Analysis of epithelial-mesenchymal transition markers in the histogenesis of hepatic progenitor cell in HBV-related liver diseases.

BACKGROUND: The origin and heterogeneity of hepatic progenitor cells (HPCs) remain unclear. This study aimed to investigate the involvement of epithelial-mesenchymal transition (EMT) in the histogenesis of HPCs. METHODS: Surgical liver specimens from patients with HBV-related hepatitis and cirrhosis were investigated with double immunofluorescence labeling to detect antigens associated with HPCs and EMT. Ductular reactions were subjected to quantitative reverse transcription PCR following isolation by laser capture microdissection. Electron microscopic examination was performed to find an ultrastructural evidence of EMT. RESULTS: The number of EpCAM-positive HPCs was proportional to the disease severity. The S100A4 expression of HPCs was firstly observed in mild hepatitis and increased significantly in moderate hepatitis, but decreased in severe hepatitis and cirrhosis. The levels of MMP-2, Twist, and Snail increased in direct proportion to the number of HPCs. Some hepatocytes adjacent to portal tracts in cirrhosis showed positivity for MMP-2. Although CK7 and E-cadherin levels decreased in mild and moderate hepatitis, HPCs re-expressed both of them in severe hepatitis and cirrhosis. However, HPCs expressed neither vimentin nor αSMA. The relative mRNA expression levels of EpCAM and EMT-associated markers supported immunohistochemical results. Electron microscopic examination demonstrated the existence of intercellular junctions among HPCs, cholangiocytes, and intermediate hepatocyte-like cells. CONCLUSION: We provided preliminary evidence for the involvement of EMT in the histogenesis of HPCs from cholangiocytes in HBV-related liver diseases. HPCs may re-transdifferentiate into hepatocytes, and the differentiation direction depends, at least in part, on interactions between HPCs and the surrounding microenvironment, especially the non-resolving inflammation caused by HBV infection.

Genomic expression profiling and bioinformatics analysis of pancreatic cancer.

Pancreatic cancer is a polygenic disease and the fourth leading cause of cancer-associated mortality worldwide; however, the tumorigenesis of pancreatic cancer remains poorly understood. Research at a molecular level, which includes the exploration of biomarkers for early diagnosis and specific targets for therapy, may effectively aid in the diagnosis of pancreatic cancer in its early stages and in the development of targeted molecular­biological approaches for treatment, thus improving prognosis. By conducting expression profiling in para­carcinoma, carcinoma and relapse of human pancreatic tissues, 319 genes or transcripts with differential expression levels >3­fold between these tissue types were identified. Further analysis with Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes demonstrated that the translation, nucleus assembly processes and molecular functions associated with vitamin B6 and pyridoxal phosphate binding in pancreatic carcinoma were abnormal. Pancreatic cancer was additionally identified to be closely associated with certain autoimmune diseases, including type I diabetes mellitus and systemic lupus erythematosus.

Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

Peroxiredoxin 3 (Prx3) is a mitochondrial member of the antioxidant family of thioredoxin peroxidases that uses mitochondrial thioredoxin 2 as a source of reducing equivalents to scavenge hydrogen peroxide (H2O2). Here, we report that the protein levels of Prx3 are significantly reduced in VHL-deficient clear cell renal cell carcinoma (CCRCC). Furthermore, stabilization of HIF-1α protein, caused either by VHL deficiency under normoxia, or by hypoxia, significantly reduced Prx3 expression. Luciferase-reporter and chromatin-immunoprecipitation assays indicated that HIF-1α binds to the hypoxia-responsive elements of PRDX3 promoter and represses its transcription. Finally, shRNA-based assays suggested that Prx3 downregulation is required for the HIF-1α-dependent proliferation of CCRCC cells. Taken together, our results shed new light onto the mechanism of HIF-1α-dependent proliferation in CCRCC cells.

Density of tumor-associated macrophages correlates with lymph node metastasis in papillary thyroid carcinoma.

