Imaging of Liver Tissues Using a Combination of Silver Impregnation and Low-Vacuum Scanning Electron Microscopy; A Simple Method for the High-Resolution Visualization of Reticulin Structures with Applicability to a Quantitative Analysis.

Background: Low-vacuum scanning electron microscopy (LVSEM) enables the detailed three-dimensional imaging of archival tissues without special pretreatments. The clinical utility of LVSEM in the assessment of liver diseases has not yet been defined. So, we investigated the utility of LVSEM imaging in morphological assessments of normal and diseased liver tissues, with a focus on reticulin structures. Methods: Formalin-fixed tissue samples of two normal livers and two hepatocellular carcinomas with background regenerative nodules/areas were stained with platinum blue stain or silver-impregnated using Watanabe's method and then comparatively observed under LVSEM. We also evaluated the applicability of LVSEM imaging of liver tissues to a quantitative analysis using a digital image analysis technique. Results: Optimal high-resolution images of reticulin structures were obtained using 10-µm-thick silver-impregnated sections. Reticulin fibers were clearly observed to run dendritically around sinusoids in normal livers, and markedly increased in regenerative nodules/areas. Normal reticulin frameworks were lost in hepatocellular carcinoma, leaving a few fragments of reticulin fibers within tumors. Moreover, when a quantitative analysis was applied to these images, we successfully demonstrated a significantly higher reticulin fiber density in regenerative nodules/areas than in the normal liver (P < 0.05). Conclusion: We not only obtained detailed three-dimensional images of reticulin structures in various liver tissues by LVSEM combined with silver impregnation but also showed their applicability to a quantitative analysis. The method presented herein may be applied to future studies for the more accurate diagnosis and better classification/risk stratification of various liver diseases.

Decreased H3K27me3 Expression Is Associated With Merkel Cell Polyomavirus-negative Merkel Cell Carcinoma, Especially Combined With Cutaneous Squamous Cell Carcinoma.

BACKGROUND/AIM: Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer, frequently infected with Merkel cell polyomavirus (MCPyV). H3K27me3 acts as a repressive histone modification that epigenetically controls gene transcription. The aim of this study was to examine H3K27me3 expression in MCC. MATERIALS AND METHODS: H3K27me3 expression levels were immunohistochemically analyzed in 20 MCPyV-positive MCCs, 15 MCPyV-negative MCCs with squamous cell carcinoma (SCC) (combined MCCs), and six MCPyV-negative pure MCCs. RESULTS: Reduced H3K27me3 expression was variously observed in MCCs. H3K27me3 H-score was significantly lower in MCPyV-negative MCCs than in MCPyV-positive MCCs (p=0.002). H3K27me3 expression was significantly lower in MCPyV-negative combined MCC component than in MCPyV-positive MCCs (p<0.001), MCPyV-negative pure MCCs (p=0.036), or pure MCC histology (p<0.001). Kaplan-Meier analysis showed no association of H3K27me3 with outcome. CONCLUSION: Differential reduction in H3K27me3 expression was observed based on MCPyV status and morphological type. These results implicate H3K27me3-mediated epigenetic changes in tumorigenesis of MCC, especially in MCPyV-negative MCC combined with SCC.

Detection of Disease-specific Fusion Genes of Soft Tissue Tumors Using Formalin-fixed Paraffin-embedded Tissues; Its Diagnostic Usefulness and Factors Affecting the Detection Rates.

BACKGROUND: Recent rapid advances in molecular biology have led the discovery of disease-specific novel fusion genes in a variety of soft tissue tumors. In this study, we attempted to detect these fusion genes using formalin-fixed paraffin-embedded (FFPE) tumor tissues and investigated their clinical utility and factors that affect the results of examination. METHODS: Reverse transcription polymerase chain reaction for the detection of tumor-specific fusion genes was performed using 41 FFPE tumor samples obtained from 37 patients representing nine histological types of soft tissue tumors that were diagnosed from 2006 to 2017 in our laboratory. RESULTS: Fusion genes in 19 (51.3%) out of 37 cases were detected successfully. Relatively high detection rates were observed in synovial sarcomas (100%, 4/4) and alveolar rhabdomyosarcomas (75%, 3/4). The detection rates of fusion genes were inversely correlated with the storage period of FFPE blocks. Decalcification by Plank-Rychlo solution significantly affected detection rates of the internal control gene (P = 0.0038). In contrast, there was no significant difference in detection rates between primary and metastatic lesion, or biopsy and resection material, or presence and absence of treatment history. CONCLUSION: In certain histological types, detection of disease-specific fusion genes of soft tissue tumors using FFPE tissues showed high sensitivity and thus had diagnostic utility. However, due to the diversity of fusion patterns and the low-quality of nucleic acid, the detection rate as a whole was sluggish and required further improvement. For factors affecting the detection results, our results suggested that it was impossible to detect fusion genes by decalcified FFPE tissues, but it may be not necessary to consider factors such as the type of specimen (biopsy or resection) and treatment history of the patients when selecting the FFPE tissues.

