Yoshihiko Hoshida

Prognostic Significance of Activated Akt Expression in Pancreatic Ductal Adenocarcinoma

Purpose: Akt is a serine/threonine kinase that plays a central role in tumorigenesis. Among the members of Akt family, Akt2 is associated with the development of human cancers. The present study was designed to clarify the prognostic significance of Akt2 and activated Akt expression in pancreatic ductal adenocarcinoma (PDAC). In addition, activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the proliferation activity of tumor cells detected by Ki-67 immunohistochemistry were examined. Experimental Design: Immunohistochemical analysis was performed on paraffin-embedded specimens from 65 patients with PDAC; 36 males and 29 females with ages ranging from 48 to 79 years (median, 66 years) of age. Expression levels of Akt2, phosphorylated Akt (p-Akt), and phosphorylated ERK 1/2 (p-ERK 1/2) were categorized as either weaker (low intensity) or equal to stronger (high intensity) compared with those in the endothelial cells of the same specimens. For Ki-67 immunohistochemistry, cases were divided into two groups: level 1, Ki-67 labeling index (LI), <20%; level 2, Ki-67 LI, ≥20%. Results: Twenty-six (42.6%), 28 (45.9%), 39 (63.9%), and 46 (75.4%) of the tumors showed high intensity of Akt2, p-Akt, and p-ERK 1/2 expression, and Ki-67 LI level 2, respectively. A significant positive correlation was observed between Akt2 and p-Akt expression (P < 0.01). Multivariate analysis revealed that p-Akt expression, Ki-67 LI, and histological differentiation are independent prognosticators for PDAC. Conclusions: p-Akt expression is a significant prognostic indicator for PDAC. Inhibition of Akt is a possible molecular approach for treatment of PDAC.

Pyothorax-Associated Lymphoma: A Review of 106 Cases

PURPOSE Pyothorax-associated lymphoma (PAL) is a non-Hodgkins lymphoma developing in the pleural cavity after a long-standing history of pyothorax. Full details of PAL are provided here. PATIENTS AND METHODS Clinical and pathologic findings were reviewed in 106 patients with PAL collected through a nationwide survey in Japan. RESULTS Age of the patients with PAL was 46 to 82 years (median, 64 years), with a male/female ratio of 12.3:1. All patients had a 20- to 64-year (median, 37-year) history of pyothorax resulting from artificial pneumothorax for treatment of pulmonary tuberculosis (80%) or tuberculous pleuritis (17%). The most common symptoms on admission were chest and/or back pain (57%) and fever (43%). Laboratory data showed that the serum neuron-specific enolase level was occasionally elevated (3.55 to 168.7 ng/mL; median, 18.65 ng/mL), suggesting a possible diagnosis of small-cell lung cancer. Histologically, PAL usually showed a diffuse proliferation of large cells of B-cell type (88%). In situ hybridization study showed that PAL in 70% of the patients was Epstein-Barr virus (EBV)-positive. PAL was responsive to chemotherapy, but the overall prognosis was poor, with a 5-year survival of 21.6%. CONCLUSION This study established the distinct nature of PAL as a disease entity. PAL is a non-Hodgkins lymphoma of exclusively B-cell phenotype in the pleural cavity of patients with long-standing history of pyothorax, and is strongly associated with EBV infection. Development of PAL is closely related to antecedent chronic inflammatory condition; therefore, PAL should be defined as malignant lymphoma developing in chronic inflammation.

VCP (p97) Regulates NFKB Signaling Pathway, Which Is Important for Metastasis of Osteosarcoma Cell Line

In order to identify genes associated with metastasis, suppression subtractive hybridization (SSH) was performed using murine osteosarcoma cell line Dunn and its subline with higher metastatic potential, LM8. SSH revealed expression of the gene encoding valosin‐containing protein (VCP; also known as p97) to be constitutively activated in LM8 cells, but it declined in Dunn cells when the cells became confluent. Because VCP is known to be involved in the ubiquitination process of Inhibitor‐αBα (IαBα), an inhibitor of nuclear factor‐αB (NFαB), whether VCP influences NFαB activation or not was examined by using VCP‐transfected Dunn cells (Dunn/VCPs). When stimulated with tumor necrosis factor‐α (TNFα), Dunn/VCPs showed constantly activated NFαB, although in the original Dunn cells and control vector transfectant (Dunn/Dunn‐c) NFαB activation ceased when the cells became confluent. Western immunoblot analysis showed an increase of phosphorylated IαBα (p‐IKBα) in the cytoplasm of confluent Dunn/Dunn‐c cells compared to that of Dunn/VCPs. Therefore, decrease of p‐IKBα degrading activity might be responsible for the decrease in NFKB activation. In vitro apoptosis assay demonstrated increased apoptosis rates of Dunn/Dunn‐c cells after TNFα stimulation compared to those of Dunn/VCPs and LM8 cells. In vivo metastasis assay showed increased incidences of metastatic events in Dunn/VCP‐1 inoculated male C3H mice compared to those in Dunn/Dunn‐c inoculated mice. These findings suggested that VCP expression plays an important role in the metastatic process. Anti‐apoptotic potential in these cells owing to constant NFKB activation via efficient cytoplasmic p‐IKBα degrading activity may explain the increased metastatic potential of these cells.

