CD79A, BCL2, BCL6 and MUM1 and negative for CD3, CD10, CD30 and CD138. The nucleoli of lymphoma cells were positive for human herpesvirus-6 (HHV-6), detected with rat monoclonal antibody 2002 (recognizing the 60/110 kDa envelope glycoprotein of HHV-6) (Thermo Fisher Scientific, Waltham, MA, USA) (Fig 2B), and also positive for human herpesvirus-8 (HHV-8), detected with rat monoclonal antibody LN53 (recognizing the latent nuclear antigen [LNA-1] open reading frame 73 of HHV-8) (Diagnostic BioSystems, Pleasanton, CA, USA) (Fig 2C). Epstein–Barr-encoded RNA in situ hybridization was negative. Southern blot analysis showed clonal rearrangement of the IGH@ gene. Computerized tomography scans revealed lymphadenopathy of the left side of the neck and left inguinal region. Bone marrow aspiration and biopsy yielded normal results. These findings were consistent with the features of PCLBCL, leg type and non-germinal centre B-cell-like subtype (Hans et al, 2004; Swerdlow et al, 2008). Following treatment with two courses of R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone), lymphadenopathy disappeared and the patient’s skin lesions showed improvement. Recent studies have demonstrated that HHV-6 and -8 participate in the pathogenesis of a wide range of malignant lymphomas. HHV-6 is a member of the genus Roseolovirus, subfamily Betaherpesvirinae. It is a human lymphotropic virus that has often been detected in acquired immunodeficiency syndrome-related lymphomas and CD30-positive lymphomas, such as Hodgkin lymphoma and angioimmunoblastic lymphadenopathy with dysproteinemia (Luppi et al, 1993; Valente et al, 1996). A possible pathogenic role of HHV-6 in lymphoproliferative disorders has been emphasized by the ability of HHV-6 DNA to transform established NIH 3T3 cells, which in turn form rapidly growing and metastasizing tumours when injected into nude mice (Razzaque, 1990). A gene homologous to the so-called rep gene of human adeno-associated virus type 2 has been identified in the HHV-6 genome (Thomson et al, 1991). The HHV-6 expression of a gene acting as a mode of heterologous gene expression and cellular transformation is likely to have important consequences for infected host cells (Araujo et al, Fig 1. Clinical presentation of the tumour on the left lower limb on admission. Correspondence

An approach for diagnosing plasma cell myeloma by three-color flow cytometry based on kappa/lambda ratios of CD38-gated CD138+ cells

BackgroundWorld Health Organization (WHO) criteria are commonly used to diagnose plasma cell myeloma (PCM); however, these criteria are complex and require several laboratory parameters. For differentiating reactive plasmacytosis from clonal plasma cell (PC) neoplasms such as PCM, it is important to accurately determine the expression of cytoplasmic immunoglobulin light chains.MethodsWe retrospectively analyzed the records of 27 selected patients with PCM who underwent bone biopsies for confirmative diagnosis according to WHO criteria. Twenty-three controls were also investigated. In the present study, all the samples were analyzed using flow cytometry (FC) in the side scatter vs. CD38 histogram mode, and the CD38-gated PC population was identified. Bivariate histograms of CD138/kappa and CD138/lambda were assessed, and the ratios of dual-positive cells to the CD138+ PC population were calculated. The kappa/lambda ratio was defined as the ratio of CD138/kappa to CD138/lambda.ResultsPCM cells were distinguished from normal PCs using cutoff levels between 0.76 and 1.5, at a sensitivity of 96.3% and specificity of 95.7%.ConclusionsThree-color FC analysis is simple to perform and inexpensive, with clinically relevant data obtained soon after the completion of FC measurements. The detection of the cytoplasmic kappa/lambda ratio of CD38-gated CD138+ PCs may be a useful tool in the diagnosis of PCM. To the best of our knowledge, this report represents the first diagnostic assessment of the cytoplasmic kappa/lambda ratio in CD38-gated CD138+ PCs using FC analysis. This method may help in more simple, efficient, rapid, and accurate diagnosis of PCM.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1568085959771735

Hypercalcaemia induced by tumour-derived parathyroid hormone-related protein and multiple cytokines in diffuse large B cell lymphoma, not otherwise specified

