Journal of Pathology and Translational Medicine

Original Articles

Correlation of Heregulin mRNA and Her-2/neu Protein Expression with Node Metastasis and DNA Ploidy Pattern in Human Invasive Breast Carcinoma.: Yee Jeong Kim, Woo Hee Jung, Hyde Lee, Sung Kong Lee, In Gul Moon, Kwang Gil Lee; Korean J Pathol. 1998;32(8):563-573.

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Abstract: The Her-2/neu protooncogene encodes a transmembrane tyrosine kinase that is structurally homologous to the receptor for epidermal growth factor. Its amplification and overexpression are associated with poor prognosis in breast cancer patients. Neu differentiation factor is a ligand for Her-2/neu protooncogene and was detected in ras-transformed rat fibroblasts. Heregulin (human homologue of neu differentiation factor) is a 44-kilodalton glycoprotein that stimulates tyrosine phosphorylation and induces growth arrest or stimulation and differentiation in human breast cancer cell lines. In this study we examined the expression of heregulin mRNA by nested reverse transcription (RT) PCR with fresh tissue, Her-2/neu protein, ICAM-1 and steroid receptors by immunohistochemistry, and DNA ploidy pattern by flow cytometry with paraffin-embedded tissue in invasive breast carcinoma. We compared the data with nodal status, lymphovascular invasion, steroid receptor status and DNA ploidy pattern. For RT-PCR to heregulin mRNA, 38 cases of fresh breast cancer tissue were obtained. Total 68 cases of invasive breast carcinoma tissue were fixed in formalin, which were used for routine histology, immunohistochemistry and flow cytometry. The results are as follows; 1) Heregulin mRNA was expressed in 86.1% of patients with invasive breast carcinoma and 100% of patients with benign breast lesion using nested RT-PCR analysis. 2) Her-2/neu protein was overexpressed in 50.0% of tumors using immunohistochemistry. The expression of Her-2/neu protein was significantly correlated with high counts of lymph nodes with metastasis (p<0.05), and high nuclear grade (p<0.05). 3) Her-2/neu protein overexpression was significantly correlated with a high DNA index(p<0.05). All of the tumors showing Her-2/neu protein overexpression and no heregulin mRNA expression revealed near tetraploid DNA content. However, both Her-2/neu overexpression and heregulin mRNA expressing tumors revealed near tetraploidy in 38.9% and diploidy in 50.0%. Based on these results, heregulin mRNA expression rate was 86.1% in human invasive breast carcinoma. Her-2/neu protein overexpression is associated with high positive lymph node number and DNA index. Statistically significant reverse correlation with lymph node metastasis is not present.

Sequential Changes of Extracellular Matrix mRNA in Anti-GBM Antibody Induced Crescentic Glomerulonephritis in the Rabbit.: Moon Hyang Park, Unn Wha Lee, In Sup Han, Rho Won Chun, Jung Woo Noh; Korean J Pathol. 1998;32(9):627-637.

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Abstract: Progressive renal fibrosis is considered to be the final common pathway leading to chronic renal insufficiency, however, the mechanism regarding renal fibrosis in renal injury is not well understood. Recently, several kinds of cytokines have been known to be related to fibrosis after renal injury. The interaction between elements regulating fibrogenesis would be better understood by looking at the effect of TGF-beta1 on the synthesis and accumulation of extracellular matrix, especially collagenous proteins. Crescentic glomerulonephritis (CGN) was induced in New Zealand White rabbits by administration of guinea pig anti-GBM IgG after sensitization with guinea pig IgG; and their kidneys were analyzed for the development of crescents and fibrosis through sequential renal biopsies. Serum creatinine levels in a time course progressively increased until day 15. We semi-quantitatively assayed the levels of the expression of alpha1(I) collagen mRNA and TGF-beta1 mRNA factored for GAPDH mRNA using RT-PCR. We observed a progressive interstitial fibrosis and the expression of collagen I both in the cortex and medulla. The effect of repeated renal biopsy itself on pathology and on the expression of alpha1(I) collagen mRNA and TGF-beta1 mRNA in a time course were not significant, but a very mild increase of the expression of alpha1(I) collagen mRNA was noted at day 15. Histology showed a progressive crescent formation and interstitial fibrosis in a time course that roughly paralleled the expression of alpha1(I) collagen mRNA in both cortex and medulla. TGF-beta1 mRNA was hardly expressed at day 0 in cortex as well as in medulla. It was elevated from day 1, peaked at day 7, and then decreased. In medulla, TGF-beta1 mRNA was noticeably expressed at day 1, peaked at day 4, and then decreased. The expression of alpha1(I) collagen mRNA was seen even before inducing CGN. It was gradually and continuously increased until day 15 both in cortex and medulla. These results suggest that the expression of TGF-beta1 mRNA precedes that of alpha1(I) collagen mRNA in the early stage of CGN and has a central role for provoking the accumulation the collagen I, the most representative interstitial extracellular matrix, in the rabbit model CGN induced by anti-GBM antibody. We conclude that the measurement of the expression of TGF-beta1 mRNA and/or alpha1(I) collagen mRNA in a biopsy sample can be a useful predictor for renal outcome.

Type IV Collagen mRNA Expression in Human Membranous Nephropathy.: Tae Sook Kim, Jung Yeon Kim, Hye Kyoung Hong, Hyun Soon Lee; Korean J Pathol. 1999;33(11):1047-1054.

