Uta Nishiwaki

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.

Role of mast cells in fibrosis of classical Hodgkin lymphoma

The underlying mechanism of fibrosis in classical Hodgkin lymphoma (CHL) remains uncertain. This study aimed to investigate the association of fibrosis in the lymph nodes of patients with CHL through histological examination of the expression of cytokines associated with fibrosis and mast cell proliferation. Additionally, we sought to determine the degree of mast cell infiltration in a nodular sclerosis subtype of CHL (NSCHL) compared with that in non-NSCHL. We analyzed lymph nodes from 22 patients with CHL, of which eight were of the NSCHL and 14 of the non-NSCHL subtype, using immunohistochemical staining of forkhead box P3 (FOXP3), transforming growth factor (TGF)-β, interleukin (IL)-3, IL-13, and stem cell factor (SCF). Mast cells were positive for TGF-β and IL-13, and FOXP3-positive cells were negative for TGF-β. Only the expression of IL-13 in Hodgkin and Reed–Sternberg (HRS) cells was significantly more frequently observed in NSCHL than that in non-NSCHL (P = 0.0028) and was associated with a higher rate of fibrosis (P = 0.0097). The number of mast cells was significantly higher in NSCHL than that in non-NSCHL (P = 0.0001). A significantly positive correlation was observed between the rate of fibrosis and the number of mast cells (correlation coefficient, 0.8524; 95% CI, 0.6725–0.9372) (P <0.0001). The number of mast cells was significantly higher in the group with IL-13-positive HRS cells than that in the group with IL-13-negative HRS cells (P = 0.0157). Based on these findings, we hypothesize that IL-13 production by HRS cells may lead to fibrosis, and furthermore, promote mast cell proliferation and infiltration. This in turn might further produce the fibrotic cytokines IL-13 and TGF-β, resulting in fibrosis typical of NSCHL.

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.

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.

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.

Transforming growth factor β– and interleukin 13–producing mast cells are associated with fibrosis in bone marrow ☆

Although bone marrow fibrosis is a lethal condition, its underlying mechanism is not fully understood. This study aimed to investigate the pathogenesis of fibrosis in the bone marrow through histologic examination of mast cell infiltration and the expression of fibrosis-associated cytokines. We analyzed 22 bone marrows with fibrosis (8 primary myelofibrosis [PMF], 5 post-essential thrombocythemia [ET], myelofibrosis, and 9 myelodysplastic syndrome [MDS] with bone marrow fibrosis [BMF]). Immunohistochemical and immunofluorescence stainings were performed using anti-mast cell tryptase, interleukin (IL) 13, transforming growth factor β (TGF-β), CD34, and CD42b antibodies. The number of mast cells in bone marrows with fibrosis was significantly higher than that in controls (P<.0001 for all cases with fibrosis versus control, P=.0470 for PMF versus control, P<.0001 post-ET myelofibrosis versus control, and P=.0005 for MDS with BMF versus control). Moreover, bone marrows with higher fibrotic grades exhibited greater amounts of infiltrating mast cells. Mast cells were positive for TGF-β and IL-13 in bone marrows with fibrosis of all 3 groups. Megakaryocytes were negative for TGF-β in post-ET and MDS with BMF, but some megakaryocytes in PMF were weakly positive for TGF-β. Megakaryocytes were negative for IL-13 in all 3 groups. Blasts were negative for both TGF-β and IL-13 in all 3 groups. Thus, TGF-β- and IL-13-producing mast cells might be key players in the development of BMF. Therefore, mast cells could be potential therapeutic targets for the treatment of BMF.

TNF-α receptor 1 expression predicts poor prognosis of diffuse large B-cell lymphoma, not otherwise specified.

We have previously shown that in tumor specimens from patients with diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS), the tumor necrosis factor-&agr; (TNF-&agr;)-positive type correlates with a poorer prognosis compared with the TNF-&agr;-negative type. In the present study, we further evaluated 60 lymphoma tissue specimens from patients with DLBCL, NOS by immunohistochemical staining with antibodies against TNF-&agr; receptor 1 (TNFR1) and TNF-&agr; receptor 2 (TNFR2). Our results demonstrated that 31 cases (52%) were positive and 29 (48%) were negative for TNFR1 and that the TNFR1-positive cases were significantly correlated with a poorer overall survival (OS; P=0.0006, log rank test) than the TNFR1-negative cases. The TNFR2-positive cases tended to have a poorer OS than the TNFR2-negative cases, although the difference was not significant. TNFR1 expression in tumor cells was a significant prognostic factor for OS and was independent of the International Prognostic Index (IPI). Among 31 TNF-&agr;-positive DLBCL, NOS cases, 27 (87%) were positive and 4 (13%) were negative for TNFR1. Both TNF-&agr;-positive and TNFR1-positive cases were significantly correlated with a poorer OS compared with the TNF-&agr;-positive but TNFR1-negative cases. Twenty-seven cases (45%) with the TNF-&agr;-positive and TNFR1-positive subtype of DLBCL, NOS had a poorer prognosis for OS and progression-free survival compared with the 33 cases (55%) with the remaining subtypes, and the TNF-&agr;-positive and TNFR1-positive subtype of DLBCL, NOS was also shown to be independent of the IPI. In addition to the IPI, the prognosis of patients can be more accurately identified by evaluating both TNF-&agr; and TNFR1 expression.

