Akira Komiyama

Expression of vascular endothelial growth factor (VEGF) in human thyroid neoplasms

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is an angiogenic factor that plays important roles in tumor growth. Angiogenesis studies on VEGF deal with various types of malignant tumors, but very little is known about the role or significance of VEGF in human thyroid neoplasms. Therefore, in the current study, we determined whether VEGF is found in normal and neoplastic thyroids and whether its expression is altered in different histological types of thyroid neoplasms. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that all specimens of thyroid tumors expressed bands corresponding to 121-, 165-, and 189-amino acid forms of VEGF. Northern blot analysis showed an increase in VEGF mRNA levels in neoplastic tissues in comparison with normal thyroid samples. By nonisotopic in situ hybridization, most of the tumor cells in follicular adenomas expressed VEGF mRNA, whereas VEGF mRNA expression was identified only in epithelium of isolated follicles in normal thyroid tissues. In papillary thyroid carcinomas, an intense labeling with VEGF probe was often found in overlying tumor cells of neoplastic papillae. VEGF expression was distinctly intensified in undifferentiated carcinoma cells that were immediately adjacent to necrotic foci. The immunohistochemical localizations of VEGF protein were comparable to the localization of VEGF mRNA. In conclusion, our results suggest that the histological types of thyroid tumor may determine the vascular pattern through a paracrine mechanism involving VEGF.

Poorly differentiated desmin-negative and vimentin-positive leiomyosarcoma of the stomach examined by the immunohistochemical and quick-freezing and deep-etching methods

Abstract A poorly differentiated leiomyosarcoma of the stomach in a 41-year-old woman is reported. The diagnosis was confirmed by the diffuse immunohistochemical reaction to HHF35, and the presence of focal density and caveolas in some of the tumour cells by conventional electron microscopy. Immunohistochemically, most tumour cells had an undifferentiated nature, in which negative immunostaining for desmin, alpha-smooth muscle actin, and type IV collagen, and positive immunostaining for vimentin were observed. By the quick-freezing and deep-etching (QF-DE) method, these tumour cells revealed the loss of bundled actin and myosin filaments, which constitute desmin associated structures (focal densities and dense patchy areas). Their cytoplasm had many mitochondria and other cell organelles. The intermediate filaments (IFs), which were determined to be vimentin by immunohistochemistry, were observed in the inter-organellar spaces, and connected with these cell organelles. Actin filaments formed a meshwork structure and were distributed mainly in subplasmalemmal regions. Although a basal lamina was not detected by conventional electron microscopy, basal lamina-like structures, an association between the extracellular matrices and the cell membrane, were observed. Using the QF-DE method, three dimensional ultrastructural alterations of the cytoskeleton and extracellular matrix of the leiomyosarcoma were observed.

Alterations of basement membrane in di-isopropanolnitrosamine-induced carcinogenesis of the rat thyroid gland : An immunohistochemical study

Abstract Alterations of basement membrane (BM) in di-isopropanolnitrosamine (DIPN)-induced carcinogenesis of the rat thyroid gland were examined by means of immunohistochemical localization of collagen type IV (CN-IV), laminin (LN), and fibronectin (FN) in pre-nodular and nodular thyroid lesions, correlating with the morphogenesis and proliferative activity of these lesions. Adult male rats of the Wistar strain were injected s.c. in the back with DIPN, and the thyroid glands were removed at the 15th and 30th week of treatment. Each of 133 thyroid lesions was histochemically analyzed. The follicular epithelial BM as revealed by CN-IV and LN was discontinued or completely lost during the progression of thyroid lesions from pre-nodular to nodular lesions and finally overt carcinomas. At the same time, the BM of vascular endothelial cells demonstrated a loss of dense capillary networks of follicles, a sinusoidal dilatation and, predominantly in carcinomas, development of interstitial-type blood vessels. However, FN, which was hardly stained in the normal thyroid tissue, was remarkably deposited in the interstitium of invasive carcinomas. These observations strongly suggested that alterations of BM structure play a key role in the morphogenesis of rat thyroid tumors, and that the expression of FN is an important step in the invasive growth of thyroid tumors.

Different organization of intermediate filaments in columnar cells of rat large intestinal mucosa as revealed by confocal laser scanning microscopy and quick-freezing and deep-etching method.

The relationship between cell differentiation and ultrastructural changes of intermediate filaments (IF) was studied in columnar cells of large intestinal mucosa of rats by confocal laser scanning microscopy and quick-freezing and deep-etching method. A feature of the IF in immature columnar cells was minibundle formation with prominent branching, which organized the meshwork structures. The minibundles, which appeared to be formed by the attachment of two or more IF in side-to-side fashion, were loosely distributed throughout the cytoplasm. In contrast, in mature columnar cells, the IF were densely distributed under the terminal web in the cytoplasm and beneath the upper part of the lateral membrane regions, whereas the other areas of the cytoplasm contained only a small number of IF. Minibundle formation was not observed, and the branching was rarely identified. The changes in the distribution and density of IF, which are expressed in specific areas of mature columnar cells, apparently represent a characteristic of intracellular differentiation. It is suggested that the dissociation of minibundled IF, which was often observed in the immature columnar cells, is an important step in the acquisition of functional polarity in cells of this type.