Expression of the IDO1/TDO2-AhR pathway in tumor cells or the tumor microenvironment is associated with Merkel cell polyomavirus status and prognosis in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer, with approximately 80% of cases related to Merkel cell polyomavirus (MCPyV). Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) are the key rate-limiting enzymes of the tryptophan-to-kynurenine metabolic pathway. With aryl hydrocarbon receptor (AhR), an intracellular transcription factor, they play a role in escaping the immunosurveillance process in several cancers. IDO1/TDO2/AhR expression associated with the MCPyV status and prognosis in MCC was investigated. Samples included 24 MCPyV-positive MCCs, 12 MCPyV-negative MCCs with squamous cell carcinoma, and 7 MCPyV-negative pure MCCs. They were stained immunohistochemically with IDO1, TDO2, and AhR antibodies and analyzed. Higher IDO1 expression in MCC tumor cells was found in MCPyV-negative than in MCPyV-positive MCC (P < .001). The tumor microenvironment (TME) in MCPyV-negative MCC expressed higher TDO2 than in MCPyV-positive MCC (P < .001). Kaplan-Meier and log-rank tests showed that MCC with lower IDO1 expression in tumor cells and with lower TDO2 and AhR expressions in TME had better overall survival than otherwise (P = .043, .008, and .035, respectively); lower TDO2 expression in TME was also associated with longer disease-specific survival (P = .016). This suggests that IDO1, TDO2, and AhR express differentially in tumor cells or TME and play different roles in tumorigenesis between MCPyV-positive and MCPyV-negative MCC that may affect the MCC biology. Evaluating IDO1/TDO2/AhR expression is important for selecting the most likely patients with MCC for immunotherapies targeting the IDO1/TDO2-AhR pathway.

Expression of Notch 3 and Jagged 1 Is Associated With Merkel Cell Polyomavirus Status and Prognosis in Merkel Cell Carcinoma.

BACKGROUND/AIM: Merkel cell carcinoma (MCC) is a rare, aggressive, neuroendocrine skin cancer and most MCCs are related to infection with Merkel cell polyomavirus (MCPyV). Notch signaling modulates cell fate in various tissues including the skin during development and homeostasis, and its aberrant activity relates to onset and progression of various malignancies. Therefore, association of NOTCH1/ NOTCH2/NOTCH3/jagged 1 (JAG1) expression with MCPyV status and prognosis in MCC was investigated. MATERIALS AND METHODS: A total of 19 MCPyV-positive and 19 MCPyV-negative MCC samples from patients were stained immunohistochemically with antibodies against NOTCH1, NOTCH2, NOTCH3, and JAG1 and analyzed. RESULTS: Expression of NOTCH1 and NOTCH2 was not associated with MCPyV status or prognosis. However, higher JAG1 expression was found in MCPyV-negative than in MCPyV-positive MCC (p<0.001), and NOTCH3 expression was higher in MCPyV-positive MCC (p=0.062). Kaplan-Meier and multivariate analyses showed that patients with MCC with higher NOTCH3 expression had better overall survival than otherwise (p=0.001 and p=0.033, respectively). CONCLUSION: Expression of NOTCH3, as a tumor suppressor, is an independent predictor of MCC outcome.

Higher Expression of Activation-induced Cytidine Deaminase Is Significantly Associated with Merkel Cell Polyomavirus-negative Merkel Cell Carcinomas.

