Lymphatic vessel density in primary melanomas predicts sentinel lymph node status and risk of metastasis

Shayan R, Karnezis T, Murali R, Wilmott J S, Ashton M W, Taylor G I, Thompson J F, Hersey P, Achen M G, Scolyer R A & Stacker S A
(2012) Histopathology  61, 702–710 Lymphatic vessel density in primary melanomas predicts sentinel lymph node status and risk of metastasis Aims: Important prognostic factors in patients with cutaneous melanoma include primary tumour thickness/depth of invasion, ulceration and mitotic rate, and the presence of tumour cells in regional lymph nodes. More recently, features of stromal components, such as blood and lymphatic vessel density, have been suggested as additional indicators of metastatic potential. Our aim was to investigate the relationship between tumour lymphatic vessels and lymph node metastasis. Methods and results: Metastasizing (n = 11) and non‐metastasizing (n = 11) primary melanoma samples matched for depth/thickness, mitotic rate and ulceration were examined for lymphatic vessel density (LVD) in the primary tumour, using an antibody to podoplanin. Significant differences were found between LVD (vessels/unit area) in the peripheral (5.73 ± 0.67) versus central (1.72 ± 0.42) regions of the metastasizing tumour group (P < 0.001), and between LVD in the peripheral areas of metastasizing (5.73 ± 0.67) versus non‐metastasizing (4.21 ± 0.37) tumours (P < 0.01). No overall difference was found between total average LVD in the two tumour groups or between their vessel morphology. Conclusions: Our results show that LVD is associated with risk of lymph node metastasis. Furthermore, the ratio of peripheral LVD:central LVD is a useful marker of primary melanomas that are likely to metastasize to lymph nodes.     

The role of VEGF-C staining in predicting regional metastasis in melanoma

Sentinel lymph node status is the most important prognostic factor in primary melanoma. The number of melanoma-associated lymphatic vessels has been associated with sentinel lymph node status and survival. Vascular endothelial growth factor-C (VEGF-C) is found to promote tumour-associated lymphatic vessel growth. In many human neoplasms, VEGF-C expression in neoplastic cells or tumour-associated macrophages (TAMs) has been linked to lymphatic dissemination of tumour cells. Recent studies have suggested a correlation between VEGF-C expression in primary melanoma and the presence of lymph node metastasis. We performed VEGF-C immunohistochemical staining on melanoma tissues of 113 patients with known sentinel lymph node status. We showed that both high VEGF-C expression in melanoma cells and TAMs are positively associated with the presence of a positive sentinel lymph node. No correlation with Breslow thickness, Clark invasion level or ulceration could be detected. VEGF-C expression in melanoma cells was predictive of a shorter overall and disease-free survival, without being an independent predictor of survival. Our results confirm that VEGF-C expression in primary cutaneous melanoma plays a role in the lymphatic spread of the tumour.     

Prognosis in human melanoma: PAR-1 expression is superior to other coagulation components and VEGF

Aims: Two hundred and four accessible cases of malignant melanoma from the Grampian region of Scotland, collected over a period of 4 years, with minimum follow‐up of 2 years, were studied for coagulation factors and vascular endothelial growth factor (VEGF) expression as potential prognostic markers. The aim was to allow comparison with previous work using microvessel density on the same cases. Methods and results: Immunohistochemistry for VEGF, tissue factor (TF), fibrin and protease‐activated thrombin receptor (PAR)‐1 in 204 cases of melanoma was performed, and intensity of expression scored. Chalkley microvessel counts (MVD) were obtained for the tumour edge. TF expression and presence of fibrin correlated well with Breslow thickness and ulceration, reaching statistical significance, but surprisingly not for metastatic recurrence. Fibrin was variably present in over half the cases, located at the invasive edge, ulcerated surface and between tumour cell surfaces. In a few cases fibrin was within tumour cells, typically co‐located with melanin and confirmed by electron microscopy. In contrast, immunohistochemistry for PAR‐1 produced statistically significant results, correlating expression with Breslow thickness (P ≤ 0.001), ulceration (P = 0.001) and recurrence (P ≤ 0.005). Intensity of reactivity of VEGF correlated significantly with Breslow thickness, Clark level, ulceration and MVD, but not for metastatic recurrence. Conclusions: It appears paradoxical that VEGF expression is not more predictive of recurrence, but even low expression may be sufficient for tumour angiogenesis and other factors must govern tumour aggression. Antagonism of VEGF may still prove a successful adjunct in future therapeutic trials. Both MVD and PAR‐1 can be used as adjuncts to Breslow thickness and ulceration as prognostic indicators for melanoma, as they appear to give independent information for all thicknesses. PAR‐1 expression is the best antibody marker of recurrence risk from those studied. It remains to be seen whether this methodology can predict response to novel antiangiogenic therapies currently entering trial.     

