Intratumoral Lymphatic Density Research Articles

Intratumoral lymphatic vascular density is an independent factor for disease-free and overall survival in advanced stage oral squamous cell carcinoma

The presence of lymphatic and blood vessels in oral squamous cell carcinoma (OSCC) should play a key role in progression and dissemination. This study aimed to evaluate the correlation between the lymphatic and blood vessel densities with prognostic outcomes in advanced stage OSCC. Immunohistochemical reactions for D-240, CD34, and CD105 were performed in 88 advanced stage OSCC cases located at the oral tongue and the floor of the mouth. The lymphatic vascular density (LVD), blood vascular density (BVD), and neoformed vascular density (NVD) were assessed by counting positive reactions in 4 hotspot areas, both intratumoral (IT) and peritumoral (PT), at high magnification (× 40). High IT LVD was associated with extracapsular spread of lymph node metastasis (P=.03). Recurrence rates were correlated with IT LVD (P < .0001), IT BVD (P=.036), and IT NVD (P=.047), and overall survival was associated with high IT LVD (P=.0016) and IT NVD (P=.009). Yet, IT LVD was an independent factor for disease-free survival and for overall survival based on the Cox proportional hazards model. Our results suggest that high IT LVD has a strong impact on survival outcomes in advanced stage OSCC.

Analyses of VEGFC/VEGF-D expressions, density and endothelial lymphatic proliferation in salivary gland neoplasms

ObjectiveThe aim of this study was to assess vascular endothelial growth factor C (VEGF-C) and VEGF-D expressions, tumor lymphatic density (D2–40) and endothelial lymphatic proliferation (D2–40/Ki-67 double labeling) in a series of salivary gland neoplasm cases. Materials and methodsTwenty pleomorphic adenomas (PA), 20 adenoid cystic carcinomas (ACC) and 20 mucoepidermoid carcinomas (MEC) were assessed, as well as 10 normal minor salivary gland (SG) tissues for comparison. ResultsAll cases presented positive VEGF-C expression in the peritumoral and intratumoral regions, and no differences in immunoexpression were detected between groups. However, the ACC group presented a significant difference in VEGF-C immunoexpression according to the predominant histological pattern. Most cases presented poor VEGF-D labeling in the peritumoral and intratumoral regions. Concerning peritumoral, intratumoral and total lymphatic endothelial density, the assessed groups revealed an increasing gradient, with lower values for PA, followed by MEC and ACC. ConclusionNo correlation was detected between VEGF-C and VEGF-D immunoexpression in relation to lymphatic tumor density and endothelial lymphatic proliferation. Western blotting (WB) revealed no difference between VEGF-C and VEGF-D expression among the lesions, corroborating the immunohistochemistry findings.

Participation of hypoxia-inducible factor-1α and lymphangiogenesis in metastatic and non-metastatic lower lip squamous cell carcinoma

This study evaluated the lymphatic density and HIF-1α immunoexpression in lower lip squamous cell carcinoma (LLSCC) and their correlation with clinicopathological (nodal metastasis, clinical stage, histological grade, recurrence and disease outcome) and survival parameters in 20 metastatic and 20 non-metastatic LLSCCs. Lymphatic density was established by counting microvessels (D2-40+) at the tumor core (intratumoral lymphatic density, ILD) and at the invasive front (peritumoral lymphatic density, PLD) and percentages of immunopositive cells for HIF-1α were established. No statistically significant differences in lymphatic densities in relation to clinicopathological parameters were observed (P > 0.05). All cases exhibited nuclear and cytoplasmic HIF-1α immunoexpression, with relatively high percentages of positivity, but this expression was not statistically different in relation to clinicopathological variables (P > 0.05). Positive correlations were observed between ILD and PLD (P = 0.002), and between nuclear HIF-1α immunoexpression at the tumor core and ILD (P = 0.001). The results suggest ILD and PLD are not directly related to the development of lymph node metastasis in LLSCC. The striking expression of HIF-1α suggests the involvement of this protein in the etiopathogenesis of LLSCCs, possibly stimulating lymphangiogenesis at the tumor core. However, this protein does not seem to exert a determining influence on the biological aggressiveness of these tumors.

