Shuji Momose

Expression of Epstein-Barr Virus–Encoded Proteins in Extranodal NK/T-cell Lymphoma, Nasal Type (ENKL): Differences in Biologic and Clinical Behaviors of LMP1-Positive and -Negative ENKL

Purpose: Extranodal NK/T-cell lymphoma, nasal type (ENKL) is closely associated with Epstein-Barr virus (EBV). To elucidate its pathogenetic role, we examined the expression profiles of EBV-encoded proteins, especially focusing on latent membrane protein 1 (LMP1). Experimental Design: Immunohistochemistry was carried out using clinical samples from ENKL cases, which were diagnosed between 1996 and 2010 at our institution. We statistically assessed the correlation between LMP1 positivity and the clinicopathologic data and further examined phosphorylation status of NF-κB RelA and Akt in ENKL cell lines. Results: Most of the 30 examined cases showed pleomorphic morphology, natural killer cell immunophenotype, and a localized disease. Immunohistochemistry detected EBERs, but not EBNA2, in all cases. LMP1 and LMP2A were positive in 22 (73.3%) and 12 cases (40.0%), respectively. LMP1-positive cases tended to show a localized disease (P = 0.060, the Fisher exact test). Nuclear localization of phosphorylated RelA and detection of phosphorylated Akt were predominantly observed in LMP1-positive cases (P = 0.002 and P < 0.001, respectively, the Fisher exact test). RNA silencing experiments of LMP1 in Hank1 cells suggested a positive correlation between LMP1 expression and phosphorylation of RelA and Akt. With a median follow-up period of 26.7 months (range, 0.2–142.3 months), the 2.5-year overall survival rates for LMP1-positive and -negative cases were estimated at 78.3% and 12.5%, respectively (P = 0.001, log-rank test). Conclusions: LMP1 expression shows correlations with phosphorylation of RelA and Akt and possibly has a favorable impact on clinical outcome in ENKL. Clin Cancer Res; 18(8); 2164–72. ©2012 AACR.

Hyperactivated STAT3 in ALK-positive diffuse large B-cell lymphoma with clathrin-ALK fusion.

Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma is a rare subtype of diffuse large B-cell lymphoma (DLBCL). Although a few cases of ALK-positive large B-cell lymphoma harbor nucleophosmin-ALK chromosomal translocation similar to ALK-positive anaplastic large cell lymphoma, most reported cases are characterized by t(2;17)(p23;q23) involving the clathrin gene. Here, we report 2 cases of ALK-positive DLBCL. The 2 cases presented similar morphologic features and immunohistochemical characteristics, that is, positivity for ALK, IgA, CD138, and MUM1; weak positivity for CD30 and CD79a; and negativity for CD20. The clathrin-ALK transcript was identified by reverse transcription-polymerase chain reaction, and the sequence was determined by direct sequencing. Recently, the essential role of STAT3 activation as well as STAT 5 activation in nucleophosmin-ALK fusion protein-mediated lymphomagenesis was reported. However, differential effects of ALK-fusion variant proteins on proliferation, transformation, and invasion properties were reported. Thus, we evaluated the phosphorylation status of STAT 3 and STAT 5, and found highly hyperphosphorylated STAT 3 on tyrosine 705 but not STAT 5 in our 2 cases of ALK-positive DLBCL with clathrin-ALK fusion. Furthermore, STAT 5A expression was not detected in either of the ALK-positive DLBCL cases, although 11 of the 36 ALK-negative DLBCL cases revealed STAT 5A expression. Expression of the antiapoptotic proteins survivin and BCL-X(L), which were believed to be the targets of STAT 3, was investigated. However, there were no significant associations between expression of survivin or BCL-X(L) and ALK positivity among the diffuse large B-cell lymphomas. In summary, similar signaling transduction mechanism involving STAT proteins seems to underlie DLBCL harboring the clathrin-ALK or nucleophosmin-ALK fusion gene.

Prepro-orexin mRNA expression in the rat brain is increased during pregnancy

The purpose of this study was to investigate whether orexin expression in the rat brain was changed during pregnancy. Brain samples were obtained from 5 nonpregnant rats and 10 pregnant rats (5; day 10 of gestation, and 5; day 20 of gestation). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to investigate the expression of prepro-orexin mRNA and the housekeeping gene in the rat brain. The signals were quantified by the densitometric analysis. The distribution and expression of orexin-A and orexin-B were determined using immunohistochemistry. The ratio of the prepro-orexin mRNA expressions to the housekeeping gene expression in pregnant rat brain were significantly higher than that in nonpregnant control. There was no significant difference between prepro-orexin mRNA levels of day 10 and day 20 of gestation. Immunohistochemical staining for orexin-A and orexin-B was present in neurons within and around the lateral and posterior hypothalamic areas in both nonpregnant and pregnant rats. These results suggest that increased prepro-orexin mRNA levels at early gestational age in the maternal rat has a role on energy metabolism during pregnancy.