BACKGROUND: Tumor-associated macrophages (TAMs) have recently been recognized as being important players in the tumoriogenesis of many cancers, including advanced thyroid cancer. However, a role in papillary thyroid carcinoma (PTC), the most prevalent thyroid cancer, has not been established. We hypothesized that TAMs also facilitate tumor progression in PTC. METHODS: We investigated TAMs density in both benign thyroid lesions and PTC tumors by CD68 immunostaining. CD68-positive cell density was further associated with the clinicopathological characteristics of PTC patients. Finally, TAMs were isolated from PTC tumors and phenotyped by cytokine and receptor profiling. RESULTS: The overall density of TAMs was found to be significantly higher in PTC tumors, compared with thyroid goiter and follicular adenoma. The density of TAMs was positively associated with lymph node metastasis in TNM (tumor-node-metastasis) stages III/VI compared with stages I/II. No association was observed in other common tumor features, including the BRAF mutation. The isolated TAMs presented with high levels of M2-associated cytokine and receptors, making M2 the predominant TAM phenotype. CONCLUSIONS: TAMs may play a functional role in the progression of PTC.

Expression of deleted in malignant brain tumours 1 (DMBT1) relates to the proliferation and malignant transformation of hepatic progenitor cells in hepatitis B virus-related liver diseases.

AIMS: The molecular mechanisms underlying proliferation and malignant transformation of hepatic progenitor cells (HPCs) remain largely unknown. The purpose of this study was to evaluate the correlation between the expression of deleted in malignant brain tumours 1 (DMBT1) and the biological behaviour of HPCs in different hepatitis B virus (HBV)-related human liver diseases. METHODS AND RESULTS: Expression of DMBT1 in HPCs was investigated by double immunofluorescence labelling in control-group and HBV-related liver diseases, including hepatitis, hepatocellular carcinoma (HCC), non-tumoral liver tissue away from HCC, non-tumoral cirrhotic tissue adjacent to HCC, and non-HCC cirrhosis. DMBT1-positive HPCs were isolated by laser capture microdissection and subjected to duplex polymerase chain reaction in order to detect homozygous deletion of DMBT1. The number of DMBT1-positive HPCs increased in direct proportion to inflammation severity. Loss of heterozygosity for DMBT1 was more frequent in HCC tumour area and non-tumoral cirrhotic tissue adjacent to HCC, compared with other HBV-related liver diseases (P < 0.05). CONCLUSIONS: DMBT1 may play an important role in the proliferation of HPCs in HBV-related liver diseases. Moreover, down-expression of DMBT1 might enhance the risk of malignant transformation of HPCs.

Paranuclear dot-like immunostaining for CD99: a unique staining pattern for diagnosing solid-pseudopapillary neoplasm of the pancreas.

Solid-pseudopapillary neoplasm of the pancreas is a rare tumor of uncertain histogenesis that is characterized by a cystic and solid growth pattern with pseudopapillary structures. The differentiation of solid-pseudopapillary neoplasm from other pancreatic tumors is of great importance. However, it is sometimes difficult because of similarities in morphologic features and immunophenotype. CD99 is a diagnostically useful marker for Ewing sarcoma/primitive neuroectodermal tumor. The aim of this study was to investigate the diagnostic value of CD99 in solid-pseudopapillary neoplasm. We investigated immunohistochemical staining for CD99 in tissue microarray blocks from 55 cases of pancreatic solid-pseudopapillary neoplasm, 51 cases of pancreatic neuroendocrine tumor, and 54 cases of pancreatic adenocarcinoma. Biopsy specimens from 7 solid-pseudopapillary neoplasms, 6 acinar cell carcinomas, and 1 pancreatoblastoma were also investigated. All the solid-pseudopapillary neoplasm cells exhibited paranuclear dot-like immunoreactivity for CD99 regardless of the clinicopathologic or morphologic features. Forty of the 51 pancreatic neuroendocrine tumors were positive for CD99. Staining here was membranous, or membranous and cytoplasmic. Four of the 54 pancreatic adenocarcinomas and 1 pancreatoblastoma showed faint membranous staining. None of the acinar cell carcinomas was reactive for CD99. Our study has identified for the first time that pancreatic solid-pseudopapillary neoplasm exhibits a unique dot-like staining pattern for CD99. This could prove to be the most useful aspect of its immunoprofile for the definitive diagnosis of solid-pseudopapillary neoplasm and differentiation from other pancreatic tumors.

