• Korean Journal of Clinical Laboratory Science
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• v.48 no.4
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• pp.371-377
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• 2016

Decalcification is routinely performed to obtain a pathological diagnosis using bone marrow biopsy. During the decalcification process using a conventional acidic solution, such as HCl, the antigenicity of tissue is damaged. Especially DNA and RNA in the bone marrow are impaired. Hence, there is the need for a standardized decalcification protocol that preserves the antigenicity of tissue. To this end, we compared the effects of two commonly used decalcifiers: Commercial decalcifier (Calcl-Clear Rapid, HCl) and the EDTA (12.5%, pH 7.0). Bone marrow biopsies sampled from 71 patients were decalcified in accordance with the protocols of respective groups-HCI versus EDTA. The differences of decalcification protocols were analyzed with respect to Hematoxylin & Eosin staining, Gomori'sreticulum staining, and immunohistochemical staining and molecular analysis. Immunohistochemical staining used Ki-67, CD20 and CD138 as primary antibodies and molecular analysis was conducted through the DNA concentration analysis, in situ hybridization (ISH) and immunoglobulin heavy chain (IGH) gene rearrangement. On the routine histopathology analysis, there was no difference between HCl and EDTA. Moreover, in case of immunohistochemical staining, the cytoplasmic membrane or cytoplasmic CD markers was well preserved. However, nuclear proteins, such as Ki-67, were stained with low quality. Conversely, according to the molecular analysis, the EDTA protocol preserved the DNA and RNA compared with the HCI. The differences of DNA quantity and quality were statistically significant between protocols of HCl and EDTA. We used 38 cases in HCl and 12 cases in EDTA. Consequently, the EDTA protocol maintains the antigenicity of the protein on tissue and is acceptable for examination with molecular base analysis. Decalcification of bone marrow biopsy by EDTA is highly recommended for the examination of immunohistochemical staining and molecular analysis.

• Journal of Gastric Cancer
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• v.2 no.1
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• pp.5-11
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• 2002

Purpose: The purpose of this study is to identify immunohistochemical evidence of lymph-node micrometastasis in histologic node-negative gastric cancer patients and to evaluate the prognostic significance of lymph-node micrometastasis.Materials and Methods: A retrospective study of 50 gastric cancer patients who underwent curative resections from October 1990 to November 1994 was performed. Two consecutive sections were prepared: one for ordinary hematoxylin and eosin staining, and the other for immunohistochemical staining with Pan cytokeratin antibody (Novocastra, UK). In the univariate analysis, the survival rate was calculated using the Life Table Method, and the multivariate analysis was determined using a Cox Proportional HazardsModel. The statistical analyses of the relationships between the clinicopathologic factors and micrometastases were performed by using a Chi-square test. Results: Of 2522 harvested lymph nodes, 81 ($4.1\%$) nodes and 19 ($38\%$) of 50 patients were identified as having lymphnode micrometastases by using immunohistochemical staining for cytokeratin. The incidence of lymph-node micrometastases was significantly higher in diffuse type carcinomas ($54\%$, P=0.024) and in patients with serosal invasion ($52.2\%$, P=0.05). For patients with lymph-node micrometastases (n=19), the 5-year survival rate was significantly decreased ($73.7\%$, P=0.015). The Lauren's classirication (P=0.021) and the depth of invasion (P=0.035) were shown by multivariate analysis to have a significant relationship with the presence of micrometastases. Multivariate analysis revealed that lymph-node micrometastasis was independently correlated with survival in histologic node-negative gastic cancer patients. Conclusion: The presence of cytokeratin detected lymphnode micrometastases correlates with the worse prognosis for patients with histologic node-negative gastric cancer.

