Transforming growth factor ß- 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 ß (TGF-ß), 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-ß and IL-13 in bone marrows with fibrosis of all 3 groups. Megakaryocytes were negative for TGF-ß in post-ET and MDS with BMF, but some megakaryocytes in PMF were weakly positive for TGF-ß. Megakaryocytes were negative for IL-13 in all 3 groups. Blasts were negative for both TGF-ß and IL-13 in all 3 groups. Thus, TGF-ß- 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.

Infiltration of effector regulatory T cells predicts poor prognosis of diffuse large B-cell lymphoma, not otherwise specified.

Regulatory T cells (Tregs) specifically express the transcription factor forkhead box P3 (FOXP3) and contribute to tumor progression. FOXP3-positive cells have been recently proven to be heterogeneous in phenotype and function, including effector Tregs (eTregs), naïve Tregs, and non-Tregs, which harbor no suppressive function. Therefore, it is crucial to investigate the "true Treg (eTreg)" population, rather than the entire FOXP3 population, with regards to their effect on tumor immunity. In particular, in diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS), FOXP3-positive cells correlated with a better prognosis. The present study sought to evaluate the relationship between the prognosis of DLBCL, NOS patients and the infiltration of true Tregs by employing dual immunostaining with FOXP3 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). CTLA-4 is a negative immunomodulatory known to be expressed by eTregs, but not by non-Tregs. Lymph nodes from 82 nodal DLBCL, NOS patients were stained with anti-FOXP3 and anti-CTLA-4 antibodies. A high infiltration of FOXP3-positive cells was associated with a significantly better prognosis than patients with low levels of FOXP3-positive cells for overall survival (OS) (P = .0233). In sharp contrast, a high infiltration of FOXP3/CTLA-4 double-positive cells was significantly associated with a poor prognosis than patients with low levels of FOXP3/CTLA-4 double-positive cells for OS (P = .0121) and progression-free survival (P = .0171), independent of the international prognostic index. FOXP3/CTLA-4 double-positive cells, eTregs, play an important role in DLBCL, NOS progression.

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)-ß, interleukin (IL)-3, IL-13, and stem cell factor (SCF). Mast cells were positive for TGF-ß and IL-13, and FOXP3-positive cells were negative for TGF-ß. 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-ß, resulting in fibrosis typical of NSCHL.

Dermatopathic Lymphadenopathy With Increased IgG4-Positive Plasma Cells.

Both dermatopathic lymphadenopathy (DL) and immunoglobulin G4-related disease (IgG4-RD) are frequently complicated with allergic diseases. However, the relationship between DL and IgG4-RD is not well known. To clarify this relationship on the basis of clinical and pathological findings, including IgG4-positive (IgG4+) plasma cell infiltration in lymph nodes (LNs) of DL patients, we analyzed LNs of 11 DL patients using immunostaining of IgG, IgG4, forkhead box P3 (FOXP3), transforming growth factor (TGF)-ß, interferon (IFN)-γ, and matrix metalloproteinase (MMP)-1, MMP-8, and MMP-13. Toluidine blue staining was also performed to identify mast cells. Of 3 patients with a high ratio of IgG4+/IgG+ cells (>40%) and elevated serum IgG4 levels, 2 developed IgG4-RD, whereas the other patient did not. Of 8 patients with a low ratio of IgG4+/IgG+ cells (<40%) or no infiltration of IgG4+ cells, 5 who could be followed did not develop IgG4-RD. The numbers of mast cells were similar to those of TGF-ß-positive cells, and serial sections showed that mast cells possibly produce TGF-ß. LNs of DL patients with a high ratio of IgG4+/IgG+ cells had significantly more mast cells and TGF-ß-positive cells than those of patients with a low ratio of IgG4+/IgG+ cells or no infiltration of IgG4+ cells. However, no fibrosis was observed in LNs of both groups. IFN-γ was positive in interdigitating dendritic cells, Langerhans cells, and macrophages. MMP-1, MMP-8, or MMP-13 was expressed in macrophages. The lack of fibrosis in LNs may have been due to the production of IFN-γ, MMP-1, MMP-8, or MMP-13. Thus, DL with increased IgG4+ cells seems to be a phenotype of IgG4-RD in LNs.

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-α (TNF-α)-positive type correlates with a poorer prognosis compared with the TNF-α-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-α receptor 1 (TNFR1) and TNF-α 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-α-positive DLBCL, NOS cases, 27 (87%) were positive and 4 (13%) were negative for TNFR1. Both TNF-α-positive and TNFR1-positive cases were significantly correlated with a poorer OS compared with the TNF-α-positive but TNFR1-negative cases. Twenty-seven cases (45%) with the TNF-α-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-α-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-α and TNFR1 expression.

Purine analog-like properties of bendamustine underlie rapid activation of DNA damage response and synergistic effects with pyrimidine analogues in lymphoid malignancies.

Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies.

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-α (TNF-α) 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-α antibody. Cytoplasmic TNF-α 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-α positive (38 cases, 61%) and TNF-α negative (24 cases, 39%)--and that TNF-α 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-α negativity. Cox regression analysis showed that TNF-α 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-α expression in tumor cells was an independent prognostic factor for the International Prognostic Index (IPI). Therefore, TNF-α-positive DLBCL, NOS may constitute a unique subtype of DLBCL, NOS with an aggressive clinical course. The addition of TNF-α 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-α expression levels and the IPI.

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

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.