Importance of lymph vessels in gastric cancer: a prognostic indicator in general and a predictor for lymph node metastasis in early stage cancer

BACKGROUND: Metastasis to regional lymph nodes (LNs) through lymphatic vessels is common in cancer progression and is an important prognostic factor in many cancers. Recent evidence suggests that tumour lymphangiogenesis promotes lymphatic metastasis. AIMS: To study the role of lymph vessel density (LVD) in gastric cancer and investigate whether LVD is associated with LN metastasis/prognosis. METHODS: Lymphatics of 117 primary human gastric cancer cases were investigated by quantitative immunohistochemical staining for podoplanin. The relation between LVD and LN metastasis and other established clinicopathological parameters was analysed. The relation between LVD and prognosis was also studied. RESULTS: Mean LVD of "hot spots" was 11.6/case. LVD significantly correlated with LN and podoplanin positive lymphatic invasion. High LVD was associated with worse overall survival. In multivariate analysis, positive LVD was a significant independent predictor of overall survival, depth of invasion, and TNM stage. LVD significantly correlated with LN metastasis at surgery and podoplanin positive lymphatic invasion. In multivariate analysis, positive LVD was an independent significant predictor of LN metastasis. CONCLUSIONS: Increased podoplanin expression is significantly associated with LN metastasis, and may play an important role in detecting LN metastasis in gastric cancer. Furthermore, LVD may be a significant prognostic factor in gastric cancer at any stage. In addition, LVD and lymph vessel invasion detected by podoplanin immunohistochemistry are associated with LN metastasis in T1 early gastric cancer. LVD assessment by podoplanin immunohistochemistry may become a useful predictor of LN metastasis in T1 early gastric cancer and may influence the decision making process for additional surgery.     

Lymphatic spread is related to VEGF-C expression and D2-40-positive myofibroblasts in intrahepatic cholangiocarcinoma.

Lymph node metastasis via lymphatic vessels is related with an adverse outcome in many tumors. It is unclear whether lymphatic spread needs the development of the new lymphatic vessels or the expression of lymphangiogenetic factor in intrahepatic cholangiocarcinoma. The aim of this study was to assess the role of lymphangiogenesis, vascular endothelial growth factor-C (VEGF-C) expression, and D2-40-positive myofibroblastic cells for lymphatic spread and patient outcome in 88 cases of intrahepatic cholangiocarcinoma. We also assessed VEGF-C expression in 15 cases of metastatic lymph nodes. There was a significant correlation between lower lymphatic vessel density in the tumor center and positive lymphatic invasion (P=0.0100). Poorly differentiated cholangiocarcinoma showed higher lymphatic vessel density in the tumor periphery and in the peritumoral area (P=0.0315 and P=0.0360, respectively). Lymphatic invasion was observed higher in the peritumoral area (63%, 24/38) and in the tumor periphery (79%, 30/38) than in the tumor center (27%, 9/38). There was no significant correlation between the proliferative lymphatic vessels and pathologic features; however, lymphatic invasion was significantly associated with VEGF-C expression (P=0.0006), and the VEGF-C expression was seen in 12 of 15 cases (80%) of metastatic lymph node. Nodal metastasis was correlated with D2-40-positive myofibroblasts (P=0.0161). VEGF-C expression was an independent prognostic factor by multivariate survival analysis (P=0.0131). Our findings suggest that VEGF-C has an important role in lymphatic invasion via the preexisting lymphatic vessels in the tumor margin, and that lymphangiogenesis does not play a direct role in lymphatic metastasis. D2-40-positive myofibroblasts may contribute to lymphatic metastasis.     