• Korean Journal of Head & Neck Oncology
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• v.27 no.1
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• pp.59-65
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• 2011

Background and Objectives : Papillary thyroid carcinoma(PTC) frequently metastasize to the regional neck, however, lateral neck lymph node metastasis is less common. The aim of this study is to investigate clinical and immunohistochemical features of PTC with lateral LN metastasis, and determine the predictive factors for lateral LN metastases. Material and Methods : We undertook a retrospective study of 83 patients treated between January 2007 and December 2009 for PTC by thyroidectomy with or without lateral neck dissection. The following criteria were used to study the clinical predictive value of lateral LN. metastases : sex, age, tumor size, multifocality, extracapsular spread(ECS) and lymphovascular emboli. Immunohistochemical staining for VEGF-A, VEGF-C, Bax, Bcl-2, Cyclin D1, Cyclin E, $p27^{kip1}$ and $p57^{kip2}$ was performed, and quantified blindly by three pathologists who had no clinical information of the patients. Immunohistochemical expression was scored as high(>50% of cells stained) or low(0-49%). Results : With use of univariate and multivariate analysis, tumor size(>2cm) and ECS were independent correlates of lateral LN metastasis in PTC. Expression of VEGF-C, Bax, and Cyclin D1 in the PTC with lateral LN metastasis was scored higher than in PTC without lateral LN metastasis(p<0.05). Conclusion : The important risk factors for lateral LN metastasis in PTC are primary tumor size and the presence of ECS. And expression of VEGF-C, Bax and cyclin D1 may be considered of lateral LN metastatic potential in PTC.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5811-5815
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• 2012

Background: Non-invasive oral verrucous carcinoma (OVC) and invasive well-differentiated oral squamous cell carcinoma (OSCC) have similar histopathologic findings but different biological behavior. These two malignancies must be correctly differentiated by pathologists. The aim of this study was to determine immunohistochemical (IHC) expression of Ki67 in OVC and well-differentiated OSCC. Methods: Expression of Ki67 was evaluated by IHC in 15 cases of epithelial hyperplasia with no dysplasia (A group), 15 cases of OVC (B group), 12 cases of microinvasive OSCC(C group) and 15 cases of well-differentiated OSCC (D group). Results: There was a significant difference in Ki67 expression based on pattern distribution of immunostaining positive cells, with quantitative and semi-quantitative analyses, among the four groups ; also, between A group and each of the other three groups (P=0.0001). But there was no significant difference between B and C, C and D, and B and D groups (P>0.05). Conclusions: The three evaluation methods of Ki67 expression showed Ki67 (Mib-1) is not a good immunohistochemical marker to assess invasion status and differentiate OVC from well-differentiated OSCC; also, it cannot be used as a diagnostic tool to distinguish between variants of OSCC with similar grade.

• Korean Journal of Clinical Laboratory Science
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• v.39 no.3
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• pp.217-222
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• 2007

In the last 5 years the Epidermal Growth Factor Receptor (EGFR) has emerged as one of the most important targets for drug development in oncology. Monoclonal antibodies targeting the external domain of EGFR have been shown to have clinical benefits in colorectal and head and neck cancer when combined with chemotherapy and/or radiation. Also the targeting of the epithelial growth factor receptor (EGFR) kinase domain using the closely related inhibitors gefitinib and erlotinib has generally been ineffective against solid tumors, many of which over express the receptor. We found that there were some differential expressions according to primary antibodies of the EGFR protein which being used as one of the histological tumor markers for non-small cell lung cancer (NSCLC). We also found that there are some differential expressions according to antibodies, the pH of the antigen retrieval (AR) buffer solutions and kinds of enzymes. There were some differential expressions according to the secondary antibodies and the detection systems. We analyzed the correlations between the immunohistochemical expressions of the EGFR protein and the gene mutations of the EGFR. The differences between automatic stainers and manual staining methods were also evaluated.

