D2-40 Antibody Research Articles

Decoding lymphangiogenesis in oral squamous cell carcinoma: Emphasis on clinical and histopathological determinants of regional metastasis.

The growth and metastasis of solid epithelial tumors is lymphangiogenesis dependent. The most important lymphangiogenic inducers facilitating this progression is Vascular endothelial Growth Factor C (VEGF-C). The recent D2-40 (Podoplanin) antibody is specific for lymphatic epithelium and allows its objective assessment. Also, lymphovascular invasion (LVI) is a risk factor for lymph node metastases (LNM) and indicates a significant influx of tumor cells into the lymphatics, causing regional metastasis. Thus, the following study was conducted to assess and correlate VEGF-C and D2-40 immunoexpressions with clinical and histopathologic lymph node status in Oral Squamous Cell Carcinoma (OSCC) cases and also to estimate the impact of intratumoral (ILVD) and peritumoral lymphatic vessel density (PLVD) in such cases. A total of 128 OSCC cases, divided as Group I: Cases with clinically and histopathologically negative lymph nodes (n = 64) and Group II: Cases with clinically negative but histopathologically positive lymph nodes (n = 64) were immunoscored for VEGF-C (Anti VEGF-C antibody) (PA5-29772, Invitrogen) and D2-40 (Anti D2-40 antibody) (IR072/8072, Dako) using standard protocols. The data was statistically evaluated using STATA 18.0 with p≤0.05 considered statistically significant throughout the study. 21.88% Group I cases and 40.62% Group II cases showed highest immuno-positivity for VEGF-C (p = 0.00). The mean D2-40 score for Intratumoral Lymphatic Vessel Density (ILVD) and Peritumoral Lymphatic Vessel Density (PLVD) was higher for group II cases (i.e., 41.5±13.73 and 35.95±8.27 respectively at 95% CI, p = 0.00) suggesting a direct correlation between Lymphatic Vessel Density (LVD) and LNM. Lymphangiogenesis is a true determinant of the biologic potential of OSCC and obtaining an objective data in terms of LVD through D2-40 could be impactful in OSSC diagnosis and guiding treatment decisions by clinicians.

Detailed study of collagen, vasculature, and innervation in the human cardiac conduction system

BackgroundThe cardiac conduction system (CCS) creates and propagates electrical signals generating the heartbeat. This study aimed to assess the collagen content, vasculature, and innervation in the human sinoatrial and atrioventricular CCS, and surrounding tissue. Materials and methodsTen sinoatrial and 17 atrioventricular CCS samples were collected from 17 adult human autopsied hearts. Masson trichrome stain was used to examine collagen, cardiomyocytes, and fat proportions. Immunohistochemically, vessels and lymphatics were studied by CD31 (pan-endothelial marker) and D2-40 (lymphatic endothelium marker) antibodies. General nerve densities were assessed by S100, while sympathetic nerves were studied using tyrosine hydroxylase, parasympathetic nerves with choline acetyltransferase, and GAP43 (neural growth marker) antibodies looked at these components. All components were quantified with QuPath software (Queens University, Belfast, Northern Ireland). ResultsInterstitial collagen was more than two times higher in the sinoatrial vs. atrioventricular CCS (55% vs. 22%). The fat content was 6.3% in the sinoatrial CCS and 6.5% in the atrioventricular CCS. The lymphatic vessel density was increased in the sinoatrial and atrioventricular CCS compared to the surrounding tissue and was lower in the sinoatrial vs. atrioventricular CCS (P=.043). The overall vasculature density did not differ between the SA and AV CCS. The overall innervation and neural growth densities were significantly increased in the CCS compared to the surrounding tissue. The overall innervation was higher in the atrial vs. ventricular CCS (P=.018). The neural growth was higher in the atrial vs. ventricular CCS (P=.018). The sympathetic neural supply was dominant in all the studied regions with the highest density in the sinoatrial CCS. ConclusionsOur results provide new insights into the unique morphology of the human CCS collagen, fat, vasculature, and innervation. A deeper understanding of the CCS anatomical components and morphologic substrates’ role will help in elucidating the causes of cardiac arrhythmias and provide a basis for further therapeutic interventions.