Intratumoral Lymphatics and Lymphatic Vessel Invasion Detected by D2‐40 Are Essential for Lymph Node Metastasis in Bladder Transitional Cell Carcinoma

Bladder cancer is frequently associated with regional lymph node metastasis at the time of diagnosis or after initial treatment, and lymph node metastasis is crucial for clinical therapeutic strategies. Lymphangiogenesis, detected by antibodies specific for lymphatic endothelial cells, is correlated with cancer spread, but the mechanisms that underlie lymphatic spread and the role of lymphangiogenesis in cancer metastasis has been less clear. The aim of this study was to investigate the association of vascular endothelial growth factor (VEGF)‐D expression, intratumoral lymphatics, and lymphatic invasion associated with lymph node metastasis as well as the prognostic analysis in patients with bladder transitional cell carcinoma (TCC). The VEGF‐D expression was evaluated by immunohistochemistry in 72 specimens, and tumoral lymphatic vessels were measured by D2‐40. Counts of lymph vessels were taken in intratumoral and peritumoral areas. Survival analyses and their independent roles were investigated using univariate and multivariate analysis models. The high expression of VEGF‐D was closely associated with the intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Higher lymphatic vessel density, intratumoral lymphatics, and lymphatic invasion showed a significant association with lymph node metastasis. Univariate analysis indicated that VEGF‐D, intratumoral lymphatics, and lymphatic invasion were associated with overall survival, but they were not independent prognostic factors for bladder TCC in multivariate analysis. We conclude that VEGF‐D plays an essential role in tumoral lymphangiogenesis. Intratumoral lymphatics and lymphatic invasion are important predictive factors of pelvic lymph node metastasis in patients with bladder cancer. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Peritumoral lymphatic invasion is associated with regional lymph node metastases in prostate adenocarcinoma.

Lymphangiogenesis, detected by antibodies specific for lymphatic endothelial cells, has been associated with regional lymph node metastases and poor prognosis in carcinomas of head and neck, breast and uterine cervix, but remains largely uninvestigated in prostate adenocarcinoma. We evaluated the lymphatic vessel density and lymphatic vessel invasion by prostate cancer cells in the intratumoral, peritumoral and normal prostate tissue compartments in cancer-bearing prostate glands and correlated them with lymph node metastases, Gleason score and other pathological parameters. Lymphatic vessels were detected by immunohistochemical stain using an antibody specific for the lymphatic endothelial cells (clone D2-40) on 33 radical prostatectomies. In all, 26 patients had lymph node dissection, and 14 of them had lymph node metastasis. The lymphatic vessel density and lymphatic vessel invasion were then recorded for each of the three compartments microscopically. Lymphatic vessel density in the intratumoral, peritumoral and normal prostate compartments was 0.91+/-0.80, 1.54+/-0.68 and 1.58+/-0.96/mm2, respectively. The intratumoral lymphatic vessel density was significantly lower than that of the peritumoral and normal prostate compartments, and the latter two were not significantly different. The lymphatic vessel density of the three compartments was not significantly different between cases with and without lymph node metastasis. The peritumoral lymphatic vessel density correlated inversely with the Gleason score. Lymphatic vessel invasion was present in significantly higher percentage of cases with lymph node metastasis (9/14, 62.3%), as compared to those without lymph node metastasis (1/12, 8.3%, P<0.01). The peritumoral lymphatic vessel invasion had a better correlation with the presence of lymph node metastases than intratumoral lymphatic vessel invasion. There is no evidence of lymphangiogenesis in prostate adenocarcinoma. Peritumoral lymphatic vessel invasion correlates with regional lymph node metastases, suggesting that the peritumoral lymphatic vessels are functionally important and identification of lymphatic vessel invasion in this compartment implies a high probability of regional lymph node metastases.     

Histopathological predictors of regional lymph node metastasis at the invasive front in early colorectal cancer