oedema. The pathogenesis of ARDS is believed to involve diffuse alveolar capillary injury. Activated neutrophils are the major cellular elements that mediate acute inflammation and have been implicated in the pathogenesis of microvascular injury that occurs in ARDS. Interactions between polymorphonuclear neutrophils (PMN) and cytokines play an important role in this process. Proinflammatory cytokines such as IL-6 and TNF-a have been found to prime or activate certain PMN functions. IL-6 is a multifunctional cytokine that is produced during acute inflammatory response. It stimulates neutrophilia and thrombopoiesis and induces the synthesis of acute-phase proteins. Sustained elevations of IL-6 in the plasma and bronchoalveolar lavage of patients with ARDS have been demonstrated and negatively correlated with disease outcome and patient survival. The presence of IL-6 in the lung alone is sufficient to promote PMN infiltration and pulmonary oedema. Recent studies have demonstrated that IL-6 contributes to tissue recruitment of PMN by chemokine induction; however, little is known about the mechanisms involved in IL-6 mediated local chemokine production. IL-6 activated Stat3 may contribute to local chemokine production through transcriptional activation of chemokine genes such as those for IL-8. TNF-a is a monocyte/macrophage-derived cytokine that is known to have a broad range of activities, including tumour cytotoxicity, antiviral effects, and potent inflammatory effects. During the course of ARDS, high levels of TNF-a are present in the blood and alveolar lining fluid. TNF-a affects the functions of PMN and vessel endothelial cells, which play a major role in the pathogenesis of ARDS. TNF-a also activates endothelial phosphodiesterase 2 (PDE2), which sensitises endothelial cells to thrombin, thereby leading to endothelial hyperpermeability. Furthermore, TNF-a increases neutrophil degranulation, superoxide production, and lysozyme release, thereby causing tissue injury. To the best of our knowledge, this is the first case report of ARDS with DLBCL, NOS, showing the lymphoma cells to be immunostained for IL-6 and TNF-a. These proinflammatory cytokines produced by the lymphoma cells might play an important role in the pathogenesis of ARDS associated lung injury in some cases of DLBCL, NOS.

Primary effusion lymphoma of T-cell origin with t(7;8)(q32;q13) in an HIV-negative patient with HCV-related liver cirrhosis and hepatocellular carcinoma positive for HHV6 and HHV8

Dear Editor, A 67-year-old man with chronic hepatitis C since 1981 was found to have a few mass lesions that were detected in an abdominal echogram performed in 2006. Hepatitis C virus (HCV) viremia was confirmed by a quantitative assay of viral load of 7.8 log IU/mL in the plasma (Cobas TaqMan HCV, Roche, Branchburg, NJ). Computed tomography (CT) detected one mass in S6 and another mass in S8 in his liver. On the basis of angiographic findings and liver biopsy, he was diagnosed with HCV-associated liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Lipiodol-transcatheter arterial embolization and percutaneous ethanol injection therapy were administered for the lesions, and HCC relapse was not detected. The patient was admitted in 2008 because of an enlarging abdomen and early satiety. CT scan showed massive fluid accumulation within the peritoneal cavity but there were no new HCC lesions (Fig. 1a). A gallium-67 scintigram revealed abnormal accumulation of the isotope in the abdominal cavity. The results of serological tests for human immunodeficiency virus (HIV), Epstein–Barr virus, and human T-lymphotropic virus 1 were negative. DNA of human herpesvirus (HHV) 6 (>2.0×10 copies/10 cells) and HHV8 DNA (>2.0× 10 copies/10 cells) were detected in peripheral blood leukocytes. The smear preparations showed noncohesive large lymphoma cells with abundant cytoplasm and prominent nucleoli (Fig. 1b). Most lymphoma cells were positive for CD45RO (Clone UCHL 1, DAKO, Carpinteria, CA; Fig. 1c) and negative for CD79a and CD20. The nucleoli of lymphoma cells were positive for latent HHV6 (2002, Thermo Fisher Scientific, Wattham, MA; Fig. 1d) and HHV8 (LN53, Diagnostic BioSystems, Pleasanton, CA; Fig. 1e) infections. Southern blotting revealed a clonal rearrangement of the T cell receptor Jγ chain gene. No clonal rearrangement of the immunoglobulin heavy chain gene was found by Southern blotting. Cytogenetic analysis of GTG banding was performed, where the specimen was cultured at 37°C for 24 h in an RPMI 1640 medium containing 10% fetal calf serum and antibiotics. After adding 0.04 μg/mL colcemide for 16 h, the cell suspension was exposed to 75 mM KCL and fixed with a mixture of methanol and acetic acid (3:1). Spreads of chromosomes were made by dropping the cell suspension onto glass slides S. Nakayama-Ichiyama (*) : T. Yokote :K. Kobayashi : Y. Hirata :N. Hiraoka :K. Iwaki :A. Takayama : T. Akioka : S. Oka : T. Miyoshi : T. Hanafusa Department of Internal Medicine (I), Osaka Medical College, 2-7 Daigakumachi, Takatsuki City, Osaka 569-0801, Japan e-mail: in1304@poh.osaka-med.ac.jp

Primary cutaneous diffuse large B-cell lymphoma, leg type, with features simulating POEMS syndrome.

A 91‐year‐old woman presented with a rapidly proliferative cutaneous lesion on the left lower limb, which was identified as a primary cutaneous diffuse large B‐cell lymphoma (PCLBCL), leg type, on biopsy. The patient also showed complications of hepatomegaly, endocrinopathy, edema, skin change, and polyneuropathy without monoclonal plasma cell proliferative disorder, and was therefore diagnosed with POEMS‐like syndrome owing to the lack of monoclonal plasma cell proliferative disorder. Levels of serum vascular endothelial growth factor (VEGF) and interleukin‐6 (IL‐6) were high with the lymphoma cells immunostained positively for VEGF and IL‐6. To the best of our knowledge, this is the first case report of PCLBCL, leg type, with POEMS‐like syndrome. The findings in this case suggest that the symptoms of POEMS‐like syndrome might be caused by the cytokines produced by the lymphoma cells. Furthermore, a wider range of diagnostic criteria associated with the result of abnormal secretion of cytokine may have to be considered for the diagnosis and evaluation of patients with possible POEMS syndrome, as against the present criteria specifying monoclonal plasma cell proliferative disorder as the essential criterion.