Clinicopathological evaluation of cancer-associated fibroblast markers in colorectal cancer (histopathological and immunohistochemical study)

Background and objectives Worldwide, colorectal cancer is considered one of the most common cancer mortality causes. Recent studies have documented that cancer-associated fibroblasts (CAFs) evoke neoangiogenesis and tumor growth for different tumors. Our study analyzed CAF markers, including α-smooth muscle actin (α-SMA), D2-40 (antibody recognizing podoplanin), and vessel markers, including CD31, for 44 colorectal cancer cases. The association between CAF markers and vessel markers with clinicopathological factors was investigated. Furthermore, the association between CAF markers with each other and their association with vessel markers was analyzed and correlated with different clinical parameters like tumor size, grade, and staging. Materials and methods Forty-four paraffin-embedded colectomy specimens were evaluated pathologically for α-SMA, D2-40 (antibody recognizing podoplanin), and CD31 (histopathological and immunohistochemical study). Results Out of forty-four cases studied, 21 (47.7%) males and 23 (52.3%) females with a mean age of 46.20 (SD ±10.619) years old. D2-40 positivity was localized to the peritumoral area, while α-SMA was detected in both intratumoral and peritumoral areas. α-SMA ahd low expression (scores 0 and 1) in three (6.8%) cases and high expression (scores 2 and 3) in 41 (93.2%) cases. Neoangiogenesis and microvessel density were estimated and calculated in intratumoral and peritumoral areas by CD31 expression. The mean±SD of CD31 was 38.34 ± 14.12. Lymphatic vessel density was also estimated and calculated by the D2-40 expression, and the mean±SD of D2-40 was 48.52 ± 13.72. Significant relationships were found between α-SMA expression, D2-40 expression, CD31 expression, and stromal fibroblast proliferation with each other and with tumor size, tumor stage, tumor grade, lymph node metastasis, and deposits. Conclusions Our study results indicated that individual CAFs might have different expression patterns and strength effects for tumor invasion and vascular invasion in colorectal cancer stroma. These findings may evoke novel therapeutic strategies for colorectal cancer treatment and prognosis.

Detection of Lymphatic Vessels in the Superficial Fascia of the Abdomen.

Recently, the superficial fascia has been recognized as a specific anatomical structure between the two adipose layers-the superficial adipose tissue (SAT) and the deep adipose tissue (DAT). The evaluation of specific characteristics of cells, fibers, blood circulation, and innervation has shown that the superficial fascia has a clear and distinct anatomical identity, but knowledge about lymphatic vessels in relation to the superficial fascia has not been described. The aim of this study was to evaluate the presence of lymphatic vessels in the hypodermis, with a specific focus on the superficial fascia and in relation to the layered subdivision of the subcutaneous tissue into SAT and DAT. Tissue specimens were harvested from three adult volunteer patients during abdominoplasty and stained with D2-40 antibody for the lymphatic endothelium. In the papillary dermis, a huge presence of lymphatic vessels was highlighted, parallel to the skin surface and embedded in the loose connective tissue. In the superficial adipose tissue, thin lymphatic vessels (mean diameter of 11.6 ± 7.71 µm) were found, close to the fibrous septa connecting the dermis to the deeper layers. The deep adipose tissue showed a comparable overall content of lymphatic vessels with respect to the superficial layer; they followed the blood vessel and had a larger diameter. In the superficial fascia, the lymphatic vessels showed higher density and a larger diameter, in both the longitudinal and transverse directions along the fibers, as well as vessels that intertwined with one another, forming a rich network of vessels. This study demonstrated a different distribution of the lymphatic vessels in the various subcutaneous layers, especially in the superficial fascia, and the demonstration of the variable gauge of the vessels leads us to believe that they play different functional roles in the collection and transport of interstitial fluid-important factors in various surgical and rehabilitation fields.

IMMUNOHISTOCHEMICAL ASSESSMENT OF LYMPHATIC VESSELS IN HUMAN LIVERS WITH CHRONIC HEPATITIS C - RELATION TO HISTOLOGICAL VARIABLES.