Prognostic factors in cutaneous malignant melanoma. A comparative study of long term and short term survivors

The clinical presentation and histologic characteristics of primary melanomas of a consecutive series of 100 short term (fatal within five years) and 100 long term survivors (disease-free for ten years or longer) were studied. The clinical parameters analyzed included the history of pre-existent mole, duration of changes before diagnosis, site, elevation, ulceration, extent of pigmentation, and satellitosis around the primary lesion. Study of histologic features included analysis of exact measurements of the primary melanoma, depth of dermal invasion, tumor cell morphology, mitotic rate, intracellular pigmentation, presence or absence of giant tumor cells, blood vessel or lymphatic invasion, lateral junctional activity, and presence and extent of lymphoid response to the primary lesion.Statistically significant differences were found between the long term and short term survivors with respect to several variables. It is one of the conclusions of this study that an elective node dissection would seem to be indicated in all patients with stage I melanoma who present with poor prognostic signs, that is, patients who belong to any combination of the following categories: male, primary lesions>1 cm., which has ulcerated, with a deeper level of dermal penetration, with dermal lymphatic or blood vessel invasion, and a lack of lymphoid reaction around the primary lesion.     

Importance of tumour cell invasion in blood and lymphatic vasculature among patients with endometrial carcinoma

Aims:  Vascular invasion is a prognostic marker for many cancers, including endometrial carcinoma. Immunohistochemistry using D2-40 and CD31 antibodies makes it possible to differentiate between lymphatic vascular invasion (LVI) and blood vessel invasion (BVI). The aim was to examine lymphatic and blood vessel invasion separately and their associations with clinicopathological characteristics and prognosis.
Methods and results:  Immunohistochemistry was performed on a retrospective population-based series of endometrial carcinoma. Altogether, 31% of the 276 tumours showed lymphatic involvement, whereas 18% showed blood vessel invasion. LVI and BVI were associated with histopathological variables such as histological grade and tumour growth pattern. Patients without vascular invasion had the best prognosis and those with BVI (with or without LVI) had the worst outcome, whereas patients with LVI had an intermediate survival on univariate analysis. Multivariate models indicated that both LVI and especially BVI had independent prognostic importance. Among endometrioid carcinomas, BVI was still significant.
Conclusions:  Our findings support the biological importance of vascular spread through the haematogenic and lymphatic routes in endometrial cancer. The significant correlation found with clinical phenotype indicates that these markers may be relevant for patient management.     

The use of LYVE-1 antibody for detecting lymphatic involvement in patients with malignant melanoma of known sentinel node status

