Periostin directly and indirectly promotes tumor lymphangiogenesis of head and neck cancer

Background

Metastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis.

Methods and Findings

Periostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed.

Conclusions

Our findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients.     

Decreased tumstatin-mRNA is associated with poor outcome in patients with NSCLC

Tumstatin is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. The purpose of this study was to evaluate the correlation between tumstatin-mRNA expression and the clinicopathologic characteristics, tumor angiogenesis, outcome of patients with non-small cell lung cancer (NSCLC). Specimens from 68 patients with NSCLC were recruited in this study. Tumstatin-mRNA expression and protein level in tumor tissues were quantified respectively by quantitative RT-PCR and ELISA. Microvessel density (MVD) was determined by CD34 immunostaining. The correlation of tumstatin-mRNA expression levels with clinicopathologic variables, tumor angiogenesis, and prognosis was analyzed. Tumstatin-mRNA expression levels were decreased in tumor. Tumstatin-mRNA expression level was significantly correlated with its protein level in tumor (r = 0.562; P = 0.001). Tumstatin-mRNA expression levels in poorly differentiated tumor tissues were significantly lower than in well-differentiated tumor tissues (P < 0.004). Furthermore, tumor tumstatin-mRNA expression were also significantly related to tumor pathologic stage (P = 0.032) and MVD (r = -0.77, P < 0.0001). Overall survival (OS) and disease-free survival (DFS) analysis indicated that NSCLC patients with low tumstatin-mRNA expression had poorer OS and DFS than those with high expression (P = 0.015 and 0.037; respectively). Multivariable Cox regression analysis revealed that the tumstatin-mRNA expression could be an independent prognostic indicators in both DFS and OS. Tumstatin-mRNA expression levels are down-regulated in NSCLC tissues. Tumstatin-mRNA expression level correlates with prognosis, which suggests that tumstatin-mRNA is a new potential independent marker of favorable prognosis in NSCLC.     

卵巢上皮肿瘤淋巴转移与血管内皮生长因子C的表达

The aim of the present study was to explore the role of vascular endothelial growth factor-C (VEGF-C) in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign cystadenomas. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density (MVD) were assessed by image analysis. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than that in borderline tumors and benign cystadenomas (p < 0.05 or p < 0.01). In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage III-IV and with lymphatic metastasis than those of clinical stage I-II and without lymphatic metastasis respectively (p < 0.05 or p < 0.01), VEGFR-3 positive vessels and MVD was significantly higher in the VEGF-C protein positive tumors than negative tumors (p < 0.05), VEGFR-3 positive vessels was significantly correlated with MVD(p < 0.01). These data suggest that VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian carcinomas, and VEGF-C could be used as a biologic marker of metastasis in ovarian epithelial carcinomas.     

Overexpression of Periostin in Stroma Positively Associated with Aggressive Prostate Cancer

Background

Periostin is an important extracellular matrix protein involved in cell development and adhesion. Previously, we identified periostin to be up-regulated in aggressive prostate cancer (CaP) using quantitative glycoproteomics and mass spectrometry. The expression of periostin was further evaluated in primary radical prostatectomy (RP) prostate tumors and adjacent non-tumorous prostate tissues using immunohistochemistry (IHC). Our IHC results revealed a low background periostin levels in the adjacent non-tumorous prostate tissues, but overexpressed periostin levels in the peritumoral stroma of primary CaP tumors.

Methods

In this study, periostin expression in CaP was further examined on multiple tissue microarrays (TMAs), which were conducted in four laboratories. To achieve consistent staining, all TMAs were stained with same protocol and scored by same image computation tool to determine the total periostin staining intensities. The TMAs were further scored by pathologists to characterize the stromal staining and epithelial staining.

Results

The periostin staining was observed mainly in peritumoral stromal cells and in some cases in tumor epithelial cells though the stronger staining was found in peritumoral stromal cells. Both periostin stromal staining and epithelial staining can differentiate BPH from CaP including low grade CaP (Gleason score ≤6), with significant p-value of 2.2e-16 and 0.001, respectively. Periostin epithelial staining differentiated PIN from low grade CaP (Gleason score ≤6) (p=0.001), while periostin stromal staining differentiated low grade Cap (Gleason score ≤6) from high grade Cap (Gleason score ≤6) (p=1.7e-05). In addition, a positive correlation between total periostin staining and Gleason score was observed (r=0.87, p=0.002).

Conclusions

The results showed that periostin staining was positively correlated with increasing Gleason score and the aggressiveness of prostate disease.     

