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.

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.

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.

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.

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.

Differential expression of aquaporins and its diagnostic utility in thyroid cancer.

BACKGROUND: Aquaporin3 (AQP3) and Aquaporin4 (AQP4) play a major role in transcellular and transepithelial water movement as water channel membrane proteins. Little is known of their expression and significance in human thyroid tissues. Thus, we examined the expression of AQP3 and AQP4 in normal, hyperplastic and neoplastic thyroid tissues in conjunction with human thyroid cancer cell lines. METHODS AND RESULTS: Immunohistochemical analyses demonstrated AQP3 in the cytoplasmic membrane of normal C cells, but not in follicular cells. In contrast, AQP4 was not found in C cells but was identified in normal follicular cells. AQP4 was positive in 92% of Graves' disease thyroids and 97% of multinodular goiters, and we failed to demonstrate AQP3 in these hyperplastic tissues. In neoplastic thyroid lesions, we observed AQP3 in 91% of medullary thyroid carcinomas but in no other follicular cell tumors. AQP4 was demonstrated in 100% of follicular adenomas, 90% of follicular carcinomas, and 85% of papillary carcinomas, while it was negative in all medullary carcinomas and undifferentiated carcinomas. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed AQP3 mRNA expression only in medullary carcinomas and AQP4 mRNA expression in follicular cell-derived tumors except for undifferentiated carcinomas. In thyroid cancer cell lines, using RT-PCR and western blotting, AQP3 mRNA and protein were only identified in the TT cell line (human medullary carcinoma cell line) and AQP4 in the other cell lines. In addition, AQP3 mRNA expression was up-regulated by FBS and calcium administration in both a dose and time dependent manner in TT cells. CONCLUSION: The differential expressions of AQP3 and AQP4 may reflect the biological nature and/or function of normal, hyperplastic, and neoplastic thyroid cells and additionally may have value in determining differential diagnoses of thyroid tumors.

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.

Neuroendocrine cells associated with neuroendocrine carcinoma of the breast: nature and significance.

BACKGROUND: The developmental mechanisms of breast neuroendocrine carcinoma (B-NEC) have not been sufficiently analysed and are not well understood. AIMS: To investigate NE cells in the background tissues surrounding B-NECs. METHODS: Three cases (four breasts) having many NE cells in the background tissues of multifocal B-NECs were identified at the University of Yamanashi Hospital and St Luke's International Hospital, Japan. These patients were, respectively, 28-, 31- and 38-year-old women with no familial history of NE tumour. The totally-resected breasts were serially studied by immunohistochemistry for specific NE markers (chromogranin A/synaptophysin) and the morphologies and/or localisation of NE cells were investigated. RESULTS: Immunohistochemical examination showed extensively-distributed NE cells in the background mammary ducts/lobules of the NECs in all breasts. These NE cells were classifiable into three emerging patterns: isolated/scattered, clustered and circumferential. Their distributions were intermingled and were not clearly related to B-NEC foci. NE cells were morphologically polygonal, oval or columnar with sometimes eosinophilic and/or fine-granular cytoplasm and round-to-ovoid nuclei lacking atypia. Some cells were located between epithelial and myoepithelial cells. Apical snouts were occasionally observed in NE cells forming luminal structures. CONCLUSIONS: Benign-appearing NE cells in the parenchyma of a breast with NEC could be regarded as hyperplastic from their emerging patterns and distribution; this NE cell hyperplasia may be associated with the histogenesis of B-NEC as a precancerous condition. These observations might raise questions about the treatment for B-NEC.

Intracardiac varix of the left atrium.

A 68-year-old female patient with a history of hyperlipidaemia and fatty liver was referred for evaluation of an incidentally detected asymptomatic cardiac mass. Computed tomographic scan imaging showed a large calcified mass in the left atrium. Echocardiography revealed a 2.4 1.5 cm, well-circumscribed, round, high echoic mass with severe calcification and low mobility attached to the lower rim of the fossa ovalis. The cardiac mass along with part of the fossa ovalis and left atrial wall were excised. Histological diagnosis was compatible with intracardiac varix.

High prevalence of neuroendocrine carcinoma in breast lesions detected by the clinical symptom of bloody nipple discharge.