Gastric adenocarcinoma of pyloric gland type with high-grade malignancy.

To the Editor: Recent advances in mucin histochemistry and immunohistochemistry have clarified the differentiation of gastric adenocarcinoma as gastric type and intestinal type. Gastric type is further divided into foveolar, pyloric gland and fundic gland phenotypes. Here, we report a case of gastric adenocarcinoma with pyloric gland phenotype demonstrated by mucin histochemistry and immunohistochemistry. A 70-year-old Japanese man received distal gastrectomy with lymph node dissection for primary gastric adenocarcinoma. The patient was alive at the time of our review, however, there was local recurrence of the tumor confirmed by endoscopic examination after 5 years from the distal gastrectomy. Gross examination showed a superficial depressed lesion (type 0-IIc), 28 × 15 mm, in the lesser curvature of the middle third of the stomach (Fig. 1a). Histological examination identified proliferation of adenocarcinoma cells which invaded into the subserosal layer. Accurate tumor size was 60 × 40 mm. Microscopic lymph node metastases were detected in 10 of 15 resected lymph nodes. The tumor was composed of acinar, trabecular and insular growth patterns with cuboidal to low columnar cells having mildly basophilic to occasionally coarse granular eosinophilic cytoplasm (Fig. 1b-d). The nuclei were larger than those from normal pyloric gland and were markedly hyperchromatic (Fig. 1b-d). Nuclear inclusions and prominent nucleoli were found intermittently. The mitotic index was high (12 per 10 high power fields). The background mucosa was what would be found in a fundic gland area having incomplete intestinal metaplasia. Samples from the specimen were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4 μm thick and stained with hematoxylin and eosin. Additionally, histochemical stainings of periodic acid Schiff with diastase (positive for neutral mucin in the gastric foveolar cells, pyloric glands and gastric mucous neck cells) and Alcian blue pH 2.5 (positive for acid mucin in the intestinal goblet cells), and immunohistochemical staining were performed on representative slides. Immunoreactivity was detected using an ENVISION kit protocol (Dako, Glostrup, Denmark) or a Ventana BenchMark GX automated immunostaining system (Tucson, AZ, USA). The neoplastic population was positive for periodic acid Schiff with diastase staining (Fig. 2a) and negative for Alcian blue pH 2.5 staining. Immunohistochemistry demonstrated a strong, diffuse cytoplasmic stain for MUC-6 (Fig. 2b) and a nuclear stain for p53 (Fig. 2c). Over 20% of the neoplastic cells were reactive for Ki-67 (Fig. 2d). There were no immunoreactivities for MUC-2, MUC-5AC, CD10, pepsinogen-I, proton pump/H, K-ATPase α subunit, chromogranin A, α-fetoprotein and trypsin. With reference to a molecular genetic study of pyloric gland adenomas by Matsubara et al., we analyzed the mutation statuses of GNAS and KRAS in the present case. Amplifications of DNAs were performed using HotStarTaq DNA Polymerase (QIAGEN, Tokyo, Japan) by polymerase chain reaction (PCR). Analyses by direct sequencing were performed by BEX Co., Ltd. (Tokyo, Japan). Additional genetic study demonstrated no mutation for GNAS exon 9 or KRAS codons 12/13. Although primary and nested polymerase chain reaction (PCR) reactions were performed, the amplification of GNAS exon 8 was unsuccessful. Based on histology of the current tumor, differential diagnoses includes gastric adenocarcinoma of fundic gland type (chief cell predominant type), neuroendocrine neoplasm, hepatoid adenocarcinoma and pancreatic-type acinar cell carcinoma. However, negative staining for pepsinogen-I (a marker for gastric chief cells and mucous neck cells), proton pump/H, K-ATPase α subunit (for parietal cells), chromogranin A (for neuroendocrine cells), α-fetoprotein (for hepatoid adenocarcinoma cells) and trypsin (for pancreatic acinar cells) excluded these histological subtypes. In addition, neither MUC2 nor CD10 showed intestinal differentiation. The present case was shown to have advanced gastric cancer (pT3 and pN3a), we have demonstrated a gastric adenocarcinoma of pyloric gland type having a high-grade malignancy. The gastric adenocarcinoma of pyloric gland type may appear to be more aggressive than that of fundic gland type, although, further large studies will be required to evaluate the accurate malignant potential of gastric adenocarcinoma of pyloric gland type. Therefore, we must be careful when diagnosing low-grade malignancy with a pathological diagnosis of gastric adenocarcinoma of fundic gland type, especially when biopsy is performed by endoscopy. Gastric type adenocarcinomas occur predominately in old age (69.8 ± 6.4 years); the age of that patient was identical to that of the present case. The lower third of the stomach, which was the most frequent occurred location of gastric type adenocarcinomas, was different from the location in which the present tumor occurred. Furthermore, gastric type adenocarcinomas showed MUC-5AC expression. Accordingly, the present tumor is likely to be different from a gastric type adenocarcinoma. In our speculation, the present case is a malignant counterpart to the pyloric gland adenoma. Pyloric gland adenomas occurred predominately in old age (73 ± 12.8 years) and the gastric corpus, which were identical with the age and the Pathology International 2015; 65: 148–150 doi:10.1111/pin.12241 bs_bs_banner