不同剂量的塞来昔布对C57BL/6小鼠移植瘤微淋巴管密度抑制作用的研究(英文)

目的:观察不同剂量的塞来昔布对C57BL/6小鼠肺癌移植瘤生长、COX-2表达和微淋巴管密度影响,探讨塞来昔布对C57BL/6小鼠肺癌移植瘤淋巴管生成可能作用机制及量效关系。方法:将Lewis肺癌细胞株接种于C57BL/6小鼠左侧腹股沟皮下建立移植瘤模型,随机分为4组:对照组、塞来昔布低剂量、中剂量、高剂量组。观察荷瘤小鼠生存状态,瘤体积变化,种瘤42天后牺牲小鼠,western blot半定量检测COX-2表达及微淋巴管密度。结果:Western blot半定量显示:塞来昔布高、中剂量组COX-2的表达水平及免疫组织化学染色微淋巴管密度计数均明显减低,差异有统计学意义(P0.05),低剂量组略有减低但差异无统计学意义(P0.05)。抑制程度呈明显的剂量依赖性。结论:塞来昔布抑制Lewis肺癌移植瘤的生长及淋巴转移,可能与下调COX-2的表达,阻遏了淋巴管生成的信号通路,抑制微淋巴管生成有关,该抑制作用呈一定的剂量相关性。     

食管鳞癌VEGF—C mRNA和CD31表达及其意义

To investigate the expression of vascular endothelial growth factor-C (VEGF-C) mRNA and CD 31 in esophageal squamous cell carcinoma (ESCC) and its promotion of lymphatic metastasis. The expression of VEGF-C mRNA was examined in 43 ESCC by in situ hybridization. Intratumoral microvessel density (MVD) was assessed by immunostaining endothelial cells, using anti-CD31 antibody. The positive rate of VEGF-C mRNA expression was 41.86%. The average rank of MVD was 76.36 +/- 20.30/mm2. VEGF-C mRNA expression correlated significantly with lymph node metastasis, TNM stage and depth of invasion (p < 0.05 or p < 0.01) in statistic, but not with histological grade (differentiation) (p > 0.05). MVD correlated significantly with lymph node metastasis and TNM stage (p < 0.05) in statistic, but not with depth of invasion and histological grade (differentiation) (p > 0.05). MVD was significant higher in the VEGF-C positive tumors than negative tumors (p < 0.05). The present study indicated that VEGF-C might play a role in lympatic metastasis via lymphangiogenesis and angiogenesis in ESCC.     

Lymphatic microvessel density as a prognostic factor in non-small cell lung carcinoma: a meta-analysis of the literature

The role of lymphatic microvessel density (LVD) as a prognostic factor for survival of patients with non-small cell lung carcinoma (NSCLC) remains controversial. To evaluate this potential role, we performed a systematic review of the electronic databases PubMed and EMBASE for relevant literature to review and compile available survival results. To be eligible, a study had to assess LVD in patients with NSCLC and to compare survival based on LVD stratification. Among 12 eligible trials, all dealt with NSCLC, and 10 trials provided results for the meta-analysis of survival data (evaluable trials). In terms of survival, high LVD was reported to be an unfavorable prognostic factor for overall survival in 8 studies, whereas it was not in 4 studies. The overall survival hazard ratio for the 10 evaluable studies (1,426 patients) was calculated to be 1.41 (95% CI: 1.14–1.75) using a random effects model, indicating a poorer survival for NSCLC patients with high LVD. The hazard ratio was 1.52 (95% CI: 1.10–2.11) in 5 NSCLC studies where LVD was assessed based on D2-40 and 1.31 (95% CI: 1.08–1.60) in 4 studies where LVD was measured based on vascular endothelial growth factor receptor-3. This study supports the hypothesis that the lymphatic microvessel count or LVD, which reflects levels of lymphangiogenesis, is a poor prognostic factor for patient survival in surgically treated NSCLC. However, the present findings may overestimate the prognostic capacity of LVD because of publication and report bias. In addition, the standardization of lymphangiogenesis assessment by the lymphatic microvessel count is necessary.     

