Peritumoral Lymphatic Vessel Density Research Articles

Multivariate analysis of metastasis‑related risk factors for patients with gastroenteropancreatic neuroendocrine tumors based on clinicopathological and endoscopic features.

Gastroenteropancreatic neuroendocrine tumors (GEP‑NETs) are relatively uncommon. Unfortunately, epidemiological studies on the incidence of GEP‑NETs worldwide have reported a marked increase in the detection of these tumors. Although they often exhibit relatively indolent clinical courses, GEP‑NETs have the potential for lethal progression, especially in patients who present with advanced disease. Early detection and surgical removal is currently the only reliable curative treatment for GEP‑NET patients. The objective of this study was to analyze the clinicopathological characteristics of GEP‑NETs and explore the metastasis‑related risk factors of patients with GEP‑NETs. One hundred and forty‑six patients diagnosed pathologically with GEP‑NETs from January 2001 to January 2015 at the Second Xiangya Hospital of Central South University were retrospectively evaluated. We retrieved and analyzed information concerning clinical characteristics and metastasis‑related risk factors, and used Chi‑square test and logistic regression analysis to analyze the clinicopathological characteristics of GEP‑NETs and explore the association between tumor metastasis and possible related risk factors. The results revealed that the most common clinical manifestations were abdominal pain (n=88), alteration in the character of stool (n=58) and melaena (n=33). Rectum (91/146, 62.3%) and stomach (19/146, 13.0%) were the main sites of metastasis. Both Chi‑square test and logistic regression analysis showed that tumor size (P<0.05), tumor type (P=0.008) and peritumoral lymphatic vessel density(LVD) (P=0.004) were significantly correlated with tumor metastasis. Neither Chi‑square test nor logistic regression analysis indicated that gender (P>0.05), age (P>0.05), tumor location (P>0.05), tumor number (P>0.05), chromaffin granule proteinA[chromograninA(CgA), P>0.05], synaptophysin(Syn, P>0.05) or intratumoral LVD (P>0.05) had a significant correlation with tumor metastasis. Chi‑square test revealed that tumor grade was significantly correlated with tumor metastasis. In conclusion, GEP‑NETs may occur in multiple sites of the digestive system and lack specific clinical manifestations. Tumor size, tumor type, peritumoral LVD, total LVD and tumor grade are metastasis‑related risk factors for GEP‑NET patients.

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.

Abstract IA15: Lymphangiogenesis and metastasis of postpartum breast cancer

Abstract Young women diagnosed with breast cancer have a 2-3 fold increased risk of metastatic disease if they are diagnosed within ten years of a completed pregnancy. We find a postpartum diagnosis to be a poor prognostic indicator independent of tumor stage and biologic subtype, leading to the investigation of whether postpartum breast involution is a mediator of metastasis. We show that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. In addition, we show that breast cancer cells exposed to the involuting mammary microenvironment acquire pro-lymphangiogenic properties that contribute to peritumor lymphatic expansion, invasion, and distant metastases. This pro-lymphatic phenotype appears to be due to tumor cells hijacking developmentally-regulated programs normally restricted to the actively involuting mammary gland. Specifically, we find upregulation of lymphangiogenesis with weaning in the normal mammary glands of rodents and humans. Further, in rodent models of postpartum breast cancer, COX-2 inhibition limited to the involution window decreases normal mammary gland lymphangiogenesis, mammary tumor associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Finally, we find that COX-2 inhibition also targets fibroblast activation and collagen production during mammary gland involution, raising the possibility that COX-2 orchestrates a whole tissue response that is supportive of tumor cell escape in the involuting gland. In summary, we show that physiologic COX-2 dependent fibroblast activation, collagen deposition, and lymphangiogenesis are coordinated in the postpartum mammary gland and suggest that tumors evolving in this unique microenvironment acquire enhanced pro-lymphangiogenic and pro-metastatic activities. These data provide further impetus for considering the use of COX-2 inhibitors in the treatment of postpartum breast cancer, and suggest novel prevention strategies targeted to recently pregnant women as well. Citation Format: Pepper J. Schedin, Virginia F. Borges. Lymphangiogenesis and metastasis of postpartum breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA15.