Akishima‐Fukasawa Y, Ishikawa Y, Akasaka Y, Uzuki M, Inomata N, Yokoo T, Ishii R, Shimokawa R, Mukai K, Kiguchi H, Suzuki K, Fujiwara M, Ogata K, Niino H, Sugiura H, Ichinose A, Kuroda Y, Kuroda D & Ishii T
(2011) Histopathology 59 , 470–481 Histopathological predictors of regional lymph node metastasis at the invasive front in early colorectal cancer Aims: In early colorectal cancer (ECC), prediction of lymph node (LN) metastasis is vital for the decision of additional surgical treatment after endoscopic mucosal/submucosal resection. The aim of this study was to determine the relationship between LN metastasis and comprehensive histopathological findings including the cancer microenvironment in ECC. Methods and results: Using 111 ECC cases, including 36 cases with LN metastasis, histopathological observations and immunohistochemistry for lymphatic vessel endothelial hyaluronan receptor‐1 (LYVE‐1), von Willebrand factor, matrix metalloproteinase‐7 (MMP‐7), CXC chemokine ligand‐12 (CXCL12) and angiopoietin‐like‐4 (ANGPTL4) were conducted. Relationships between LN metastasis and growth pattern, status of muscularis mucosae, depth of cancer invasion, overall histopathological type, histopathological type at the invasive front, tumour budding, neutrophil infiltration in cancer cells (NIC), fibrotic cancer‐stroma type, Crohn’s‐like lymphoid reaction, microscopic abscess formation and lymphatic invasion were determined. In addition, the expression of MMP‐7, CXCL12 and ANGPTL4 in cancer cells at the invasive front were also considered in the context of LN metastasis. By multivariate analysis, lymphatic invasion, NIC and MMP‐7 expression at the invasive front were independent predictors of LN metastasis. Conclusions: LN metastasis is regulated not only by the characteristics of cancer cells but also by microenvironmental factors of lymphatics and neutrophils, especially at the invasive front.     

Lateral peritumoral lymphatic vessel invasion can predict lymph node metastasis in esophageal squamous cell carcinoma.

Lymph node metastasis is an important prognostic factor in many types of cancer. We investigated the clinical significance of lymphangiogenesis and lymphatic vessel invasion in esophageal squamous cell carcinoma. We evaluated lymphatic vessel density and lymphatic vessel invasion in the intratumoral, peritumoral and normal compartments using D2-40 immunostaining. In addition, the peritumoral compartment was divided into the lateral peritumoral compartment and the non-lateral peritumoral compartment. The lymphatic vessel density was higher in the peritumoral and intratumoral compartments than in the normal compartment. However, the lymphatic vessel density did not correlate with any pathological parameters including lymph node metastasis. Intratumoral and peritumoral lymph vessels were small and collapsed while normal lymphatic vessels and lymphatic vessels with lymphatic vessel invasion were dilated and large. The presence of lymphatic vessel invasion, in the lateral peritumoral compartment but nowhere else, significantly correlated with lymph node metastasis. These results suggest that lymphangiogenesis might occur with esophageal cancer, but it does not play a direct role in lymphatic vessel invasion and lymph node metastasis. Peritumoral lymphatic vessel invasion, especially in the lateral peritumoral compartment, should imply a high probability of regional lymph node metastasis.     

Correlation of vascular endothelial growth factor-D expression and VEGFR-3-positive vessel density with lymph node metastasis in gastric carcinoma

Lymph node metastasis is an important prognostic factor in gastric cancer. Vascular endothelial growth factor-D (VEGF-D) is a lymphangiogenic growth factor that activates VEGF receptor (VEGFR)-3, a receptor expressed in the lymphatic endothelium. We investigated the clinical value of VEGF-D expression and VEGFR-3 positive vessel density in gastric carcinoma with regard to lymphangiogenesis. Immunohistochemical staining was used to determine the expression of VEGF-D and VEGFR- 3 in specimens from 104 cases of resected gastric cancer. VEGF-D expression was observed in 62.5% of the gastric cancers and in 9.6% of the non-neoplastic gastric tissue. The VEGFR-3-positive vessel density was significantly greater in the VEGFD positive group than the negative group. VEGF-D expression was significantly associated with lymph node metastasis, increased serum CEA levels, and the nonsignet ring cell type. The VEGFR-3-positive vessel density was correlated with tumor size, lymphatic invasion, and lymph node metastasis. The VEGF-D expression and high VEGFR-3-positive vessel density were significant poor prognostic factors for relapse-free survival. These results suggest that VEGF-D and VEGFR-3-positive vessel density are potential molecular markers that predict lymphatic involvement in gastric carcinoma.     