Viral hepatitis C is a significant public health challenge. The disease may remain clinically silent in both acute and chronic forms, and chronic infections may progress to advanced disease such as cirrhosis and hepatocellular carcinoma, requiring costly treatment, compromising the patient's quality of life and even leading to death. For this reason, it is one of the most frequent indications for liver transplantation. Although treatment with direct-acting antivirals represents remarkable progress, many patients are still infected and even those who cleared the viral infection must be followed due to their previous hepatic lesions, especially regarding the disturbances of lobular architecture and the sanguineal and lymphatic vessels. To assess immunohistochemical aspects of lymphatic sprouts and mature lymphatic vascularity with histological variables of liver injury attributable to hepatitis C virus (HCV) and fatty disease. The present study included 72 liver biopsies of cases with chronic hepatitis C. Morphologic changes reflecting "staging" and "activity" were analyzed. Immunohistochemical reactions were performed with monoclonal antibody D2-40 anti-podoplanin. Major histological variables were also semiquantified so as to enable the search for possible associations among histological and Immunohistochemical criteria, as well as with genotypes 1 and 3 of HCV. Histological findings showed that the different degrees of strutural changes were well represented in this casuistic. Intralobular/parenchymal necro-inflammatory activity was predominantly mild to moderate. Most cases did not show major evidences of fatty disease, which was found significantly higher in cases infected with HCV genotype 3. The amount of portal lymphatic sprouts increased along with the progression of structural changes, maximal at cirrhosis. Portal lymphatic sprouts as well as portal mature lymphatic vessels also showed an increase parallel to the increase in the degree of portal/septal inflammatory infiltrate. In the present study, no significant association was found between the proportion of portal lymphatic sprouts or portal mature lymphatic vessels and the degree of periportal/periseptal activity. No significant relations were detected between lymphatic sprouts/mature vessels and periportal or parenchymal inflammatory activity, nor with infections due to HCV genotype 1 or 3. Visualization and semiquantitation of sprouts and mature lymphatic vessels were clearly yielded by Immunohistochemical staining with monoclonal antibody D2-40. The amount of lymphatics was increased along fibrogenic process, significantly related to progression of liver disease and maximal at cirrhosis. No significant relations were detected with necro-inflammatory activity at interface or in the parenchyma.

Immunophenotypic Differences in Tumor-Infiltrating Lymphocytes and Neovascularization Between Primary Cutaneous Melanoma With and Without Metastasis: An Immunohistochemical Study of 80 Cases.

The prognostic implications of the immunophenotype of the tumor-infiltrating lymphocytes (TILs) in primary cutaneous melanoma are well known. In recent years, the study of this immunophenotype has also resulted in immunotherapeutic consequences. The aims of this study were to characterize the subpopulations of TILs in primary cutaneous melanoma, in cases with and without metastasis, as well as the neovascularization associated with the primary neoplasm, and its influence on the development of metastasis. To this end, the immunophenotype of TILs and the neovascularization of 80 patients with primary cutaneous melanoma (40 each with metastatic and non-metastatic melanoma) were analyzed by immunohistochemistry for CD3, CD4, CD8, FOXP3, PD-1, CD31, and D2-40 antibodies. We found that higher frequencies of TILs with brisk pattern, and CD4+, CD8+, and CD20+ cells in TILs, and a lower frequency of CD31+ vessels were histopathological features associated with better prognosis in primary cutaneous melanoma. Our results support the notion that the immunohistochemical study of TILs and neovascularization in primary cutaneous melanoma may be helpful tools for identifying patients at increased risk of metastasis development.

Expression of podoplanin in tumor cells and lymphatic vessels in both tumoral and peritumoral areas and correlation with metastatic potential of oral squamous cell carcinoma.