BACKGROUND: Merkel cell carcinomas (MCCs), clinically aggressive neuroendocrine skin cancers, are divided into Merkel cell polyomavirus (MCPyV)-positive and -negative tumors, which show different clinicopathological features and may develop through different mechanisms of carcinogenesis. Aberrant expression of activation-induced cytidine deaminase (AID) as a genomic modulator was demonstrated through pathogen-related NF-κB signal in Helicobacter pylori-associated gastric cancer, adult T cell leukemia/lymphoma (HTLV-1), hepatoma (HCV), and Burkitt lymphoma (EBV). METHODS: To elucidate the relation of aberrant AID expression in MCPyV-positive and -negative MCCs, we evaluated immunohistochemical expressions of AID and AID-regulating factors between 24 MCPyV-positive and 17 MCPyV-negative MCCs. RESULTS: AID expression was significantly higher in MCPyV-negative MCCs than MCPyV-positive ones (P = 0.026), although expression of NF-κB p65 (phospho S536) (AID-enhancer) was significantly higher in MCPyV-positive MCCs than MCPyV-negative ones (P = 0.034). Expressions of PAX5 and c-Myb were not significantly different between these subgroups. Expressions of AID and AID-regulating factors were not correlated to prognosis of MCC patients. CONCLUSION: Our findings suggest that although pathogen-induced AID expression through upregulation of NF-κB may be relevant to carcinogenesis of MCPyV-positive MCCs, the significantly higher aberrant AID expression in MCPyV-negative MCCs is consistent with the fact that MCPyV-negative MCCs have an extremely higher mutation burden than MCPyV-positive ones.

Association of expression of the hedgehog signal with Merkel cell polyomavirus infection and prognosis of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that mostly occurs in the elderly. Merkel cell polyomavirus (MCPyV) is detected in approximately 80% of MCCs and is associated with carcinogenesis. Hedgehog signaling pathway plays a role in human embryogenesis and organogenesis. In addition, reactivation of this pathway later in life can cause tumors. Twenty-nineMCPyV-positive and 21 MCPyV-negative MCCs were immunohistochemically stained with primary antibodies for hedgehog signaling (SHH, IHH, PTCH1, SMO, GLI1, GLI2, and GLI3) and evaluated using H-score. Polymerase chain reaction and sequence analysis for SHH and GLI1 exons were also performed. Expression of SHH was higher in MCPyV-positive MCCs than in MCPyV-negative MCCs (P<.001). Higher expression of GLI1, MCPyV infection, male sex, and Japanese ethnicity were associated with better overall survival (P=.034, P=.001, P=.042, and P=.036, respectively). Higher expression of SHH and MCPyV infection were associated with improved MCC-specific survival (P=.037 and P=.002, respectively). The mutation analysis of prognosis-related GLI1 and SHH genes in our study revealed a low frequency of mutations in the 10 exons examined, except GLI1 exon 5 (18/22 cases), all having the same silent mutation of c.576G>A. Only 2 mutations with amino acid changes were detected in MCPyV-negative MCCs only: 1 missense mutation in GLI1 exon 4 and 1 nonsense mutation in SHH-3B. Expression of SHH and GLI1 may be useful prognostic markers of MCC because increased expression was associated with better prognosis. The high rate of c.576G>A silent mutation in GLI1 exon 5 was a feature of MCC.

Multiple Skin Cancers in a Renal Transplant Recipient: A Patient Report with Analyses of Human Papillomavirus and Human Polyomavirus Infection.

Skin cancer is an important complication in renal transplant recipients. Associations of transplant-related skin tumor with ultraviolet radiation, age at transplantation, type of immunosuppressant drug administered, and viral infection have been reported; however, the details remain unclear. We report a 61-year-old man who had underwent renal transplantation at 38 years of age and developed multiple skin tumors or squamous cell carcinomas (SCCs). Polymerase chain reaction (PCR) analyses of the patient's 12 tumors for viral DNAs of cutaneous or mucosal human papillomavirus (HPV) and 6 human polyomaviruses (MCPyV, trichodysplasia spinulosa-associated, BK, JC, KI and WU polyomaviruses) only detected cutaneous HPV-DNA in only 5 of the tumors; no other viruses were detected. Real-time PCR showed high loads of cutaneous HPV in 3 SCCs and very low loads of MCPyV in 9. Immunohistochemistry revealed no tumor cell expression for MCPyV-large T-antigen or mucosal HPV. Our report not only reconfirmed the association of cutaneous HPV5 with skin cancer in renal transplant recipients in previous studies but also showed no relevant association of 6 human polyomaviruses and mucosal HPV with skin tumors.