• Biomedical Science Letters
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• v.16 no.4
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• pp.349-357
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• 2010

We have examined the histological changes of skin during hair follicle growth after depilation in C57BL/6N mice. We first studied on histological changes of number of mast cells and thickness of skin during hair follicle growth periods (telogen, 1 day, 3 day, 5 day, 10 day, 14 day, 17 day and 21 day after depilation) by toluidine blue, Giemsa and H&E staining methods. We second studied immunoreactive density of cytokines and Brdu labeled cells in skin during hair follicle growth periods after depilation in C57BL/6N mice by immunohistochemical methods. The histological changes on skin thickness was increased from telogen to 14 day. The number of mast cells was decreased in 3,5 and 10 day and increased in 14, 17 and 20 day after depilation. Immunoreactive density of cytokines [protein kinase C-${\alpha}$ (PKC-${\alpha}$), c-kit, and vascular endothelial growth factor (VEGF)] in 1, 3, 5, 10, and 14 day after depilation was mildly stained in bulge and cutaneous trunci m., but immunoreactive density of cytokines in 17 and 21 day was heavily stained in epidermis, bulge, outer root sheath (ORS), inner root sheath (IRS) and cutaneous trunci m.. Immunoreactive density of Brdu labeled cells in skin in 1 and 3 day was heavily stained in bulge, epidermis and connective tissue under the cutaneous trunci m.. In all periods, immunoreactive density of Brdu labeled cells in skin was heavily stained in bulge, subcutaneous tissue, cutaneous trunci m, ORS and IRS. These experiments suggest that histological changes related to hair follicle growth elevated mast cell counts, skin thickness and epidermis thickness and heavily stained immunoreactive density of cytokines and Brdu labeled cutaneous trunci m. and connective tissue under the cutaneous trunci m. after depilation in C57BL/6N mice.

• Archives of Reconstructive Microsurgery
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• v.8 no.2
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• pp.97-107
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• 1999

The osteogenic capacity of the vascularized periosteum autograft has been extensively demonstrated by experimental works. The objective of this study was to characterize the behavior of experimental model of vascularized periosteal flap(VPF) by observing sequential stages of osteogenesis after simulated VPF in rabbits. In experimental group, segmental resection of bone including the periosteum was performed in 22 radii of 22 New Zealand white rabbits preserving the periosteal circulation of median artery to the periosteum. In order to simulate the transplantation of VPF, the vascular pedicle consisting of median artery and veins was dissected from adjacent soft tissue and the periosteum was longitudinally incised to remove the bone followed by repair of the periosteum. From the first to sixteenth week after the simulated VPF, the changes in VPFs were observed by radiological, light microscopical, scanning electron microscopical methods and the activity of osteocalcin was measured by immunohistochemical method. In control group, the bone tissue and periosteum were completely removed from the mid-shaft of radius and the findings were observed by radiological and light microscopical methods. From the results of this study, it is demonstrated that the experimental model of VPF is vigorously and uniformly osteogenic. Therefore it is thought that VPF can be used as a measure to treat bone defect of shaft of long bone.

• Radiation Oncology Journal
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• v.28 no.2
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• pp.57-63
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• 2010

Purpose: We performed an immunohistochemical study with pre-treatment biopsy specimens to evaluate the prognostic significance of four biomolecular markers which can be used as a predictive assay for radiotherapy (RT) treatment of nasopharyngeal carcinoma (NPC). Materials and Methods: From January 1998 through December 2006, 68 patients were histologically diagnosed as non-metastatic NPC and treated by RT. Only 38 patients had the paraffin block for the immunohistochemical study. Thirty-one patients had undifferentiated carcinoma and 7 patients had squamous cell carcinoma. Thirtytwo patients (84%) had advanced stage NPC (2002 AJCC Stage III~IV). Immunohistochemical staining was performed for Met, COX-2, nm23-H1, and epidermal growth factor receptor (EGFR) expression using routine methods. Results: The median follow-up time was 30 months (range, 11 to 83 months) for all patients, and 39 months (range, 19 to 83 months) for surviving patients. The 5-year overall survival (OS) rate of the patients with high Met extent (${\geq}50%$) was significantly lower than that of the patients with low Met extent (48% vs. 84%, p=0.02). In addition, Met extent was also a significant prognostic factor in multivariate analysis (p=0.01). No correlation was observed between Met extent and T stage, N stage, stage group, gender, age, and the response to chemotherapy or RT. Met extent showed moderate correlation with COX-2 expression (Pearson coefficient 0.496, p<0.01), but COX-2 expression did not affect OS. Neither nm23-H1 or EGFR expression was a prognostic factor for OS in this study. Conclusion: High Met extent (${\geq}50%$) might be an independent prognostic factor that predicts poor OS in NPC treated with RT.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.2
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• pp.118-125
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• 2010

