D2-40 Antibody Research Articles

Prognostic Role Of The Microenvironment In Follicular lymphoma

BackgroundFollicular lymphoma (FL) is characterized by clinical heterogeneity. There is a need for easily quantifiable prognostic biomarkers. No consensus has been achieved regarding the prognostic significance of the microvessel density, number of macrophages and cytotoxic lymphocytes within the tumor. ObjectivesTo characterize the microvessel density, number of macrophages and cytotoxic lymphocytes within tumor tissue using immunohistochemical (IHC) analysis of sections from paraffin blocks of lymph node biopsies in two groups of patients with different clinical courses of FL. Patients and methodsThe study included 59 patients with FL: 39 women (67%) and 20 men (33%). The median age was 53 years (range: 27–83 years). Forty nine patients were observed in the National Research Center for Hematology (Moscow), and 10 patients in Cancer Research Center (Moscow), from April 2001 until May 2011. Group 1, “good outcome”, included 28 patients who were followed for 2 or more years after the therapy, had remission, or developed relapse in five or more years from the start of treatment (late relapse). Group 2, “poor outcome”, included 31 patients who died due to tumor progression in the first 1–2 years from the time of diagnosis, had primary tumor resistance, or developed FL relapse in the first year from the start of treatment (early relapse). Patients in both groups received the same initial treatment with rituximab. Five year OS rate in group 1 was significantly higher than in group 2 (83±7% vs 28±13%; ð=0.03). In all 59 cases the samples for IHC analysis were selected by using a table of random numbers, without knowing to which study group they belonged. Each study parameter (microvessel density, number of cytotoxic lymphocytes and macrophages within a tumor) was evaluated and then assigned to prognostic group 1 or 2.The vascularization of tumor tissue was assessed by IHC on sections from paraffin blocks of tumor lymph node biopsies. CD34 and D2-40 (podoplanin) antibodies were used for the visualization of blood and lymphatic vessels, respectively. CD68 antibody was used to detect activated macrophages. Granzyme B antibody was used to visualize cytotoxic lymphocytes.Morphometric analysis was performed using light microscopy and a Leica digital camera (400x). The pictures were processed by the computer program “VideoTesT-Morphology 5.2” in order to estimate the percentage of vessels in the tumor tissue. IHC specimen evaluation was carried out using a table of random numbers. Number of macrophages and cytotoxic lymphocytes in nodular and nodular-diffuse types of tumor growth was evaluated by light microscopy (400x): the cells were counted in 1 mm2 of tumor tissue, and the number of positive cells was determined in the intrafollicular and interfollicular space. ResultsBlood vessel density and lymph vessel density in group 2 were significantly higher than in group 1 (p=0.03). The cut-off point for blood and lymphatic vessel density in 1 mm2, which differentiates group 1 from group 2, was 0.04. The number of cytotoxic lymphocytes in intrafollicular space, interfollicular space and diffuse area of tumor nodules in group 1 was significantly higher than in group 2 (ð=0.05). The cut-off point for tumor-associated cytotoxic lymphocytes in 1 mm2, which differentiates group 1 from group 2, was 100 (Figure 1). The number of macrophages within tumors with nodular and diffuse growth in group 2 was significantly higher than in group 1 (ð=0.01) (Figure 2). The cut-off point for macrophages in 1 mm2, which differentiates group 1 from group 2, was 250. Statistical analysis were done using JMP ver. 10.0 (SAS, Cary, NC). [Display omitted] [Display omitted] ConclusionOur results demonstrate an association between higher angiogenic activity in the tumor and poor prognosis. A low number of cytotoxic lymphocytes and a high number of macrophages within the tumor were also associated with a poor prognosis. We suggest that these results will help to predict clinical response in FL using rituximab. Disclosures:No relevant conflicts of interest to declare.