Cancer risk after renal transplantation in Japan

Excess of cancer in patients receiving renal transplantation is well‐known in Western countries, but information in Japan remains limited. Our study examined whether excess risk is found in patients receiving renal transplantation in Japan. Between 1970 and 1995, 1155 males and 589 females underwent renal transplantation in 6 hospitals, and a total of 12,982 person‐years of observation was accumulated. Malignancies developed in 2.6% of patients; O/E ration was 2.78. Median interval from renal transplantation to tumor development was 58 months. The interval in the patients receiving medication with cyclosporine‐A (CyA) (median, 42.5 months) was significantly shorter than that with non‐CyA (median, 95.5 months). Median age at the diagnosis of malignancy was 40 years, which is much younger than that in the general population. Relative risk was highest in renal cancer, followed by thyroid cancer, malignant lymphoma and uterine cancer. A distribution of malignancies was different from that reported from Western countries. These findings showed the excess risk of malignancies in Japan with renal transplants, especially in male patients, similar to that observed in Western countries, though the types of malignancy were different. Int. J. Cancer 71:517‐520, 1997.

Lymphoproliferative disorders in renal transplant patients in Japan

Post‐transplantation lymphoproliferative disorders (PT‐LPD) are characterized by a clinically and morphologically heterogeneous group of lymphoid proliferation occurring after organ or bone marrow transplantation. The immunodeficient state provides a basis for lymphomagenesis probably through activation of oncogenic viruses. Twenty‐four patients in whom PT‐LPD developed after renal transplantation in Japan were analyzed. They received hemodialysis for 4 to 226 (median 13) months before transplantation. In situ hybridization was performed to detect Epstein‐Barr virus (EBV). Polymerase chain reaction and Southern hybridization with primers in the tax and pol regions of human T‐cell leukemia virus type I (HTLV‐1) were performed on DNA extracted from paraffin‐embedded specimens. Immunohistochemical analysis revealed that 12 cases were B‐cell type, 10 cases (42%) T‐cell type and 2 NK‐cell type. Five of the T‐cell cases were classified as adult T‐cell lymphoma with proven HTLV‐1 genome in the tumor and seropositivity for the virus. These cases were classified as adult T‐cell lymphoma (ALT). More than 80% of B‐cell, 30% of T‐cell and both NK/T‐cell lymphomas were EBV‐positive. Co‐infection of EBV and HTLV‐1 was found in 2 cases with ATL. These findings showed that ATL is common among Japanese renal transplant patients, which might be due to transmission of HTLV‐1 via blood transfusion during hemodialysis.

Elevated Expression of Valosin-Containing Protein (p97) in Hepatocellular Carcinoma Is Correlated With Increased Incidence of Tumor Recurrence

PURPOSE Valosin-containing protein (VCP; also known as p97) has been shown to be associated with antiapoptotic function and metastasis via activation of the nuclear factor-kappaB signaling pathway. In this study, association of VCP expression with recurrence of hepatocellular carcinoma (HCC) and patient survival was examined. PATIENTS AND METHODS VCP expression in 170 patients (139 male and 31 female) with ages ranging from 31 to 81 years (median, 61 years) was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, in which staining intensity in tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells. RESULTS Immunohistochemically, 57 patients (35.2%) showed level 1, and 105 patients (64.8%) showed level 2, VCP expression. Quantitative RT-PCR analysis revealed higher VCP mRNA expression in level 2 patients (n = 7) than level 1 (n = 4) (P <.05). Patients with VCP-level 2 HCC showed higher rate of portal vein invasion in the tumor (P <.01) and poorer disease-free and overall survival (P <.0001 and P <.05, respectively) compared with level 1 patients. Multivariate analysis revealed VCP expression level, tumor multiplicity, and degree of fibrosis in the noncancerous liver tissue to be independent prognosticators for disease-free and overall survival. VCP level was an indicator for disease-free survival in each early- (I and II) and advanced- (III and IV) stage group of pathologic tumor-node-metastasis classification (P <.001 and P <.01, respectively). CONCLUSION VCP expression level has prognostic significance for disease-free and overall survival of patients with HCC.

Expression of Hepatoma-Derived Growth Factor Is Correlated with Lymph Node Metastasis and Prognosis of Gastric Carcinoma

Purpose: Hepatoma-derived growth factor (HDGF) is a unique nuclear/growth factor and might play an important role in the development and progression of carcinomas. In the present study, association of HDGF expression with recurrence and prognosis of gastric carcinoma was examined. Patients and Methods: HDGF expression in 317 patients with gastric carcinoma (233 males and 84 females) with ages ranging from 26 to 81 years (median, 60 years) was analyzed by immunohistochemistry. Samples with >90% of tumor cells to express positive immunoreactivity similar to or stronger than that in endothelial cells both for nucleus and cytoplasm were regarded as HDGF index level 2, and others as HDGF index level 1. Results: One hundred and eighty-two cases showed level 1 HDGF expression, whereas 135 cases showed level 2 HDGF expression. Patients with level 2 expression showed higher rates of proximal tumor location (P < 0.0001), large tumor size (P < 0.0001), infiltrative tumor growth (P < 0.0001), presence of vascular and lymphatic invasion (P < 0.0001 for both), presence of lymph node metastasis (P < 0.0001), deep tumor invasion (P < 0.0001), and poorer disease-free and overall survival (P < 0.0001 for both) compared to those with level 1 expression. Multivariate analysis revealed HDGF expression level as an independent prognosticator for disease-free and overall survival. Conclusion: HDGF expression level was shown to be a prognostic factor for gastric carcinoma.