Multiple cytokine- and chemokine-producing primary testicular diffuse large B-cell lymphoma, not otherwise specified

Primary testicular lymphoma is a very aggressive malignancy nd a poor outcome is confirmed for most patients with diffuse arge B-cell lymphoma (DLBCL); long-term survival curves have ot shown clear evidence of a substantial proportion of cured atients [1]. It has been suggested that primary testicular lymhoma is protected by the blood–testis barrier [2], which includes n immunological barrier [3]. Developing germ cells must be proected against the immune system because they express proteins hat can serve as foreign antigens. B7-H1 and interferon (IFN)ave been associated with this immunological barrier. Constitutive xpression of B7-H1 was confined to peritubular smooth musle cells, a contractile monolayer that surrounds the seminiferous pithelium. Peritubular cells (that constitutively express B7-H1) nd Sertoli cells (that express IFN-induced B7-H1), which are oth involved in barrier functions, may provide serial lines of efense against immune attack. B7-H1 effectively contributes to he inhibition of immune responses by negatively interfering with D8+ T-cell proliferation [4]. In addition, IFNinduces the expresion of major histocompatibility complex class II in Sertoli cells, hich may mediate the increase in regulatory T cells (Tregs) that an suppress other bystander T cells in barrier functions [4]. A 64-year-old man was admitted to our hospital with bilateral esticular swelling. The results of blood analysis were as follows: hite blood cell count, 6.97 × 109/L; hemoglobin, 148 g/L; platelet ount, 339 × 109/L; lactate dehydrogenase, 208 IU/L; and C-reactive rotein, 1.29 mg/dL. Magnetic resonance imaging of the testes evealed swelling of both testes with low signal intensity of the ight whole testis and some parts of the left testis on T2-weighted maging. Whole-body computed tomograhy revealed swelling of he para-aortic lymph nodes and invasion of left adrenal gland. he patient underwent right orchidectomy for evaluation of the iagnosis. Histological examination showed a diffuse proliferation f large lymphoid cells that totally effaced the normal architecure (Fig. 1A). A portion of the seminiferous tubules remained ntact, but lymphoma cells (Fig. 1B), which were positive for CD20 Fig. 1C), had infiltrated the rest. Immunohistochemical analyis of tumor cells showed them to be positive for CD20 and D79a, but negative for CD3, CD5, and CD10. The lymphoma cells ere positive for IFN(H145; Santa Cruz Biotechnologies, Santa ruz, CA, USA; Fig. 1D), tumor necrosis factor(TNF) (52B83; anta Cruz Biotechnologies; Fig. 1E), thymus and activationegulated cytokine (TARC) (6SN; Novocastra, Newcastle Upon Tyne, K; Fig. 1F) and macrophage-derived chemokine (MDC) (Sigmaldrich, St. Louis, MO, USA; Fig. 1G). TARCand MDC-positive ymphoma cells were surrounded by reactive lymphocytes, which ere positive for CCR4 (Fig. 2A) and CD4 (Fig. 2B). Epstein–Barrncoded RNA in situ hybridization was negative. Southern blotting evealed clonal rearrangement of the immunoglobulin heavy chain

Syndrome of inappropriate antidiuretic hormone secretion associated with primary cutaneous anaplastic large cell lymphoma.

A 40-year-old Japanese woman with a history of red, itchy skin since the age of 6 years was diagnosed with atopic dermatitis and treated with topical steroids. She presented with a 2-month history of systemic erythema and a right shoulder tumour, which was increasing in size and showed ulceration. The results of her blood analysis were as follows: white blood cell count 6·9 9 10/l (87·0% neutrophils and 7·0% lymphocytes), red blood cell count 4·09 9 10/l, haemoglobin concentration 91 g/l, platelet count 331 9 10/l, Na 138 mmol/l, K 3·7 mmol/l, lactate dehydrogenase 298 iu/l and C-reactive protein 37·4 mg/l. Serology for human immunodeficiency virus and human T-cell lymphotrophic virus was negative. A biopsy of the shoulder lesion showed a dense, monomorphous infiltrate composed of large anaplastic cells (left). No epidermotropism or microabscesses were observed. More than 75% of the infiltrating cells were large CD30+ lymphoid cells; immunostaining showed CD30+, CD3 , CD8 , CD20 and anaplastic lymphoma kinase+. Monoclonal rearrangement of the TRB@ and TRG@ genes was identified. Computed tomography scans showed hepatosplenomegaly and lymphadenopathy of the left neck and axillary and inguinal regions. Bone marrow aspiration and biopsy were normal. These findings were consistent with primary cutaneous anaplastic large cell lymphoma, and the tumour was classified as stage T3bN3M0. The patient received cyclophosphamide, doxorubicin, vincristine and prednisolone-like multi-agent chemotherapy. On the 10th day of chemotherapy, the serum sodium level had decreased to 129 mmol/l with a plasma osmolarity of 265 mOsm/kg per H2O, while her urine osmolarity was 678 mOsm/kg per H2O. The patient was euvolaemic and renal function tests were normal. The plasma antidiuretic hormone (ADH) concentration was 7·1 pg/ml (normal range, 1·3–4·1 pg/ml), with no evidence of thyroid, adrenal or anterior pituitary dysfunction. The patient was diagnosed with syndrome of inappropriate antidiuretic hormone (SIADH) and was treated intravenously with 1000 ml of 0·9% saline/ day, along with restricted fluid intake. Her sodium levels increased to 138 mmol/l within 10 days of starting treatment. However, she later died due to disease progression. Retrospective immunohistochemical analysis of lymphoma cells was positive for ADH protein (right). The onset of SIADH during treatment was attributed to tumour lysis.