Neither expression of VEGF-C/D nor lymph vessel density supports lymphatic invasion as the mechanism responsible for local spread of recurrent salivary pleomorphic adenoma

Recent research suggests that multinodular recurrent pleomorphic adenoma (PA) might result from cell migration through lymphatics. Lymphangiogenesis in malignancies is mediated by vascular endothelial growth factors C and D (VEGF-C/D). We studied the expression of VEGF-C/D in PA by immunohistochemistry as well as lymphatic vessel density (LVD). In 6 non-recurrent, 4 primary-to-recur, and 10 recurrent PAs, VEGF-C/D expression was assessed by immunohistochemistry. Staining was scored in terms of staining intensity (0 = absent to 3 = strong), and the percentage of positive tumor cells (scored as 0 (0-19 %), 1 (20-39 %), 2 (40-50 %), and 3 (60-100 %)) and a sum score were calculated. Intra- and peritumoral LVD was assessed by counting of LV after immunostaining, using the D2-40 antibody. All but one sample were VEGF-C negative. The differences in VEGF-D expression between non-recurrent, primary-to-recur, and recurrent PAs were not significant (p>0.05). VEGF-D expression did not correlate with peritumoral LVD (p>0.05). Our study revealed a significant difference between intra- and peritumoral LVD values when comparing individual and all sample groups (p=0.01). The lack of VEGF-C expression and of significant differences in VEGF-D expression and peritumoral LVD between patients with non-recurrent, primary-to-recur, and recurrent PAs does not support the lymphangiogenic local spread hypothesis

The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas

BackgroundPancreatic cancer is a rare tumor with an extremely low survival rate. Its known risk factors include the chronic use of tobacco and excessive alcohol consumption and the presence of chronic inflammatory diseases, such as pancreatitis and type 2 diabetes. Angiogenesis and lymphangiogenesis, which have been the focus of recent research, are considered prognostic factors for cancer development. Knowing the angiogenic and lymphangiogenic profiles of a tumor may provide new insights for designing treatments according to the different properties of the tumor. The aim of this study was to evaluate the density of blood and lymphatic vessels, and the expression of VEGF-A, in pancreatic adenocarcinomas, as well as the relationship between blood and lymphatic vascular density and the prognostically important clinical-pathological features of pancreatic tumors.MethodsParaffin blocks containing tumor samples from 100 patients who were diagnosed with pancreatic cancer between 1990 and 2010 were used to construct a tissue microarray. VEGF expression was assessed in these samples by immunohistochemistry. To assess the lymphatic and vascular properties of the tumors, 63 cases that contained sufficient material were sectioned routinely. The sections were then stained with the D2-40 antibody to identify the lymphatic vessels and with a CD34 antibody to identify the blood vessels. The vessels were counted individually with the Leica Application Suite v4 program. All statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA) software, and p values ≤ 0.05 were considered significant.ResultsIn the Cox regression analysis, advanced age (p=0.03) and a history of type 2 diabetes (p=0.014) or chronic pancreatitis (p=0.02) were shown to be prognostic factors for pancreatic cancer. Blood vessel density (BVD) had no relationship with clinical-pathological features or death. Lymphatic vessel density (LVD) was inversely correlated with death (p=0.002), and by Kaplan-Meyer survival analysis, we found a significant association between low LVD (p=0.021), VEGF expression (p=0.023) and low patient survival.ConclusionsPancreatic carcinogenesis is related to a history of chronic inflammatory processes, such as type 2 diabetes and chronic pancreatitis. In pancreatic cancer development, lymphangiogenesis can be considered an early event that enables the dissemination of metastases. VEGF expression and low LVD can be considered as poor prognostic factors as tumors with this profile are fast growing and highly aggressive.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5113892881028514

Decreased Lymphatic Vessel Density Is Associated With Postoperative Endoscopic Recurrence in Crohn’s Disease

Increased lymphatic vessel (LV) density has been found in uninflamed intestinal wall of patients with Crohn's disease (CD). The goal of the study was to search for an association between LV density in the proximal ileal resection margin at the time of surgery and endoscopic recurrence. Ileocolonic resection specimens were obtained from 28 CD patients and 10 control subjects. The ileal proximal uninflamed section was used for the histological quantification of LV using immunohistochemistry with D2-40 antibody in the mucosa and submucosa. Quantification of LV was performed in 8 consecutive fields and was blinded to recurrence score. Patients were divided into 2 groups based on the presence (Rutgeerts score, i3/i4) (R+) or absence (Rutgeerts score, i0/i1) (R-) of endoscopic recurrence 1 year after the surgery. All patients were free of immunomodulators or biologics between surgery and postoperative endoscopy. Median LV density was lower in control subjects than in CD patients in the mucosa (4.5%; interquartile range [IQR], 3.6-5.3 versus 5.9%; IQR, 4.2-8.5; P = 0.04) and submucosa (2.4%; IQR, 1.9-3.6 versus 5.7%; IQR, 4.3-6.9; P < 0.01). R- patients had a higher LV density in the proximal resection margin at surgery than R+ patients, both in the mucosa (8.5%; IQR, 6.5-10.3 versus 4.4%; IQR, 3.1-6.1; P < 0.01) and in the submucosa (6.3%; IQR, 5.5-9.3 versus 5.3%; IQR, 3.4-5.9; P = 0.03). Mucosal LV density greater than 7% predicted the absence of endoscopic recurrence at 1 year, with a sensitivity of 81% and a specificity of 75%. Decreased LV density is associated with high risk of endoscopic recurrence after surgery. Therapies that improve lymphatic flow in the gut may reduce the incidence of endoscopic recurrence.