Aim:This study aims to study the expression of podoplanin in tumor cells as well as the lymphatic vessels (LVs) in both tumoral and peritumoral areas and correlate the importance of the lymphatic microvascular density (LMVD) in oral squamous cell carcinoma (OSCC) and its metastatic potential.Materials and Methods:D2–40 expression and LV density (LVD) were assessed using antibody D240, in 45 diagnosed cases of all the three grades of OSCC. D2–40 expression was evaluated in both epithelial cells as well as the LVs.Results:D2–40 expression in OSCC showed two different patterns - diffuse and focal. LMVD was calculated and difference in peritumoral and intra tumoral LVs was also assessed. A marked increase was seen we progressed from well-differentiated tumor to poorly differentiated ones, but this difference was found to be statistically nonsignificant. D2–40 immunostaining also highlighted the presence of lymphatic invasion present within the tumors which was detected by the presence of tumor emboli within the LVs. Overall, no significant correlation was found between D240 epithelial positivity and LVD.Conclusion:The expression of podoplanin in tumor cells and lymphatics when correlated with histopathological status and clinically with the lymph node status can definitely help in the adjuvant therapies used in OSCC.

Vanishing bone disease: Decoding the rare entity of Gorham–Stout disease

Vanishing bone disease is a rare entity with an obscure cause and undetermined pathophysiology and characterized by spontaneous and progressive resorption of bones. To date, the etiology of Gorham–Stout syndrome is still controversial. However, LYVE-1, a receptor, and podoplanin, a transmembrane glycoprotein identified by the antibody D2-40, are two extensively used molecular markers of lymphatic endothelial cells. These markers do exist in the medullary and cortical portions of Gorham–Stout disease (GSD) patients' bones. The following case report describes a case of 32-year-old male with GSD involving the maxilla with a brief discussion on etiopathogenesis, clinical features, radiological and histological findings, and diagnosis with treatment options.

Microvessels in Epithelial Ovarian Tumors: High Microvessel Density Is a Significant Feature of Malignant Ovarian Tumors.

Examine features of blood and lymphatic vessels in ovarian tumors and their significance to prognosis of ovarian cancer. A total of 139 women with epithelial ovarian tumors were included: 86 malignant, 17 borderline and 36 benign. Density, percentage, mean size and number of blood microvessels in tumors were measured by immunohistochemistry with antibodies against CD34 and CD105. Lymphatic vessel density was assayed using the D2-40 antibody against podoplanin. Angiogenesis was most profuse in malignant tumors. Small size of lymph vessels predicted 26% shorter 5-year survival of ovarian cancer patients. Further, high percentage of lymphatic vessels in tumors was associated with lymph node metastasis, and high density with cancer recurrence. Lower number of microvessels, as assessed by CD34 staining, predicted shorter progression-free survival. Additionally, the large size of microvessels assessed by CD34 and the high number of vessels assessed by CD105 were related to residual tumor >1 cm at primary surgery and also, large vessel size was associated with stage III, as assessed by CD105 staining. CD34 and CD105 define different characteristics of microvessels. Parameters of lymph vessels may predict the prognosis of ovarian cancer.

Peri-tumoral Lymphovascular density by antipodoplanin antibody D2-40, as a predictor of nodal metastasis in oral squamous cell carcinoma

BACKGROUND:
 The involvement of regional lymph nodes by cancer is a major determinant of prognosis in oral squamous cell carcinoma (OSCC) and is a significant predictor of patient survival. Peri-tumoral lymphangiogenesis is being studied as an important tool in predicting neck lymph node metastasis. Podoplanin, a transmembrane glycoprotein is expressed on lymphatic vessel endothelium and considered as a marker of lymphangiogenesis. The high podoplanin expression has been associated with invasion, progression, and metastasis of oral cancer. D2-40, an anti-podoplanin monoclonal antibody, is a specific marker of lymphatic endothelial cells. The present study was designed to investigate the peritumoral lymphovascular density (PT-LVD) by evaluating D2-40 expression and its potential utility in predicting lymph node metastasis in OSCC.
 METHODS: Seventy-two patients with OSCC were enrolled (36 with early and 36 with advanced-stage tumors). Differential expression of peritumoral lymphovascular density (PT-LVD) was evaluated using D2-40 expression by immunohistochemistry (IHC). Expression was categorized as low and high, derived from the mean of a number of vessels per 10 high power fields in hot-spots. Statistical methods were employed to correlate expression with age, gender, tumor site, stage, and neck lymph node involvement. 
 RESULTS: We observed a significant association between peritumoral lymphovascular density (PT-LVD) with clinical neck lymph node status (p=0.001) and tumor stage (p=0.033). There was no significant correlation of D2-40 expression with age, gender, tumor site, T stage, and tumor differentiation.
 CONCLUSION: PT-LVD is a positive predictor of neck lymph node metastasis. It has translational potential, along with other clinical parameters for decision making in the treatment of the neck in oral cancer.