BACKGROUND: Sentinel node (SN) status is the most important prognostic indicator in patients with cutaneous melanoma without clinically evident metastatic spread, but the procedure is associated with considerable morbidity. The LYVE-1 lymphatic marker offers the possibility of studying lymphangiogenesis and tumour metastasis within the primary excision. AIMS: To establish whether lymphatic vessel numbers/distribution within the primary tumour correlated with SN status. To assess whether tumour cells were easily demonstrable within lymphatics and could be used as a surrogate for SN status. METHODS: Double immunostaining for LYVE-1 and S100 in cutaneous biopsies from 18 SN+ patients with no lymphatic/vascular involvement on routine histology and 18 SN- patients matched for tumour thickness and ulceration.RESULTS: Lymphatic vessels were detected in all cases. Vessels within the tumour mass were suggestive of active lymphangiogenesis; those outside were mainly mature vessels with well defined walls. Tumour cells within lymphatics were detected in one of 18 SN- and five of 18 SN+ patients. Lymphatics containing tumour cells were all outside the tumour mass in well formed vessels, suggesting melanoma cell invasion into preformed lymphatics. There was no significant difference in lymphatic counts between SN+ and SN- patients. Although peritumorous lymphatic counts were higher in ulcerated than non-ulcerated melanomas, they did not vary with Breslow thickness. CONCLUSION: LYVE-1 staining can reliably demonstrate lymphatic vessel distribution, but lymphatic counts cannot predict melanoma metastatic potential and cannot substitute for SN biopsy. LYVE-1 immunostaining can detect melanoma cells within lymphatics, but is unreliable in predicting melanoma metastasis, failing to detect metastatic spread in more than two thirds of patients with regional node metastasis.     

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 lymph vessel invasion identified by the endothelial lymphatic marker D2-40 in node negative breast cancer.

Monoclonal antibody D2-40, a marker of lymphatic endothelium, identifies tumor emboli in lymph vessels. The aim of the study was to assess whether D2-40+ lymph vessel invasion (LVI) correlates with clinicopathologic factors including lymphovascular invasion (LVI) as assessed by haematoxylin and eosin-stained sections (H&E+ or H&E-) and to assess the prognostic significance in node-negative breast cancer. The study group consisted of 303 node-negative breast cancer patients that had a median follow-up of 7.6 years. Clinical and pathological data were retrieved from the Henrietta Banting database. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections of the primary invasive carcinoma using D2-40. Immunostaining with CD31 was performed on the discordant cases that were H&E+/D2-40-. D2-40+ lymph vessel invasion was detected in 82/303 (27%) cases. The foci of lymphatic invasion occurred predominantly at the invasive front of the tumor. The absence of D2-40 and CD31 in 13/17 discordant cases was suggestive of retraction artefact. D2-40+ lymph vessel invasion correlated significantly with age (P=0.0003), tumor size (P=0.005), histological grade (P=0.0001), H&E+ (P=<0.0001) and estrogen receptor status (P=0.005) but not with histological type or progesterone receptor status. Multivariate analysis revealed that D2-40+ lymph vessel invasion was the only significant predictor of distant recurrence. There was no significant association between D2-40 status and local recurrence (P=0.752) or regional recurrence (P=0.13). Both D2-40+lymph vessel invasion (P=0.009) and H&E+LVI cases (P=0.02) were associated with overall shorter survival in univariate analysis. These data indicate that D2-40 identifies lymphatic invasion in breast tumors and is a significant predictor of outcome in breast cancer.     

Lymphatic microvessel density as prognostic marker in colorectal cancer.

Lymph node metastases is an important prognostic indicator for disease progression and crucial for therapeutic strategies in the work-up of colorectal carcinoma. In this study, we investigated tumor lymphangiogenesis and vascular endothelial growth factor (VEGF) expression as predictive markers for the risk of lymph node metastasis and their relation to other prognostic parameters in colorectal carcinoma. Resected colorectal carcinomas from 90 patients were examined, including 30 patients without lymph node metastases, 30 with only lymph node metastases, and 30 with liver metastases. Cases were immunostained for CD31, D2-40, and VEGF. Positivity stained microvessels were counted in densely vascular/lymphatic foci (hot spots) at x 400 field (=0.17 mm2). Intensity of staining for VEGF was scored on a two-tiered scale. D2-40 lymphatic microvessel density demonstrated significant correlation with CD31 counts (20+/-9 vs 18+/-6/0.17 mm2 field, P<0.05) and VEGF expression (P<0.01). VEGF was expressed in 61/90 (67%) cases. D2-40 identified lymphatic tumor invasion in 48/90 patients, which was greater than CD31 (37/90) and hematoxylin and eosin (H&E) (31/90). There was a positive significant correlation of D2-40, CD31 counts, and VEGF expression with the presence of lymphovascular invasion and lymph node metastases (P<0.05). D2-40 lymphatic microvessel density correlated significantly with depth of invasion (pT), positive vascular pedicle lymph nodes and liver metastases (P<0.05). In conclusion, D2-40 lymphatic microvessel density showed prognostic significance with positive correlation with lymphovascular invasion, pT, and metastases to lymph nodes and liver. Immunostaining with D2-40 enhances the detection of lymphatic invasion relative to H&E staining and the endothelial marker, CD31.     