Immunoglobulin expressions are only associated with MCPyV-positive Merkel cell carcinomas but not with MCPyV-negative ones: comparison of prognosis.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer, often associated with Merkel cell polyomavirus (MCPyV). Recently, immunoglobulin (Ig) expression was reported in MCC, thereby suggesting that B cells might be their cellular ancestors. We tested 30 MCCs (20 MCPyV-positive and 10 MCPyV-negative) using immunohistochemistry for the expressions of IgG, IgA, IgM, Igκ, Igλ, terminal desoxynucleotidyl transferase, paired box gene 5 (PAX5), octamer transcription factor-2 (Oct-2), and sex-determining region Y-box 11 (SOX11). We performed in situ hybridization for Igκ-mRNA or Igλ-mRNA and Ig heavy chain (IgH) gene rearrangement (IgH-R) analyses. The expressions of PAX5, TdT, Oct-2, and SOX11 were not significantly different between MCPyV-positive and MCPyV-negative MCCs. At least 1 of IgG, IgA, IgM, or Igκ was expressed in MCPyV-positive (14/20, 70%) and none in MCPyV-negative MCCs (P=0.0003). There was a higher tendency for Igκ-mRNA expression (7/19, using in situ hybridization) and IgH-R (10/20, using polymerase chain reaction) in MCPyV-positive than in MCPyV-negative MCCs (0/10 and 2/10, respectively), thus suggesting a different Ig production pattern and pathogenesis between the 2 types of MCC. Ig expression or IgH-R in MCPyV-positive MCCs might be associated with MCPyV gene integration or expression in cancer cells but do not necessarily suggest a B-cell origin for MCCs. IgH expression or IgH-R nonsignificantly correlated with improved prognosis. However, these might be important factors that influence the survival of neoplastic cells and might allow the development of novel therapies for patients with MCPyV-positive MCCs.

A new in situ hybridization and immunohistochemistry with a novel antibody to detect small T-antigen expressions of Merkel cell polyomavirus (MCPyV).

BACKGROUND: Approximately 80% of Merkel cell carcinomas (MCCs) harbor Merkel cell polyomavirus (MCPyV) which monoclonally integrates into the genome and has prognostic significance. The presence or absence of MCPyV is usually diagnosed using CM2B4 immunohistochemistry (IHC) for MCPyV-large T antigen (LT) protein. However, this method poses a risk of misdiagnosis. METHODS: In this study, we determined MCPyV infection in MCCs using real-time PCR for MCPyV-LT DNA and prepared 16 cases of MCPyV-DNA-positive and -negative groups. Diagnostic sensitivity and specificity of conventional PCR for MCPyV-small T antigen (MCPyV-ST), IHC using a newly developed polyclonal antibody (ST-1) for MCPyV-ST protein (MCPyV-ST) (aa: 164-177), and in situ hybridization (ISH) as well as real-time PCR for MCPyV-ST mRNA were compared against CM2B4-IHC for sensitivity (0.94, 15/16) and specificity (0.94, 15/16). RESULTS: The followings are the respective sensitivity and specificity results from examinations for MCPyV-ST gene: conventional PCR for the MCPyV-ST (0.94, 1.0), ST-1-IHC (0.69, 1.0), real-time PCR for ST mRNA (1.0, no data), ST mRNA ISH (0.94, 1.0). Each of the MCPyV-pseudonegative (1/16) and -pseudopositive (1/16) diagnoses evaluated using CM2B4-IHC were accurately corrected by examinations for MCPyV-ST or its expression as well as real-time PCR for MCPyV-LT. Sensitivity of CM2B4-IHC (0.94) was superior to that of ST-1-IHC (0.69) but equal to that of ST mRNA-ISH (0.94). Specificities of ST-1-IHC (1.0) and ST mRNA-ISH (1.0) were superior to that of CM2B4-IHC (0.94). CONCLUSIONS: Therefore, combined application of ST mRNA-ISH and ST-IHC as well as CM2B4-IHC is recommended and will contribute to the diagnostic accuracy for MCPyV infection in MCCs. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9966295741144834.

Merkel cell polyomavirus (MCPyV) strains in Japanese merkel cell carcinomas (MCC) are distinct from Caucasian type MCPyVs: genetic variability and phylogeny of MCPyV genomes obtained from Japanese MCPyV-infected MCCs.

