High Peritumoral Lymphatic Vessel Density Research Articles

Significance of oral cancer-associated fibroblasts in angiogenesis, lymphangiogenesis, and tumor invasion in oral squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) are recognized as a pivotal promoter in cancer initiation and development. However, the role of CAFs in the progression and metastasis of oral squamous cell carcinoma (OSCC) has not been fully elucidated. Lymphatic vessel density (LVD) and microvessel density (MVD) and the expression of α-smooth muscle actin (α-SMA) and matrix metalloproteinase-9 (MMP-9) were evaluated by immunohistochemistry in 86 cases of OSCC. The correlations between α-SMA expression and MMP-9 expression, LVD, MVD, and other clinicopathological parameters were analyzed. In vitro invasion assay was performed to assess the effect of CAFs on the invasion of OSCC cells. We also investigated the effect of CAFs on the angiogenesis and lymphangiogenesis by inoculating CAFs with OSCC cells into nude mice subcutaneously. Positive expression of α-SMA protein was detected in 69.8% of the tumors. Increased α-SMA expression was correlated strongly with enhanced tumor invasion, higher tumor grade, increased risk of recurrence, lymph node involvement, and higher peritumoral lymphatic vessel density and microvessel density (P < 0.05). CAFs induced more cancer cells to invade relative to normal fibroblasts (NFs) (P < 0.05). Compared with co-injection of OSCC cells and NFs or injection of tumor cells alone, co-injection of OSCC cells and CAFs resulted in earlier tumor formation and bigger tumor volume accompanied with increased angiogenesis and lymphangiogenesis (P < 0.05). CAFs may play critical roles in OSCC progression as an inducer of tumor invasion, angiogenesis, and lymphangiogenesis. Therapeutic strategies targeting CAFs against OSCC is promising and need further exploration.

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.

The lymphatic system in clinically localized urothelial carcinoma of the bladder: Morphologic characteristics and predictive value

ObjectiveTo assess the lymphatic vessel density (LVD) and lymphangiogenesis in urothelial carcinoma of the bladder (UCB) and to identify predictors of progression in patients treated by transurethral resection (TUR). Materials and methodsOne hundred eleven patients who underwent TUR for UCB were retrospectively included. Lymphatic endothelial cells were stained immunohistochemically [D2-40 (podoplanin) antibody in all samples; Prox-1, LYVE-1, and VEGFR-3 (Flt-4) in subgroups]. LVD was measured in representative intratumoral (ITLVD), peritumoral (PTLVD), and nontumoral (NTLVD) areas using standardized criteria. Double-immunostainings with D2-40/CD-34 were performed to distinguish between blood and lymphatic vessels, and D2-40/Ki-67 stainings were done to detect lymphangiogenesis. Lymph-specific parameters were correlated with pathologic and clinical characteristics. In patients with non-muscle-invasive UCB (n = 76) univariable and multivariable analyses were performed to identify predictors of progression. ResultsThe PTLVD was significantly higher than ITLVD and NTLVD (P < 0.001). Proliferating lymphatic vessels were observed in all specimens assessed with D2-40/Ki-67. Characteristic suburothelial D2-40 positivity was observed in noninvasive pTa tumors. LYVE-1-stainings revealed the existence of tumor-associated macrophages. The presence of intratumoral lymphatic vessels was significantly associated with higher tumor stage, high grade, and sessile growth (all P < 0.001). Muscle-invasive tumors (P = 0.020), higher grade (P = 0.026), the presence of lymphovascular invasion (P < 0.001), and concomitant carcinoma in situ (CIS) (P = 0.020), sessile growth (P = 0.004), and loss of suburothelial D2-40 positivity (P = 0.031) were associated with disease progression in univariable analysis. LVD values in any area were not significantly associated with progression despite detection of proliferating lymphatic vessels. The presence of concomitant CIS was identified as an independent predictor of progression on multivariable analysis (P = 0.041; hazard ratio 4.620). ConclusionsA high peritumoral LVD is present in clinically localized UCB. The presence of intratumoral lymphatic vessels correlates with characteristics of aggressive disease. Lymphangiogenesis occurs; however, the lymph-specific parameters tested in this study cannot be used to predict progression following TUR. The presence of concomitant CIS is an important risk factor for later disease progression in patients with non-muscle-invasive UCB. Our results contribute to the understanding of metastatic tumor spread in UCB.