Hepatic progenitor cell represents a transitioning cell population between liver epithelium and stroma.

Following an acute injury, the liver may maintain its structure and function through mitotic division of mature hepatocytes (i.e. hepatic regeneration). However, the regeneration ability of hepatocytes can be impaired in chronic liver diseases including chronic viral infection and alcohol abuse. Hepatic progenitor cells/oval cells (HPCs/OCs), capable of differentiation into both hepatocytes and cholangiocytes, occur and proliferate during chronic injury. Unfortunately, a use of HPCs for clinical therapy is blocked by the difficulty of exact identity of HPCs in liver. Focusing on the links between phenotype of HPCs and real stem cells originating from fetal liver or bone marrow (BM), the recent studies of HPCs neglect functional analysis and the close relationship between activation of HPCs and extracellular matrix (ECM) remodeling. It is currently widely accepted that mesenchymal-epithelial transition (EMT) and epithelial-mesenchymal transition (MET) play important roles not only in liver development but also in healing of chronic injured adult liver. Co-expression of epithelial/mesenchymal and HPCs markers has been demonstrated in cells undergoing EMT/MET. These cells led to hepatic regeneration after transplanted into rats with chronic liver injury. Notably, there is an increased expression of mesenchymal markers in HPCs after exposure to transforming growth factor-beta1 (TGF-ß1). Based on these evidences, we hypothesize that HPCs represent a transitioning cell population undergoing EMT/MET, both parenchymal and mesenchymal cells of liver may be the direct sources of HPCs.

Preliminary study on the clinical and pathological relevance of gastric hepatoid adenocarcinoma.

OBJECTIVE: To appraise the histological characteristics and clinical features of gastric hepatoid adenocarcinoma (GHAC) and their relevance with prognosis. METHODS: From January 2001 to December 2003, six patients were diagnosed and confirmed pathologically in Ruijin Hospital as having a gastric hepatoid carcinoma. All these six patients, together with 30 randomly selected patients with gastric poorly differentiated adenocarcinoma (GPDA) and 30 with a primary hepatocellular carcinoma (HCC) who served as controls, were studied and analyzed clinically, histologically and immunohistologically. RESULTS: The average age of the six patients with GHAC was 66.8 years, and their serum alpha fetoprotein (AFP) level was 84-2230 ng/mL. Of these six patients, two had a recurrence of cancer and two had liver metastasis. Their average survival period was 17 months. Morphologically, the histological appearance was similar to that of HCC, with glycogen granules and hyaline globules arranged in a solid or trabecular pattern and an abundance of blood vessels and sinusoids, while blood vessel or lymphatic invasion were more often seen. Immunohistological staining showed that the tumor cells were positive for AFP and negative for Hepatocyte paraffin 1 (HepPar1), and the tumor cells were separated by CD34-positive blood vessels into a small trabecular pattern. The GPDA had a solid nest or diffuse distribution pattern and was negative for both AFP and HepPar1. In the HCC, the histological feature was manifested as solid, small and large trabecular patterns with abundance of blood vessels and sinusoids; immunohistochemical staining showed the tumor cells were positive for HepPar1. CONCLUSION: Serum AFP-positive GHAC occurred more frequently in older patients with higher rates of recurrence and liver metastasis and a poor prognosis. Histologically, GHAC was similar to HCC. The tumor cells that were positive for AFP but negative for HerPar1 could be used to differentiate with GPDA (both AFP and HerPar1 negative) and primary HCC (most AFP and HerPar1 positive).

[BCL-XL expression and mutation in non-Hodgkin's lymphoma].