Most of merkel cell carcinomas (MCC), a rare, aggressive skin cancer with neuroendocrine features, harbor merkel cell polyomavirus (MCPyV). Seroepidemiological studies suggested high prevalence of MCPyV in the human population. More than ten sequence data on MCPyV strains in Japan have been available, whereas most sequence data were detected from patients living in Europe or European ancestry. Analysis of nine almost complete and 19 partial sequences from two oncogenes, small T antigen (ST) and large T antigen (LT) genomes obtained from 32 Japanese MCPyV-infected MCC revealed that each Japanese MCPyV-infected MCC harbored a specific MCPyV strain with some synonymous or, silent mutations and stop codons or deletions, but functional domains of T antigen had no amino acid changes. All stop codons were localized after retinoblastoma protein-binding domain. These Japanese MCPyV strains were very closely interrelated to themselves and a consensus sequence of Japanese strain was generated. Phylogenetic analysis of our nine sequences and 70 other sequences for ST and LT gene registered in GenBank indicated that Japanese or Asian MCPyV strains formed distinct clades from Caucasian clade, and phylogenetic tree of our nine and 75 other sequences for ST gene formed characteristic three clades and showed that all Japanese or Asian strains were included in the dominant clade. These suggested the possibility of geographically related genotypes of MCPyV. The genomic characterization of MCPyV variants will provide an important database and insights for illuminating their evolutional and biological differences.

Detection of Merkel cell polyomavirus in the human tissues from 41 Japanese autopsy cases using polymerase chain reaction.

It has recently been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus (MCPyV). MCPyV has been detected in human tissue samples. However, detailed distribution of MCPyV in non-neoplastic Japanese human tissues remains unclear. To address this, we used single or real-time quantitative polymerase chain reaction (PCR) for 41 autopsy cases. PCR revealed MCPyV-DNA in non-neoplastic samples: total, 29/41 (71%); adult, 29/39 (74%); fetus or infant, 0/2; men, 24/28 (86%); women, 5/13 (38%); total human tissues, 66/572 (12%); skin, 8/15 (53%); adrenal gland, 9/33 (27%), and other 16 organs (4-25%). This study first reported the presence of MCPyV-DNA in non-neoplastic tissues of thyroid gland, adrenal gland, spleen, bone marrow, stomach, gallbladder, pancreas, heart, and aorta. PCR revealed that viral load ranged from 0.00026 to 0.22 in all MCPyV-positive tissues compared with Merkel cell carcinoma samples. These detailed PCR data showed higher prevalence of MCPyV infection in Japanese men than women (p = 0.004) and broad distribution of MCPyV with low viral load in more non-neoplastic human tissues than in the previous reports. These data provide valuable insights for further studies of MCPyV infection and MCPyV-related diseases.

CD1a expression in PEComas.

According to the World Health Organization classification, neoplasms with perivascular epithelioid cell differentiation (PEComas) are mesenchymal tumors composed of histologically and immunohistochemically distinctive PEC. Generally, nearly all PEComas have immunoreactivity for both melanocytic (HMB-45 and/or melan A) and smooth muscle (actin (SMA) and/or desmin) markers. Recently the authors reported that benign clear cell sugar tumor of the lung, one of the PEComas, expressed CD1a. Therefore the purpose of the present study was to investigate the relationship between PEComas and CD1a expression. Nineteen PEComas were obtained, which included angiomyolipoma of the kidney or liver, lymphangiomyomatosis of the uterus or lung and clear cell sugar tumor of the lung. Eighteen tumors had alpha-SMA and HMB-45 expression and 16 had melan A expression. In contrast, all 19 tumors had CD1a expression. The present study confirms CD1a expression in many cases of PEComa. These data suggest that CD1a expression can be an additional new marker for PEComas and also supports the distinct and integrated disease entity of PEComas.

A new animal model for primary and persistent Epstein-Barr virus infection: human EBV-infected rabbit characteristics determined using sequential imaging and pathological analysis.

Most adults have persistent Epstein-Barr virus (EBV)-infection. Adolescents and young adults with primary EBV-infection frequently develop infectious mononucleosis. Latent EBV-infection is associated with various diseases, neoplasms, and hematological disorders. In vivo animal models of human EBV infection, such as non-human primates, have had limited success. A new rabbit model for primary human EBV-infection is described in this study. Seven male rabbits inoculated intravenously with EBV were sequentially imaged by ultrasonography and computed tomography, and examined for anti-EBV-VCA titer and EBV-DNA levels in blood. Six rabbits demonstrated transient splenomegaly, increased anti-EBV-VCA titers and/or EBV-DNA in blood. Transient infiltration of some EBER1-positive lymphocytes was observed in biopsied liver tissues. After splenomegaly, two rabbits tested continuously negative, two alternatively positive and negative, and one consistently positive EBV detection in blood for 470 days. One tested negative for both EBV DNA and splenomegaly. On the 14th day, mild to moderate numbers of EBER1-positive lymphocytes expressing LMP1, EBNA2, or ZEBRA infiltrated mainly in enlarged white pulps of two splenectomized materials. These cells included both B and T cells. EBV clonality analysis revealed an oligoclonal pattern. These indicate that EBV-inoculated rabbits exhibiting heterogenous host reactions are a good model for primary and persistent human EBV infection.