Lymphatic vessel density as a prognostic indicator for patients with stage I cervical carcinoma

Lymphatic vessel density (LVD) was recently considered important for spread of several malignant tumors. However, there are no reports describing the situation in cervical carcinoma. The purpose of this study was to investigate whether LVD could serve as a risk factor for nodal metastasis and recurrence of cervical carcinoma in 147 cases of stage I patients. Other questions were if depth of invasion, proliferation rate, and tumor size could be used as predictive markers for Chinese patients with cervical carcinoma. The lymphatics were determined by immunohistochemistry with the antibody to LYVE-1, a specific lymphatic endothelium marker, and average LVD was calculated. Double immunohistochemistry and double immunofluorescence staining for LYVE-1/CD34 were used to distinguish between lymphatic and blood vessels. The results showed that average LVD in cervical carcinoma was statistically associated with inflammatory cell infiltration of carcinoma tissues, but not associated with other pathological parameters. Average LVD of the cases with nodal metastasis or recurrence was significantly higher than the cases without metastasis and recurrence in both stage IA and stage IB cervical carcinomas. The correlation between both depth of invasion and tumor size with nodal metastasis and recurrence of cervical carcinoma was also statistically significant. Ki-67 labeling index was found to be correlated with the recurrence of disease, but not to be correlated with nodal metastasis. We concluded that for the patients with stage I cervical carcinoma, increased LVD could serve as a high-risk factor for nodal metastasis and recurrence. Depth of invasion and tumor size could also be useful indicators.     

The extent of retraction clefts correlates with lymphatic vessel density and VEGF-C expression and predicts nodal metastasis and poor prognosis in early-stage breast carcinoma

Although the earliest feature of disseminated disease in breast cancer is regional lymph node involvement, little is known about the mechanisms whereby cancer cells interact with lymphatic endothelial cells and enter the lymphatic system. We have previously reported that the extensive presence of retraction clefts in breast carcinomas highly significantly correlates with lymphatic tumor spread and predicts poor outcome, suggesting that retraction clefts are not just fixation artifacts, but real potential spaces that are exaggerated by tissue processing and may reflect an early stage of lymphatic invasion. In this study, we examined the correlation between the extent of retraction clefts and lymphangiogenesis, as assessed by lymphatic vessel density and vascular endothelial growth factor-C (VEGF-C) expression in a series of 256 early-stage breast carcinomas. The presence and extent of retraction clefts around tumor cell nests was determined by review of all hematoxylin- and eosin-stained tumor sections. Lymphatic vessels were detected by podoplanin immunohistochemistry and lymphatic vessel density was measured using the hot-spot method. The expression of VEGF-C in the tumor cells was determined by immunohistochemistry and analyzed semiquantitatively on a four-tiered scale. High levels of retraction clefts, peritumor lymphatic vessel density and VEGF-C expression at the invasive edge in breast carcinomas significantly correlated with tumor size, histological grade, lymphatic invasion and nodal metastasis. Breast carcinomas showing extensive retraction clefts (>20% of tumor volume) were found to have significantly higher lymphatic vessel density and VEGF-C expression levels compared to tumors without this feature. High retraction clefts, peritumor lymphatic vessel density and VEGF-C expression predicted poor outcome in breast carcinomas. Our results support the hypothesis that retraction clefts are real potential spaces that may represent 'pre-lymphatic spaces' facilitating initial lymphatic invasion and that growth factors secreted by the tumor cells may stimulate tumor-associated lymphangiogenesis by promoting the endothelialization of these 'pre-lymphatic channels'.     

The aggressiveness of urothelial carcinoma depends to a large extent on lymphovascular invasion – the prognostic contribution of related molecular markers