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.     

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.     

Prognostic significance of intratumoral and peritumoral lymphatic density and blood vessel density in invasive breast carcinomas

We studied tumor lymphatic and vascular densities and lymphovascular invasion (LVI) as prognostic markers in 48 cases of invasive breast cancer treated with partial or total mastectomy and lymph node dissection. All cases were immunostained with D2-40 and CD31. Positively stained microvessels were counted in densely vascular/lymphatic foci (hot spots) at x400. The mean+/-SD peritumoral lymphatic microvessel density (LMD) was significantly higher than intratumoral LMD (9+/-7 vs 4+/-6; P< .01). There was a positive correlation of D2-40 LMD (peritumoral and intratumoral) and CD31 microvessel density counts with lymph node metastasis (r=0.35, 0.5, and 0.38), nuclear grade (r=0.36, 0.28, and 0.3), and stage (r=0.42, 0.56, and 0.49), respectively. Peritumoral and intratumoral D2-40 LMD correlated significantly with the presence of angiolymphatic invasion (detected by D2-40; r=0.54 and 0.54, respectively). D2-40 detected more LVI than H&E- and CD31-detectable vascular invasion (18/48, 5/48, 11/48, respectively). Increased D2-40 detected LVI, and high CD31 microvessel counts showed significant adverse effect on survival status.     

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.     

Stromal expression of vascular endothelial growth factor C is relevant to predict sentinel lymph node status in melanomas

The dissemination of tumour cells to the lymph nodes is a complex process involving the formation of new lymph vessels, or lymphangiogenesis, produced by the tumour itself. The main growth factor involved in lymphangiogenesis is vascular endothelial growth factor C (VEGF-C), which is secreted by several different malignant tumours, including melanoma. Not only has VEGF-C expression been found in tumour cells, it has also been detected in tumour stromal cells like macrophages and fibroblasts. This study aimed to determine whether the expression of VEGF-C in tumour and stromal cells in cutaneous melanoma determines lymphangiogenesis and neoplastic dissemination to lymph nodes. We examined cases from 50 patients with melanoma who underwent selective biopsy of the sentinel lymph node. Immunohistochemical study was done with D2-40 to label lymph vessels, and the expression of VEGF-C was evaluated in tumour and stromal cells. Lymph vessel density was greater in sentinel lymph node-positive than in sentinel lymph node-negative cases, though the difference was not significant (P = 0.075). A significant correlation was seen between lymph vessel density and tumour thickness and the presence of ulceration. The main finding was that the expression of VEGF-C in fibroblasts was highly associated with the presence of metastasis in the sentinel node and with the Clark level. However, VEGF-C expression showed no relation in either tumour cells or macrophages with node status or other prognostic factors, such as the Breslow index or Clark level. Our results highlight the relevance of the stroma in tumour progression in cutaneous melanoma and its role in the spread to lymph nodes.     