Prognostic Significance of KAI1/CD82 and its Relation Tod2-40 Labeled Lymphatic Vessel Invasion (LVI) and Lymphatic Vessel Density (LVD) in Esophageal Squamous Cell Carcinoma (ESCC)

Objectives: The KAI1/CD82 appears to inhibit multiple steps of cancer metastatic. D2-40 labeled LVI and LVD are also closely associated with cancer metastasis. We investigated the expression levels of KAI1/CD82, D2-40 labeled LVI and LVD and their correlation with clinicopathological factors in ESCC. Methods: Immunohistochemistry and Western Blot were used to detect KAI1/CD82 expression levels in the peritumoral tissue and ESCC. LVI and LVD detected by D2-40 immunohistochemical staining. The relationships between the KAI1/CD82 expression levels, LVI and LVD were analyzed. The prognosis of ESCC was analyzed by Kaplan-Meier survival analysis and Cox’s proportional hazards model. Results: KAI1/CD82 expression was markedly lower in ESCC than in the para-carcinoma tissue (P<0.05). Positive peritumoral LVI and high mean peritumoral LVD were positively correlated while KAI1/ CD82 expression was negatively correlated with tumor invasion, lymph node metastasis, and clinical stage. Kaplan-Meier analysis revealed that high mean peritumoral LVD and positive LVI was negatively correlated with overall survival (OS) and disease-free survival (DFS) time, while KAI1/CD82 expression was positively correlated with OS and DFS time. Low KAI1/CD82 expression, high mean peritumoral LVD, positive LVI was associated with a poor prognosis in ESCC. Multivariate Cox regression analysis indicated that the positive LVI and KAI1/CD82 positive expression were independent predictors for OS in ESCC. Positive LVI and high mean peritumoral LVD were independent predictors for DFS in ESCC Conclusions: KAI1/CD82 expression, LVI and LVD were significantly correlated with some clinicopathological factors of ESCC including lymph node metastasis, differentiation, and clinical stage. Combined detection of these factors may be of significant value in predicting the prognosis and metastasis in ESCC patients.

Prognostic Significance of Lymphatic Vessel Density Detected by D2-40 and Its Relation to Claudin-4 Expression in Prostatic Adenocarcinoma.

Background Lymphovascular invasion is an important pathway of metastatic spread and regional lymph node metastasis is the major prognostic factor in prostatic adenocarcinoma. D2-40 is used to identify the lymphatic vessels and to assess the lymphatic vessel density (LVD). Expression of claudin-4 may be related to invasion and progression of carcinoma cells in several primary tumors. Aim To evaluate intra- and peritumoral LVD through immunohistochemical expression of D2-40 in relation to claudin-4 expression and clinicopathological parameters in prostatic adenocarcinoma. Materials and Methods Immunohistochemical staining procedure was performed on 53 paraffin-embedded blocks of radical prostatectomy specimens for prostatic adenocarcinoma using anti D2-40 and claudin-4 antibodies. Sections were evaluated for mean LVD in intratumoral and peritumoral tissues assessed by D2-40 expression. Results LVD in intratumoral tissues was significantly lower compared with peritumoral areas (P = .0001). Peritumoral mean LVD was significantly higher in cases with lymphovascular invasion (P = .041) and in cases with positive lymph node metastasis (P = .003) than intratumoral mean LVD. High claudin-4 expression was significantly correlated with high tumor grade (P = .0001), lymphovascular invasion (P = .006), and positive lymph node metastasis (P = .004). High claudin-4 expression was significantly associated with increased mean LVD in peritumoral tissues. Conclusion Increased peritumoral mean LVD in prostatic adenocarcinoma is associated with lymphovascular invasion and positive lymph node metastasis. High claudin-4 expression is associated with high tumor grade, lymphocascular invasion, positive lymph node metastasis, and high mean peritumoral LVD suggesting that D2-40 and claudin-4 may represent different mechanisms of lymphatic vessel invasion with both biomarkers is related to poor prognosis.

