Motoaki Shiratsuchi

Expression of programmed cell death ligand 1 is associated with poor overall survival in patients with diffuse large B-cell lymphoma

Programmed cell death ligand 1 (PD-L1) is expressed on both select diffuse large B-cell lymphoma (DLBCL) tumor cells and on tumor-infiltrating nonmalignant cells. The programmed cell death 1 (PD-1)/PD-L1 pathway inhibits host antitumor responses; however, little is known about how this pathway functions in the tumor microenvironment. The aim of this study was to determine the clinicopathological impact of PD-L1(+) DLBCL. We performed PD-L1/PAX5 double immunostaining in 1253 DLBCL biopsy samples and established a new definition of PD-L1(+) DLBCL. We also defined the criteria for microenvironmental PD-L1(+) (mPD-L1(+)) DLBCL (ie, PD-L1(-) DLBCL in which PD-L1(+) nonmalignant cells are abundant in the tumor microenvironment). Of the 273 patients whose clinical information was available, quantitative analysis of PD-1(+) tumor-infiltrating lymphocytes (TILs) was performed. The prevalence rates of PD-L1(+) and mPD-L1(+) DLBCL were 11% and 15.3%, respectively. Both PD-L1(+) and mPD-L1(+) DLBCL were significantly associated with non-germinal center B-cell (GCB) type and Epstein-Barr virus positivity. The number of PD-1(+) TILs was significantly higher in GCB-type tumors and lower in mPD-L1(-) and PD-L1(+) DLBCL. Patients with PD-L1(+) DLBCL had inferior overall survival (OS) compared with that in patients with PD-L1(-) DLBCL (P = .0009). In contrast, there was no significant difference in OS between mPD-L1(+) and mPD-L1(-) DLBCL (P = .31). The expression of PD-L1 maintained prognostic value for OS in multivariate analysis (P = .0323). This is the first report describing the clinicopathological features and outcomes of PD-L1(+) DLBCL. Immunotherapy targeting the PD-1/PD-L1 pathway should be considered in this distinct DLBCL subgroup.

Clinicopathologic Analysis of 22 Cases of Subcutaneous Panniculitis-Like CD56- or CD56+ Lymphoma and Review of 44 Other Reported Cases

In 22 histologic cases of subcutaneous panniculitis-like lymphoma, we studied the clinicopathologic differences between CD56- and CD56+ cases (11 each). CD56- cases had skin ulcers (1 [9%]); tumor invasion in the superficial dermis (1 [9%]); erythrophagocytosis (10 [91%]); and medium-sized (11 [100%]), CD8+ (10 [91%]), T-cell receptor (TcR)betaF1+ (10 [91%]), and CD95 (Fas)- tumor cells. CD56+ cases had skin ulcers (9 [82%]); tumor invasion in the superficial dermis (8 [73%]); erythrophagocytosis (1 [9%]); and pleomorphic large (10 [91%]), CD8+ (2/10 [20%]), TcRbetaF1 + (3/10 [30%]), and CD95 (Fas)+ (7/10 [70%]) tumor cells. These 7 factors were significantly different between groups (P < .01). Median survival rates were 96 and 12 months for the CD56- and CD56+ groups, respectively. Age younger than 40 years, no skin ulcers, no tumor invasion in the superficial dermis, and CD8+, TcRbetaF1 +, CD95 (Fas)-, and CD56- tumor cells were significantly better prognostic factors (P < .01). The CD56- and CD56+ groups showed different tumor cell characteristics, clinicopathologic findings, and prognosis. In the CD56+ group, 1 was gamma/delta T-cell phenotype, 3 were alpha/beta T-cell, and 6 were TcRbetaF1- and gamma/delta- NK/T-cell, and 3 NK/T-cell cases had nuclear signals of Epstein-Barr virus-encoded RNA. Cases of CD56+ T- and NK/T-cell lymphoma had similar clinicopathologic findings and prognosis.

Inhibition of intracellular bacterial replication in fibroblasts is dependent on the perforin-like protein (perforin-2) encoded by macrophage-expressed gene 1.

Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a perforin-like mRNA previously described as macrophage-expressed gene 1 (Mpeg1). The presence and level of the perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knockdown of the perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous perforin-like molecule has been knocked down with a red fluorescent protein-tagged version restores bactericidal activity. The perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria, including Gram-positive and -negative, and acid fast bacteria. The perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the perforin-like protein. MEF damage cell walls of intracellular bacteria by insertion, polymerization, and pore formation of the perforin-like protein, analogous to pore formers of complement and perforin-1 of cytolytic lymphocytes. We propose the name perforin-2.

Clinical significance of telomerase activity in multiple myeloma

The clinical course of patients with multiple myeloma varies, and therefore it is important to evaluate the disease state. We studied the telomerase activity of myeloma cells as a possible prognostic factor in such patients.

Different Risk Factors Related to Adenovirus- or BK Virus-Associated Hemorrhagic Cystitis following Allogeneic Stem Cell Transplantation

Virus-associated hemorrhagic cystitis (HC) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Although numerous studies have attempted to identify factors that predispose patients to viral HC, its causes remain controversial. We analyzed retrospectively the results of 266 allogeneic HSCTs to identify factors associated with HC. Of this group, 42 patients (15.8%) were diagnosed with viral HC, because of either adenovirus (ADV; n = 26; 9.8%) or BK virus (BKV; n = 16; 6.0%). ADV-HC was frequently associated with T cell purging, and was less common in patients with acute graft-versus-host-disease (GVHD). Conversely, BKV-HC was more frequently observed in patients with excessive immune reactions such as GVHD, preengraftment immune reaction, and hemophagocytic syndrome. These observations indicate that ADV- and BKV-HC may differ significantly in their risk factors and pathogenesis. Profound immune deficiency is more likely to be associated with ADV-HC, whereas immune hyperactivity might play a key role in BKV-HC.

Subcutaneous panniculitis by Epstein‐Barr virus‐infected natural killer (NK) cell proliferation terminating in aggressive subcutaneous NK cell lymphoma

We describe here a case involving a patient presenting initially with subcutaneous panniculitis, which developed after 12 years into aggressive subcutaneous natural killer (NK) cell lymphoma with peripheral blood involvement and hemophagocytosis. The surface marker of lymphoid cells in peripheral blood was CD2+3–7+8–16+56+. Skin biopsies were taken in May 1986 and June 1998. The initial biopsy revealed a diffuse proliferation of atypical lymphoid cells in the subcutaneous tissue with panniculitis, while the second biopsy revealed the presence of large lymphoid cells in the subcutaneous tissue with necrotic changes, consistent with a diagnosis of malignant lymphoma (diffuse pleomorphic type). The lymphoid cells from these two specimens were positive for CD56 and such cytotoxic molecules as T‐cell intracellular antigen‐1 (TIA‐1), granzyme B, and, interestingly, also positive for Epstein‐Barr (EB) virus by in situ hybridization. This suggests that chronic EB virus infections play an important role in the early stages of tumorigenesis and in the progression of NK cell lymphoproliferative disorders. Am. J. Hematol. 64:221–225, 2000.

Imatinib induces demethylation of miR-203 gene : An epigenetic mechanism of anti-tumor effect of imatinib

MicroRNA (miRNA) is an important regulator of cellular proliferation, differentiation and death. Leukemia-specific signature of miRNAs suggests that epigenetic dysregulation of miRNAs is important for leukemogenesis. We focused on the role of DNA methylation of miR-203 which targets BCR-ABL1 mRNA. The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment. Imatinib induced the demethylation of the miR-203 promoter region, resulting in low expression of targeted BCR-ABL1 gene, and loss of proliferation of leukemic cells. In conclusion, demethylation of miR-203 is one of the molecular mechanisms of imatinib-induced inhibition of BCR-ABL1-positive leukemic cells.

