Subcutaneous porocarcinoma clinically presenting as a soft tissue tumor.

Porocarcinoma is a rare malignancy with glandular adnexal differentiation. A 38-year-old Japanese man noticed a subcutaneous mass in right inguinal region about 20 years prior to being examined. Radiological examinations demonstrated the mass, 11 × 10 cm in size, was in the subcutaneous fat tissue. Recently, the mass grew rapidly, and it was biopsied by an orthopedist based on clinical diagnosis of primary soft tissue tumor. Histopathological examination of the resected specimens also revealed that the tumor lacked involvement to the skin. Microscopically, the tumor was mainly composed of poroid cells with partially obvious squamous differentiation, accompanied by focal ductal structures immunoreactive for CEA and EMA. The tumor contained a low-grade area consisting of poroid cells and high-grade area with squamous differentiation. This histopathological heterogeneity suggested malignant transformation from poroma. The patient had the tumor in almost same size over the period of 20 years, which is the longest in the previous reports. This unique case of subcutaneous porocarcinoma is reported.

Immature squamous metaplasia (focal atypical epithelial hyperplasia) of the pancreatic duct-immunohistochemical distinction from intraductal carcinoma.

AIMS: Immature squamous metaplasia of the pancreatic duct (ISMPD) can be difficult to differentiate from an intraductal carcinoma of the pancreas (ICP), and little is known about the pathological nature of ISMPD. The aim of this study was to analyse 20 ISMPD and 10 ICP tissue samples. METHODS AND RESULTS: ISMPD shares some characteristics with ICP. Seven of 20 ISMPD samples were covered by a layer of pancreatic duct epithelium, whereas this was not seen in the ICP samples. Immunohistochemistry of ISMPD revealed positivity for p63 (100%), cytokeratin 5/6 (95%), cytokeratin 7 (95%), cytokeratin 20 (10%), and MUC-1 (95%), and the samples were negative for p53, carcinoembryonic antigen (CEA), and bcl-2. In contrast, ICP was positive for p63 (40%), p53 (10%), cytokeratin 7 (90%), cytokeratin 20 (20%), CEA (30%), and MUC-1 (80%), and negative for cytokeratin 5/6. However, in 84% (16) of the ISMPD samples, cytokeratin 7 was expressed only by an epithelial layer at the apical surface; this expression pattern was not found in any of the 10 ICP samples. The mean Ki67 labelling index was 1.0% in ISMPD and 18.5% in ICP. CONCLUSIONS: Our study suggests that immunohistochemical staining for cytokeratin 5/6 and Ki67 constitutes the best combination for differentiating ISMPD from ICP.

Transcription factor Runx2 is a regulator of epithelial-mesenchymal transition and invasion in thyroid carcinomas.

Runx2/Cbfa1 is a member of the Runt-related transcription factor family and is an essential regulator of osteoblast/chondrocyte differentiation. Recently, aberrant expression of Runx2 and its oncogenic functions have been identified in the progression and metastasis of human cancers. In this study, we investigated the expression profile of Runx family genes in normal thyroid tissue, non-neoplastic but abnormal thyroid tissue, various types of thyroid tumors and representative human thyroid carcinoma cell lines. Using reverse transcriptase-PCR and western blotting, we found that Runx2 was consistently upregulated in papillary carcinomas (PCs) and thyroid carcinoma cell lines compared with normal thyroid tissue. With immunohistochemistry, we observed negative or focal immunoreactivity of Runx2 in the nuclei of normal thyroid follicular cells. None of the non-neoplastic thyroid tissues, including Graves' thyroid and adenomatous goiter, had diffuse positivity of Runx2. Expression of Runx2 in benign follicular adenomas varied from negative to diffusely positive. Meanwhile, all malignant thyroid tumors showed some Runx2 immunopositivity. It was diffuse and intense in 83% (19/23) of PCs, 71% (5/7) of follicular carcinomas (FCs) and 40% (4/10) of undifferentiated carcinomas (UCs). In thyroid carcinoma cell lines, the MEK inhibitor U0126 suppressed Runx2, suggesting an association of the MAPK/ERK pathway with Runx2 regulation. Effective silencing of Runx2 by short interfering RNA (siRNA) demonstrated downregulation of EMT-related molecules (SNAI2, SNAI3 and TWIST1), MMP2 and vasculogenic factors (VEGFA and VEGFC) in thyroid carcinoma cells. We also confirmed that Runx2 silencing suppresses thyroid carcinoma cell invasion in transwell assays. In conclusion, this study provides insight into the potential molecular mechanism of thyroid cancer invasion. Our data suggest that enhanced Runx2 is functionally linked to tumor invasion and metastasis of thyroid carcinoma by regulating EMT-related molecules, matrix metalloproteinases and angiogenic/lymphangiogenic factors.