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Abstract PDF: Human membranous nephropathy (MN) is morphologically characterized by subepithelial immune complex deposits and progressive thickening of glomerular basement membranes (GBM). Studies have suggested that the enhanced secretion of classical and novel type IV collagen chains in MN contributes to spike formation and the novel type IV collagen chain is particularly related to thickening of GBM. It is unclear whether the increased accumulation of extracellular matrix (ECM) proteins in GBM is due to the increased mRNA expression for type IV collagen in glomerular visceral epithelial cells (GECs). To answer this question, we analyzed seven renal biopsies of patients with idiopathic MN using in situ hybridization. In MN, the number of GECs expressing mRNA for alpha1(IV) collagen was 2.82+/-1.80/glomerular cross section (gcs), and the number expressing mRNA for alpha4(IV) collagen was 8.42+/-2.85/gcs. The number of GECs expressing mRNA for alpha4(IV) collagen was significantly larger than that of alpha1(IV) collagen mRNA. The expression of mRNA for these ECM proteins in normal controls was negligible. These results suggest that subepithelial immune complexes stimulate the gene expression of alpha1(IV) collagen and alpha4(IV) collagen in glomerular GECs which, in turn, increase the secretion of ECM proteins and contribute to the thickening of GBM in MN.

In Situ Detection of mRNA and RNA Component of Human Telomerase in Proliferative Lesions of the Stomach.: Mi Sook Kim, Sang Woo Juhng; Korean J Pathol. 2001;35(4):299-305.

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Abstract PDF: BACKGROUND
Proliferative lesions of the stomach were investigated by in situ hybridization using RNA probes for telomerase components and compared with the results by TRAP (telomeric repeat amplification protocol) assay.
METHODS
RNA probes for hTR (human telomerase RNA component) and hTERT (mRNA coding for a catalytic subunit of human telomerase) were made by cloning and in vitro transcription. The probes were applied for in situ hybridization in 23 cases of adenocarcinoma of the intestinal type and adjacent dysplasia, and in the normal and metaplastic mucosa of the stomach.
RESULTS
Telomerase activity by TRAP was positive in all cases of adenocarcinoma, most cases of dysplasia, and many cases of normal mucosa. hTR in situ hybridization showed positive staining in the adenocarcinoma cells, dysplastic cells, a few cells in the proliferation zone of the normal mucosa, and a few infiltrated lymphocytes. hTERT showed positive staining in the same cells.
CONCLUSIONS
Telomerase is expressed in most cases of dysplastic lesions and is thought to be acquired in the early steps of carcinogenesis. The expression is noted in a few cells of the normal proliferative zones and the infiltrated lymphocytes, emphasizing the importance of in situ detection of telomerase at the cell level.

Application of Epstein-Barr Virus Cell Lines (CCL85 EB-3) in Performing the EBER mRNA In Situ Hybridization as a Positive Control.: Sung Sook Kim, Woon Sup Han, Joo Young Suh, Joo Ryung Huh; Korean J Cytopathol. 1996;7(1):38-43.

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Abstract PDF: Epstein-Barr virus(EBV) is associated with a wide spectrum of benign and malignant disorders including leukoplakia, Hodgkin's lymphoma, central nervous system lymphoma, peripheral T cell lymphoma and nasopharyngeal undifferentiated carcinoma. There are several distinctive aspects of biology of the virus that are important in investigation of virus in clinical specimens. The abundant expression of the EBER mRNA transcripts makes possible the sensitive detection of latent expression in EBV-associated tumors. Although there has been a dramatic increased interest in the direct characterization of EBV in clinical specimens, there have been few studies about the effective and reliable positive controls in performing in situ hybridization technique for EBV, especially on paraffin-embedded tissue. We applied Burkitts lymphoma cell line as positive control in EBV in hydridization using Oncor Kit. The cell block of Burkitt lymphoma cell line(CCL85 EB-3) showed strong and specific positivity for EBER in situ in nuclei of EBV infected cells.

In Situ mRNA Hybridization and an Immunohistochemical Study of EGFR in Uterine Cervix Cancer.: Hyang Mi Ko, Chang Soo Park, Sang Woo Juhng; Korean J Pathol. 1995;29(3):343-351.

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Abstract PDF: Epidermal growth factor receptor (EGFR) is an intergral membrane protein. Overexpression or mutation of EGFR may play a role in careinogenesis. Recently, many molecular biologic techniques have been used to study expression of oncogenes. One of them, in situ mRNA hybridization, using paraffin embedded blocks, offers a unique means to allow precise localization within histological preparations, and also overcomes problems relating to translation defects and abnormal translation. In order to confirm the usefulness of epidermal growth factor receptor as a tumor marker, and to compare the expression of EGFR between in situ MRNA hybridization and an immunohistochemical study, in situ MRNA hybridization was performed along with an immunohistochemical study for EGFR in paraffin sections of 84 uterine cervix carcinomas. A positive reaction for EGFR was observed mairdy in the cytoplasm of tumor cells. The vascular muscle layer and uterine muscle tissue around the cancer nest revealed a positive reaction in immunohistochemical stain for EGFR, with a negative reaction for EGFR mRNA. In the cancer nests, the immunohistochemical positive reaction for EGFR was strong in differentiated cells and keratin pearls, but a strong positive reaction for EGFR mRNA was localized in undifferentiated cells. The overall positive of immunostaing for EGFR was 77% for uterine cervix carcinoma; 71 % for carcinoma in situ, 71 % for microinvaseve carcinoma, and 89% for invasive carcinoma. The overall positivity of EGFR from in situ MRNA hybridization was 94% of the uterine cervix carcinoma; 93% for carcinoma in situ, 93% for microinvasive carcinoma, and 96% for invasive carcinoma. From these results, EGFR is a useful tumor marker for uterine cervix carcinoma, and in situ mRNA hybridization has greater sensitivity and specificity than immunohistochemistry.

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