AIM: Bloody nipple discharge (BND) is an important clinical symptom in breast disorders, especially cancers. However, the association between this symptom and breast neuroendocrine carcinomas (NECs) has not been sufficiently investigated or well understood. METHODS: We clinicopathologically studied 89 cases using biopsy and/or resection in 144 patients who came to the hospital for a thorough examination of symptomatic BND. RESULTS: Of these 89 cases examined histologically, 24 (27%) were neuroendocrine carcinomas (NECs) in which >50% of cells immuno-expressed chromogranin A and/or synaptophysin. Moreover, NECs made up 44% (24/55) of the mammary cancers found because of the BND. The frequency of diagnosing malignancy preoperatively in 24 NECs was 4% by nipple discharge cytology, 40% by fine needle aspiration cytology, 62% by core needle biopsy and 67% by mammotome biopsy. There were neither postoperative recurrences nor metastases in the NEC cases during a mean follow-up of 83.7 months. The 24 NECs were subclassified into neuroendocrine ductal carcinoma in situ (NE-DCIS) (9 cases) and microinvasive (7 cases) and invasive (8 cases) NECs with extensive NE-DCIS components. Most NECs had early-stage and low-grade pathological parameters: pTis or pT1 (96%), pN0 (96%), low nuclear grade (83%), absence of necrosis (88%), immuno-positivity of estrogen and progesterone receptors (100%) and absence of HER2 protein overexpression (100%). CONCLUSIONS: NECs predominantly with NE-DCIS lesions, often under-diagnosed preoperatively, accounted for an important share of breast conditions associated with BND. It is, therefore, worth keeping this type of breast cancer in mind when performing medical examinations on patients with BND.

Global histone modification of histone H3 in colorectal cancer and its precursor lesions.

Chromatin remodeling through histone modification is an important mechanism of epigenetic gene dysregulation in human cancers. However, little is known about global alteration of histone status during tumorigenesis and cancer progression. Histone H3 status was examined in benign and malignant colorectal tumors by immunohistochemistry and Western blotting. For immunohistochemical evaluation, 4 anti-histone H3 antibodies, specific to dimethylation at lysine 4 (H3K4me2), acetylation at lysine 9 (H3K9ac), dimethylation at lysine 9 (H3K9me2), and trimethylation at lysine 27 (H3K27me3), were used. On immunohistochemistry, H3K4me2, H3K9ac, and H3K27me3 showed no significant changes between normal and colorectal tumors. On the other hand, the global level of H3K9me2 was distinctly higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells. In addition, it was significantly higher in adenocarcinoma than in adenoma. Correspondingly, Western blotting confirmed that H3K9me2 expression was significantly higher in adenocarcinomas than in normal colorectal mucosa. No alteration of H3K9me2 was observed with tumor differentiation and with the histological subtypes of colorectal cancers. These results suggest that aberration of the global H3K9me2 level is an important epigenetic event in colorectal tumorigenesis and carcinogenesis involved with gene regulation in neoplastic cells through chromatin remodeling.

Unusual thyroid carcinoma with excessive extracellular hyaline globules: a case of "hyalinizing papillary carcinoma".

We present an unusual case of papillary thyroid carcinoma in a 47-year-old Japanese woman. The tumor, 0.8 cm in diameter, was located in the upper left lobe of the thyroid. Histologically, we observed a microfollicular-like and trabecular arrangement of the tumor cells with marked hyalinized stroma and hyaline globules. Immunohistochemically, tumor cells were positive for thyroglobulin and thyroid transcription factor 1. Hyaline stroma and globular bodies were immunopositive for laminin and type IV collagen. MIB-1 index was approximately 1% without membranous immunoreactivity. Under the electron microscope, hyaline stroma and globules showed electron-dense, complex meshwork structures composed of granular and fibrous elements similar to the structure of the lamina densa. Genetic analysis demonstrated a BRAF(V600E) mutation. Based on these findings, we diagnosed the present tumor as a rare morphological variation of papillary thyroid carcinoma with excessive hyaline globules consisting of basal membrane materials.

Fine-needle aspiration biopsy of low-grade cribriform cystadenocarcinoma of the salivary gland.