Aims:  Bladder cancer is the second most common malignancy of the urogenital region. The majority of bladder cancer deaths occur as a consequence of metastatic disease. Blood vessel density (BVD), a surrogate marker for angiogenesis, has been shown to be predictive of progression and poor prognosis, as well as lymphatic vessel density (LVD). The aim of this study was to evaluate, in human urothelial bladder cancer (UBC), the clinical and prognostic significance of angiogenesis, lymphangiogenesis and lymphovascular invasion, assessed with the use of specific immunohistochemical markers.
Methods and results:  Immunohistochemistry for CD31 (a blood vessel endothelial cell marker), D2-40 (a lymphatic vessel endothelial cell marker), vascular endothelial growth factor (VEGF)-C and VEGF-receptor 3 antibodies was performed in 83 patients with urothelial carcinoma who underwent radical cystectomy. The classic histopathological characteristics, associated with lymphovascular invasion and loco-regional dissemination, had a negative influence on 5-year overall survival (OS) rates. BVD and LVD were correlated with advanced and poorly differentiated UBC with lymphovascular invasion. Blood vessel invasion (BVI) by malignant emboli assessed by CD31 staining, and lymphatic vessel invasion (LVI) by isolated malignant cells assessed by D2-40 staining significantly affected OS. VEGF-C overexpression was correlated with both BVI and LVI by single malignant cells assessed by CD31 and D2-40, respectively. BVI by malignant emboli assessed by CD31 staining remained as an independent prognostic factor.
Conclusions:  Patients with UBC with embolic BVI assessed by CD31 and LVI by isolated malignant cells assessed by D2-40 have a worse prognosis and may benefit from adjuvant therapies.     

Protein expression status in mucosal and submucosal portions of early gastric cancers and their predictive value for lymph node metastasis

We aimed to find out predictive markers for lymph node (LN) metastasis of early gastric carcinoma (EGC) by separating evaluation of protein expression in mucosa and submucosa considering tumor heterogeneity. We selected 37 pN1–3 EGCs and depth‐ and size‐matched 31 pN0 EGCs as training set and 72 EGCs including 14 pN1–3 EGCs as test set. Protein expression for β‐catenin, E‐cadherin, N‐cadherin, galectin‐3, c‐MET, TrkB, and Ki‐67 was assessed by immunohistochemistry in mucosal (‐m) and submucosal (‐sm) portions of tumor. In the training set, Ki67‐m was higher than in Ki67‐sm (mean ± SD: 82.67 ± 11.99% vs 61.79 ± 22.53%, p < 0.001). Altered E‐cadherin‐sm, high Ki67‐m, and high Ki67‐sm were correlated with LN metastasis (p < 0.05) and Ki67‐sm was independent with lymphatic invasion and desmoplasia (p = 0.015 by multivariate logistic analysis). The test set confirmed Ki67‐sm and E‐cadherin‐sm as predictors of LN metastasis (p < 0.05). Submucosal EGCs with ≥2 predictive factors out of high Ki67‐sm, altered E‐cadherin‐sm, large tumor size (≥3 cm), diffuse type histology, and present lymphatic invasion yielded 100% sensitivity and 90.9% specificity for prediction of LN metastasis in 21 submucosal EGCs of test set. The proliferative activity of tumor in submucosa is suggested to be an independent predictor for LN metastasis in EGC.     

Lymphatic invasion revealed by multispectral imaging is common in primary melanomas and associates with prognosis

Lymphatic invasion by tumor cells has been noted infrequently in primary melanomas. Our primary hypotheses were that using immunohistochemical markers of lymphatic vessels and of tumor cells would improve detection of lymphatic invasion and that lymphatic invasion would correlate with regional nodal metastatic disease. This study included 106 patients who were diagnosed between 1972 and 1991 and who had 10 years or more of follow-up. We performed dual immunohistochemical stains for podoplanin (for lymphatic vessels) and S-100 (for melanoma cells). Lymphatic invasion was identified by light microscopy and confirmed by multispectral imaging analysis. Lymphatic invasion was detected by morphology alone in 5 cases (4.7%) in contrast to immunohistochemical staining augmented by multispectral imaging analysis where 35 cases (33%) were identified (P < .0001). Lymphatic invasion was significantly associated with time to regional nodal metastatic disease, as well as first metastasis and melanoma-specific death. "Local metastasis," defined by immunohistochemistry-detected lymphatic invasion, satellites, or neural invasion, identified 64% of those who had regional nodal metastatic disease within 5 years of diagnosis. Lymphatic invasion is an underobserved phenomenon in primary melanomas that can be better detected by immunohistochemical staining. The presence of lymphatic invasion may be a clinically useful predictor of regionally metastatic disease.     