Inconsistent immunohistochemical expression of lymphatic and blood endothelial cell markers in cutaneous lymphangiomas

We sought to describe expression of the immunohistochemical markers, CD31, D2-40 and LYVE-1 (lymphatic vessel hyaluronan receptor-1), that may assist in differentiating cutaneous lymphangioma and cutaneous hemangioma. Immunohistochemical staining of lymphangioma and hemangioma specimens was completed with CD31, D2-40 and LYVE-1 antibodies. Thirty-three examples of lymphangioma and 13 examples of hemangioma were evaluated. CD31 was positive in 91% of lymphangioma and 100% of hemangioma (sensitivity 0.91, 95% confidence interval 0.75-0.98; specificity 0, 95% confidence interval 0-0.28). D2-40 was positive in 54% of lymphangioma and 23% in hemangioma (sensitivity 0.50 95% confidence interval 0.32-0.68; specificity 0.50 95% confidence interval 0.28-0.72). LYVE-1 was positive in 6% in lymphangioma and 15% hemangioma (sensitivity 0.06 95% confidence interval 0.01-0.22, specificity 0.85 95% confidence interval 0.54-0.97). Due to the similar embryonic origins, it is unlikely that a single marker may accurately differentiate the lymphangioma from hemangioma. A combination of immunohistochemical stains such CD31, D2-40 and LYVE-1 testing may potentially increase the diagnostic accuracy in differentiating lymphangioma from hemangioma. However, our findings suggest that there is no advantage in performing immunohistochemical staining to differentiate lymphangioma from hemangioma.

Tumor‐associated lymphatic endothelial cell promotes invasion of cervical cancer cells

The most common way for cervical cancer to spread is through the lymphatic system. Tumor-associated lymphatic endothelial cell (TLEC) has been considered to play a crucial role in metastasis of certain cancers. The aim of this study was to isolate TLEC from human cervical cancers and explore its involvement in metastasis-associated behaviors in vitro. Lymphatic vessels in 62 cervix tissue specimens ranging from cervical intraepithelial neoplasia (CIN) to advanced invasive cancer were detected using immunochemical staining with D2-40 antibody. Relation of lymphatic vessel density (LVD) to clinicopathological characters was analyzed. Primary TLECs were isolated by LYVE-1 immuno-magnetic beads from cervical cancer tissues and verified through expression of LEC markers Prox-1 and D2-40, and then cultured in vitro. Invasiveness and viability of cells were assessed by transwell assay and typan blue exclusion, respectively. Our results showed that higher LVD was significantly associated with advanced FIGO stage, pelvic lymphatic nodal metastasis (LNM), and poorer cell differentiation. TLECs were successfully primarily isolated and cultured in vitro. Supernatant of TLEC enhanced invasiveness of Hela cell, but did not significantly affect cell viability. In conclusion, TLECs might actively promote lymphatic metastasis of cervical cancer. Further studies are needed to demonstrate the underlying mechanisms.