Podoplanin as Key Player of Tumor Progression and Lymph Vessel Proliferation in Ovarian Cancer.

Podoplanin plays a key role in tumor progression and metastasis. We evaluated lymphatics proliferation rate and podoplanin expression in tumor cells of ovarian carcinoma. Seventy-five paraffin-embedded specimens of ovarian cancer were immunohistochemically assessed in order to quantify peritumoral (LMVDP) and intratumoral (LMVDT) lymphatic microvessel density of proliferating lymphatics and for podoplanin variability in tumor cells. LMVDT correlated with proliferating tumor vessels located in the peritumoral area (p=0.024) and with the number of mature vessels located in the intratumoral area (p<0.0001), while LMVDP correlated with peritumoral mature vessels (p<0.000l). Proliferating tumor cells at the invasive front were highly positive for podoplanin. To the best of our knowledge, this study represents the first assessment of lymphatic endothelial cell proliferation correlated with podoplanin expression in tumor cells from ovarian cancer. Our data support podoplanin as a potential target that may help reduce ovarian cancer dissemination and lymphatic metastasis.

Correlation of intratumoral lymphatic microvessel density, vascular endothelial growth factor C and cell proliferation in salivary gland tumors.

Lymphatic dissemination is one of the most important pathways for metastasis in many solid tumors, including head and neck carcinomas. The lymphatic growth of cancer has been used as a significant independent adverse prognostic factor and provides information about tumor progression. Salivary gland tumors present different prognoses and have the ability to develop metastases; however, this information regarding the lymphatic spread is scarce. This paper quantifies the lymphatic microvessel density (LMD) in benign and malignant salivary gland tumors and analyzes the relationship between LMD and tumor expression of vascular endothelial growth factors C (VEGF-C) and the proliferative index. The results show that there is no correlation between LMD, VEGF-C and the proliferative index in the majority of salivary gland tumors analyzed, apart from polymorphous low-grade carcinoma which exhibits statistical correlation between LMD and the proliferative index (p<0.05). This correlation probably does not indicate a poor prognosis for this PLGA, since this is a low metastasizing carcinoma of the salivary glands. Different from other solid tumors, such as breast or prostatic carcinomas, there is no correlation between VEGF-C and LMD in salivary gland tumors, and so these traits are not able to estimate the metastatic risk or the prognosis of these tumors.

The role of intratumoral lymphovascular density in distinguishing primary from secondary mucinous ovarian tumors

OBJECTIVE:Ovarian mucinous metastases commonly present as the first sign of the disease and are capable of simulating primary tumors. Our aim was to investigate the role of intratumoral lymphatic vascular density together with other surgical-pathological features in distinguishing primary from secondary mucinous ovarian tumors.METHODS:A total of 124 cases of mucinous tumors in the ovary (63 primary and 61 metastatic) were compared according to their clinicopathological features and immunohistochemical profiles. The intratumoral lymphatic vascular density was quantified by counting the number of vessels stained by the D2-40 antibody.RESULTS:Metastases occurred in older patients and were associated with a higher proportion of tumors smaller than 10.0 cm; bilaterality; extensive necrosis; extraovarian extension; increased expression of cytokeratin 20, CDX2, CA19.9 and MUC2; and decreased expression of cytokeratin 7, CA125 and MUC5AC. The lymphatic vascular density was increased among primary tumors. However, after multivariate analysis, the best predictors of a secondary tumor were a size of 10.0 cm or less, bilaterality and cytokeratin 7 negativity. Lack of MUC2 expression was an important factor excluding metastasis.CONCLUSIONS:The higher intratumoral lymphatic vascular density in primary tumors when compared with secondary lesions suggests differences in the microenvironment. However, considering the differential diagnosis, the best discriminator of a secondary tumor is the combination of tumor size, laterality and the pattern of expression of cytokeratin 7 and MUC2.