Expression of aquaporin3 in human neoplastic tissues.

AIMS: Aquaporin3 (AQP3) is distributed widely in mammalian tissues and plays an important role in fluid homeostasis. The aim of this study was to investigate the pattern of expression of AQP3 in a variety of human neoplastic tissues and to explore its diagnostic implications. METHODS AND RESULTS: We studied 798 neoplastic tissues using immunohistochemistry with anti-AQP3 antibody. We demonstrated a high positive frequency of AQP3 immunoreactivity in pituitary adenomas, salivary gland tumours, thymic tumours, adenocarcinoma of the lung and prostate, squamous cell carcinomas of the skin, oesophagus and uterine cervix, apocrine carcinoma of the breast, germinal cell tumours of the ovary and testis and urothelial carcinoma of the bladder. None of the sarcomas or central nervous system tumours showed AQP3 immunoreactivity. Most tumours with a high frequency of AQP3 positivity had corresponding or surrounding normal cells that also expressed AQP3. AQP3 was not a specific marker for benign or malignant epithelial neoplasms. CONCLUSION: AQP3 protein is expressed in a variety of epithelial tumours limiting its use as a diagnostic marker. Furthermore, AQP3 expression in tumour cells reflected the expression status of AQP3 in the corresponding normal cells. Our data suggest that water metabolism through AQP3 is maintained during neoplastic transformation in most human tissues.

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis.

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease-2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm(2)) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm(2)) (P < 0.01). (3)H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.

RET rearrangements and BRAF mutation in undifferentiated thyroid carcinomas having papillary carcinoma components.

AIMS: To elucidate the genetic background of anaplastic transformation, RET rearrangements and BRAF mutation were studied in composite undifferentiated carcinomas (UCs) of the thyroid, which are UCs having papillary carcinoma (PC) components. METHODS AND RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) was performed for RET rearrangements and PCR for BRAF mutation in UC and PC components that were microdissected separately from seven composite UCs. Forty-two thyroid cancers with single component histology (14 UCs and 28 PCs) were also studied in the same manner. RET/PTC1 was undetectable in both components from all seven composite UCs, and RET/PTC3 was identified in both components of one composite UC. BRAF mutation was identified in both components from three composite UCs and only in the PC components from two composite UCs. In contrast, in thyroid carcinomas with single component histology, RET/PTC1 was detected in 11% of PCs and in none of the UCs, and RET/PTC3 was not found in any of the tumours studied. BRAF mutation was identified in 82% of PCs and in 21% of UCs. CONCLUSIONS: The high frequency of BRAF mutation and the absence of RET rearrangements in UC components from composite UCs supports the hypothesis that UCs may actually represent progressive malignant degeneration of a BRAF-mutated, well-differentiated thyroid carcinoma.

Epigenetic silencing of TTF-1/NKX2-1 through DNA hypermethylation and histone H3 modulation in thyroid carcinomas.

Thyroid transcription factor-1 (TTF-1), also known as NKX2-1, is a homeodomain containing transcriptional factor identified in thyroid, lung and central nervous system. In the thyroid, TTF-1 is essential for thyroid organogenesis and governs thyroid functions by regulating various thyroid-specific genes. We previously demonstrated that most differentiated thyroid neoplasms, including follicular adenomas/carcinomas and papillary carcinomas, express TTF-1 at both protein and mRNA levels. However, certain subtypes of thyroid cancers have shown low or negative expression of TTF-1. The aim of our study was to investigate the function of epigenetic modification in dysregulation of TTF-1 in thyroid carcinoma cells. We evaluated the expression of TTF-1 in primary thyroid tissues (normal thyroid, papillary carcinoma and undifferentiated carcinoma) and in thyroid carcinoma cell lines using immunohistochemistry and RT-PCR. Methylation-specific PCR targeting CpG islands of TTF-1 and chromatin immunoprecipitation (ChIP) for histone H3 lysine 9 (H3-lys9) were applied to clarify the correlation of the TTF-1 expression profile and epigenetic status. We also explored whether epigenetic modifiers, including 5-aza-deoxycytidine, could restore TTF-1 expression in thyroid carcinoma cells. In our current study, immunohistochemistry and RT-PCR showed positive expression of TTF-1 in normal thyroids and papillary carcinomas. Meanwhile, most of the undifferentiated carcinomas and the cell lines lost TTF-1 expression. No methylation in the CpG of TTF-1 promoter was detected in normal thyroids or papillary carcinomas. In contrast, DNA methylation was identified in 60% of the undifferentiated carcinomas (6/10) and 50% of the cell lines (4/8). ChIP assay demonstrated that acetylation of H3-lys9 was positively correlated with TTF-1 expression in thyroid carcinoma cells. Finally, DNA demethylating agents could restore TTF-1 gene expression in the thyroid carcinoma cell lines. Our data suggest that epigenetics is involved with inactivation of TTF-1 in thyroid carcinomas, and provide a possible means of using TTF-1 as a target for differentiation-inducing therapy through epigenetic modification.