Immunohistochemical Assessment of Lymphovascular Invasion in Stage I Colorectal Carcinoma

Several studies have suggested that the presence of occult nodal metastases (micrometastases) is related to adverse clinical course in stage I colorectal carcinoma. Herein we analyzed the correlation between nodal micrometastases and lymphovascular invasion (LVI) or lymphatic vessel density (LVD) in a series of stage I colorectal carcinomas; the cohort included cases characterized or not characterized by disease progression during the follow-up. In these cases, LVI and LVD were evidenced through the immunohistochemical detection of the specific marker for lymphatic vessels, D2-40. LVI was significantly more frequent in colorectal carcinomas characterized by the presence of micrometastases (P<0.0001), high peritumoral LVD (P<0.0001), and disease progression (P<0.0001). The analysis for progression risk indicated that nodal micrometastases and LVI were significant, negative, independent prognostic parameters associated with shorter disease-free survival of stage I colorectal cancer (P=0.0001; P=0.0242). In conclusion, in this study we demonstrated for the first time that LVI is significantly associated with nodal occult metastases in stage I colorectal carcinoma. In the light of its significant, independent, prognostic value in this neoplasia, the detection of LVI may represent a faster and cheaper tool compared with the time-consuming evaluation of micrometastases to select high-risk patients who may benefit from adjuvant systemic treatment. Furthermore, the assessment of LVI may be applied to establish the likelihood of nodal involvement from carcinomas treated with conservative local excision techniques, which provide no regional nodes for histologic examination.

Intratumoral c-Met expression is associated with vascular endothelial growth factor C expression, lymphangiogenesis, and lymph node metastasis in oral squamous cell carcinoma: implications for use as a prognostic marker

Hepatocyte growth factor has been identified as a lymphangiogenic factor in experimental animal models. However, the correlation between hepatocyte growth factor or c-Met expression and lymphangiogenesis in human spontaneous tumors has been rarely reported, and the distribution pattern of c-Met on tumor-related lymphatic vessels remains to be further investigated. Lymphatic vessel density, lymphatic invasion, the expression of hepatocyte growth factor, c-Met, and vascular endothelial growth factor C proteins were evaluated by immunohistochemistry in 76 cases of oral squamous cell carcinoma. The distribution of c-Met on lymphatic endothelium was examined. High expression of c-Met in tumor cells was significantly associated with advanced clinical stage (P = .045), high expression of vascular endothelial growth factor-C (P < .001), higher peritumoral lymphatic vessel density (P = .003), higher incidence of peritumoral lymphatic invasion (P = .032), and positive lymph node status (P = .005), in spite of its negative expression on most lymphatic vessels. Patients with high-c-Met expression tumors exhibited shorter overall survival and disease-free survival (P < .001 and P = .010, respectively). Taken together, our results provide indirect evidence for an association and possible regulatory link of c-Met with the lymphangiogenic factor, vascular endothelial growth factor C, and, by extension, with lymphangiogenesis and lymph node metastasis, suggesting important prognostic significance of c-Met for patients with oral squamous cell carcinoma.

Expressions of COX-2 and VEGF-C in gastric cancer: correlations with lymphangiogenesis and prognostic implications