Computed Tomography Perfusion Imaging of Renal Cell Carcinoma

The aim of this study was to systematically analyze the correlation between computed tomography (CT) perfusion and histopathological angiogenic and prognostic markers in patients with renal cell carcinoma (RCC). Fifteen patients (12 men; mean age, 64.5 ± 9.4 years) with RCC underwent contrast-enhanced CT perfusion imaging (scan range, 10 cm; scan time, 40 seconds; dual-source 128-section CT) 1 day before surgery. The procedure for surgical specimen processing was modified to obtain an exact match with CT images. Microvessel density (MVD) was quantified by CD34 staining, and lymphatic vessel density (LVD) was stained with D2-40 antibodies. The CT perfusion values blood flow (BF), blood volume (BV), and flow extraction product (K(Trans)) were calculated using the maximum-slope and a delay-corrected modified Patlak approach and were correlated to MVD and LVD. The relationship between CT perfusion and the prognostic markers pT stage, Fuhrman grade, and tumor necrosis was evaluated. Histopathology revealed varying high MVD but low or absent intratumoral LVD. The BF and BV of RCC, both including and excluding necrotic regions, showed significant correlations with MVD (r = 0.600-0.829, P < 0.05 each). Significant correlations between MVD and K(Trans) were found only in small tumor areas exhibiting no necrosis (r = 0.550, P < 0.05). No significant correlation was found between BF, BV, and K(Trans) with intratumoral LVD (P = 0.35-0.82). With higher pT stage and Fuhrman grade, BF, BV, and K(Trans) were lower, similar to the MVD, but without reaching statistical significance. Blood flow, BV, and K(Trans) were significantly higher in RCCs with less than 50% necrosis than in those with 50% or grater necrosis (P < 0.05 each). Our study indicates that BF and BV from CT perfusion reflect blood vessels of RCC. Computed tompgraphic perfusion parameters differ significantly depending upon the degree of tumor necrosis.

구강암 마우스모델에서 림프관형성 인자가 생존율에 미치는 영향

Purpose: Vascular endothelial growth factor (VEGF)-C and its tyrosine kinase receptor, VEGF receptor (VEGFR)-3 are recently known to have lymphangiogenic activities in various tumor types. In this study, we determined whether the expression of lymphangiogenic factors correlate with nodal metastasis or survival in a nude mouse model of oral squamous cell carcinoma (OSCC). Methods: Three OSCC cells (KB, SCC4, SCC9) were xenografted into the right mandibular gland of athymic nude mice. The mice were followed for tumor development and growth, and the mice were sacrificed when they had lost more than 20% of their initial body weight, or the diameter of the induced tumor exceeds 20 mm. After necropsy, the murine tumors were examined histologically and radiologically (micro-positron emission tomography computed tomography) for regional or distant metastasis. We performed immunohistochemical assays with anti-VEGF-C, VEGFR-3, CD105, and D2-40 antibodies. Immunofluorescence double staining for LYVE-1/CD31 was also performed. To quantify the VEGF-C and VEGFR-3 level in the cancer tissue, Western blotting was performed. Finally, we determined the correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time. Results: OSCC tumor cells into the mandibular gland of the nude mice successfully resulted in the formation of recapitulating orthotopic tumor. Tumor cells of the induced tumor did not express VEGF-C. VEGF-C/VEGFR-3 expression was mainly distributed in the endothelial cells of the stromal area. There were no correlation between the degree of expression of VEGF-C/VEGFR-3 and the mean survival time of mice injected with different OSCC cell lines. Conclusion: An recapitulating orthotopic model of OSCC in nude mice was established, which copies the cervical nodal metastasis of human OSCC. Overexpression of lymphangiogenic factors seems to have no effect on survival of hosts in this in vivo experiment.

M2-Polarized Tumor-Associated Macrophage Infiltration of Regional Lymph Nodes Is Associated With Nodal Lymphangiogenesis and Occult Nodal Involvement in pN0 Pancreatic Cancer

Tumor-associated macrophages (TAMs) are reportedly involved in lymphangiogenesis in primary tumors, playing a crucial role in lymphatic metastasis. Furthermore, nodal lymphangiogenesis precedes and promotes regional lymph node (RLN) metastasis. We investigated the relationship of M2-polarized TAM infiltration of the RLNs, nodal lymphangiogenesis, and occult nodal involvement in pN0 pancreatic cancer. Hematoxylin-eosin-stained primary tumor and regional LN specimens from 40 patients diagnosed with pN0 pancreatic cancer according to the pathological TNM classification were assessed. To evaluate lymphangiogenesis, lymphatic vessel density was measured by using D2-40 antibody. CD163 and cytokeratin AE1/AE3 antibodies were used to detect M2-polarized TAMs and isolated tumor cells in the RLNs, respectively. The nodal lymphatic vessel density had a strong association with the M2-polarized TAM density in the RLNs (P < 0.0001). Most of these TAMs expressed vascular endothelial growth factor C. Furthermore, in the RLNs, the M2-polarized TAM density was significantly associated with the incidence of isolated tumor cells (P = 0.0477). M2-polarized TAM infiltration of RLNs is significantly associated with nodal lymphangiogenesis and occult nodal involvement in pN0 pancreatic cancer. Node-infiltrating M2-polarized TAMs may facilitate nodal lymphangiogenesis via the production of vascular endothelial growth factor C and thus promote RLN metastasis.