Gene expression in endothelial cells and intimal smooth muscle cells in atherosclerosis-prone or atherosclerosis-resistant regions of the human aorta.

BACKGROUND/AIMS: We compared the atherogenic gene expression in the intimas of atherosclerosis-prone regions (proximal walls), which are exposed to disturbed shear stress, and atherosclerosis-resistant regions (apices), which are exposed to unidirectional laminar shear stress, at the orifices of the intercostal arteries of human aortas. METHODS AND RESULTS: Expression of mRNAs, detected by in situ RT-PCR, for IL-1 beta, TNF-alpha, VCAM-1, PAF receptor and GRP in endothelial cells (ECs), and of PDGF receptor beta (PDGFR-beta), MCP-1, GRP and collagen type-1 by smooth muscle cells (SMCs) in the proximal walls, was significantly enhanced, while seldom observed in the elastic-hyperplastic layer of the apices. Protein expression of PDGFR-beta, IL-1 beta and TNF-alpha was also observed on the proximal walls. SMC growth in the apices was inhibited. Cultured SMC growth and their expression of PDGFR-beta were also significantly inhibited by elastin. CONCLUSION: These results suggest that the construction of the elastic-hyperplastic layer and the subsequent inhibition of SMC growth by elastin, with stabilized ECs under unidirectional laminar shear stress, resulted in atherosclerosis-resistant regions at the apices of human aortas, and that the continuous induction of atherogenic gene expression by ECs activated by disturbed shear stress inhibits the formation of atherosclerosis-resistant intima along the proximal walls.

Activation of EDTA-resistant gelatinases in malignant human tumors.

Among the many proteases associated with human cancer, seprase or fibroblast activation protein alpha, a type II transmembrane glycoprotein, has two types of EDTA-resistant protease activities: dipeptidyl peptidase and a 170-kDa gelatinase activity. To test if activation of gelatinases associated with seprase could be involved in malignant tumors, we used a mammalian expression system to generate a soluble recombinant seprase (r-seprase). In the presence of putative EDTA-sensitive activators, r-seprase was converted into 70- to 50-kDa shortened forms of seprase (s-seprase), which exhibited a 7-fold increase in gelatinase activity, whereas levels of dipeptidyl peptidase activity remained unchanged. In malignant human tumors, seprase is expressed predominantly in tumor cells as shown by in situ hybridization and immunohistochemistry. Proteins purified from experimental xenografts and malignant tumors using antibody- or lectin-affinity columns in the presence of 5 mmol/L EDTA were assayed for seprase activation in vivo. Seprase expression and activation occur most prevalently in ovarian carcinoma but were also detected in four other malignant tumor types, including adenocarcinoma of the colon and stomach, invasive ductal carcinoma of the breast, and malignant melanoma. Together, these data show that, in malignant tumors, seprase is proteolytically activated to confer its substrate specificity in collagen proteolysis and tumor invasion.

Expression of aquaporin 3 (AQP3) in normal and neoplastic lung tissues.