BackgroundCyclooxygenase-2 (COX-2) has recently been considered to promote lymphangiogenesis by up-regulating vascular endothelial growth factor-C (VEGF-C) in breast and lung cancer. However, the impact of COX-2 on lymphangiogenesis of gastric cancer remains unclear. This study aims to test the expression of COX-2 and VEGF-C in human gastric cancer, and to analyze the correlation with lymphatic vessel density (LVD), clinicopathologic features and survival prognosis.MethodsUsing immunohistochemistry, COX-2, VEGF-C and level of LVD were analyzed in 56 R0-resected primary gastric adenocarcinomas, while paracancerous normal mucosal tissues were also collected as control from 25 concurrent patients. The relationships among COX-2 and VEGF-C expression, LVD, and clinicopathologic parameters were analyzed. The correlations of COX-2, VEGF-C and level of LVD with patient prognosis were also evaluated by univariate tests and multivariate Cox regression.ResultsThe expression rates of COX-2 and VEGF-C were 69.64% and 55.36%, respectively, in gastric carcinoma. Peritumoral LVD was significantly higher than that in both normal and intratumoral tissue (P < 0.05). It was significantly correlated with lymph node metastasis and invasion depth (P = 0.003, P = 0.05). VEGF-C was significantly associated with peritumoral LVD (r = 0.308, P = 0.021). However, COX-2 was not correlated with VEGF-C (r = 0.110, P = 0.419) or LVD (r = 0.042, P = 0.758). Univariate analysis showed that survival time was impaired by higher COX-2 expression and higher peritumoral LVD. Multivariate survival analysis showed that age, COX-2 expression and peritumoral LVD were independent prognostic factors.ConclusionsAlthough COX-2 expression was associated with survival time, it was not correlated with VEGF-C and peritumoral LVD. Our data did not show that overexpression of COX-2 promotes tumor lymphangiogenesis through an up-regulation of VEGF-C expression in gastric carcinoma. Age, COX-2 and peritumoral LVD were independent prognostic factors for human gastric carcinoma.

Expression of VEGF‐C and VEGF‐D as Significant Markers for Assessment of Lymphangiogenesis and Lymph Node Metastasis in Non‐Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF)-C and VEGF-D induce lymphangiogenesis through activation of VEGF receptor 3 (VEGFR-3) and have been implicated in tumor spread to the lymphatic system. Lymph node dissemination critically determines clinical outcome and therapeutic options of patients with non-small cell lung cancer (NSCLC). However, the relationship of VEGF-C, VEGF-D, and lymph node metastasis in cancers, including NSCLC, is still controversial. To evaluate the relationship between lymphangiogenesis and lymph node metastasis, the expression of VEGF-C and VEGF-D in NSCLC tumors were detected by immunohistochemistry and quantitative real-time polymerase chain reaction (QRT-PCR). QRT-PCR revealed that in marginal region VEGF-C and VEGF-D mRNA was significantly higher than in tumor center, and VEGF-D mRNA was also higher than that in peritumoral lung tissue. Immunohistochemically, we observed the same heterogeneous expression of VEGF-C and VEGF-D proteins. The group with high expression of VEGF-C and VEGF-D in marginal region had a higher incidence of lymph node metastasis compared with the group with low expression. Furthermore, the group with high expression of VEGF-D in marginal region had a higher incidence of lymphatic invasion. The group with high peritumoral lymphatic vessel density (LVD) had higher expression of VEGF-C and VEGF-D mRNA compared with the group with low peritumoral LVD. Our studies suggested that the expression of VEGF-C and VEGF-D at invasive edge was significantly associated with lymph node metastasis or lymphatic invasion in patients with NSCLC and may be involved in regulation of lymphangiogenesis and lymph node metastasis in NSCLC.

Lymphatic Vessel Density and VEGF-C Expression are Significantly Different Among Benign and Malignant Thyroid Lesions

Thyroid cancer is the most frequent endocrine neoplasia worldwide. The route for metastasis and loco-regional invasion preferentially occurs by lymphatic vessels. For this reason, the assessment of lymphatic vessel density (LVD) is supposed to represent both a prognostic parameter and also a potential therapeutic target. In order to evaluate the value of LVD in benign and malignant thyroid lesions, we analyzed 110 thyroidectomy specimens using D2-40, a specific marker for lymphatic vessels and vascular endothelial growth factor C (VEGF-C), the most potent molecule of lymphatic proliferation. LVD was significantly different between papillary and follicular carcinomas in total (p = 0.045) and peritumoral area (p = 0.042). Follicular adenoma and follicular carcinoma showed an important difference of intra- (p = 0.019) and peritumoral (p = 0.033) LVD. VEGF-C was more markedly expressed in malignancies than in benign lesions (p = 0.0001). Almost all cancers with high positive VEGF-C expression also exhibited increased peritumoral LVD (p = 0.049) when compared with the benign lesions. Indeed, the high peritumoral LVD of malignant thyroid lesions is an important finding for surgery planning and supports the practice of total thyroidectomy in malignant thyroid neoplasm's since the lymphatic peritumoral vessels definitely are an escape path for tumor cells.