An Analysis of the Expression of Bcl-2, Podoplanin and Lymph Angiogenesis in Benign and Malignant Salivary Gland Tumours

Background: Salivary gland tumours comprise a significant proportion of oral tumours and are the next common neoplasm of the mouth after squamous cell carcinoma. These neoplasm’s have widely variable histopathologic and biologic characteristics, which makes it difficult to determine the pathogenesis and selection of therapeutic modalities. Alteration in some proto-oncogenes and tumour suppressor genes may lead to the development and progression of these tumours. Aim: The purpose of this study was to analyze the immune histochemical expression of Bcl-2 and epithelial podoplanin and lymph angiogenesis in benign and malignant salivary gland tumours. Material and method: The sample consisted of 20 formalin-fixed paraffin embedded blocks of benign and malignant salivary glands tumours. Immunohistochemical staining procedure was performed using monoclonal anti Bcl-2 antibody and D2-40 antibody. The sections were evaluated for Bcl-2 and epithelial podoplanin expression and D2-40 positive lymphatic vessels. Results and conclusion: Bcl-2 showed positive immune expression in all benign and malignant salivary gland tumours with mucoepidermoid carcinomas showing most intense expression. Podoplanin immunostaining was also assessed, highest recorded score was in peripheral mucoepidermoid carcinoma (10.4? 3.05). Lymphatic micro vessel density expressed by podoplanin showed intense score in central mucoepidermoid carcinoma (5.05? 5.0). Present investigation demonstrated positive Bcl-2, podoplanin response and Lymphatic micro vessel density in both benign and malignant salivary gland tumours suggesting that Bcl-2 and podoplanin alteration may be associated with the progression of these neoplasms.

High density of peritumoral lymphatic vessels measured by D2-40/podoplanin and LYVE-1 expression in gastric cancer patients: an excellent prognostic indicator or a false friend?

BackgroundOne of the most important prognostic indicators in gastric cancer is the presence of metastases in lymph nodes. Even now, little is known about lymphangiogenesis in neoplastic tissue, and little is also known about the transmission of a neoplastic cell from the tumor mass into a lymphatic vessel.MethodsThis study examined the relationships between the density of lymphatic vessels (LVD) stained immunohistochemically with lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and D2-40 (podoplanin) antibodies, the expression of vascular endothelial growth factor (VEGF)-C/D, selected clinical and pathomorphological factors, and the 5-year overall survival of gastric cancer patients.ResultsStatistical analysis showed no impact of increased intratumoral or peritumoral LVD on gastric cancer patient survival, irrespective of the protein used to stain lymphatic vessels. Analysis showed that the probability of overall survival was decreased in the cases with enhanced VEGF-D immunoreactivity (P = 0.0045).ConclusionThe study showed that the studied markers cannot be used to determine the required extent of the surgical procedure, as they have no statistically significant correlation with the degree of progression of the cancer, the stage of the disease assessed according to the TNM 5th classification of malignant tumors, clinicopathological features, and patient survival. VEGF-D is the only marker that can be regarded as an unfavorable prognostic indicator for patients with advanced gastric cancer.

Lymphangiogenesis and Podoplanin Expression in Oral Squamous Cell Carcinoma and the Associated Lymph Nodes

The objective of this study was to evaluate lymphangiogenesis in oral squamous cell carcinoma and in the associated lymph nodes and podoplanin expression in neoplastic cells at the invasive front. In addition, the association of the above parameters with lymph node metastasis was also investigated. We used immunohistochemistry to examine primary tumors and lymph nodes, regardless of metastasis. Lymphatic vessel density (LVD) and microvessel density (MVD) were assessed by antibodies D2-40 and CD105, respectively, in intratumoral and peritumoral areas and in lymph node regions. Vascular endothelial growth factor-C and vascular endothelial growth factor receptor-3 expression was evaluated in tumor cells and D2-40 (podoplanin) expression in parenchymal cells found at the invasive front. The majority of cases with nodal involvement presented a high peritumoral LVD. In addition, a strong association of LVD with size and site of primary tumors could also be identified. MVD was statistically associated with metastasis, and a significant association between the lymphangiogenic factors and the density of vessels in the intratumoral region was also seen. The well-differentiated tumors did not express podoplanin. LVD and MVD were higher in metastatic lymph nodes than in nonmetastatic lymph nodes. The enhanced vascular network in metastatic lymph nodes reinforces the previous reports of lymphangiogenesis occurrence in lymph nodes. Moreover, the expression of podoplanin by more undifferentiated tumor cells suggests that this protein could be an indicator of tumor aggressiveness.