Aquaporin 3 (AQP3) acts as the membrane channel of water and other small solutes and plays a major role in fluid homeostasis. To investigate the expression of AQP3 in normal and neoplastic lung tissues, we studied a series of 149 lung carcinoma tissues and 2 cell lines by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction. In normal lung tissues, immunohistochemical expression of AQP3 was demonstrated in bronchial basal cells, alveolar type II cells, bronchiolar epithelial cells, and secretory cells of submucosal glands. In lung carcinomas, AQP3 expression was observed in 59 (70.2%) of 84 adenocarcinomas. Squamous cell carcinoma and large cell carcinoma had rather low positive ratios (35.8% and 13.4%, respectively). No AQP3 expression was demonstrated in small cell carcinoma, pleomorphic carcinoma, or metastatic colon adenocarcinoma. In adenocarcinomas, AQP3 was detected in all tumors of bronchioloalveolar subtype. Papillary subtype also showed a higher positive ratio of AQP3 compared with that in acinar and solid with mucin subtypes. In addition, AQP3 expression was related to tumor differentiation and clinical stage in adenocarcinomas. Western blotting and reverse transcriptase-polymerase chain reaction analyses confirmed the expression of AQP3 protein and messenger RNA in cell lines and tissues of lung adenocarcinoma. We conclude that AQP3 is widely expressed in the normal respiratory tract and can play an important role in the maintenance of water homeostasis. In addition, lung carcinomas, especially adenocarcinomas, can produce AQP3, possibly in connection with their functional and/or biological nature, although the detailed mechanism of AQP3 expression in lung carcinomas remains to be clarified.

Estimation of prognoses for cervical intraepithelial neoplasia 2 by p16INK4a immunoexpression and high-risk HPV in situ hybridization signal types.

The present study used immunohistochemical staining and in situ hybridization (ISH) to examine whether progression of cervical intraepithelial neoplasia, grade 2 (CIN 2) can be predicted by p16INK4a immunoexpression and high-risk human papilloma virus (HPV) ISH signal types. We studied 52 cases histologically diagnosed with CIN 2: dysplasia regressed in 28 cases; 13 cases progressed to CIN 3; and CIN 2 persisted in 11 cases. Expression of p16INK4a and high-risk HPV signal both related to grade of CIN. Stronger p16INK4a immunoexpression and a higher frequency of expression of a punctate nuclear signal were observed in CIN 2 lesions before progression compared with those before regression. CIN 2 cases in which moderate to strong immunoexpression of p16INK4a and a punctate signal were observed simultaneously progressed to CIN 3 in 10 (91%) of 11 cases. CIN 2 cases with moderate to strong immunoexpression of p16INK4a and a high-risk HPV punctate signal should be treated because of the great risk of progression.

Conservation and alteration of chromosome territory arrangements in thyroid carcinoma cell nuclei.

Chromosome territories (CTs) are intranuclear subregions occupied by individual chromosomes in an interphase cell. In this study, we investigated intranuclear CT positionings of chromosomes 10 (CS10), 18 (CS18), and 19 (CS19) in epithelial cells from four normal thyroid tissue (NT), four adenomatous goiters (AGs), six papillary carcinomas (PCs), and two undifferentiated carcinomas (UCs) using the multicolor fluorescence in situ hybridization method. In the NT and AGs, the radial positionings of CS10 and CS18 were detected at the periphery of nuclei in more than 60% and 80% of cells, respectively, whereas the radial positioning of CS19 was in the central region of the nuclei in more than 80% of cells. In the PCs, radial positioning pattern of CS10 and CS18 were similar to that in the NT. The nuclei with centrally located CS19 in PCs were less frequent than those in NT cells (p < 0.01). On the other hand, UCs with cells having DNA amplification demonstrated the locational abnormalities of the CS10, CS18, and CS19 radial positions. These findings indicate that alteration of CT positioning could be related to DNA amplification and, morphologically, may explain the nuclear atypia that accompanies the abnormal chromatin feature.

Topoisomerase IIalpha gene amplification in gastric carcinomas: correlation with the HER2 gene. An immunohistochemical, immunoblotting, and multicolor fluorescence in situ hybridization study.

Topoisomerase IIalpha (topoIIalpha) is an enzyme required for DNA replication and a molecular target for drugs called anthracyclines. The topoIIalpha gene (TOP2A) is located close to the HER-2/neu oncogene (HER2). We assessed gastric cancers to (1) clarify the relationship between gene amplification and protein overexpression of topoIIalpha and HER2; (2) evaluate the correlation between gene amplification and protein overexpression of topoIIalpha; and (3) examine the relationship between the results of immunohistochemistry and Western blot analysis for topoIIalpha. In a combined analysis of immunohistochemistry and fluorescence in situ hybridization on 552 formalin-fixed and paraffin-embedded gastric cancer tissues, 38 cases were found to have HER2 amplification. Further examination by fluorescence in situ hybridization revealed amplification of TOP2A in 13 of the 38 cases. No aberrations in the TOP2A gene were observed in cases without HER2 overexpression, except for one containing a gene deletion. The TopoIIalpha protein-labeling index was not correlated with TOP2A amplification. Fluorescence in situ hybridization was performed on nuclear imprint specimens obtained from 9 cases using simultaneous probes for TOP2A, HER2, and centromere 17. Of these 9 cases, 3 displayed coamplification of TOP2A and HER2, and only 1 of the 3 cases revealed a high expression of topoIIalpha in Western blot. Although patients having gastric adenocarcinoma with TOP2A amplification could be considered suitable for clinical trials, information involving anthracycline therapy is not firmly understood in regards to the status of TOP2A amplification or protein overexpression. Therefore, results of the current study will provide further insight for the clinical application of anthracycline in gastric cancers.