Low-grade cribriform cystadenocarcinoma (LGCCC) is a rare tumor, defined in the 2005 WHO classification as a primary salivary duct tumor. Previously, the neoplasm had been recognized as a variant of salivary duct carcinomas. A 56-year-old Japanese woman noticed a mass in the left subaural region. On radiological examinations, a multicystic tumor was seen in the left parotid gland. Fine-needle aspiration biopsy was performed. The smears revealed several characteristic cytologic features. The tumor cells were arranged in irregular overlapping and showed inconspicuous nuclear atypia with variable-sized and irregularly shaped cytoplasmic vacuoles. Based on these findings, a cystic tumor with uncertain malignancy was diagnosed. A parotidectomy was performed, because the tumor was slowly growing and contained solid components on the radiological images. Based on the histologic findings, along with immunohistochemistry, LGCCC was diagnosed based on resemblance to breast low-grade ductal carcinoma in situ and intraductal proliferation of tumor cells. This is the first report of the cytomorphological findings of LGCCC.

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.

Hypoxia-inducible adenosine A2B receptor modulates proliferation of colon carcinoma cells.

Extracellular adenosine regulates a wide variety of physiological processes by interacting with 4 adenosine receptor subtypes: A1, A2A, A2B, and A3. However, little is known of their pathophysiological roles in human cancers. In this study, we examined the expression pattern of adenosine receptors in various colorectal tissues and human colon carcinoma cell lines and investigated the biologic functions regarding colon carcinogenesis. Using reverse transcriptase polymerase chain reaction and Western blotting, we found that adenosine receptor A2B (ADORA2B) was consistently up-regulated in colorectal carcinoma tissues and colon cancer cell lines compared with normal colorectal mucosa. In immunohistochemistry, we observed diffuse immunopositivity of ADORA2B in 67% of colorectal adenocarcinomas (39/58), 17% of tubular adenomas (5/30), and 0% of normal colon glands (0/62). During a hypoxic state, there was also a significant induction of ADORA2B expression in the messenger RNA level at 8 hours of incubation and in the protein level at 24 hours of incubation in colon carcinoma cell lines. To examine the function of ADORA2B, we applied an ADORA2B-selective antagonist (MRS1754) to the colon carcinoma cells, which significantly inhibited cell growth in a dose-dependent manner as demonstrated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay. In conclusions, ADORA2B was overexpressed in colorectal carcinomas grown under a hypoxic state, presumably promoting cancer cell growth. Our data suggest that this adenosine receptor is a potential therapeutic target for colorectal cancer.

Polyclonal origin of hormone-producing cell populations evaluated as a direct in situ demonstration in EGFP/BALB/C chimeric mice.

We report the first demonstration of the embryonal patch patterns of endocrine organs and the polyclonality of hormone-producing cell populations using chimeric mice produced by aggregation of C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb transgenic mice and BALB/C mice. Confocal laser scanning microscopy (CLSM) analysis for enhanced green fluorescent protein (EGFP) and immunohistochemistry with anti-EGFP antibody revealed that all endocrine organs of chimeric mice had a mosaic appearance of EGFP-positive patches and EGFP-negative patches. The patches composed of EGFP-positive cells were distinctive in their size and shape. The pituitary patches were large and irregular, representing a geographical pattern. In contrast, parathyroid, pancreatic islet, and adrenal medulla patches were small and demarcated, representing an island-like pattern. Thyroid follicles and adrenal cortex cords showed a mixture of monophenotypia and polyphenotypia, indicating polyclonal embryonic origin. Furthermore, we studied the tissue clonality of hormone-producing cell populations in the pituitary, thyroid, and pancreatic islets using a combination method of CLSM for EGFP and immunohistochemistry for hormones. All the pituitary cell populations of GH, prolactin, TSH, FSH, LH, and ACTH, the calcitonin-producing cell population in the thyroid, and the insulin- and glucagon-producing cell populations in pancreatic islets had mosaic patterns in EGFP expression in the chimeric mice, suggesting polyclonal embryonic origin. In conclusion, the different patch patterns of the endocrine organs could contribute to the understanding of embryonic development and organization of endocrine organs. Furthermore, we clearly demonstrate that all hormone-producing cell populations are of polyclonal embryonic origin, derived from more than two progenitor cells.