Peritumoral lymphangiogenesis induced by vascular endothelial growth factor C and D promotes lymph node metastasis in breast cancer patients

ABSTRACT: BACKGROUND: Mounting clinical and experimental data suggest that the migration of tumor cells into lymph nodes is greatly facilitated by lymphangiogenesis. Vascular endothelial growth factor (VEGF)-C and D have been identified as lymphangiogenic growth factors and play an important role in tumor lymphangiogenesis. The purpose of this study was to investigate the location of lymphangiogenesis driven by tumor-derived VEGF-C/D in breast cancer, and to determine the role of intratumoral and peritumoral lymphatic vessel density (LVD) in lymphangiogenesis in breast cancer. METHODS: The expression levels of VEGF-C/D were determined by immunohistochemistry, and intratumoral LVD and peritumoral LVD were assessed using immunohistochemistry and the D2-40 antibody in 73 patients with primary breast cancer. The associations of intratumoral LVD and peritumoral LVD with VEGF-C/D expression, clinicopathological features and prognosis were assessed. RESULTS: VEGF-C and D expression were significantly higher in breast cancer than benign disease (P < 0.01). VEGF-C (P < 0.001) and VEGF-D (P = 0.005) expression were significantly associated with peritumoral LVD, but not intratumoral LVD. Intratumoral LVD was associated with tumor size (P = 0.01). Peritumoral LVD was significantly associated with lymph node metastasis (LNM; P = 0.005), lymphatic vessel invasion (LVI; P = 0.017) and late tumor,node,metastasis(TNM) stage (P = 0.011). Moreover, peritumoral LVD was an independent risk factor for axillary lymph node metastasis, overall survival and disease-free survival in multivariate analysis. CONCLUSIONS: This study suggests that tumor-derived VEGF-C/D induce peritumoral lymphangiogenesis, which may be one mechanism that leads to lymphatic invasion and metastatic spread. Peritumoral LVD has potential as an independent prognostic factor in breast cancer patients.     

Expression of periostin in the serum of NSCLC and its function on proliferation and migration of human lung adenocarcinoma cell line (A549) in vitro

Periostin is over expressed in many epithelial malignant cancers, including lung cancer, breast cancer, ovarian cancer and colon cancer. It is related with the progression and migration of breast and ovarian cancer cells in vitro. The aim of this study was to investigate the serum level of periostin in non-small cell lung cancer (NSCLC) and its relationship with established biological and prognostic factors by enzyme-linked-immunosorbent serologic assay. We also observe the function of periostin on the proliferation and migration of human lung adenocarcinoma cell line (A549) and discuss the mechanism. The mean value for serum periostin (POSTN) was elevated in NSCLC patients (242.84 ± 5.33 pg/ml) compared to the normal healthy volunteers (215.66 ± 11.67 pg/ml) (p = 0.030). The serum level of periostin of NSCLC patients had no connection with gender, age, pathological type, TNM stage, lymph node status, tumor size and invasiveness. We constructed a plasmid named pEGFP-N1/POSTN expressing full-length human periostin. Transfecting the plasmid to A549 cells and periostin was efficiently expressed in transfected A549 cells. Our data showed that periostin could promote the proliferation and migration of A549 cells by inducing vimentin and N-cadherin expression and downregulating E-cadherin expression. These results strongly suggest that periostin is a novel molecular which play an important role during the progression and development of NSCLC.     

PEDF inhibits lymphatic metastasis of nasopharyngeal carcinoma as a new lymphangiogenesis inhibitor

Nasopharyngeal carcinoma (NPC) is one of the most malignant tumors in southern China and Asia, and lymph node metastasis is an important cause for treatment failure. Lymphangiogenesis is a crucial step in lymphatic metastasis of NPC, while little is known about lymphangiogenesis in NPC. Similar to angiogenesis, lymphangitic neovascularization is a process of balance between pro-lymphangiogenesis and anti-lymphangiogenesis factors, but there are few studies on endogenous lymphangiogenesis inhibitors. Pigment epithelium-derived factor (PEDF) is a well-known effective endogenous angiogenesis inhibitor. However, the relationship between PEDF and lymphangiogenesis remains unknown. Our present study reveals that PEDF is lowly expressed in human NPC tissues with poor prognosis and is negatively correlated with lymphatic vessel density (LVD). Consistently, PEDF inhibits lymphangiogenesis and lymphatic metastasis of NPC in vivo experiments. Mechanistically, PEDF inhibits the proliferation, migration, and tube formation of lymphatic endothelial cells and promotes cell apoptosis. On the other hand, PEDF reduces the expression and secretion of vascular endothelial growth factor C (VEGF-C) of NPC cells through the nuclear factor-κB (NF-κB) signaling pathway. Our findings indicate that PEDF plays a vital role in lymphatic metastasis by targeting both lymphatic endothelial cells and NPC cells, and PEDF may represent a novel therapeutic target for NPC.Subject terms: Metastasis, Head and neck cancer     

Identification of Gene Expression Differences between Lymphangiogenic and Non-Lymphangiogenic Non-Small Cell Lung Cancer Cell Lines