Increased vascular endothelial growth factor-C expression is insufficient to induce lymphatic metastasis in human soft-tissue sarcomas.

Unlike carcinomas, soft-tissue sarcoma (STS) rarely exhibit lymphatic spread. Consequently, we examined expression and function of vascular endothelial growth factor (VEGF)-C and STS-associated lymphatic vessel density (LVD) components of this process. VEGF-C and VEGF-A mRNA and VEGF-C protein expression were evaluated in STS, STS cell lines, and breast cancers (reverse transcription-PCR, quantitative reverse transcription-PCR, and ELISA). STS cell conditioned medium after VEGF-C knockdown was examined for endothelial cell proliferation and migration effects (MTS and migration assays). Paraffin-embedded human lymph node-negative and lymph node-positive STS and lymph node-negative and lymph node-positive breast cancers were examined for VEGF-C, D2-40, and CD31 expression (immunohistochemistry). LVD differences were analyzed by Wilcoxon rank-sum tests. STS and breast cancer VEGF-C expression was comparable and higher than normal tissue levels. STS cells secreted functional VEGF-C: STS conditioned medium induced lymphatic endothelial cell proliferation and migration, which was abrogated by STS cell VEGF-C knockdown. STS and breast cancer intratumoral LVD was similar. STS peritumoral LVD (PT-LVD) was reduced versus breast cancer PT-LVD (P < 0.001). Significantly higher PT-LVD was observed in lymph node-positive versus lymph node-negative STS; lymphatic spreading STS subtypes also had higher LVD. STS VEGF-C expression and PT-LVD lacked correlation, and many lymph node-negative STS had high PT-LVD, suggesting complexity in this metastatic process. Compared with breast cancers, STS exhibited lower PT-LVD independent of VEGF-C expression, which may underlie STS lymph node metastasis rarity. Moreover, lymphatic vessels appear necessary but not sufficient to sustain STS lymphatic spread. Examining STS "nonlymphatic" dissemination may help elucidate mechanisms of lymphatic spread, insights critically important to cancer metastasis control.

O-93 ALCAM is an independent predictor of survival in unselected breast cancer

Lymphatic spread of gastroenteric tumor cells to regional lymph nodes is one of the early events in metastatic cancers and is often associated with distant metastatic spread and poor prognosis. Expression levels of newly described lymphatic endothelial markers, LYVE-1, VEGF-C, VEGF-D and the VEGF receptors VEGFR-3 were assessed in our study.Paired (tumor and corresponding normal tissue) samples were obtained. The expression level of each factor was determined using RT-PCR and quantified by using a real-time quantitative PCR (RT-QPCR) technique, with respective cloned cDNA plasmids as internal standards.The expression of VEGF-C and lymphatic endothelial marker VEGFR-3 was significantly greater in patients with lymph node metastasis than in those without metastasis, but no different expression level of VEGF-D and LYVE-1 was detected in both groups of patients. Lymphatic vessel density (LVD), which was assessed by immunohistochemistry for LYVE-1, was correlated with lymphangiogenesis factors and lymph node metastasis. Expression of VEGF-C and VEGFR-3 was significantly associated with higher peritumoral LVD than normal group, and LVD was found greater in the node-positive group than in the node-negative group.These results indicate that quantitative analysis of lymphangiogenic marker VEGF-C and VEGFR-3 in gastroenteric specimens may be useful in predicting metastasis of gastroenteric cancer to regional lymph nodes, but the role of LYVE-1 in predicting metastasis of gastroenteric cancer requires further analysis.