Background and Purpose: Bone metastases rarely occur in patients with oral squamous cell carcinoma (OSCC), so the molecular mechanisms of bone metastasis of OSCC remains unclear. Studies with animal models allow progresses in understanding the molecular events for bone metastasis and provide new targets for therapy. So we tried to establish a murine model for bone metastasis of oral squamous cell carcinoma. Materials and Methods: Human OSCC cells (KB cell line) were xenografted to nude mice via direct inoculation into the tibial marrow. Mice with tibial tumors were sacrificed once a week, until seven weeks after the injection of human tumor cells. Growth of tibial tumors were observed by histology. Expression of TGF-$\beta$ and CXCR-4 in bone OSCC (experimental) and subcutaneous tumor (control) was also evaluated by immunohistochemical staining. Results: Bone OSCC was successfully induced by intra-tibial injection of KB cells. Tumor mass was developed in the marrow tissues of tibia and finally invade the endosteum of tibia. Immunohistochemical staining showed higher expression of TGF-$\beta$ in bone tumors than in subcutaneous tumors. Conclusion: A murine model of bone metastasis of OSCC was suggested that imitated the clinical findings of distant vascular metastasis. This bone tumor model should facilitate understanding of the molecular pathogenesis of OSCC bone metastasis, and aid in the developement of treatment strategies against OSCC bone metastasis.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3963-3967
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• 2013

Background: The presence of Epstein-Barr virus (EBV) in Non-Hodgkin's lymphoma can be identified by immunohistochemistry for detection of EBV latent membrane protein (LMP). The role of EBV as an etiologic agent in the development of non-Hodgkin lymphoma has been supported by detection of high levels of latent membrane protein 1 (LMP-1) expression in tumors. However, no study has been conducted in a Pakistani population up till now to determine the frequency of Epstein-Barr virus positivity. The objective of our study was to determine a value for non-Hodgkin lymphoma patients using EBV LMP-1 immunostaining in our institution. Materials and Methods: This study was carried out at the Department of Histopathology, Armed Forces Institute of Pathology (AFIP), Pakistan from December 2011 to December 2012. It was a cross sectional study. A total of 71 patients who were diagnosed with various subtypes of NHL after histological and EBV LMP-1 immunohistochemical evaluation were studied. Sampling technique was non-probability purposive. Statistical analysis was achieved using SPSS version 17.0. Mean and SD were calculated for quantitative variables like patient age. Frequencies and percentages were calculated for qualitative variables like subgroup of NHL, results outcome of IHC for EBV and gender distribution. Results: Mean age of the patients was $53.6{\pm}16$ years (Mean${\pm}$SD). A total of 50 (70.4%) were male and 21 (29.6%) were female. Some 9 (12.7%) out of 71 cases were positive for EBV-LMP-1 immunostaining, 2 (22.2%) follicular lymphoma cases, 1 (11.1%) case of T-cell lymphoblastic lymphoma, 4 (44.4%) cases of diffuse large B cell lymphomas, 1 (11.1%) mantle cell lymphoma and 1 (11.1%) angioimmunoblastic T cell lymphoma case. Conclusion: In our study, frequency of EBV in NHL is 12.7% and is mostly seen in diffuse large B cell lymphoma. This requires further evaluation to find out whether this positivity is due to co-infection or has a role in pathogenesis.