Silencing of Yap Gene Inhibits Metastasis in a Severe Combined Immune Deficiency Mice Orthotopic Implantation Gastric Cancer Model

YAP signaling has been proved to be involved in cell growth, invasion and metastasis in multiple malignancies. But little has been reported about the effects of YAP transduction on gastric cancer metastases. The present study was aimed to explore the effects of shRNA-mediated targeting of YAP gene on human gastric cancer metastases in severe combined immune deficiency (SCID) mice. Human gastric carcinoma cell line SGC7901 was implanted into the stomach of SCID mice, which were randomly divided into 3 groups (n=10). Via intraperitoneal injection, mice received YAP-shRNA, negative control, or normal saline respectively. The results of TUNEL and FQ-PCR showed an obvious induction of apoptosis in the YAP-shRNA group. Intratumoral vascular density and lymphatic density were suppressed in the YAP-shRNA group. In conclusion, silencing of YAP gene effectively induces the cell apoptosis and inhibits microvascular angiogenesis and lymphagiogenesis of gastric cancer cells in vivo, suggesting that YAP may serve as a promising therapeutic target for treatment of cancer.

Immunoexpression of HIF-1A and Measurement of Density in Lower Lip Squamous Cell Carcinoma in Metastatic and Non-Metastatic Cases: A Relationship With Prognosis Parameters

This study evaluated the immunoexpression of HIF-1α and correlated it with intratumoral and peritumoral lymphatic density in metastatic and non-metastatic lower lip squamous cell carcinoma (LLSCC). Study Design: This study evaluated the expression pattern and the percentage of cells immunostained for HIF-1α in 50 cases of LLSCC; 25 had regional nodal metastasis and 25 did not. Lymphatic microvessel density was determined. Results: HIF-1α exhibited predominantly diffuse cytoplasmic staining in tumors.

Inducible nitric oxide synthase expression in gastric adenocarcinoma: impact on lymphangiogenesis and lymphatic metastasis

BackgroundLymphatic metastasis is the most important parameter in the spread of gastric carcinomas. Nitric oxide (NO) is a signaling molecule that plays an important role in inflammation and carcinogenesis. In this study, the possible link between inducible nitric oxide synthase (iNOS) expression with lymphangiogenesis and the clinicopathological parameters of gastric carcinomas was investigated.MethodsIn this study, iNOS expression and D2-40 (lymphatic endothelium-specific marker monoclonal antibody) reactivity were examined immunohistochemically in 41 gastric adenocarcinoma and 20 non-neoplastic gastric tissues. iNOS expression was scored semiquantitatively in the tumor parenchyma and stroma. D2-40-positive lymphatic vessels were used in the determination of lymphatic invasion and intratumoral and peritumoral lymphatic vascular density.ResultsiNOS expression was higher in gastric carcinoma tissue compared with non-neoplastic tissue. Particularly, iNOS expression in tumor cells was found to be closely related to lymphangiogenesis and lymphatic metastasis. The density of lymphatic invasion as well as intratumoral and peritumoral lymphatic vascular density were positively correlated with lymph node metastasis.ConclusionsOur results suggest that iNOS-mediated NO formation plays an important role in gastric carcinogenesis, tumor lymphangiogenesis, and the development of lymphatic metastases. Inhibition of the NO pathway may be an alternative treatment of gastric carcinomas.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1713572940104388.

Metastasis associated protein 1 correlates with Hypoxia inducible-factor 1 alpha expression and lymphangiogenesis in esophageal cancer.

Metastasis to regional lymph nodes represents the first step of dissemination in esophageal cancer and serves as an unfavorable prognostic indicator for disease progression. The formation of tumor lymphatic microvessels is dependent on the production of lymphangiogenic growth factors by tumor cells, such as vascular endothelial growth factor C (VEGF-C), metastasis-associated gene 1 (MTA1), and hypoxia inducible-factor l alpha (HIF-1α). The degree of intratumoral lymphatic microvessel density (LMVD) by immunohistochemistry is thought to influence tumor lymph node metastasis (LNM) and prognosis in various solid tumors. The aim of this study was to investigate the expression of MTA1 and HIF-1α in human esophageal cancer and to explore the relationship between their expression and lymphangiogenesis. The expression of MTA1 and HIF-1α, and LMVD marked by D2-40 were detected using immunohistochemical staining in eighty-two human esophageal cancer cases. The positive rates of MTA1 and HIF-1α expression were 69.5% and 59.8%, respectively. The expression of MTA1 and HIF-1α were both correlated with LNM. In addition, MTA1 and HIF-1α expression has a positive correlation in human esophageal cancer; LMVD correlated significantly with LNM. LMVD in MTA1 and HIF-1α over-expression cases was higher than that in none or weak cases, and this difference has statistical significance. Our findings suggest that MTA1 plays an important role in lymphangiogenesis and LNM by stabilizing HIF-1α in esophageal cancer. The inhibition of lymphangiogenesis, MTA1, or HIF-1α activity may have an important therapeutic benefit in the control of esophageal cancer.