Significance of Vascular Endothelial Growth Factor (VEGF)-C and VEGF-D in the Progression of Cutaneous Melanoma.

Induction of tumor lymphangiogenesis by vascular endothelial growth factor (VEGF)-C and VEGF-D promotes metastasis in many human cancers. The aim of this study was to examine the role of VEGF-C and VEGF-D in lymphangiogenesis and lymph node metastasis in patients with cutaneous melanoma. Fifty-four melanoma specimens (18 with lymph node metastasis, 36 nonmetastatic) were investigated by immunostaining for VEGF-C, VEGF-D, and for lymphatic endothelial marker D2-40. VEGF-C and VEGF-D expression was assessed as a percentage and intensity of stained tumor cells, tumor-associated macrophages and fibroblasts. The quantification of lymphangiogenesis was conducted by computer-assisted morphometric analysis. The expressions of both VEGF-C and VEGF-D in tumor cells were significantly higher in lymph node metastatic melanomas compared with nonmetastatic melanomas (P = .015 VEGF-C; P = .005 VEGF-D). There was no statistically significant difference between metastatic and nonmetastatic melanomas regarding the expression of VEGF-C and VEGF-D in macrophages and fibroblasts. Metastatic melanomas showed a significantly higher intratumoral and peritumoral lymphatic vessel density (LVD) compared with nonmetastatic melanomas (P = .000 intratumoral, P = .000 peritumoral). Melanomas with VEGF-C positive tumor cells showed a significantly higher intratumoral and peritumoral LVD compared with VEGF-C negative tumor cells group of melanomas (P = .006 intratumoral, P = .010 peritumoral). VEGF-C expression in macrophages, fibroblasts, as well as VEGF-D expression in tumor cells, macrophages, and fibroblasts, showed no correlation with the intratumoral and peritumoral LVD. Our findings show the significance of VEGF-C in tumor cells in the induction of intratumoral and peritumoral lymphangiogenesis. This study suggests that both VEGF-C and VEGF-D in tumor cells promote lymph node metastasis, and that the immunohistochemical analysis of expression can be a useful tool for predicting clinical behavior of cutaneous melanoma.

Effects of forkhead box C2 on carcinogenesis and lymphatic metastasis in endometrial carcinoma.

We investigated the distribution of endometrial lymphatic vessels and expression of forkhead box C2 (FOXC2) in normal endometrium during menstrual cycle and in endometrial adenocarcinoma. Full-thickness uterine samples and endometrial adenocarcinoma samples were collected for immunohistochemical analysis using D2-40 and FOXC2 mouse monoclonal antibodies. The lymphatic vessel density (LVD) of the endometrium was significantly reduced compared with the myometrium during the cycle. Intra-tumoral LVD was significantly decreased in both stages of endometrioid adenocarcinoma compared with normal endometrium and myometrium. Intra-tumoral LVD significantly decreased from stage IA to stage IIIC. Peri-tumoral LVD for stage IA and stage IIIC tumors was significantly increased compared with normal endometrial LVD, but decreased compared with normal myometrial LVD. Stage IIIC showed increased peri-tumoral LVD when compared with stage IA. The positive rate of FOXC2 was 73.3% in proliferative endometrium and 80% in secretory endometrium. Secretory endometrium showed significantly increased FOXC2 expression compared with proliferative endometrium. Endometrioid adenocarcinoma showed significantly increased FOXC2 expression compared with normal endometrium, both in the epithelium and stroma. FOXC2 expression in the stroma significantly increased when pelvic and/or para-aotic lymph nodes were involved. FOXC2 was immunolocalized in low-risk endometrial carcinoma in endometrioid adenocarcinoma, but not in normal endometrium. Endometrial lymphatic vessels were located in normal endometrium and myometrium across the menstrual cycle and in intra-and peri-endometrioid adenocarcinoma, and increased in endometrial adenocarcinoma. Peri-tumoral lymphatics were associated with increased lymphatic metastasis. FOXC2 may be associated with the genesis of endometrial carcinoma and lymphangiogensis in endometrial adenocarcinoma in intra- and peri-tumoral lymphatics.