Peritumoural,but not intratumoural,lymphatic vessel density and invasion correlate with colorectal carcinoma poor-outcome markers

To evaluate whether lymphatic vessel density (LVD) and lymphatic vessel invasion (LVI) are useful markers of worse outcome in colorectal carcinoma and if LVD and LVI correlate to the classical clinical-pathological parameters, we analysed 120 cases of colorectal carcinomas selected from the files of Division of Pathology, Hospital das Clinicas, São Paulo University, Brazil. Assessment of LVD and LVI was performed by immunohistochemical detection of lymphatic vessels, using the monoclonal antibody D2-40. Higher LVD was found in the intratumoural area, when comparing with normal and peritumoural areas (p?p?=?0.037) and liver metastasis (p?=?0.012). Remarkably, LVI was found associated with local invasion (p?=?0.016), nodal metastasis (p?=?0.022) and hepatic metastasis (p?相似文献   

血管内皮生长因子C及其受体3在人恶性黑色素瘤组织内的表达及意义

目的 观察人恶性黑色素瘤组织内血管内皮生长因子C(VEGF-C)及其受体3(VEGFR-3)的表达,探讨VEGF-C和VEGFR-3在恶性黑色素瘤淋巴管生成及淋巴道转移中的作用.方法 取人恶性黑色素瘤组织48例(石蜡标本30例,术后新鲜组织18例),应用免疫组织化学和RT-PCR技术,观察VEGF-C和VEGFR-3蛋白及mRNA在恶性黑色素瘤组织内的表达情况.以淋巴管内皮透明质酸受体(LYVE-1)标记淋巴管,计数恶性黑色素瘤组织淋巴管数密度.结果 VEGF-C和VEGFR-3蛋白主要表达于恶性黑色素瘤细胞胞浆内,在肿瘤周围的血管和淋巴管内皮上也可见VEGFR-3蛋白表达,VEGF-C和VEGFR-3蛋白在淋巴结转移组恶性黑色素瘤组织内的表达水平明显高于无淋巴结转移组(P<0.05).在18例新鲜恶性黑色素瘤中,淋巴结转移组VEGF-C和VEGFR-3mRNA的表达明显高于无淋巴结转移组(P<0.01).LYVE-1表达于肿瘤间质内的淋巴管内皮细胞,淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度(LMVD)为9.845±2.454,无淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度为6.534±2.193,淋巴结转移组恶性黑色素瘤组织内的淋巴管数密度明显高于无淋巴结转移组(P<0.01).结论恶性黑色素瘤组织内VEGF-C表达明显增高,并通过上调其受体VEGFR-3的表达促进恶性黑色素瘤组织内淋巴管的生成,从而促进恶性黑色素瘤的淋巴道转移.     

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.     

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.     

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'.     

In human invasive breast ductal carcinoma, tumor stromal macrophages and tumor nest macrophages have distinct relationships with clinicopathological parameters and tumor angiogenesis

Tumor-associated macrophages play a crucial role in breast cancer progression and tumor angiogenesis. However, evaluation of tumor-associated macrophages incorporating their histological locations is lacking. The aim of this study was to clarify whether macrophages in tumor stroma and macrophages in tumor cell nests have distinctive properties in relation to pertinent breast cancer clinicopathological parameters and tumor angiogenesis. In 94 human invasive breast ductal carcinomas, tumor-associated macrophages were immunostained with anti-CD68 antibody and counted or graded according to these histological locations. Microvessels were immunostained with anti-CD34 antibody and counted for microvessel density. We found that the presence of tumor stromal and tumor nest macrophages was closely correlated (p?=?0.001). Both tumor stromal and tumor nest macrophages were associated with mitotic count (p?=?0.001 and p?=?0.037, respectively). However, only higher tumor stromal macrophage grades were associated with higher tumor grades (p?=?0.004) and negative estrogen receptor status (p?=?0.007). Multivariate analysis showed that tumors with a high mitotic count score (score 3 vs. scores 1 and 2) had a higher tumor stromal macrophage density (Grades III and IV) when adjusted for tumor size, tubule formation, and estrogen receptor status (odds ratio 3.41, p?=?0.010). The tumor nest macrophage count significantly correlated with the microvessel density (p?<?0.001). These results imply that tumor stromal macrophages and tumor nest macrophages residing in different tumor microenvironments have distinctive roles.