It is well established that lung tumors induce the formation of lymphatic vessels. However, the molecular mechanisms controlling tumor lymphangiogenesis in lung cancer have not been fully delineated. In the present study, we identify a panel of non-small cell lung cancer (NSCLC) cell lines that induce lymphangiogenesis and use genome-wide mRNA expression to characterize the molecular mechanisms regulating tumor lymphangiogenesis. We show that Calu-1, H1993, HCC461, HCC827, and H2122 NSCLC cell lines form tumors that induce lymphangiogenesis whereas Calu-3, H1155, H1975, and H2073 NSCLC cell lines form tumors that do not induce lymphangiogenesis. By analyzing genome-wide mRNA expression data, we identify a 17-gene expression signature that distinguishes lymphangiogenic from non-lymphangiogenic NSCLC cell lines. Importantly, VEGF-C is the only lymphatic growth factor in this expression signature and is approximately 50-fold higher in the lymphangiogenic group than in the non-lymphangiogenic group. We show that forced expression of VEGF-C by H1975 cells induces lymphangiogenesis and that knockdown of VEGF-C in H1993 cells inhibits lymphangiogenesis. Additionally, we demonstrate that the triple angiokinase inhibitor, nintedanib (small molecule that blocks all FGFRs, PDGFRs, and VEGFRs), suppresses tumor lymphangiogenesis in H1993 tumors. Together, these data suggest that VEGF-C is the dominant driver of tumor lymphangiogenesis in NSCLC and reveal a specific therapy that could potentially block tumor lymphangiogenesis in NSCLC patients.     

Thrombocytes Correlate with Lymphangiogenesis in Human Esophageal Cancer and Mediate Growth of Lymphatic Endothelial Cells In Vitro

Recent data provide evidence for an important role of thrombocytes in lymphangiogenesis within human malignant disease. The aim of this study was to investigate the role of thrombocytes in lymphangiogenesis in human esophageal cancer. Perioperative peripheral blood platelet counts (PBPC) were evaluated retrospectively in 320 patients with esophageal cancer, comprising 184 adenocarcinomas (AC), and 136 squamous cell carcinomas (SCC). Data on lymphangiogenesis evaluated by anti-podoplanin immunostaining were available from previous studies, platelets within the tumor tissue were assessed by CD61 immunostaining. For in vitro studies, human lymphatic endothelial cells (LECs) were isolated and co-cultured with peripheral blood platelets. Stromal thrombocytic clusters (STC) were evident in 82 samples (25.6%), and vascular thrombocytic clusters (VTC) in 56 (17.5%). STC and VTC were associated with a significantly higher PBPC at investigation of all cases. The presence of STC was associated with higher lymphatic microvessel density (p<0.001), PBPC and STC were associated with lymphovascular invasion of tumor cells in a regression model. The presence of STCs was associated with shorter DFS of all patients (p = 0.036, Breslow test), and VTC with shorter DFS in in SCC (p = 0.025, Breslow test). In cell culture, LEC proliferation was enhanced by co-culture with human platelets in a dose- and time-dependent manner mediated by the release of PDGF-BB and VEGF-C. Platelets play an important role in lymphangiogenesis and lymphovascular invasion in esophageal cancer, influencing prognosis. So the disruption of signaling pathways between platelets, tumor cells and lymphatic endothelium might be of benefit for patients.     

lncRNA DGCR5在非小细胞肺癌组织中的表达及其与临床病理特征的相关性分析

摘要 目的：探究lncRNA DGCR5在非小细胞肺癌(NSCLC)组织中的表达及其与临床病理特征的相关性。方法：选取2020年1月至2021年12月在我院肿瘤科收治的进行手术治疗的NSCLC患者86例,在手术期间从患者获得肿瘤和非肿瘤的肺癌旁组织样本。采用qRT-PCR测定肿瘤组织及癌旁组织中lncRNA DGCR5表达水平。分析lncRNA DGCR5表达水平与NSCLC患者性别、年龄、临床分期、T分期、N分期等临床病理参数的关系,lncRNA DGCR5表达水平与患者预后总生存期（OS）和无进展生存期（PFS）的关系。结果：与癌旁组织相比,lncRNA DGCR5在NSCLC肿瘤组织中的表达水平相对较低,差异具有统计学意义（P<0.01）。lncRNA DGCR5表达与肿瘤分化程度、TNM分期、肿瘤体积、淋巴转移和远处转移之间存在明显相关性,差异具有统计学意义（P<0.05）。采用Kaplan-Meier法进行生存分析,研究发现lncRNA DGCR5高表达组中位OS及中位DFS分别显著高于lncRNA DGCR5低表达组（P<0.05）。低分化程度、II+ IIIa临床分期、N1-N3淋巴转移、远处转移、及lncRNA DGCR5 低表达均与NSCLC患者总生存率和无进展生存率相关。结论：LncRNA DGCR5在NSCLC患者肿瘤组织中的表达量降低,NSCLC患者血LncRNA DGCR5表达水平与分化程度、TNM分期、淋巴转移、远处转移及预后具有相关性。LncRNA DGCR5可作为早期诊断和治疗NSCLC的新型生物标志物。     