Cyclooxygenase-2-dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer.

Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2-dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.

Improved computer-assisted analysis of the global lymphatic network in human cervical tissues

Lymphatic dissemination is a key event in cervical cancer progression and related tumor lymphatic markers are viewed as promising prognostic factor of nodal extension. However, validating such parameters requires an objective characterization of the lymphatic vasculature. Here, we performed a global analysis of the lymphatic network using a new computerized method applied on whole uterine cervical digital images. Sixty-eight cases of cervical neoplasia (12 CIN3, 10 FIGO stage 1A and 46 stage IB1) and 10 cases of normal cervical tissue were reacted with antibodies raised against D2-40, D2-40/p16 and D2-40/Ki67. Immunostained structures were automatically detected on whole slides. The lymphatic vessel density (D2-40), proliferating lymphatic vessel density (D2-40/ki67) and spatial lymphatic distribution in respect to the adjacent epithelium were assessed from normal cervix to early cervical cancer and correlated with lymphovascular space invasion and lymph node status. Prominent lymphatic vessel density and proliferating lymphatic vessel density are detected under the transformation zone of benign cervix and no further increase is noted during cancer progression. Notably, a shift of lymphatic vessel distribution toward the neoplastic edges is detected. In IB1 cervical cancer, although intra- and peritumoral lymphatic vessel density are neither correlated with lymphovascular space invasion nor with lymph node metastasis, a specific spatial distribution with more lymphatic vessels in the vicinity of tumor edges is predictive of lymphatic dissemination. Herein, we provide a new computerized method suitable for an innovative detailed analysis of the lymphatic network. We show that the transformation zone of the benign cervix acts as a baseline lymphangiogenic niche before the initiation of neoplastic process. During cancer progression, this specific microenvironment is maintained with lymphatic vessels even in closer vicinity to tumor cells.

Microvascular Invasion of Testicular Nonseminomatous Germ Cell Tumors: Implications of Separate Evaluation of Lymphatic and Blood Vessels

We separately evaluated the lymphatic and blood vascular systems to assess the diagnostic accuracy of microvascular invasion and identify predictive markers for occult metastasis of testicular nonseminomatous germ cell tumors. Tissue samples of 86 patients treated for testicular nonseminomatous germ cell tumors (stage 1 in 48 and stage greater than 1 in 38) were stained using the lymphatic endothelial cell specific marker LYVE-1 and the blood vessel endothelial cell marker von Willebrand factor. We assessed lymph vessel density in LYVE-1 stained sections and blood vessel density in von Willebrand factor stained sections. Lymphovascular invasion in LYVE-1 stained sections and blood vascular invasion in von Willebrand factor stained sections were documented. Parameters were correlated with standard clinicopathological data. Blood vessel density in von Willebrand factor sections was significantly greater than lymphatic vessel density in LYVE-1 sections (p<0.001). Peritumor and nontumor lymphatic vessel density in LYVE-1 sections was associated with metastasis at diagnosis (OR 1.277/U, p=0.020 and OR 1.113/U, p=0.095). Lymphovascular invasion in LYVE-1 sections was significantly associated with metastasis (OR=4.517, p=0.002) but blood vascular invasion in von Willebrand factor sections was only slightly significant (OR 2.261, p=0.071). Only lymphovascular invasion in LYVE-1 stained sections was significantly associated with metastasis in a multiple logistic regression model. Microvascular invasion in hematoxylin and eosin stained sections was not associated with metastasis but microvascular invasion evaluated in LYVE-1 and von Willebrand factor stained sections was associated with metastasis (OR 3.506, p=0.016). Lymphovascular invasion in LYVE-1 stained sections was the most important predictive parameter for metastasis at diagnosis, suggesting greater relevance of the lymphatic system in metastatic dissemination of testicular nonseminomatous germ cell tumors. Vascular endothelial cell specific markers provide higher diagnostic accuracy for microvascular invasion. Our results may impact the current concept of microvascular invasion used for risk stratification of clinical stage 1 testicular nonseminomatous germ cell tumors.