Lymphatic Invasion Predicts Aggressive Behavior in Melanocytic Tumors of Uncertain Malignant Potential (MELTUMP)

Lymphatic invasion (LI) identified by immunohistochemical (IHC) staining is common in primary cutaneous melanoma, and LI has been shown to be an independent prognostic factor in melanoma. Its prognostic significance in melanocytic tumors of uncertain malignant potential (MELTUMPs) has not been well characterized. This study included 32 patients with provisional diagnoses of MELTUMP. Lesions were evaluated for tumor thickness, the presence of ulceration, mitotic figures, mitotic figures at the base, tumor infiltrating lymphocytes, as well as peritumoral and intratumoral lymphatic density. Dual IHC staining was used to microscopically detect lymphatic endothelium (podoplanin) containing melanoma cells (S100), with the aid of multispectral imaging in select cases. Univariate analysis was performed to identify associations between clinical and pathologic variables and melanoma-related events. The 32 patients had a median follow-up of 111 months. Two patients subsequently died of melanoma-related disease, 1 died of unknown causes, 5 developed nodal metastases, and the remainder showed no evidence of progressive disease. LI was identified in 8/32 patients (25%) by dual IHC staining, which included the 2 patients who died of melanoma-related disease, 1 patient with bulky nodal metastasis, 1/4 patients with microscopic nodal metastases, and 4 patients who showed no evidence of progressive disease. The presence of LI was associated with melanoma metastases or melanoma-related death (P=0.05). The presence of LI by dual IHC in MELTUMPs is associated with a poorer prognosis, specifically with melanoma metastasis, and may therefore serve as a useful prognostic factor for risk stratification of patients with these diagnostically challenging lesions.

Inhibition of Metastatic Potential in Breast CarcinomaIn VivoandIn Vitrothrough Targeting VEGFRs and FGFRs

Angiogenesis and lymphangiogenesis are considered to play key roles in tumor metastasis. Targeting receptor tyrosine kinases essentially involved in the angiogenesis and lymphangiogenesis would theoretically prevent cancer metastasis. However, the optimal multikinase inhibitor for metastasis suppression has yet to be developed. In this study, we evaluated the effect of NSTPBP 0100194-A (194-A), a multikinase inhibitor of vascular endothelial growth factor receptors (VEGFRs)/fibroblast growth factor receptors (FGFRs), on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of the highly invasive breast cancer cell line 4T1-Luc+. We investigated the biologic effect of 194-A on various invasive breast cancer cell lines as well as endothelial and lymphatic endothelial cells. Intriguingly, we found that 194-A drastically reduced the formation of lung, liver, and lymph node metastasis of 4T1-Luc+ and decreased primary tumor growth. This was associated with significant reductions in intratumoral lymphatic vessel length (LVL) and microvessel density (MVD). 194-A blocked VEGFRs mediated signaling on both endothelial and lymphatic endothelial cells. Moreover, 194-A significantly inhibited the invasive capacity induced by VEGF-C or FGF-2 in vitro in both 4T1 and MDA-MB231 cells. In conclusion, these experimental results demonstrate that simultaneous inhibition of VEGFRs/FGFRs kinases may be a promising strategy to prevent breast cancer metastasis.