Distribution of Tsc1 protein detected by immunohistochemistry in various normal rat tissues and the renal carcinomas of Eker rat: detection of limited colocalization with Tsc1 and Tsc2 gene products in vivo.

We and others previously demonstrated that hereditary mutation and a subsequent second hit in the rat homolog of tuberous sclerosis gene (Tsc2) are responsible for Eker renal carcinomas (RC). In humans, alteration in the TSC2 gene is known to cause the tuberous sclerosis complex (TSC) that results in hamartomatous lesions in multiple organs, but the function of TSC2 is not fully understood. In recent years, a second gene (TSC1) responsible for human TSC has been cloned, and binding between TSC1 and TSC2 proteins was reported. In this study, to clarify associations between Tsc proteins in vivo, the expression of Tsc1 protein was detected by immunohistochemistry, and compared with Tsc2 expression. Tsc1 protein was expressed in the nervous system and in many endocrine tissues, including pancreatic islets, the parathyroids, testis, and ovary. Tsc1 was also detected in the many epithelial tissues of organs, such as kidney, uterus, small and large intestine, and liver. Our results indicate overlapping, but not identical, organ distributions of Tsc1 and Tsc2 proteins. At the intracellular distribution, double fluorescent immunolabeling allowed the determination that only a partial portion of Tsc1 signals overlapped with Tsc2 in some organs. These results suggest the existence of co-localizing and independent forms of Tsc proteins in endogenous expressions. Additionally, relatively high expression of Tsc1 protein was detected in RC in the Tsc2 mutant (Eker) rat.

Primary splenic diffuse large B-cell lymphoma manifesting in red pulp

We evaluated six cases of diffuse large B-cell lymphoma (DLBCL) involving the red pulp of the spleen. All had B symptoms and an aggressive clinical course. The lymphoma cells proliferated diffusely and non-cohesively in the cords of the red pulp. The lymphoma involved the bone marrow in four of the five patients and the liver in all four of the patients examined. However, lymph node (LN) involvement was rare at presentation, and systemic LN involvement was not observed even in the terminal phase. The lymphoma cells infiltrated the intrasinusoidal/intravascular and interstitial spaces of the involved tissues and were detected in the peripheral blood in two of the six patients. CD5-expressing lymphoma cells were detected in four of the five patients examined. Because these cases had some unique clinical features and occurred in distinct splenic sites, we proposed that primary splenic DLBCL manifesting in red pulp is a distinct clinicopathological entity.

Transgenic rescue from embryonic lethality and renal carcinogenesis in the Nihon rat model by introduction of a wild-type Bhd gene

We recently reported that a germline insertion of a single nucleotide in the rat homologue of the human Birt–Hogg–Dubé gene (BHD) gives rise to dominantly inherited cancer in the Nihon rat model. In this study, we constructed transgenic Nihon rats with introduction of a wild-type Bhd gene to ascertain whether suppression of the Nihon phenotype is possible. Rescue from embryonic lethality of mutant homozygotes (Nihon/Nihon) and suppression of renal carcinogenesis in heterozygotes (Nihon/+) were both observed, defining the germline Bhd mutation in the Nihon rat as an embryonal lethal and tumor predisposing mutation. This transgenic rescue system will be useful to analyse Bhd gene function, its relation to tumorigenesis in vivo, and genetic–environmental interactions in carcinogenesis.