Lymphangiogenesis and Its Correlation with the VEGF Expression and the Sentinel Lymph Node in Cutaneous Melanomas

The aim of the study is to evaluate the density of intratumoral and peritumoral lymphatic vessels in primary cutaneous melanomas and to assess their correlation with the status of sentinel lymph nodes and the VEGF expression in tumor cells and stromal cells. A total of 40 patients were enrolled in the study: the melanomas were radically excised with the extirpation of the sentinel lymph node. The study subjects were divided into two groups: 20 cases with positive and 20 cases with negative sentinel lymph node results. The density of lymphatic vessels was evaluated by the antibody D2-40 and the VEGF expression was investigated in the semiquantitative way. The VEGF expression in melanoma cells and the stromal cells was negative to variable positive at both SLN negative and SLN positive patients in all pT stages. In the group of SLN positive patients, the density of intratumoral lymphatic vessels was low up to moderate, while it was observed to be absent, somewhere on the low level in the group of SLN negative patients. On the other side, the density of peritumoral lymphatic vessels was equally numerous at both SLN negative and SLN positive patients. The lymphatic invasion was found out at 4 SLN positive patients only. The ulceration was chiefly in the group of LN positive patients. The results show that the density of lymphangiogenesis and the intensity of the VEGF expression are considered to be an unreliable predictor of melanoma metastasis to the sentinel lymph node, but the ulceration and the lymphatic invasion can predict the potential for metastasis.

Blood microvessel density, lymphatic microvessel density and lymphatic invasion in predicting melanoma metastases: systematic review and meta-analysis

In malignant melanoma (MM) there is an urgent need to identify new markers with predictive value superior to the traditional clinical and histological parameters. Angiogenesis and lymphangiogenesis have been recognized as critical processes in tumour growth and metastasis development, and numerous studies have evaluated the significance of these parameters in predicting the prognosis in solid tumours, including MM. We set out to determine whether angiogenesis, lymphangiogenesis and lymphatic invasion (LI) are valuable prognostic markers in MM. We systematically reviewed the available literature and subsequently performed a meta-analysis on the compiled data. To be eligible for the systematic review, a study had to provide the microvessel density (MVD), the lymphatic vessel density (LVD) or information about LI, assessed by immunohistochemistry on the primary site in patients with MM. To be evaluable for the meta-analysis, a study also had to provide information on clinical outcome. We approached selected studies with the Reporting recommendations for tumour marker (REMARK) criteria, verifying whether they had followed the recommendations. In total, nine angiogenesis, seven lymphangiogenesis and 10 LI studies were included in our meta-analysis, representing 419, 474 and 802 patients, respectively. Using meta-analysis, we showed that peritumoral LVD and the presence of LI have prognostic value for patients with MM. In contrast, MVD and intratumoral LVD did not have prognostic value in these patients. LVD and LI seem to have prognostic value for patients with MM.