Prognostic Significance of EpCAM-Positive Disseminated Tumor Cells in Rectal Cancer Patients With Stage I Disease

Here we evaluated the prevalence and prognostic impact of epithelial cell adhesion molecule (EpCAM)-positive disseminated tumor cells (DTCs) in stage I rectal cancer. Further we tested the association of these single tumor cells or small tumor cell groups with the extent of peritumoral lymphangiogenesis. A total of 845 regional lymph nodes (LN) of 44 patients classified as negative on conventional histopathology were retrospectively reanalyzed with immunohistochemistry (IHC) using the monoclonal antibody Ber-Ep4 directed against EpCAM for the detection of DTCs. The degree of lymphangiogenesis in the primary tumors was assessed by IHC of the primary tumor tissue using the monoclonal antibody D2-40, which reacts with the lymphatic endothelium. The IHC results were correlated with clinico-pathologic parameters and clinical follow-up data. EpCAM-positive DTCs in LNs were detected in 8 (18.2%) of the 44 patients. During a median follow-up of 59 months, 3 (37.5%) of the 8 patients with EpCAM-positive DTCs relapsed, whereas none of the DTC-negative patients developed tumor recurrence (P=0.004). Survival analysis revealed a significant effect of the prevalence of DTCs on overall survival (P=0.0009) and on recurrence-free survival (P=0.0001). Finally, the prevalence of EpCAM-positive DTCs in perirectal LNs was significantly correlated with a high density of peritumoral lymphatic vessels (P=0.015). Our results show that DTCs may occur in stage I of rectal cancer and are associated with poor prognosis. Their occurrence seems to be linked to a high density of newly formed lymphatic vessel at the primary tumor site. According to our data, patients with DTCs in their LN might benefit from adjuvant therapy.

Prognostic value of lymphangiogenesis and lymphatic vessel invasion in non-small cell lung cancer

背景与目的已有的研究表明新生淋巴管与多种肿瘤的进展和淋巴转移相关，本研究拟探讨非小细胞肺癌病灶内（旁）新生淋巴管密度及其受肿瘤侵袭状况对非小细胞肺癌的预后价值。方法选择特异性单克隆抗体D2-40标记新生淋巴管内皮细胞，以免疫组化方法检测79例Ⅱ期-Ⅲ期非小细胞肺癌病灶内及其中部分病灶旁新生淋巴管表达及其受侵状况，结合患者临床、病理及随访资料，判断其对患者预后的影响和评估价值。结果① 病灶内新生淋巴管密度：N2患者高于N0患者（P=0.015），新生淋巴管受侵患者明显高于未受侵患者（P=0.009），肺癌低分化明显高于高分化肺癌（P=0.007），腺癌高于鳞癌（P=0.025），患者生存率与之呈负性相关（P=0.007）；②病灶旁新生淋巴管密度与预后无明显相关性；③N2肺癌病灶内新生淋巴管受侵多于N0肺癌；④病灶内和病灶旁新生淋巴管受侵患者的生存率低于未受侵患者。结论病灶内新生淋巴管密度是影响非小细胞肺癌患者预后的重要因素，可作为判断预后的指标。新生淋巴管受侵的预后意义值得关注。

Lymphatic invasion identified with D2-40 immunostaining as a risk factor of nodal metastasis in T1 colorectal cancer