Abstract P6-02-04: TMEM (Tumor MicroEnvironment of Metastasis) in human breast cancer is a blood vessel associated intravasation microenvironment unrelated to lymphatics

Abstract In breast cancer, both lymph node and distant metastasis represent dissemination of tumor cells from a primary site, but the mechanism of spread and the subsequent risk of mortality may not be the same. Historically, lymphatic spread has been documented both descriptively, as presence or absence of lymphovascular invasion (LVI), and as a formal part of TNM staging. Until recently, however, there has been no way to directly assess the risk of hematogenous dissemination by the primary tumor. Observations from multiphoton-based intravital imaging of rodent models of breast cancer and the analysis of Mena function in tumor cells in vivo have characterized an intravasation microenvironment (ME) involved in the systemic dissemination of tumor cells from primary breast tumors. We have identified the corresponding structure in FFPE tissue and called it TMEM (Tumor MicroEnvironment of Metastasis). This microanatomic landmark is defined as the direct apposition of a Mena-overexpressing intravasation competent carcinoma cell, a perivascular macrophage, and an endothelial cell. In a case control study of 30 case-control pairs, where each matched pair differed only in their metastatic status – non-metastatic vs. metastatic – we found that the density of TMEM was significantly associated with development of systemic metastasis (p = 0.00006). The relationship of hematogenous- and lymphatic-mediated tumor cell spread is not understood. Using the previously described cohort in which we showed that TMEM was associated with metastasis, the purpose of this study was to 1) assess intratumoral lymphatic density, 2) determine if TMEM- lymphatic structures associated with lymphatics exist, and 3) determine if TMEM- lymphatic structures correlate with systemic metastatic risk. Cases were stained with a triple immunostain identical to that used in our earlier study except that D2-40 (a lymphatic marker) was used, rather than CD31 (a blood vessel marker). The marker for macrophages (CD68) and invasive tumor cells (Mena) remained the same. Two pathologists, blinded to outcome, evaluated the presence or absence of intratumoral lymphatics and quantitated the number of TMEM-lymphatic structures per 10 high power (400x) fields in areas of highest intratumoral lymphatic density. A TMEM-lymphatic structure was defined as the direct apposition of a lymphatic (D2-40) endothelial cell with a macrophage and invasive tumor cell. Intratumoral lymphatics were absent in a majority of tumors in each of the 2 groups (18 of 30 non-metastatic, 16 of 30 metastatic; p = 0.6). TMEM-lymphatic structures were rare and were equally present in the 2 groups (3 metastatic and 3 non-metastatic cases). Using the Wilcoxon (paired) signed-rank test, we found no significant difference in the density of these structures between the two groups (p = 0.4). Furthermore, TMEM-lymphatic structures did not correlate with the presence of lymph node metastases (p = 0.8). We conclude that lymphatic vessels do not participate in the TMEM assembly that has been associated with hematogenous metastasis. TMEM density assessment reflects a hematogenous intravasation ME and offers a novel approach to the assessment of metastatic risk. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-04.

Abstract 1410: TMEM (Tumor MicroEnvironment of Metastasis) in human breast cancer is a blood vessel associated intravasation microenvironment unrelated to lymphatics