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

Epigallocatechin-3-Gallate Inhibits Stem-Like Inflammatory Breast Cancer Cells

Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by high rates of proliferation, lymphangiogenesis and metastasis, and an overall poor survival. As regular green tea consumption has been associated with improved prognosis of breast cancer patients, including decreased risk of recurrence, here the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) were tested on two IBC lines: SUM-149 and SUM-190. EGCG decreased expression of genes that promote proliferation, migration, invasion, and survival. Consistently, growth, invasive properties, and survival of IBC cells were reduced by EGCG treatment. EGCG also reduced lymphangiogenesis-promoting genes, in particular VEGF-D. Conditioned media from EGCG-treated IBC cells displayed decreased VEGF-D secretion and reduced ability to promote lymphangiogenesis in vitro as measured by hTERT-HDLEC lymphatic endothelial cell migration and tube formation. Tumorsphere formation by SUM-149 cells was robustly inhibited by EGCG, suggesting effects on self-renewal ability. Stem-like SUM-149 cells with high aldehyde dehydrogenase (ALDH) activity, previously implicated in poor patient prognosis, were isolated. EGCG treatment reduced growth and induced apoptosis of the stem-like SUM-149 cells in culture. In an orthotopic mouse model, EGCG decreased growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells and their expression of VEGF-D, which correlated with a significant decrease in peritumoral lymphatic vessel density. Thus, EGCG inhibits the overall aggressive IBC phenotype. Reduction of the stem-like cell compartment by EGCG may explain the decreased risk of breast cancer recurrence among green tea drinkers. Recent clinical trials demonstrate the efficacy of green tea polyphenol extracts in treatment of prostate cancer and lymphocytic leukemia with low toxicity. Given the poor prognosis of IBC patients, our findings suggest further exploration of EGCG or green tea in combinatorial treatments against active IBC disease or in maintenance regimens to avoid recurrence is warranted.

Lymphatic biomarkers in primary melanomas as predictors of regional lymph node metastasis and patient outcomes

Recently developed lymphatic-specific immunohistochemical markers can now be utilized to assess intratumoral and/or peritumoral lymphatic vessel density (LVD), to detect lymphatic vessel invasion (LVI) by melanoma cells and to identify lymphatic marker expression in melanoma cells themselves. We systematically reviewed the available evidence for the expression of lymphatic markers as predictors of regional node metastasis and survival in melanoma patients. The currently available evidence suggests that LVD (particularly in a peritumoral location) and LVI are predictors of sentinel node metastasis and poorer survival. Nevertheless, adherence to international guidelines in the conduct and reporting of the studies was generally poor, with wide methodologic variations and heterogeneous findings. Larger, carefully conducted and well-reported studies that confirm these preliminary findings are required before it would be appropriate to recommend the routine application of costly and time-consuming immunohistochemistry for lymphatic markers in the routine clinical assessment of primary cutaneous melanomas.

Abstract A94: Epigallocatechin-3-gallate inhibits stem-like inflammatory breast cancer cells