Quantitative Analysis of Lymphangiogenic Markers in Human Gastroenteric Tumor

Lymphatic spread of gastroenteric tumor cells to regional lymph nodes is one of the early events in metastatic cancers and is often associated with distant metastatic spread and poor prognosis. Expression levels of newly described lymphatic endothelial markers, LYVE-1, VEGF-C, VEGF-D and the VEGF receptors VEGFR-3 were assessed in our study. Paired (tumor and corresponding normal tissue) samples were obtained. The expression level of each factor was determined using RT-PCR and quantified by using a real-time quantitative PCR (RT-QPCR) technique, with respective cloned cDNA plasmids as internal standards. The expression of VEGF-C and lymphatic endothelial marker VEGFR-3 was significantly greater in patients with lymph node metastasis than in those without metastasis, but no different expression level of VEGF-D and LYVE-1 was detected in both groups of patients. Lymphatic vessel density (LVD), which was assessed by immunohistochemistry for LYVE-1, was correlated with lymphangiogenesis factors and lymph node metastasis. Expression of VEGF-C and VEGFR-3 was significantly associated with higher peritumoral LVD than normal group, and LVD was found greater in the node-positive group than in the node-negative group. These results indicate that quantitative analysis of lymphangiogenic marker VEGF-C and VEGFR-3 in gastroenteric specimens may be useful in predicting metastasis of gastroenteric cancer to regional lymph nodes, but the role of LYVE-1 in predicting metastasis of gastroenteric cancer requires further analysis.

Expression of vascular endothelial growth factor‐C and its receptor in invasive micropapillary carcinoma of the breast

Invasion to lymphatic vessels and metastasis to lymph nodes are frequent complications in invasive micropapillary carcinoma (IMPC) of human breast cancer. Vascular endothelial growth factor-C (VEGF-C) and its receptor, VEGFR-3 have been implicated as the important factors in the formation of lymphatic vessels and recent experimental evidence strongly suggests that lymphangiogenesis in tumor promotes lymphatic metastasis. To clarify the mechanism of its occurrence, the expression of VEGF-C, VEGFR-3 and lymphatic vessel density (LVD) was examined in 40 cases of IMPC (pure and mixed type) and in 40 cases of pseudo-IMPC. Cytoplasmic expression of VEGF-C and VEGFR-3 were more frequent in tumor cells of IMPC compared to those of pseudo-IMPC. A significant positive correlation was found between the expression of VEGF-C and VEGFR-3 in both IMPC and pseudo-IMPC. The expression of VEGF-C was also significantly associated with higher peritumoral LVD, lymphatic invasion and number of lymph node metastasis in IMPC. These findings suggest that VEGF-C promotes the proliferation of peritumoral lymphatic vessels and that lymphatic invasion and metastasis to lymph nodes are frequently induced in IMPC of breast.

Peritumoral Lymphatic Vessel Density and Vascular Endothelial Growth Factor C Expression in Early-Stage Squamous Cell Carcinoma of the Uterine Cervix

Lymphatic invasion and nodal metastasis plays a major role in the spread of cervical cancer; however, little is known about the mechanisms whereby tumor cells enter the lymphatic system. We examined the intra- and peritumoral lymphatic vessel density (LVD) using D2-40 immunohistochemistry in 111 cervical squamous cell carcinomas and correlated them with vascular endothelial growth factor (VEGF)-C expression, clinicopathologic tumor features, and outcome. Compared with benign cervix, intratumoral and peritumoral LVD was significantly increased (P < 0.0001). Peritumoral LVD was significantly higher than intratumoral LVD (P = 0.009). High peritumoral, but not intratumoral, LVD showed significant correlation with high tumor stage, lymphatic invasion, and nodal metastasis. VEGF-C showed increased expression at the invasive edge compared with the center of tumors (P < 0.0001) and correlated with high peritumoral LVD, lymphatic invasion, and nodal metastasis. High peritumoral LVD and VEGF-C expression at the invasive edge of tumors were associated with poor overall and recurrence-free survival in univariate analysis. In multivariate analysis, peritumoral LVD was the only independent term predictive of overall survival. Our findings suggest a potential role for VEGF-C in tumor-induced lymphangiogenesis represented by high peritumoral LVD, which may be one of the mechanisms leading to lymphatic invasion and metastatic spread. High peritumoral LVD may be an independent prognostic factor in early-stage cervical cancer.