Immunohistological analysis in diagnosis of plasma cell myeloma based on cytoplasmic kappa/lambda ratio of CD38-positive plasma cells.

Abstract The accurate determination of cytoplasmic immunoglobulin (cIg) light chain (LC) expression is important to differentiate reactive plasmacytosis from a clonal plasma cell neoplasm such as plasma cell myeloma (PCM). Through retrospective analysis, we studied the cytoplasmic kappa/lambda ratio of CD38-positive plasma cells in the bone marrow from 19 PCM patients and 19 controls. To demonstrate cIg LC expression, the bone marrow was immunostained for IgA, IgG, IgM, kappa, and lambda. The kappa/lambda ratio was defined as the ratio of the kappa-positive cell to the lambda-positive cell in plasma cells. PCM cells were distinguished from normal plasma cells by cut-off levels between 0.59 and 4.0, a sensitivity of 94.7%, and a specificity of 94.7%. The detection of the cytoplasmic kappa/lambda ratio of CD38-positive plasma cells may be a useful tool in the diagnosis of PCM and the correct diagnosis of PCM may be achieved more simply.

A paraneoplastic neuromyelitis optica spectrum disorder associated with a mature B-cell neoplasm.

Neuromyelitis optica (NMO) is an idiopathic inflammatory emyelinating disease of the central nervous system that primarly affects the optic nerves and the spinal cord. Optic neuritis nd myelitis can occur in discrete episodes separated by years 1]. Although it has long been considered a subtype of muliple sclerosis (MS), new pathological and serological findings ave clearly defined NMO as a distinct disease because of the iscovery of NMO immunoglobulin G (NMO-IgG), which is a ighly disease-specific autoantibody often called anti-aquaporin(AQP4) antibody ([AQP4-Ab]) found in NMO and NMO spectrum isorders, but absent in the classical form of MS [1]. A 57-year-old woman was admitted to our hospital because he developed progressive paresthesia in the chest and weakness n the lower limbs. She was afebrile, conscious, and there was o lymphadenopathy. Ophthalmological examination was normal. eurological examination on admission revealed flaccid parapareis with hyperreflexia in both lower limbs, bilateral extensor lantar responses, and a sensory level to pinprick at T1–T4 level. agnetic resonance imaging (MRI) of the spinal cord revealed a T2yperintense lesion extending from C5 to T4 (Fig. 1A). Brain MRI howed T2-hyperintense lesions mostly in periventricular white atter, but also in corona radiata. On gadolinium enhancement, he T1-weighted spin-echo sequence, which is a sensitive investiation for detecting acute optic neuritis, was normal. Whole-body omputed tomography revealed no evidence of lymphadenopathy. gallium-67 scintigram revealed no abnormal accumulation. The esults of blood analysis were as follows: white blood cell count, 5.32 × 109/L (48% neutrophils, 8% lymphocytes, and 39.5% abnoral small cleaved lymphoid cells with inconspicuous nucleoli); emoglobin, 125 g/L; platelet count, 219 × 109/L; lactate dehydroenase, 209 U/L (normal: 130–250 U/L); and C-reactive protein, .01 mg/dL (normal: <0.25 mg/dL). Serum NMO-IgG measured sing an AQP4 autoantibody ELISA kit (RSR Limited, Cardiff, UK) AQP4-Ab) was 3.8 U/mL (normal: <1.0 U/mL) [2]. A bone marow aspirate showed a diffuse abnormal lymphoid infiltration of 2.6%, with the same morphology as in peripheral blood (Fig. 2A). low cytometry CD45 gating for immunophenotyping displayed he following results: positive for CD10, CD19, CD20, and cytolasmic bcl-2; negative for CD3, CD4, and CD5; and lambda light hain restriction. Immunohistochemical analysis of the abnoral cells showed that the cells were positive for CD10 (56C6, ovocastra, Newcastle Upon Tyne, England; Fig. 2B), CD20 (SL26, yowa Medex, Tokyo, Japan; Fig. 2C), and bcl-2 (HM57, DAKO, arpinteria, CA; Fig. 2D). The abnormal cells were negative for QP4 (Sigma–Aldrich, St. Louis, MO, USA). Fluorescence in situ ybridization analysis of fusion signal of IgH/bcl-2 genes was postive. Southern blot analysis showed clonal rearrangement of the mmunoglobulin heavy chain gene. Cerebrospinal fluid (CSF) examnation showed pleocytosis (44 cells/mm3; normal: <5 cells/mm3), and thoracic spinal cord at the time of presentation (A) and after a regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP therapy) (B). The scans show that the T2-hyperintense lesion and severe cord edema observed in the initial scan improved after therapy. These radiological changes correlated with clinical improvement of the patient.