Abstract Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by an unusual growth pattern with nests of tumor emboli rather than a solid mass, rapid spread via lymphatic dissemination and high proliferative rates. Multi-modality therapies have improved survival but the outcome still remains poorer for these patients. Given the potent anti-cancer properties of green tea polyphenols, including epigallocatechin-3-gallate (EGCG), here we tested the hypothesis that EGCG inhibits IBC cells. Two IBC cell lines were studied. SUM-149 cells have a triple negative receptor status (ER-, PR-, Her-2-), while SUM-190 cells are ER-, PR- and Her-2 amplified. Doses of EGCG used ranged from 5 μg/ml (10.9 μM) to 160 μg/ml (349.1 μM), depending upon the presence of serum which inactivates the polyphenol. Treatment with 40 μg/ml (87.3 μM) EGCG decreased mRNA expression of genes that promote migration and invasion (RHOC, FN1, CDH1 and VIM), lymphangiogenesis (VEGF-D), proliferation (CCND1), and survival (BCL-XL) in both lines. Consistently, EGCG treatment resulted in decreased invasive phenotype, ability for anchorage independent growth and viability, as judged by Matrigel outgrowth, soft agar colony assays and ATP assays. Substantial apoptosis was detected starting at 80 μg/ml (174.5 μM) and 160 μg/ml (349.1 μM) of EGCG for SUM-149 and SUM-190 cells, respectively. Conditioned media isolated from EGCG-treated IBC cells displayed decreased secretion of the pro-lymphangiogenic factor VEGF-D and reduced ability to promote cultured hTERT-HDLEC lymphatic endothelial cell migration in wound healing assays and tube formation in Matrigel, processes critical for lymphangiogenesis. The poor clinical outcome of IBC patients has been attributed to a stem-like cell compartment, which is characterized by increased aldehyde dehydrogenase (ALDH) activity. The SUM-149, but not the SUM-190, line has been reported to contain such a stem-like compartment. EGCG treatment inhibited SUM-149 cell spheroid formation and decreased the size of pre-formed spheroids, suggesting efficacy on the cancer stem-like cells. ALDH-positive SUM-149 cells were isolated using an ALDEFUOR Fluorescence Activated Cell Sorting-based assay. EGCG treatment of these cells led to a significant dose-dependent decrease in ATP levels. Furthermore, doses of 80 and 160 μg/ml EGCG induced apoptosis, as judged by increased Caspase-3 and -7 activities. ALDH-positive SUM-149 cancer stem-like cells were used in an orthotopic mammary fat pad mouse model (n = 6/ group). After 25 days, palpable tumors were detected in all of the mice. The experimental group was given a 0.1 ml intraperitoneal injection of 16.5 mg/kg EGCG while the control group was injected with vehicle 1X PBS. Treatment was administered five times a week for five weeks and then daily for one last week for a total of 43 days. EGCG significantly reduced the growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells such that at the end of the experiment a 37.7 ± 4.4% decrease in tumor volume was seen in the EGCG-treated vs control groups. RT-PCR and immunoblot analyses revealed substantially reduced expression of VEGF-D RNA and VEGF-D protein within tumor tissues of EGCG-treated vs control animals, which correlated with a trend toward decreased peritumoral lymphatic vessel density. Thus, EGCG has pleiotropic inhibitory effects on IBC cells, including the cancer stem-like cell compartment believed to be responsible for recurrence. Recent clinical trials using various green tea polyphenol preparations have shown efficacy in treatment of prostate cancer and lymphocytic leukemia, and low toxicity. Our studies suggest the use of EGCG or green tea in combination with standard therapeutic modalities for IBC patients warrants further exploration. Citation Format: Nora D. Mineva, K. Eric Paulson, Stephen P. Naber, Amy S. Yee, Gail E. Sonenshein. Epigallocatechin-3-gallate inhibits stem-like inflammatory breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A94.

THORACIC SURGERY Intratumoral lymphatic vessel density, and intratumoral and peritumoral lymphatic vessel invasion as predictive factors of lymph node metastasis and prognostic factors in esophageal cancer

In esophageal cancer, metastases in regional lymph nodes occur early and are a poor prognostic factor. We studied intratumoral and peritumoral lymphatic vessel density, and intratumoral and peritumoral lymphatic vessel invasion using D2-40 immunohistochemical staining in 74 resected esophageal cancer specimens and correlated results with patients’ clinicopathologic features and outcomes. Results were analyzed by univariate and multivariate logistic regression as well as univariate and multivariate survival analysis. The peritumoral lymphatic vessel density was significantly higher than the intratumoral lymphatic vessel density (p < 0.001). The peritumoral lymphatic vessel density and intratumoral lymphatic vessel density positively correlated with lymph node metastasis, tumor stage, depth of tumor invasion, and residual tumor. Positive intratumoral lymphatic vessel invasion and peritumoral lymphatic vessel invasion were significantly correlated with lymph node metastasis, tumor size, tumor depth, stage, and residual tumor. Multivariate logistic regression analysis identified intratumoral lymphatic vessel density (p = 0.028) and peritumoral lymphatic vessel invasion (p = 0.036) as risk factors of lymph node metastasis. Cox regression analysis showed that intratumoral lymphatic vessel invasion and peritumoral lymphatic vessel invasion are independent prognostic factors of disease-free survival (p = 0.018, p = 0.021), cancer-specific survival (p = 0.016, p = 0.025), and overall survival (p = 0.017, p = 0.02). Intratumoral lymphatics and peritumoral lymphatic vessel invasion are predictive factors of lymph node metastasis. Intratumoral and peritumoral lymphatic vessel invasion are independent prognostic factors in locally advanced esophageal cancer.

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.

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.

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.