Cytotoxic T‐cell lymphoma presenting as secondary myelofibrosis with high levels of PDGF and TGF‐β

To the Editor: Myelo®brosis is often observed in a variety of hematological malignancies including chronic myelogenous leukemia (1), acute megakaryoblastic leukemia (2) and hairy cell leukemia (3). However, malignant lymphoma with diffuse myelo®brosis is rare. We present here a case of cytotoxic T-cell lymphoma presenting as pancytopenia due to diffuse reticulo®brosis of bone marrow. Furthermore, we also discuss the pathogenesis of the ®brosis, and in particular the role of cytokines relative to the ®brosis. A 19-yr-old female was admitted to our hospital to undergo evaluation of pancytopenia in August 1998. Peripheral blood analysis showed a hemoglobin level of 51 g/L, a white blood cell count of 2.7r10/L without abnormal cells, and a platelet count of 35r10/L. Her right cervical lymph nodes were enlarged, and both the liver and spleen were palpable. A systemic computed tomography (CT) scan demonstrated multiple swellings of the bilateral cervical, supraclavicular, para-aortic and mesenteric lymph nodes and marked splenomegaly (Fig. 1). Biopsy of the right cervical lymph node revealed diffuse proliferation of small atypical lymphocytes with some multi-nuclear giant cells. Immunophenotyping revealed that both cells were positive for CD3, CD8, TCRbF1 and TIA-1 and negative for CD4, CD20, CD30 and CD56. Bone marrow aspiration resulted in a dry tap, and its biopsy revealed diffuse reticulo®brosis with lymphoid cells (Fig. 2A). Normal hematopoiesis was severely suppressed. We diagnosed peripheral cytotoxic T-cell lymphoma with myelo®brosis, and the patient subsequently underwent chemotherapy (cyclophosphamide, doxorubicin, vincristine and prednisolone). We performed six courses of chemotherapy. After three courses of chemotherapy, a bone marrow biopsy demonstrated recovery of hematopoiesis and a disappearance of ®brosis (Fig. 2B). However, small lymph nodes 1±2 cm in diameter remained in the neck and abdomen even after all six courses of chemotherapy. The patient underwent alloperipheral blood stem cell transplantation in April 1999, and is alive as of August 2000 with no progression of the disease. We determined the levels of several serum parameters at diagnosis and after three courses of treatment (Table 1). The serum concentration of soluble interleukin-2 receptor and LDH decreased from 14,800 to 502 U/mL, and from 972 to 313 U/ L, respectively. In addition, two serum markers of ®brosis, including procollagen terminal peptide III (4), which was released during the synthesis of type

Killing machines: three pore-forming proteins of the immune system.

The evolution of early multicellular eukaryotes 400–500 million years ago required a defensive strategy against microbial invasion. Pore-forming proteins containing the membrane-attack-complex-perforin (MACPF) domain were selected as the most efficient means to destroy bacteria or virally infected cells. The mechanism of pore formation by the MACPF domain is distinctive in that pore formation is purely physical and unspecific. The MACPF domain polymerizes, refolds, and inserts itself into bilayer membranes or bacterial outer cell walls. The displacement of surface lipid/carbohydrate molecules by the polymerizing MACPF domain creates clusters of large, water-filled holes that destabilize the barrier function and provide access for additional anti-bacterial or anti-viral effectors to sensitive sites that complete the destruction of the invader via enzymatic or chemical attack. The highly efficient mechanism of anti-microbial defense by a combined physical and chemical strategy using pore-forming MACPF-proteins has been retargeted during evolution of vertebrates and mammals for three purposes: (1) to kill extracellular bacteria C9/polyC9 evolved in conjunction with complement, (2) to kill virus infected and cancer cells perforin-1/polyperforin-1 CTL evolved targeted by NK and CTL, and (3) to kill intracellular bacteria transmembrane perforin-2/putative polyperforin-2 evolved targeted by phagocytic and nonphagocytic cells. Our laboratory has been involved in the discovery and description of each of the three pore-formers that will be reviewed here.

Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria

Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system. DOI: http://dx.doi.org/10.7554/eLife.06508.001