The study was aimed to investigate the BCL-XL expression and mutation, and its clinical significance in non-Hodgkin's lymphoma. Lymphoma cells were selectively isolated by laser microdissection. BCL-XL expression from lymphoma tissue and microdissected lymphoma cells was measured by using real-time quantitative reverse transcription-polymerase chain reaction. BCL-XL mutation was analyzed by using direct sequencing of PCR products. The results showed that compared to 15 patients with reactive hyperplasia, BCL-XL was overexpressed in follicular lymphoma (n = 30), both in lymphoma tissue (P = 0.0064) and in microdissected lymphoma cells (P < 0.0001). No significant rise of BCL-XL expression was observed in patients with T-cell lymphoma (n = 24) and diffuse large B cell lymphoma (n = 24). In follicular lymphoma, high BCL-XL level was associated with multiple extranodal involvement (P = 0.0004), elevated lactate dehydrogenase level (P = 0.0019), high-risk international prognostic index (P = 0.0013) and a short overall survival time (P = 0.0451). Mutation analysis revealed one synonymous mutation (Codon 109 ACA-->ACC) in one case of follicular lymphoma patient. It is concluded that BCL-XL expression is closely correlated with progress of follicular lymphoma and prognosis of patients with follicular lymphoma. The value of BCL-XL expression as a prognostic marker in follicular lymphoma should be considered.

[Significance of galectin-3 and CD44v6 expression in differential diagnosis of thyroid nodules].

OBJECTIVE: To investigate the difference of galectin-3 and CD44v6 expression between benign and malignant thyroid nodules, and to evaluate their clinical value in distinguishing thyroid cancer from benign thyroid nodules. METHODS: The expression of galectin-3 and CD44v6 was immunohistochemically detected by the ABC method in 143 benign and malignant thyroid nodule samples. RESULTS: Expression of these two markers in benign thyroid nodules: galectin-3 was negative in 10 cases of para-cancer normal tissue and 14 cases of benign nodules found in the other benign thyroid disease. It was weakly positive in 4 of 52 nodular goiter (7.7%). Also it was weakly positive in 2 of 22 follicular adenomas (9.1%). But all three eosinophilic follicular adenomas were diffusely or focally positive for galectin-3. CD44v6 was negative in 10 cases of para-cancer normal tissue, but positive in 4 of 14 nodular lesions found in benign thyroid diseases (28.6%). It was also positive in 16 of 52 nodular goiters (30.8%), and weakly positive in 7 of 22 follicular adenomas (31.8%). The two markers in malignant lesions: galectin-3 was positive in 50 of 52 thyroid adenocarcinoma (96.2%), CD44v6 was positive in 42 of 52 thyroid adenocarcinoma (80.8%). The positive rate of galectin-3 and CD44v6 expression in thyroid cancer was significantly higher than that in benign thyroid nodule and normal tissue (P < 0.001). The sensitivity, specificity and accuracy of galectin-3 combined with CD44v6 in differentiating benign from malignant thyroid nodule were 80.8%, 93.4%, 88.8%; they were 96.2%, 90.1%, 92.3% for Galectin-3 alone. CONCLUSION: The immunohistochemical expression of galectin-3 and CD44v6 by the ABC method is significantly higher in thyroid cancers than in benign thyroid nodules, especially galectin-3 in thyrocyte being helpful in differentiating benign thyroid nodule from thyroid cancer.

[Pathologic diagnosis and subtyping of lymphoma in bone marrow biopsies using histologic examination, immunohistochemistry and gene rearrangement studies].