Lymphangiogenesis in Hodgkin Lymphoma and in Indirectly Related Conditions – A Review

The significance of lymphangiogenesis in malignant tumors and specifically in classic Hodgkin lymphoma has not been properly evaluated, in contrast with that of angiogenesis in these neoplasms. In the study reviewed herein, the relevance of lymphatic vessel proliferation was explored in 19 cases of classic Hodgkin lymphoma stained with the D2-40 (anti-podoplanin) antibody. In each case, three lymphatic vessel hot spots were analyzed twice. Of the 57 hot spots thus investigated, 15 were chosen at random for image acquisition and analysis and microvessel counting. The mean perimeter, major axis length, surface area and complexity factor for each hot spot were established by morphometry, and associations with clinical and laboratory traits of classic Hodgkin lymphoma were identified. No relationships were found with the clinical features or immunostaining for typical markers of this lymphoma. However, significant inverse relations were found with BAX, pRb and IκB-α expression in tumor cells, genes believed to play roles in apoptosis in this lymphoma. The mean lymphatic major axis length was inversely correlated with the complexity factor. Clinicopathological associations were further obtained for the expression of BAX, pRb, and IκB-α in a large cohort of classic Hodgkin lymphoma patients that was previously published. Since the issue was poorly explored at this point, we have reviewed the significance of lymphangiogenesis in indirectly associated conditions.

Development of a biomarker of efficacy in second-line treatment for lymphangioma of the tongue: a pilot study.

Lymphangioma of the tongue is a rare lymphatic malformation, and various authors have reported the successful use of sirolimus for its treatment. However, the safety of sirolimus in children needs further evaluation so that those who do not respond are not necessarily exposed to its potential adverse effects. We hypothesised that assessment of lymphangiogenesis can be used to predict whether the patient will respond to sirolimus, so we organised a prospective study after ethics committee approval had been given. After clinical and histological diagnoses of lymphangioma of the tongue had been confirmed, 16 patients were given sirolimus 0.8mg/day in three divided doses. Clinical response was assessed and compared with lymphatic microvessel density (LMVD), which was calculated immunohistochemically using the monoclonal antibody D2-40 as the lymphatic endothelial marker. Nine patients responded well, five partially, and two failed to respond. Mean (SD) LVD among the good responders was 21.00 (3.74), whereas among non-responders it was 8.00 (4.24). There was a significant difference in mean LVD between good responders, partial responders, and non-responders (p=0.04). Sirolimus is effective in treating children with lymphangioma of the tongue, and lymphangiogenesis is a useful therapeutic predictive marker.

The Association of ICAM-1 Detected by Immunohistochemical Staining with Triple Negative and Non-Triple Negative Breast Carcinoma

Metastasis of tumour cell greatly contributes to the cause of mortality. Tumour-associated macrophage (TAM) and the intercellular adhesion molecule 1 (ICAM-1) were associated with metastases of breast carcinoma. However, the relationship between lymphatic vessel densities and invasion with TAM and ICAM-1 remained unclear. The aim of this study was to investigate the relationship of lymphovascular densities and invasions with patient’s clinicopathological data. The roles of TAM and ICAM-1 influencing lymphovascular invasions were also investigated. Haematoxylin and eosin (H&E) and immunohistochemical (IHC) staining on a consecutive section of 37 formalin fixed-paraffin embedded (FFPE) breast invasive carcinoma samples were carried out. The D2-40, CD34, CD163, and ICAM-1 antibodies were used to stain lymphatic vessel, blood vessel, TAM, and ICAM-1 receptor, respectively. The total lymphatic vessel density (LVD) was significantly reduced on increased tumor size (p=0.045). The increase of intra-tumoral LVD and lymphatic vessel invasion (LVI) was significantly associated with human epidermal growth factor receptor 2 (HER2) negative status (p=0.022 and p=0.05, respectively). The percentage of LVI was higher than blood vessel invasion (BVI) in 18.5%. Lymphovascular invasions detected in H&E were missed in 49.76% compared with those detected in IHC-stained tissues (206/410). ICAM-1 scores were significantly associated with non-triple negative breast cancer (non-TNBC) (p=0.008). ICAM-1 is significantly overexpressed on non-TNBC sample. Therefore, ICAM-1 might be clinically useful as a targeted molecule for non-TNBC patients. In histological reporting, in addition to H&E staining, IHC staining using D2-40 and CD34 should be considered to increase the accuracy of diagnosis.