Mesothelin (MSLN) promoter is hypomethylated in malignant mesothelioma, but its expression is not associated with methylation status of the promoter

Gene methylation leads to malignant progression in some tumors. The mechanism by which mesothelin is expressed in malignant mesothelioma (MM) is not well understood. MM is histologically divided into 3 subtypes, that is, the epithelioid, sarcomatoid, and biphasic types, and it was shown that mesothelin expression was restricted to the epithelioid type and the epithelioid component of the biphasic type of MM. However, its regulatory mechanism of expression has not been clarified. Here, we studied the expression of mesothelin by immunohistochemistry along with the methylation status of 20 CpG sites in the promoter of the mesothelin gene (MSLN) in 118 lung specimens, including 39 MM, 41 lung carcinoma, 26 nonneoplastic pulmonary lesions, and 12 normal lung tissue samples by the methylation-sensitive single nucleotide primer extension technique. We confirmed that mesothelin was expressed in the epithelioid type and epithelioid component of the biphasic type of MM but neither in the sarcomatoid type nor sarcomatous component of the biphasic type. Surprisingly, the MSLN promoter was significantly hypomethylated in the MM cases regardless of its subtype, compared with the other pulmonary lesions and normal lung tissue samples. These findings suggested that hypomethylation of the MSLN promoter may be specifically associated with the formation of MM, regardless of its expression status, and that the expression of mesothelin protein was lost in the sarcomatoid type by some unknown posttranscriptional regulatory mechanism. We also identified 4 CpG sites, among the 20 sites studied, to be more specifically hypomethylated in MM cases.

Y-box binding protein-1 expression in diffuse large B-cell lymphoma: an impact on prognosis in the rituximab era

The expression of YB-1 has been reported to predict poor clinical outcome in many human malignancies, including hematopoietic malignancies. In this study, we investigated the correlations between YB-1 expression and the clinicopathological features of patients with diffuse large B-cell lymphoma (DLBCL) in a single institution. The expression of YB-1 was analyzed in 168 cases by immunohistochemistry. Fifteen out of 168 cases (8.9%) showed cytoplasmic expression of YB-1. The expression of YB-1 was significantly associated with 5-year overall survival (OS) (p = 0.023). Rituximab plus CHOP therapy (n = 94) improved the 5-year survival rate in both YB-1-positive and -negative patients. In conclusion, the data presented in this report provide evidence that the cytoplasmic expression of YB-1 is a poor prognosis factor in DLBCL treated with CHOP therapy, whereas rituximab improves the survival of both YB-1-positive and -negative patients with DLBCL.

The G1556S-type tuberin variant suppresses tumor formation in tuberous sclerosis 2 mutant (Eker) rats despite its deficiency in mTOR inhibition

Tuberin, a tumor-suppressor protein produced by the tuberous sclerosis gene TSC2, downregulates the Rheb-mTOR-S6K pathway (mTOR axis). Comparison of the effects of human tuberin mutations, such as G1556S, suggests that pathways other than the mTOR axis might also be involved in the pathogenesis of tuberous sclerosis. Here we test this possibility using the rat G1556S-type mutation (GSM) and a transgenic Eker (Tsc2 mutant) rat system. Cells expressing GSM-tuberin failed to downregulate the mTOR axis. GSM-tuberin had an altered localization, which underlie its reduced ability to form a complex with hamartin, and a site-specific alteration in phosphorylation status indicating diverse regulation by Akt. GSM-transgenic (GSM-Tg) rats exhibited suppression of macroscopic renal tumors following N-ethyl-N-nitrosourea treatment. Intriguingly, rats with weaker GSM-Tg expression showed microscopic cystic and pre-tumorous lesions that were restricted in size and expansion, although they had hyper-phosphorylation of ribosomal protein S6. These results highlight a novel pathway involving tuberin that regulates tumor suppression independently of the mTOR inhibitory function. Identification of such a novel pathway will provide clear implications for generation of new therapeutic targets in the treatment of these tumors.

Clinicopathologic investigation of methotrexate-induced lymphoproliferative disorders, with a focus on regression

Abstract Although recent accumulative data reveal the clinicopathogenesis of regression in methotrexate-induced lymphoproliferative disorders (MTX-LPDs), the precise understanding including this category remains controversial. In this study, we analyzed 62 patients with MTX-LPD. Forty-three patients showed regression (Reg group), with high rates of Hodgkin lymphoma (HL) and LPD (90 and 88%, respectively). Among the 43 patients of the Reg group, 14 patients (33%) relapsed. The median duration before relapse in the Reg group was 10.6 months. Although the difference of OS between the Reg and Non-Reg groups was not significantly different, relapse-free patients in the Reg group had a superior overall survival (OS). MTX duration had a significant impact on Epstein–Barr virus (EBV) infection (p = .00131). Furthermore, EBV infection was significantly related to clinical manifestations, including spleen invasion, in the regression phenomenon. Some human leukocyte antigens (HLA) alleles might affect MTX-LPD development via EBV infection, although A*2402 and DRB1*0405 might be affected as fundamental factors.