Abstract Observations from intravital imaging (Wyckoff et al. Can Res 2007) and the analysis of Mena function in tumor cells in vivo (Roussos et al. JCS 2011) has characterized an intravasation microenvironment (ME) involved in the systemic dissemination of tumor cells from primary breast tumors. We have identified the corresponding structure in FFPE tissue and called it TMEM (Tumor MicroEnvironment of Metastasis). This microanatomic landmark is defined as the direct apposition of a Mena-overexpressing intravasation competent carcinoma cell, a perivascular macrophage, and an endothelial cell. In a case control study of 60 patients, where each matched pair differed only in their metastatic status - non-metastatic vs. metastatic - the density of TMEM was assessed and found to be significantly associated with development of systemic metastasis (p = 0.00006) (Robinson et al. Clin Can Res 2009). Although lymph node status is considered an important prognostic factor in breast cancer, the direct involvement of lymphatics in systemic tumor cell dissemination from primary tumors has not been demonstrated. In addition, the relationship of hematogenous- and lymphatic-mediated tumor cell spread is not understood. The purpose of this study was to 1) assess intratumoral lymphatic density in this same cohort, 2) determine if TMEM-like structures associated with lymphatics exist, and 3) determine if lymphaticTMEM-like structures correlate with systemic metastatic risk. Cases were stained with a triple immunostain identical to that used in the Robinson et al. study except that D2-40 (a lymphatic marker) was used, rather than CD31 (a blood vessel marker). The marker for macrophages (CD68) and invasive tumor cells (Mena) remained the same. Two pathologists, blinded to outcome, evaluated for the presence or absence of intratumoral lymphatics and quantitated the number of TMEM-like structures per 10 high power (400x) fields in areas of highest intratumoral lymphatic density. A TMEM-like structure was defined as the direct apposition of a lymphatic (D2-40) endothelial cell with a macrophage and invasive tumor cell. Intratumoral lymphatics were absent in a majority of tumors in each of the 2 groups (18 of 30 non-metastatic, 16 of 30 metastatic; p=0.6). TMEM-like structures were rare and were equally present in the 2 groups (3 metastatic and 3 non-metastatic cases). Using the Wilcoxon (paired) signed-rank test, we found no significant difference in the density of these structures between the two groups (p = 0.4). Furthermore, TMEM-like structures did not correlate with the presence of lymph node metastases (p=0.8). We conclude that lymphatic vessels do not participate in the TMEM assembly that has been associated with hematogenous metastasis. TMEM density assessment reflects a hematogenous intravasation ME and offers a novel approach to the assessment of metastatic risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1410. doi:1538-7445.AM2012-1410

Prognostic Significance of Tumor-Associated Lymphangiogenesis in Malignant Melanomas of the Conjunctiva

To evaluate whether tumor-associated lymphangiogenesis contributes to prognosis of conjunctival malignant melanomas and to study its association with other tumor characteristics. Nonrandomized, retrospective case series. A total of 109 consecutive patients with primary conjunctival malignant melanoma. Proliferating lymphatic vessels were identified immunohistochemically using lymphatic vascular endothelial hyaluronan receptor-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as proliferation marker. Baseline tumor characteristics included tumor location, tumor thickness, tumor diameter, tumor origin, and tumor growth pattern. Kaplan-Meier and Cox regression analyses of the risk of local recurrence, lymphatic spread, distant metastasis, and melanoma-related death were performed. Intratumoral lymphatic vascular density and its association with tumor characteristics and recurrence-free, lymphatic spread-free, distant metastasis-free, and melanoma-specific survival. Intratumoral and peritumoral proliferating lymphatic vessels could be detected in all of the 109 conjunctival melanoma samples. High intratumoral lymphatic density was significantly associated with palpebral tumor location (P<0.001), greater tumor thickness (P<0.001), larger tumor diameter (P = 0.001), tumor origin de novo (P = 0.002), and nodular tumor growth pattern (P = 0.037). Patients with high intratumoral lymphatic density revealed significantly lower recurrence-free, lymphatic spread-free, distant metastasis-free, and melanoma-specific survival rates (P<0.001 for all). By multivariate Cox regression, factors predictive of local recurrence included palpebral tumor location (hazard ratio [HR] 2.66, P = 0.014), large tumor diameter (HR 5.48, P<0.001), and high intratumoral lymphatic density (HR 2.48, P = 0.043); factors predictive of lymphatic spread included palpebral tumor location (HR 4.13, P = 0.009), high tumor thickness (HR 12.17, P<0.001), and high intratumoral lymphatic density (HR 6.79, P = 0.019); factors predictive of distant metastasis included palpebral tumor location (HR 7.63, P<0.001), high tumor thickness (HR 8.60, P<0.001), large tumor diameter (HR 0.30, P = 0.029), and high intratumoral lymphatic density (HR 8.90, P = 0.047); and factors predictive of melanoma-related death included palpebral tumor location (HR 7.74, P<0.001), high tumor thickness (HR 10.88, P<0.001), large tumor diameter (HR 0.28, P = 0.018), and, with borderline significance, high intratumoral lymphatic density (HR 8.46, P = 0.052). Tumor-associated lymphangiogenesis seems to be associated with an increased risk of local recurrence, lymphatic spread, distant metastasis, and melanoma-related death in patients with conjunctival malignant melanomas. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Abstract LB-289: Angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma of the minor salivary glands