Survival prediction in patients with cutaneous melanoma by tumour lymphangiogenesis

ABSTRACT Objectives: Melanoma induces lymphangiogenesis by secreting lymphangiogenic growth factors. The aim of this study was to examine the role of tumour lymphangiogenesis in survival of patients with cutaneous melanoma. Methods: Immunostaining of one hundred melanoma specimens was done with lymphatic-specific antibody D2-40. The quantification of tumour lymphangiogenesis – lymphatic vessel density (LVD) and lymphatic vessel area (LVA) – was calculated by computer-assisted morphometric analysis. Results: High intratumoural LVD, high peritumoural LVD, male gender, greater tumour thickness and Clark level IV/V were significantly associated with shorter disease-free survival (p= 0.001, p= 0.004, p= 0.004, p= 0.000 and p= 0.008, respectively) and melanoma-specific survival (p= 0.002, p= 0.002, p= 0.001, p= 0.000 and p= 0.017, respectively), while the trunk melanoma site was significantly associated only with shorter disease-free survival (p= 0.033). No significant association of LVA with survival was found. At multivariate analysis, peritumoural LVD [hazard ratio (HR) = 2.143, 95% confidence interval (CI) 1.097–4.189, p= 0.026)] and melanoma thickness (HR = 1.276, 95%CI 1.106–1.473, p= 0.001) were independent predictors of disease-free survival, while intratumoural LVD (HR = 3.446, 95%CI 1.465–8.109, p= 0.005), peritumoural LVD (HR = 2.742, 95%CI 1.313–5.725, p= 0.007) and gender (HR = 2.880, 95%CI 1.304–6.362, p= 0.009) were independent predictors of melanoma-specific survival. Conclusion: Тhis study shows that LVD enables better prediction of survival than melanoma thickness and other clinical-pathological parameters. Intratumoural LVD is the most significant predictor of melanoma-specific survival, while only peritumoural LVD has a significant impact on both, a disease-free survival and a melanoma-specific survival.

Correlative 3D Imaging and Microfluidic Modelling of Human Pulmonary Lymphatics using Immunohistochemistry and High-resolution \u03bcCT

Lung lymphatics maintain fluid homoeostasis by providing a drainage system that returns fluid, cells and metabolites to the circulatory system. The 3D structure of the human pulmonary lymphatic network is essential to lung function, but it is poorly characterised. Image-based 3D mathematical modelling of pulmonary lymphatic microfluidics has been limited by the lack of accurate and representative image geometries. This is due to the microstructural similarity of the lymphatics to the blood vessel network, the lack of lymphatic-specific biomarkers, the technical limitations associated with image resolution in 3D, and sectioning artefacts present in 2D techniques. We present a method that combines lymphatic specific (D240 antibody) immunohistochemistry (IHC), optimised high-resolution X-ray microfocus computed tomography (μCT) and finite-element mathematical modelling to assess the function of human peripheral lung tissue. The initial results identify lymphatic heterogeneity within and between lung tissue. Lymphatic vessel volume fraction and fractal dimension significantly decreases away from the lung pleural surface (p < 0.001, n = 25 and p < 0.01, n = 20, respectively). Microfluidic modelling successfully shows that in lung tissue the fluid derived from the blood vessels drains through the interstitium into the lymphatic vessel network and this drainage is different in the subpleural space compared to the intralobular space. When comparing lung tissue from health and disease, human pulmonary lymphatics were significantly different across five morphometric measures used in this study (p ≤ 0.0001). This proof of principle study establishes a new engineering technology and workflow for further studies of pulmonary lymphatics and demonstrates for the first time the combination of correlative μCT and IHC to enable 3D mathematical modelling of human lung microfluidics at micrometre resolution.