Hepatoma-Derived Growth Factor Is a Novel Prognostic Factor for Patients with Pancreatic Cancer

Purpose: Hepatoma-derived growth factor (HDGF) is a nucleus-targeted growth factor playing an important role in the development and progression of cancers. This study investigated the correlation of HDGF expression and prognosis in patients with pancreatic ductal carcinoma. Patients and Methods: HDGF expression in pancreatic cancer cell lines was analyzed by Western blotting. HDGF expression was analyzed by immunohistochemistry for 50 patients with primary ductal carcinoma of the pancreas (33 male and 17 female) ranging in age from 48 to 80 years (median, 65 years) receiving surgical treatment. Cancer cells showing stronger staining than the noncancerous ducts were regarded as positive. Cases showing positive staining in <90% and >90% of tumor cells were regarded as HDGF labeling index (LI) levels 1 and 2, respectively. HDGF LI was determined separately for the nucleus and the cytoplasm. Results: Western blotting showed HDGF expression in pancreatic cancer cells similar to that of hepatic cell lines. Twenty-three (46%) and 27 (54%) cases and 22 (44%) and 28 (56%) cases showed HDGF LI levels 1 and 2 for the nucleus and the cytoplasm, respectively. Patients with nuclear HDGF LI level 1 showed a significantly better 5-year survival rate (37.0%) than those with level 2 (6.8%; P = 0.023). No significant difference was observed in the cytoplasmic HDGF LI classification. Multivariate analysis revealed nuclear HDGF LI to be an independent prognosticator. Conclusions: These findings suggest that HDGF could be a novel prognostic factor for pancreatic ductal carcinoma.

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

Small cell lung cancer (SCLC) invades locally and metastasizes distantly extremely early when compared with nonsmall cell lung cancer (NSCLC). The underlying molecular mechanisms, however, have not been elucidated. Accumulating evidence suggests that downregulation of several members of tetraspanins is associated with progression of solid tumors, thus indicating poor prognosis. Here we screened 30 lung cancer cell lines for expression of tetraspanins, CD9, CD63, CD81, CD82, CD151, and NAG-2. Flow cytometry revealed that, among these proteins, CD9 is broadly expressed in NSCLC lines, but is absent or highly reduced in most SCLC lines (P<0.0001). Using the Boyden chamber and videomicroscopic cell motility assays, we showed that stable transfection of CD9 into an SCLC line, OS3-R5, reduced cell motility on fibronectin. Furthermore, by transient transfection of green fluorescent protein (GFP)-tagged CD9 into three other SCLC lines, we observed that SCLC cells expressing GFP-CD9 were uniformly less motile than untransfected cells. CD9 or GFP-CD9 was associated with β1 integrins and distributed at the tumor cell periphery and cell–cell contacts, suggesting that CD9 modifies β1 integrin function to reduce motility. These findings suggest that low expression of CD9 may contribute to the highly invasive and metastatic phenotype of SCLC.

Elevated Expression of Valosin-Containing Protein (p97) Is Associated with Poor Prognosis of Prostate Cancer

Purpose: Valosin-containing protein (VCP) has been shown to be associated with metastasis and prognosis in human cancers. In the present study, the correlation of VCP with recurrence and prognosis in patients with prostate cancer (PCA) receiving conservative therapy was examined. Experimental Design: VCP expression was analyzed immunohistochemically in 136 patients ranging from 46 to 92 years (median, 72 years), who received conservative therapy, including androgen deprivation, radiotherapy, or watchful waiting. Staining intensity of tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells. The correlation of VCP expression between the mRNA and protein levels was examined in 10 patients. Results: Thirty-two cases (23.5%) showed level 1 and 100 (76.5%) level 2 VCP expression. Quantitative reverse transcription-PCR analysis revealed greater VCPmRNA expression in level 2 (n = 5) than level 1 cases (n = 5; P < 0.05). A significant difference was observed between VCP level 1 and 2 patients in the positive rate for the digital rectal examination (P < 0.01), serum prostate-specific antigen level (P < 0.0001), cancer volume (P < 0.0001), Gleason score (P < 0.0001), stage (P < 0.0001), and progression-free and overall survival (P < 0.0001 for both). Multivariate analysis revealed VCP expression level, serum prostate-specific antigen level, and Gleason score to be independent prognosticators for progression-free and overall survival. Progression of PCA was found in 9.4% of level 1 but in 64% of level 2 patients. Conclusions: PCA with level 1 VCP expression could be treated conservatively.