The management of T1 colorectal cancer after local resection is controversial. Regional lymph node metastasis often occurs, requiring subsequent colonic resection. The aim of this study was to reevaluate the risk factors of nodal metastasis of T1 colorectal cancer, especially to examine lymphatic vessel invasion in serially prepared hematoxylin and eosin sections and D2-40 immunostained sections to determine which is a better indicator of lymph node metastasis of T1 colorectal cancer. The study investigated 120 patients who underwent bowel resection and were histologically diagnosed to have T1 colorectal cancer in Kanagawa Cancer Center Hospital from 1995 to 2005. Serially prepared paraffin sections were stained with hematoxylin and eosin, or immunostained with D2-40 antibody or von Willebrand factor, and reevaluated for lymphatic vessel invasion and other risk factors, including venous invasion, histological grade, depth of submucosal invasion, and budding. Lymphatic invasion diagnosed with either hematoxylin and eosin staining (p=0.022), or D2-40 immunostaining (p=0.001), and budding (p=0.013) were significant risk factors for lymph node metastasis in the univariate analysis. Venous involvement, histological grade, or depth of submucosal invasion was not significant. The multivariate logistic regression analysis for the three risk factors found lymphatic invasion diagnosed with D2-40 as an independent risk factor (odds ratio 6.048, p=0.018, CI 1.360-26.89). The sensitivity, specificity, positive predictive value, and negative predictive value were 58%, 88%, 35%, and 95%, respectively. Lymphatic vessel invasion diagnosed with D2-40 was a better indicator to evaluate the risk for lymph node metastasis by T1 colorectal cancer.

Lymphatic and Capillary Invasion Patterns in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) comprises approximately 15 to 20 per cent of all breast cancer cases. Many studies have detected less lymph node metastasis in TNBC than sporadic breast cancers. In this study, we studied capillary and lymphatic invasion in tumors of patients with TNBC. To differentiate the capillary invasion and lymphovascular invasion. We used the Antihuman CD34 and antihuman D2-40 antibodies. Antihuman CD34 antibodies stain the blood vessels and lymphatics. However, antihuman D2-40 antibodies stain lymphatics specifically. Two experienced pathologists blinded to clinical data evaluated capillary and lymphatic invasion existence in 39 TNBC patients' tumor samples. Tumor samples were immunohistochemically stained with CD34 (endothelial cell marker) and D2-40 (podoplanin, a membrane protein, specific for lymphatic endothelium). The CD34-positive samples were categorized into two groups depending on their reaction with D2-40: lymphatic (D2-40-positive) and capillary (D2-40-negative) invasion. We have detected vascular invasion in 15 of 39 samples (38.5%) with CD34. Among those, capillary invasion was found in 14 (35.9%) and lymphatic invasion in three (7.7%) and both in two (5.1%) tumors. We did not find any significant correlation among capillary invasion, lymphatic invasion, vascular invasion, tumor grade, menopause status, history of cancer, and TNM. Capillary invasion is more commonly observed than lymphatic invasion in patients with TNBC. This finding supports the fact that more hematogenous metastasis (spreading) and less lymph node metastasis are seen in patients with TNBC.

Ciliary Body Lymphangiogenesis in Uveal Melanoma with and without Extraocular Extension

To determine whether peritumoral ciliary body lymphatics are found in uveal melanoma in the absence of extraocular extension. Consecutive case series from 1999 to 2005. Thirty-two uveal melanoma cases involving the ciliary body from the Ophthalmic Pathology Laboratory, University of Toronto, of which 23 showed no extraocular extension. All immunofluorescence studies and quantitative analyses were performed in a masked fashion. Sections were immunostained for the presence of lymphatic endothelium using podoplanin (D2-40 antibody) and blood vessel endothelium using CD34. Identification and quantification of D2-40-positive lymphatic vessels in the ciliary body. In every case (n = 32), D2-40-positive lymphatics were detected in the peritumoral ciliary body. Lymphatic signal was significantly increased in the peritumoral ciliary body compared with the nonperitumoral ciliary body (P < 0.0001). There was no difference in lymphatic signal between cases with and without extraocular extension (P > 0.05). Lymphatics were not detected within the tumors. Peritumoral lymphangiogenesis was present in the ciliary body in uveal melanomas with and without extraocular extension, and as such, the presence of peritumoral lymphatics is not recommended as a prognostic marker in uveal melanoma.