Immunohistochemical analysis of retraction pocket pars tensa of tympanic membrane in children

AimsImmunohistochemical analysis of retraction pocket pars tensa of tympanic membrane in children. Identification of signs typical for cholesteatoma and support of retraction theory of cholesteatoma. Study design: a prospective study analysing 31 surgically removed retraction pockets. DepartmentUniversity Hospital, Children's Medical Centre Methods: Retraction pockets processed by a standard process for immunohistochemical analysis. The observed findings were specified using antibodies CD45 LCA (leukocyte common antigen), CD31 (platelet endothelial cell adhesion molecule), D2-40 (marker of lymphatic endothelium), MMP9 (marker of degradation of connective tissue extracellular matrix) and Ki67 (cellular marker of proliferation). ResultsAll observed parameters except for MMP9 had a significantly higher incidence in retraction pocket stage III compared to stage II according to Charachon. ConclusionWe described immunohistochemical signs of retraction pocket pars tensa of tympanic membrane in children resulting in cholesteatoma. All the observed signs occur in the structure of matrix and perimatrix of cholesteatoma. A significantly higher incidence of all observed parameters except from MMP9 was proved in retraction pocket stage III, unlike in stage II. This observation proves the fact that retraction pocket is a progressive disease and is a procholesteatoma stage.

The impact of examining VEGF-C expression, D2-40 based detection of LVI, and LVD on the Prediction of Lymph Node Metastasis in Endometrial Carcinoma.

Background: Background: Lymph node metastasis (LNM) is a main route of endometrial carcinoma (EC) spread and it plays a chief prognostic role. Our objective is to find LMN predictors through investigating lymph vessel invasion (LVI), and lymphatic vessel density (LVD), in addition to VEGF-C expression in the primary tumor site of the EC. Material and Methods: A retrospective Immunohistochemical study applying VEGF-C and D2-40 antibodies on 40 EC cases, in addition to 20 cases of proliferative endometrium, and 20 cases with atypical endometrial hyperplasia as control groups. The studied cases were screened for expression of VEGF-C, D2-40- LVI, and LVD. Statistical analysis with the correlation of the findings to various clinicopathological parameters were achieved. Results: Expression of VEGF-C was moderate to high in 87.5% of EC cases, only mild to moderate focal staining in atypical hyperplasia cases, and exclusively negative in proliferative endometrium cases. VEGF-C expression could independently predict LNM with sensitivity, specificity , PPV, NPV, and accuracy of (100, 51.6, 62.5, 100, 62.5%)respectively. D2-40-LVI detection system is more sensitive in predicting LNM in EC than routine H&E stained section. It was detected in 9/9of EC with LNM cases ,(sensetivity100%) compared to 7/9 cases detected by H&E staining (sensetivity77.8%). D2-40 -LVI detection system could predict LNM with sensitivity, specificity, PPV, NPV, and accuracy of (100, 67.7, 47.4, 100, 75%. )respectively. D2-40-LVI ,Peritumoral LVD, and VEGF-C expression showed a significant correlation to LNM (p

Morphometric analysis of tumor stromal lymphatic vessels and lymphangiogenesis in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) often causes lymphatic metastasis, particularly to the regional cervical lymph nodes. Although lymphatic metastasis is closely associated with lymphangiogenesis in the tumor tissues, the detailed mechanisms are not yet fully understood. The purpose of this study was to examine the morphological association of lymphangiogenesis in the tumor stromal tissue with cervical lymph node metastases in OSCC. The medical records of 13 patients treated for OSCC affecting the tongue at the Department of Oral Surgery of Nihon University School of Dentistry at Matsudo Hospital between November 2003 and March 2011 were reviewed. The 13 cases were divided into two groups, with 7 cases in the metastasis group and 6 cases in the non-metastasis group. Histopathological (hematoxylin-eosin stain) and immunohistochemical (D2-40 antibody) examinations were performed. Lymphatic vascular density and lymphatic vascular area, lymphatic vascular morphology, and the number of lymph vessels were quantified using ImageJ software. The lymphatic vessels in the metastasis group were distributed with higher density and ran irregularly, with more remarkable deformation than those in the non-metastasis group. Cervical lymph node metastasis is one of the factors affecting progression and the prognosis of OSCC patients. In the present study, lymphatic vessels were found in the OSCC stroma, which also suggests that an increased density of lymphatic vessels leads to cervical lymph node metastases.