Abstract The purpose of this study was to investigate angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma (MEC) of the minor salivary glands as potential predictors of tumor behavior. Intratumoral and peritumoral neoformed (NMVD) and lymphatic microvessel density (LMVD) were evaluated by CD105 and D2–40 immunohistochemistry, respectively. D2–40, VEGF-A, and VEGF-C immunoexpression were also investigated in tumor parenchyma. Twenty-six paraffin-embedded MEC samples were included in the study. Palate (50%) and buccal mucosa (19.2%) were the main affected sites. Sixteen (61.5%) tumors were low-grade, 6 (23.1%) intermediate-grade, and 4 (15.4%) high-grade. Twenty (77%) cases were clinically T1-T2, and 6 (23%) were T3-T4. Nodal metastasis occurred in 3 (11.5%) cases and distant metastasis in 1 (3.8%). Three (11.5%) cases recurred, and 3 (11.5%) patients died from disease. NMVD mean was 19.58±13.91 (400x field) for intratumoral evaluation and 10.90±7.79 (400x field) for peritumoral evaluation. LMVD mean was 5.66±3.43 (400x field) for peritumoral evaluation and 0.54±3.43 (400x field) for intratumoral evaluation. Just 6 (23.1%) cases reveal intratumoral lymphatic vessels. Twenty-two (84,6%) cases showed D2–40 in neoplastic cells, mainly in epidermoid type. Fifteen (57.7%) cases were positive for VEGFA, and 11 (42.3%) were positive for VEGF-C. A positive association was observed between VEGF-A positivity and intratumoral NMVD (p=0.05). Cases with nodal metastasis, altogether, were related to low intratumoral NMVD, high peritumoral LMVD, D2–40 expression and absence of VEGF-A. Distant metastasis was associated with low intratumoral NMVD, high peritumoral LMVD, D2–40 positivity and absence of VEGF-A and VEGF-C expression. Recurrent cases presented low intratumoral NMVD, and absence of VEGF-A and VEGF-C expression. Low intratumoral NMVD, and D2–40 positive cells were found in all tumors of patients that died of the disease. In conclusion, although NMVD, LMVD, D2–40, VEGF-A, and VEGF-C could not predict the prognosis of MEC, intratumoral NMVD, peritumoral LMVD, D2–40 and VEGF-A expression seems play a role in the occurrence of a worse behavior in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-289. doi:10.1158/1538-7445.AM2011-LB-289

Association between intratumoral lymphatic microvessel density (LMVD) and clinicopathologic features in endometrial cancer: a retrospective cohort study

BackgroundLymph node metastasis in endometrial cancer significantly decreases survival rate. Few data on the influence of intratumoral lymphatic microvessel density (LMVD) on survival in endometrial cancer are available. Our aim was to assess the intratumoral LMVD of endometrial carcinomas and to investigate its association with classical pathological factors, lymph node metastasis and survival.MethodsFifty-seven patients with endometrial carcinoma diagnosed between 2000 and 2008 underwent complete surgical staging and evaluation of intratumoral LMVD and other histologic variables. Lymphatic microvessels were identified by immunohistochemical staining using monoclonal antibody against human podoplanin (clone D2-40) and evaluated by counting the number of immunostained lymphatic vessels in 10 hot spot areas at 400× magnification. The LMVD was expressed by the mean number of vessels in these 10 hot spot microscopic fields. We next investigated the association of LMVD with the clinicopathologic findings and prognosis.ResultsThe mean number of lymphatic vessels counted in all cases ranged between 0 and 4.7. The median value of mean LMVD was 0.5, and defined the cut-off for low and high LMVD. We identified low intratumoral LMVD in 27 (47.4%) patients and high LMVD in 30 (52.6%) patients. High intratumoral LMVD was associated with lesser miometrial and adnaexal infiltration, lesser cervical and peritoneal involvement, and fewer fatal cases. Although there was lower lymph node involvement among cases with high LMVD, the difference did not reach significance. No association was seen between LMVD and FIGO staging, histological type, or vascular invasion. On the other hand, low intratumoral LMVD was associated with poor outcome. Seventy-five percent of deaths occurred in patients with low intratumoral LMVD.ConclusionOur results show association of high intratumoral LMVD with features related to more localized disease and better outcome. We discuss the role of lymphangiogenesis as an early event in the endometrial carcinogenesis.