'Increased expression of seprase, a membrane-type serine protease, is associated with lymph node metastasis in human colorectal cancer'.

Seprase is a membrane-bound serine proteinase with gelatinase activity, which may be involved in cancer invasion and metastasis. We examined seprase expression in colorectal cancer specimens obtained from 109 patients. Seprase immunoreactivity was found in cancer cells and adjacent stromal cells. Immunoblotting showed higher levels of seprase protein in colorectal cancer tissue than in normal colorectal tissue. A semiquantitative assessment of the immunohistochemistry results revealed a significant correlation between seprase expression and lymph node metastasis. These results suggested that an abundant expression of seprase in colorectal cancer tissue is associated with lymph node metastasis.

Expression of nuclear membrane proteins in normal, hyperplastic, and neoplastic thyroid epithelial cells.

Emerin, lamin A/C, lamin B, and lamin-associated polypeptide 2 (LAP2) are nuclear membrane proteins that play an important role in maintaining nuclear structure and coordinating cell activity. We studied the expression and significance of nuclear membrane proteins in neoplastic thyroid cells by immunohistochemistry, RT-PCR, and real-time PCR. In papillary carcinomas (PCs), the nuclear proteins most frequently expressed at high levels were emerin (82 % positive), lamin A/C (64 %), and LAP2 (82 %). Follicular carcinomas (FCs) most frequently expressed lamin B, while none of the undifferentiated carcinomas (UCs) showed strong expression of emerin or lamin A/C. In all medullary carcinomas (MCs), intermediate to high levels of expression of lamin A/C and LAP2 were found. By RT-PCR analysis, messenger RNA (mRNA) expression of all nuclear membrane proteins except emerin was higher in PC than in normal tissue. Real-time PCR analysis showed that mRNA expression of nuclear membrane protein varied between cell lines. Our findings suggest that expression of nuclear membrane proteins may be related to follicular function in normal and hyperplastic follicles, and we hypothesize that they are also involved in the proliferation and differentiation of neoplastic thyroid cells. We suggest that they reflect the biological nature and/or function of normal, hyperplastic, and neoplastic thyroid cells and may have some value in diagnosing thyroid tumors.

Induction of human inhibitor of apoptosis protein-2 by shear stress in endothelial cells.

To disclose the anti-atherosclerotic mechanisms of steady laminar shear stress, we analyzed the expression of human inhibitor of apoptosis protein-2 (HIAP-2), whose gene was selected from a cDNA library of sheared endothelial cells (ECs), on ECs. HIAP-2 was dose-independently and time-dependently induced in ECs by shear stress, regulated at the transcriptional level. HIAP-2 expression was also identified in vivo. Shear stress-mediated inhibition of EC apoptosis was associated with the inhibition of caspase-3 activity, suggesting that the shear stress prevents EC apoptosis via negative regulation of caspase-3 by the increment of HIAP-2.

Heterogeneous Immunoreactivity of Emerin, a Nuclear Envelope LEM-domain Protein, in Normal Thyroid Follicles.

Emerin is a LEM domain-containing integral membrane protein of the vertebrate nuclear envelope. Recently it has been reported that emerin regulates tissue-specific gene/protein expression. We studied the relationship between emerin expression and follicle function in normal and hyperplastic human thyroid tissues using immunohistochemistry and statistical methods. Emerin immunoreactivity was heterogeneous among follicular cells and follicles in normal thyroid tissue. It tended to be strong in the nuclei of tall follicular cells of small follicles and weak or negative in the nuclei of flat follicular cells of large follicles. Follicles with strong expression of emerin were also strongly positive for thyroglobulin (Tg) and thyroxine (T4) in follicular cells and colloid substance, suggesting active functioning follicles. In contrast, large follicles with weak expression of emerin were also weak or negative for Tg and T4. Emerin immunoreactivity was strong in almost all nuclei of hyperplastic follicular cells in Graves' disease tissues. These findings suggest that emerin expression may be related with follicular function and may contribute to the understanding of hormonogenesis in normal thyroid follicles.