Imbalance between expression of endothelin receptors A and B in terminal liver cirrhosis due to hepatitis C viral infection: immunohistochemical study of autopsy cases.

BACKGROUND AND AIM: Expression of endothelin receptors in terminal liver cirrhosis is not well investigated. The aim of this study was to investigate the expression of the endothelin type A receptor (ETAR) and endothelin type B receptor (ETBR) immunohistochemically using paraffin-embedded tissue sections from patents with terminal liver cirrhosis (TLC), non-terminal liver cirrhosis (NTLC) and non-cirrhotic liver fibrosis (NCLF) caused by hepatitis C viral infection. METHODS: Liver tissue sections from 38 autopsy cases, including 12 cases of NCLF (mild, moderate or severe liver fibrosis), 11 cases of NTLC and 15 cases of TLC, were stained using anti-ETAR and anti-ETBR antibodies after antigen retrieval. Double staining using antibodies to alpha-smooth muscle actin (ASMA) was also performed. RESULTS: There were significantly fewer ETBR-positive cells in TLC compared with NTLC and NCLF. Numbers of ASMA-positive stellate cells expressing ETBR were also significantly lower in TLC. Therefore, the ETAR/ETBR ratio of sinusoidal cells is significantly higher in TLC than in NTLC and NCLF. ASMA-positive stellate cells showed similar evidence of ETAR and ETBR expression. CONCLUSIONS: There is a difference in ETAR and ETBR expression among TLC, NTLC and NCLF: the ETAR/ETBR ratio is increased in TLC due to a relative decrease in ETBR expression. This finding may be useful for the diagnosis of TLC with regard to circulatory disturbances in the liver.

Immunohistochemical distribution of phosphatidylglucoside using anti-phosphatidylglucoside monoclonal antibody (DIM21).

The immunohistochemical distribution of phosphatidylglucoside (PhGlc) in organs obtained from human autopsy cases was investigated using the DIM21 antibody. Immunohistochemical staining was performed on formaline-fixed, paraffin-embedded sections using the simple stain peroxidase method. The sections were then subjected to antigen retrieval by microwave irradiation in citrate buffer. PhGlc expression was observed in not only the epithelial but also the non-epithelial components of several visceral organs. Squamous and glandular epithelial cells were positive for PhGlc in several organs. The surface areas of the epithelium, particularly the squamous epithelium, were positive. Mesothelial cells were also positive in some organs. Endothelial cells, polymorphonuclear (PMN) cells are positive in several organs. Macrophage is positive in many organs. Epithelial cells of the gallbladder were positive, however, the intrahepatic bile ducts were not positive. In the brain tissue, astroglial cells, the chorioide plexus, the pituitary gland, and ependymal cells were positive. Further investigation is indispensable in order to establish a relationship between cell differentiation and PhGlc expression.

Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann-Pick disease type C (NPC) fibroblasts: a potential basis for glial cell activation in the NPC brain.

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin-6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1-/- mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1-/- mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1-/- mouse brain.

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases.

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2-40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF-C), VEGF-D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF-D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF-C or VEGF-D expression. High LVD, induced by VEGF-C or VEGF-D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.

Benign clear (sugar) cell tumor of the lung with CD1a expression.

Reported herein is a case of benign clear cell tumor of the lung in a 60-year-old man. Chest X-ray and CT examination revealed an abnormal nodule with homogenous density and a clear margin in the lower lobe of the left lung. The resected tumor was 13 mm in size, well-circumscribed and was graysh-white on cut surface. Histological examination showed a diffuse growth pattern of polygonal tumor cells with indented and pleomorphic nuclei, and clear abundant cytoplasm with a distinct cell border surrounded by thin-walled vascular spaces and sinusoid-type vessels. The clear cytoplasm contained numerous glycogen granules as demonstrated by PAS staining. In the present case there was focal immunoreactivity for S-100 protein, HMB-45, neuron-specific enolase, cathepsin B and melan A, which are consistent with reported immunohistochemical staining patterns of benign clear cell tumor. Based on these findings, the tumor was diagnosed as a benign clear cell tumor of the lung. Although most clear cell tumors are considered to belong to the family of neoplasms with perivascular epithelioid cell differentiation (PEComas), histogenesis of benign clear cell tumor of the lung has remained unclear. This first report of CD1a expression in this tumor might provide a new insight into its histogenesis and diagnosis.