OBJECTIVE: To assess the value of histologic examination, immunohistochemistry and gene rearrangement studies in the diagnosis and subtyping of lymphoma with bone marrow involvement (BMI). METHODS: Sixty-two formalin fixed, paraffin embedded bone marrow biopsy specimens were studied. Immunohistochemical and immunoglobulin heavy chain (IgH) and T-cell receptor gene rearrangement studies were performed in each case. RESULTS: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) demonstrated mainly and interstitial infiltration by dysplastic lymphocytes, with intertrabecular nodular arrangement or in dispersion. Sometimes, pseudofollicles may be noted. A predominantly para- or intertrabecular infiltration by nodules of lymphoma cells was characteristic of follicle center cell lymphoma (FCL) cases. In most lymphoplasmacytoid lymphoma (LPL) cases, there was infiltration by small lymphocytes and plasma cells between bony trabeculae. In marginal zone cell lymphoma (MZL), vague inter- or para-trabecular nodules of polymorphic lymphoma cells with clear cytoplasm might be noted. Small to medium-sized dysplastic lymphocytes, with absence of paraimmunoblasts or pseudofollicles, were the most frequent findings in mantle cell lymphoma (MCL). Hairy cell leukemia (HCL) might be identified by the presence of distinct cell membrane and abundant clear cytoplasm, resulting in a "fried-egg" appearance. Tumor cells with large nuclei and eosinophilic nucleoli were characteristically seen in lymphomatosis diffusa (Hodgkin's disease, HD). In T-cell non-Hodgkin lymphoma with BMI, dispersed or clusters of intertrabecular neoplastic lymphoid cells with clear cytoplasm and gyriform nuclei were often observed. In diffuse large B-cell lymphoma (DLBL), the tumor cells were large and isolated or arranged in diffuse pattern. Immunohistochemically, a panel of markers, including CD3 CD20, and CD79 are valuable for the differential diagnosis of T- and B-cell lymphomas. The neoplastic cells in MCL were cyclin D1- and CD5-positive, while BCL2- and CD10-positivity was characteristic for FCL. CLL/SLL cells might be stained with CD5 and CD23, in addition to CD20 and CD79. CD25 expression might be noted in HCL: the positivity for CD15, CD30 and fascin suggests HD. There was a higher positivity rate for IgH gene rearrangement in CLL/SLL, LPL MZL and DLBL (80%, 60%, 66.7%, 70% respectively) and for T- cell receptor gamma gene rearrangement in T-cell lymphoma (66.7%). CONCLUSION: A combination of histopathology, immunohistochemistry and IgH / T-cell receptor gamma gene rearrangement studies may be of aid to the diagnosis and subtyping of lymphoma with BMI, especially if there is only a small number of tumor cells present in the specimen.

Hepatic progenitor cells in human liver cirrhosis: immunohistochemical, electron microscopic and immunofluorencence confocal microscopic findings.

AIM: To investigate whether hepatic progenitor cells (HPC), that reveal the features of oval cells in rodents and small epithelial cells (SEC) in certain human liver disease, were also found in human liver cirrhosis (HLC). METHODS: Surgical liver specimens from 20 cases of hepatitis B virus-positive HLC (15 cases containing hepatocellular carcinoma) were investigated by light microscopic immunohistochemistry (LM-IHC). Among them specimens from 15 cases were investigated by electron microscopy (EM) and those from 5 cases by immunofluorencence confocal laser scanning microscopy (ICLSM). Antibodies against cytokeratin 7 and albumin were used and single and/or double labelling were performed respectively. RESULTS: LM-IHC showed that at the margins of regenerating nodules and in the fibrous septae, a small number of cells in the proliferating bile ductules were positive for CK7 and albumin. At the EM level these HPC were morphologically similar to the SEC described previously, and also similar to the oval cells seen in experimental hepatocarcinogenesis. They were characterized by their small size, oval shape, a high nucleus/cytoplasm ratio, a low organelle content in cytoplasm, and existence of tonofilaments and intercellular junctions. ICLSM revealed that HPC expressed both cytokeratin 7 and albumin. CONCLUSION: HPC with ultrastructural and immunophenotypical features of oval cells, i.e., hepatic stem cell-like cells as noted in other liver diseases, were found in HLC. These findings further support the hypothesis that bipotent hepatic stem cells, that may give rise to biliary epithelial cells and hepatocytes, exist in human livers.

[Clinical and molecular research in a case of familial Carney complex].