Lack of lymphangiogenesis in human primary cutaneous melanoma. Consequences for the mechanism of lymphatic dissemination.

Cutaneous melanoma has an initial preference for lymphatic spread. Remarkably, melanoma progression toward this metastasizing phenotype is accompanied by intense blood vessel angiogenesis (hemangiogenesis), but lymphangiogenesis, the formation of new lymph vessels in the tumor, has never been reported. To investigate how primary melanoma cells interact with the existing lymphatic microvasculature, and whether lymphangiogenesis occurs, an immunostaining was developed that differentially decorates blood and lymph vessels in frozen tissue sections. The density and distribution of both these vessel types in and around thin (< or = 1.5 mm) and thick (> or = 1.5 mm) primary melanoma lesions and in normal and uninvolved skin were determined. Although especially in thick melanoma lesions a significant increase in blood vessel density was observed, lymphatic density remained unaltered, showing that lymphangiogenesis did not occur. Morphological analysis indicated, however, that melanoma progression is accompanied by a sequence of events that involves hemangiogenesis supporting tumor expansion, especially in the vertical growth phase. Often, stromal sepia are formed around the blood capillaries in the tumor neovasculature protecting them from invasion. Lymph vessels inside the tumor were infrequently observed. However, subepidermal lymph vessels often seemed to be entrapped and penetrated by the expanding tumor mass. In this way, hemangiogenesis, as the driving force behind tumor expansion, might indirectly increase the chance of lymphatic invasion in the absence of lymphangiogenesis. This model explains the paradox that, although melanoma metastasis seems to require angiogenesis, a consistent relation of prognosis with blood capillary density in primary cutaneous melanoma is lacking.     

Significance of lymphatic invasion on regional lymph node metastasis in early gastric cancer using LYVE-1 immunohistochemical analysis

It has been reported that lymphatic invasion is a predictor for lymph node metastasis in early gastric cancer (EGC); however, it has been impossible to differentiate between lymphatic invasion and blood vessel invasion using current staining techniques. We studied the significance of lymphatic invasion on regional lymph node metastasis in EGC by using human lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) antibody, specific to lymphatic vessels, and von Willebrand factor (vWF) antibody, specific to the blood vessels, to clearly distinguish these vascular tissues.EGC tissues were obtained from 66 node-positive and 66 node-negative subjects and were matched by age and sex. These tissues were immunostained with antibodies against LYVE-1 and vWF. Multivariate logistic regression analysis demonstrated that lymphatic invasion was a significant independent predictor for regional lymph node metastasis (odds ratio, 4.667; P = .0094), whereas blood vessel invasion was not. Thus, lymphatic invasion identified by LYVE-1 antibody could predict the existence of regional lymph node metastasis in EGC.     

Tumour lymphangiogenesis is a possible predictor of sentinel lymph node status in cutaneous melanoma: a case-control study

BACKGROUND: Cutaneous melanoma spreads preferentially through the lymphatic route and sentinel lymph node (SLN) status is regarded as the most important predictor of survival. AIMS: To evaluate whether tumour lymphangiogenesis and the expression of vascular endothelial growth factor C (VEGF-C) is related to the risk of SLN metastasis and to clinical outcome in a case-control series of patients with melanoma. METHODS: Forty five invasive melanoma specimens (15 cases and 30 matched controls) were investigated by immunostaining for the lymphatic endothelial marker D2-40 and for VEGF-C. Lymphangiogenesis was measured using computer assisted morphometric analysis. RESULTS: Peritumorous lymphatic vessels were more numerous, had larger average size, and greater relative area than intratumorous lymphatics. The number and area of peritumorous and intratumorous lymphatics was significantly higher in melanomas associated with SLN metastasis than in non-metastatic melanomas. No significant difference in VEGF-C expression by neoplastic cells was shown between metastatic and non-metastatic melanomas. Using logistic regression analysis, intratumorous lymphatic vessel (LV) area was the most significant predictor of SLN metastasis (p = 0.04). Using multivariate analysis, peritumorous LV density was an independent variable affecting overall survival, whereas the intratumorous LV area approached significance (p = 0.07). CONCLUSIONS: This study provides evidence that the presence of high peritumorous and intratumorous lymphatic microvessel density is associated with SLN metastasis and shorter survival. The intratumorous lymphatic vessel area is the most significant factor predicting SLN metastasis. The tumour associated lymphatic network constitutes a potential criterion in the selection of high risk patients for complementary treatment and a new target for antimelanoma therapeutic strategies.     