Peritumoral lymphangiogenesis induced by vascular endothelial growth factor C and D promotes lymph node metastasis in breast cancer patients

BackgroundMounting clinical and experimental data suggest that the migration of tumor cells into lymph nodes is greatly facilitated by lymphangiogenesis. Vascular endothelial growth factor (VEGF)-C and D have been identified as lymphangiogenic growth factors and play an important role in tumor lymphangiogenesis. The purpose of this study was to investigate the location of lymphangiogenesis driven by tumor-derived VEGF-C/D in breast cancer, and to determine the role of intratumoral and peritumoral lymphatic vessel density (LVD) in lymphangiogenesis in breast cancer.MethodsThe expression levels of VEGF-C/D were determined by immunohistochemistry, and intratumoral LVD and peritumoral LVD were assessed using immunohistochemistry and the D2-40 antibody in 73 patients with primary breast cancer. The associations of intratumoral LVD and peritumoral LVD with VEGF-C/D expression, clinicopathological features and prognosis were assessed.ResultsVEGF-C and D expression were significantly higher in breast cancer than benign disease (P < 0.01). VEGF-C (P < 0.001) and VEGF-D (P = 0.005) expression were significantly associated with peritumoral LVD, but not intratumoral LVD. Intratumoral LVD was associated with tumor size (P = 0.01). Peritumoral LVD was significantly associated with lymph node metastasis (LNM; P = 0.005), lymphatic vessel invasion (LVI; P = 0.017) and late tumor,node, metastasis (TNM) stage (P = 0.011). Moreover, peritumoral LVD was an independent risk factor for axillary lymph node metastasis, overall survival and disease-free survival in multivariate analysis.ConclusionsThis study suggests that tumor-derived VEGF-C/D induce peritumoral lymphangiogenesis, which may be one mechanism that leads to lymphatic invasion and metastatic spread. Peritumoral LVD has potential as an independent prognostic factor in breast cancer patients.

Podoplanin Lymphatic Density and Invasion Correlate With Adverse Clinicopathologic and Biological Factors and Survival in Neuroblastomas

Neuroblastoma (NB) is a challenging problem in oncology, as the majority of patients have lymphatic and/or hematogenous metastases at diagnosis. We investigated the prognostic significance of lymphatic density (LD) and invasion (LI) in NBs using the lymphatic endothelial marker podoplanin (PDPN). A total of 77 neuroblastic tumors and 9 ganglioneuromas (GNs) were immunostained for PDPN using D2-40 antibody. Intratumoral lymphatics were identified in 87% (67/77) of NBs and 7/9 GNs. The LD counts were significantly higher (P<0.01) in NBs (median=19.6, range=0.00 to 89.3) than in GNs (median=10.2, range=0 to 18.7). LI, assessed in D2-40-stained lymphatics, was present in 52/67 (78%) NBs. LDs were significantly higher in NBs from patients with adverse clinical factors (advanced-stage, high-risk group, primary abdominal compared with extra-abdominal sites), biological factors (MYCN amplification, 1p deletion, 17q gain), and distant lymph node metastases. LDs and LI were also significantly higher in NBs belonging to an unfavorable pathology prognostic group and in those with a high mitosis-karyorrhexis index. High LD and the presence of LI correlated with a shorter event-free survival in univariable analyses. High LD and the presence of LI were also associated with worse overall survival, although the association was less strong. In conclusion, increased LDs and the presence of LI correlated with adverse clinicopathologic and biological factors and survival. These findings suggest that PDPN has the potential to provide valuable prognostic information to clinicians for risk assessment in NBs.

Angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma of minor salivary glands

Mucoepidermoid carcinoma is the most common malignant tumor of salivary glands. This tumor is characterized by a great variability in clinical behavior, and little is known about the pathological mechanisms involved in its variance. Angiogenesis is an important step in tumor progression and is believed to be an essential event for metastatic dissemination. We aimed to investigate angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma measuring the density of neoformed and lymphatic vessels using CD105 and D2-40 antibodies, respectively, and by immunohistochemical evaluation of VEGF-A and VEGF-C proteins. It was also investigated the expression of D2-40 in neoplastic cells. We studied 26 cases of mucoepidermoid carcinoma, which showed great angiogenic activity measured by neoformed vessel density. However, a low density of lymphatics was observed. VEGF-A, VEGF-C, and D2-40 were commonly detected in mucoepidermoid carcinoma, but only VEGF-A expression correlated with neoformed vessel density. Recurrence and nodal metastasis were associated with low VEGF-A expression and low neoformed vessel density, indicating that impaired angiogenesis could lead to an aggressive phenotype. Angiogenesis seems important in the modulation of mucoepidermoid carcinoma pathogenesis; however, none of the parameters analyzed could predict tumor behavior.