Tumor-Associated Lymphangiogenesis in the Development of Conjunctival Squamous Cell Carcinoma

To analyze whether tumor-associated lymphangiogenesis accompanies the development from premalignant conjunctival intraepithelial neoplasia (CIN) into invasive squamous cell carcinoma (SCC) of the conjunctiva and to study its association with prognosis and other tumor characteristics. Case-controlled, matched-pair cohort study. Twenty patients with invasive SCC were closely matched with 20 patients with high-grade CIN and 20 patients with low-grade CIN regarding tumor size, tumor location, tumor extension, and patients' age. Proliferating lymphatic vessels were identified using lymphatic vascular endothelial hyaluronan receptor-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as proliferation marker. Baseline tumor characteristics included tumor size, tumor-to-limbus distance, tumor-to-fornix distance, 2009 TNM classification, tumor cell type, mitotic rate, and Ki-67 proliferation index. Kaplan-Meier and Cox regression analyses of recurrence-free survival were performed. Lymphatic vascular density (LVD) and relative lymphatic vascular area (RLVA) of proliferating lymphatic vessels within the tumor mass (intratumoral) and within an area < or = 500 microm from the tumor border (peritumoral), and its association with tumor characteristics and recurrence-free survival. Intratumoral and peritumoral proliferating lymphatic vessels could be detected in all of the 60 conjunctival tumor samples. Invasive SCC revealed significantly higher values of intratumoral and peritumoral LVD and RLVA of proliferating lymphatics than high-grade or low-grade CIN (P < or = 0.001). Higher intratumoral lymphatic densities were significantly associated with larger tumor size (P=0.001), lower tumor-to-limbus distance (P=0.002), lower tumor-to-fornix distance (P=0.003), and higher TNM categories (P<0.001). Recurrence-free survival rates decreased significantly with higher intratumoral lymphatic densities (P<0.001). By multivariate Cox regression, large tumor size (hazard ratio 1.68, P=0.002) and high intratumoral lymphatic density (hazard ratio 1.10, P=0.046) were significant prognostic predictors of local recurrence. Development of conjunctival SCC from premalignant lesions is accompanied by the outgrowth of new conjunctival lymphatic vessels. This active tumor-associated lymphangiogenesis seems to be associated with an increased risk of local recurrence in patients with CIN and conjunctival invasive SCC. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Intratumoral lymphangiogenesis in oral squamous cell carcinoma and its clinicopathological significance

Lymphatic metastasis has always been regarded as a major prognostic indicator for disease progression and as a guide for therapeutic strategies to oral squamous cell carcinoma (OSCC), but to date, how tumor cells access and spread via the lymphatics have not been fully elucidated. Whether tumor cells metastasize by expansion and invasion of pre-existing peritumoral lymphatics or by the induction and invasion of newly formed lymphatics within tumors is controversial. In order to address this issue and find out the clinicopathological significance of intratumoral lymphangiogenesis, we investigated 86 archival specimens from patients with OSCC, quantitating lymph vessels by immunostaining with D2-40. We also quantified lymphatic invasion and examined the possible associations of all the above parameters with clinicopathological features and outcome. Higher intratumoral lymphatic density (ILD) and peritumoral lymphatic density (PLD) were both significantly associated with the presence of lymph node metastasis at the time of diagnosis and the outcome of the post-operation biopsy of 77 patients (P = 0.001). Higher ILD was significantly associated with a higher incidence of intratumoral lymphatic invasion, peritumoral lymphatic invasion and recurrence of tumor (P = 0.001 and P = 0.041 and P = 0.001, respectively). Patients with higher ILD exhibited shorter 5-year cumulative and disease-free survival (P = 0.001). Thus, lymphangiogenesis indeed occurs in oral squamous cell carcinoma; ILD might be used as an index to inflect the aggression of the disease, to evaluate the status of lymphatic metastasis, to separate patients at higher risk of an adverse clinical outcome.

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.