Tumor and lymph node lymphangiogenesis--impact on cancer metastasis

The extent of lymph node (LN) metastasis is a major determinant for the staging and the prognosis of most human malignancies and often guides therapeutic decisions. Although the clinical significance of LN involvement is well documented, little has been known about the molecular mechanisms that promote tumor spread via lymphatic vessels to sentinel and distal LN and beyond. However, recent discoveries have identified novel lymphatic-specific markers, and the newly discovered lymphangiogenesis factors vascular endothelial growth factor-C (VEGF-C) and VEGF-D were found to promote tumor-associated lymphatic vessel growth in mouse tumor models, leading to enhanced tumor spread to sentinel LN. Our recent findings indicate that VEGF-A also acts as a potent tumor lymphangiogenesis factor that promotes lymphatic tumor spread. VEGF-A overexpressing primary tumors induced sentinel LN lymphangiogenesis even before metastasizing and maintained their lymphangiogenic activity after metastasis to draining LN. Our recent studies showed that primary human melanomas that later metastasized were characterized by increased lymphangiogenesis and that the degree of tumor lymphangiogenesis can serve as a novel predictor of LN metastasis and overall patient survival, independently of tumor thickness. Tumor lymphangiogenesis also significantly predicted the presence of sentinel LN metastases at the time of surgical excision of the primary melanoma. Together, these findings suggest that tumor lymphangiogenesis actively contributes to cancer dissemination, that blockade of lymphatic vessel growth might inhibit tumor metastasis to LN, and that the extent of tumor-associated lymphangiogenesis could serve as a novel, prognostic parameter for the metastatic risk of human cancers.     

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.     

D2-40 immunoreactivity in penile squamous cell carcinoma: a marker of aggressiveness

D2-40 immunohistochemical expression was investigated in tissue specimens from 39 patients with squamous cell carcinoma of the penis who underwent partial or total penectomy between 1987 and 2008. Patient age, tumor size, and grade; D2-40-positive lymphatic vessel density in intratumoral, peritumoral, and normal tissue; cell positivity for D2-40 in intratumoral and normal tissue; and D2-40 staining intensity and distribution were analyzed and correlated with disease-specific survival. Analysis of D2-40-positive lymphatics disclosed that mean lymphatic vessel density was greater in peritumoral tissue than in intratumoral and normal tissue and lower in patients with lymph node metastasis than in those without lymph node metastasis. The receiver operating characteristic curve showed that an intratumoral lymphatic vessel density greater than 2.0 had 83.3% sensitivity and 78% specificity in predicting lymph node metastasis. Analysis of cell immunoreactivity showed cytoplasmic D2-40 positivity in intratumoral and normal tissue in 89.7% and 65.5% of patients, respectively. A strong correlation emerged between grade of cell differentiation and D2-40 immunoreactivity in intratumoral tissue; in particular, 88.9% of tumors with weak podoplanin expression were G1, whereas strong cellular immunoreactivity was detected in 83.3% of G3 patients (P = .003; χ(2) test). A significant correlation was also noted between pattern of reactivity and tumor grade because the basal layer was positive in patients with undifferentiated tumors (100% of G3) and in 72.2% of G1 tumors (P = .021; χ(2) test). D2-40 seems to be a useful marker for the development of node metastasis in squamous cell carcinoma of the penis, although validation in larger series is required to confirm its predictive value.     

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2-40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF-C), VEGF-D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF-D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF-C or VEGF-D expression. High LVD, induced by VEGF-C or VEGF-D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.