OBJECTIVE: A case of primary pigmented nodular adrenal disease (PPNAD) was first diagnosed in Ruijin Hospital, Shanghai, China and molecular genetic research was then carried on the proband and his family members. METHODS: History and laboratory tests were routinely taken. Liddle's test, adrenal CT and pituitary magnetic resonance imaging were also carried out. Complete family history was obtained and eight of the family members donated their blood for DNA extraction. Polymerase chain reaction was done on all the exons of PRKAR1A gene and the product of the reaction was sequenced with ABI 3700. The right adrenal of the patient was then resected, part of the tissue was preserved in liquid nitrogen for DNA/RNA extraction and the remaining sent to Department of Pathology. RESULTS: The patient presented an atypical appearance of Cushing's syndrome. His father had a typical history of cardiac myoma. Cortisone level could not be refrained in Liddle's test for the patient. Imaging examination presented a nodular adrenal and a full pituitary. A novel mutation of PRKAR1A-S147N was found in both the patient's and his father's gene. CONCLUSIONS: This is the first patient diagnosed as PPNAD based on his clinical manifestations, laboratory tests and imaging and pathological examinations. According to the history of his father and the results of molecular genetic analysis, the diagnosis of Carney complex can be established on this patient and his father. It is also the first time that this kind of point mutation was found in Chinese people.

[The diagnosis and differential diagnosis of histiocytic necrotizing lymphadenitis. A study of histology, immunohistochemistry and electron microscopy].

OBJECTIVE: To study the diagnosis and differential diagnosis of histiocytic necrotizing lymphadenitis (Kikuchi disease, KD). METHODS: Histologic analysis and immunohistochemical study (EnVision method) was carried out in 46 cases of KD, 5 cases of nonspecific lymphadenitis (NLD), 5 cases of non-Hodgkin's lymphoma (NHL), 5 cases of Hodgkin's lymphoma (HD), 5 cases of cat-scratch disease (CSD) and 5 cases of tuberculous lymphadenitis (TBL). Electron microscopy was also performed in 6 cases of KD and 2 cases of NHL. RESULTS: Three histologic (proliferative, necrotizing and xanthomatous) patterns were noted in KD. In any of these patterns, there were some basic histologic findings: a wedge-shaped pale area at the edge of the lymph node or paracortical nodules associated with an increase in apoptotic cells or karyorrhectic debris, crescentic histiocytes, proliferative mononuclear histiocytes and absence of or very scanty neutrophils. Immunohistochemical study demonstrated focal occurrence of histiocytes expressing both CD68 and MPO. Electron microscopy confirmed the presence of apoptotic bodies, proliferative mononuclear histiocytes, crescentic histiocytes and dispersed T cells in the lesional areas. CONCLUSIONS: In general, there should not be much difficulty in differentiating KD from other types of lymphadenopathy. Sometimes, problems arise mainly because of the diversity of KD histology. Correct diagnosis can be made if one pays attention to the described characteristic morphology, peculiar immunophenotype of the histiocytes and possibly ultrastructural features.

Stem-like cells in hepatoblastoma.

BACKGROUND: The principles of tumor biology suggest that hepatoblastoma is derived from a pluripotent stem cell. Our studies were undertaken to investigate this tumor for the presence of cells with morphologic and immunophenotypic features of the oval cells of rodents that are thought to be closely related to hepatic stem cells. PROCEDURE: Hepatoblastomas of various subtypes were investigated by electron microscopy and immunoelectron microscopy with antibodies against cytokeratin 7, in the liver a marker of biliary differentiation, and albumin, a marker of hepatocytic differentiation. Immunohistochemical investigations were performed with the antibodies OV-1 and OV-6, which recognize antigens associated with oval cells. RESULTS: OV-1 stained scattered cells in seven of 12 tumors investigated and OV-6 in nine. Small epithelial cells (SEC) with the ultrastructural features of the oval cells were found by electron microscopy. They were characterized by small size (7-18 microm), often an oval shape, tonofilament bundles, and tight junctions or desmosome-like junctions. SEC were found in small numbers in areas of fetal differentiation and in moderate numbers in areas of embryonal differentiation. In small cell hepatoblastoma, nearly all the tumor cells exhibited SEC-like features. Immunoelectron microscopy revealed coexpression of cytokeratin 7 and albumin by SEC. CONCLUSIONS: SEC with ultrastructural and immunophenotypic features exhibited by oval cells, i.e., hepatic stem-like cells, are found in hepatoblastoma. Their numbers vary with the differing degrees of differentiation seen in the various subtypes. The findings further support the hypothesis that hepatoblastoma is derived from a pluripotent stem cell.