Mesenteric Lymphatic Vessel Density Is Associated with Disease Behavior and Postoperative Recurrence in Crohn’s Disease

PurposeThe aims of the present study were to examine the density of lymphatic vessels in the mesentery and to assess the predictive value of the mesenteric lymphatic vessel density for postoperative clinical recurrence. MethodsIleocolonic resection specimens were obtained from 53 patients with Crohn’s disease and 10 non-inflammatory bowel disease control subjects. Mesentery adipose tissues adjacent to the bowel wall were used for the histological quantification of lymphatic vessels using immunohistochemistry with the D2-40 antibody. The relationships between lymphatic vessel density and disease behavior, the presence of granulomas, the presence of creeping fat, and postoperative clinical recurrence were assessed. ResultsMedian lymphatic vessel density in the mesentery adjacent to inflamed or non-inflamed intestine was lower in control subjects than in Crohn’s disease patients (2.13‰; interquartile range [IQR], 1.83–2.61; 8.34‰; IQR, 6.39–10.22; 4.43‰; IQR, 3.32–5.78; P ˂ 0.001). Increased mesenteric lymphatic vessel density was significantly associated with stricturing behavior, the presence of intestinal granulomas, the presence of creeping fat, and bowel thickness. Interestingly, patients with disease recurrence had an increased mesenteric lymphatic vessel density of the proximal mesenteric margin at the time of resection compared with those who did not have disease recurrence (6.23‰; IQR, 5.43–6.75 vs. 3.28‰; IQR, 2.93–4.29; P ˂ 0.001). ConclusionsIn addition to its correlation with disease behavior, bowel thickness, and the presence of intestinal granulomas and creeping fat, increased mesenteric lymphatic vessel density in the proximal margin is predictive of early clinical recurrence after surgery in patients with Crohn’s disease.

Neoangiogenesis of gastric submucosa-invasive adenocarcinoma.

Early gastric cancer may be defined as mucosal or submucosal invasive carcinoma, and exhibits a good prognosis: 90% of patients survive >10 years. Early gastric cancer infrequently exhibits lymph node metastasis, although submucosal invasion, the presence of vascular invasion and/or lymphatic permeation are independent risk factors for lymph node metastasis in early gastric cancer. The analysis of tumor lymphangiogenesis and angiogenesis are important to determine the extent of invasive progression and metastasis in patients. Previously, the presence of vessels expressing the D2-40 antibody and the factor-VIII protein has been identified immunohistochemically. The vessels that are immunoreactive for D2-40 and factor-VIII are morphologically similar to lymphatic vessels or small-size veins, also termed venules. In the present study, the association between tumor invasion and neoangiogenesis in early gastric cancer was examined. The D2-40/factor-VIII double-stained vessel (DSV) density was analyzed, in addition to lymphatic and blood vessel (vein and artery) density, using 46 submucosa-invasive and 50 mucosal carcinomas, and 20 non-neoplastic gastric tissues. The lymphatic density and DSV density of submucosa beneath the carcinoma and submucosa of the surrounding region in submucosa-invasive carcinoma were significantly increased (P<0.001) in comparison with those in mucosal carcinoma or non-neoplastic gastric tissue. No significant difference was observed in blood vessel density between non-neoplastic gastric, mucosal carcinoma and submucosa-invasive carcinoma tissues other than that of mucosa. The present study suggests the potential for the presence of D2-40/factor-VIII DSV and the importance of this vessel for neoangiogenesis in early gastric cancer.