Maspin and c-erbB-2 expression in correlation with microvessel density in invasive ductal breast cancer

Maspin is a unique member of the serpin family involved in regulation of cell migration, apoptosis and angiogenesis in breast and prostate cancers. In this study maspin expression in comparison with c-erbB-2 (HER2/neu) oncogene expression and microvessel density was investigated. The examined material included specimens of primary invasive ductal breast cancer derived from 69 patients. They were analyzed immunocytochemically to assess maspin and c-erbB-2 expression, as well as microvessel density using endothelium marker CD31. In the studied cancers, maspin expression in cancer cells was detected in more than half of the cases (50.73%). Although statistically insignificant (p=0.27), maspin expression showed decreasing tendency with the increase of tumor grade. C-erbB-2 oncogene expression was observed in 78.26% of the examined cancers. Statistically significant positive correlation was found between c-erbB-2 expression and tumor grade (p<0.005). Analysis of the dependence between maspin and c-erbB-2 expression exhibited statistically significant inverse correlation (p<0.001). Mean microvessel density (MVD) of the studied cancers was 71.64 (SD=19.36). MVD decreased with the increase of maspin expression, whereas in the cases showing c-erbB-2 overexpression MVD was clearly higher. Both correlations were statistically significant (p<0.005). In conclusion, it could be stated that increase in maspin expression is associated with weaker expression of c-erbB-2 oncogene and lower microvessel density, which implies a significant role of maspin in tumor biology. However, the exact mechanism of maspin action (including its potential role in angiogenesis), as well as the assessment of its prognostic significance in breast cancer require further studies.     

非小细胞肺癌中NF-κ B、VEGF 的表达及其与肿瘤血管形成的研究

目的:探讨核因子-κB(NF-κB)和血管内皮生长因子(VEGF)在非小细胞肺癌中的表达及其与肿瘤血管形成的关系。方法:应用免疫组织化学方法检测56例非小细胞肺癌及20例癌旁肺组织中的NF-κB P65、VEGF的表达,并用抗CD34测定肿瘤血管的密度(MVD)。结果:(1)在非小细胞肺癌中NF-κB P65、VEGF的表达阳性率分别为83.9%(47/56)、69.6%(39/56),明显高于癌旁组织(P<0.05);(2)NF-κB P65的表达在不同的TNM分期、淋巴结及胸腔积液、吸烟的分组之间差异有统计学意义,VEGF的表达在淋巴结及胸膜转移之间差异有统计学意义;(3)NF-κB P65、VEGF、MVD三者间存在明显相关性。结论:NF-κB、VEGF异常表达与NSCLC的发生、发展及肿瘤血管的形成密切的关系。     

Tumor Invasiveness,Not Lymphangiogenesis,Is Correlated with Lymph Node Metastasis and Unfavorable Prognosis in Young Breast Cancer Patients (≤35 Years)

The morbidity rate of breast cancer is on the rise, and the age of onset appears to be trending toward a young age. Breast cancer in young women (BCYW) has a number of distinctive features that differ from breast cancer in middle-aged or elderly women (BCMEW). Lymphatic metastasis plays an important role in the spread of BCYW; however, the mechanisms of lymph node metastasis (LNM) in BCYW are not clear. This study aimed to investigate the mechanism of lymphatic metastasis in BCYW and to evaluate the relationships between lymphangiogenesis, the expression of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) expression, clinicopathological characteristics, and prognosis. Using immunohistochemistry, MMP-9, VEGF-C and the level of lymphatic microvessel density (LMVD) were analyzed in 106 cases of breast invasive ductal carcinoma and 20 cases of breast proliferative lesions. Compared with BCMEW, BCYW had higher MMP-9 expression, higher LNM, and more adverse prognoses. In BCYW, high MMP-9 expression was positively correlated with LNM and impaired survival time. However, in BCMEW, MMP-9 expression was not correlated with LNM or survival time. In addition, high VEGF-C expression was positively correlated with a high level of LMVD in both BCYW and BCMEW. Nevertheless, a high level of LMVD was not correlated with LNM or survival time in the two groups. More importantly, univariate and multivariate survival analysis showed that MMP-9 expression and LNM were independent prognostic factors in BCYW. Our present study indicates that lymphangiogenesis induced by VEGF-C is augmented in breast cancer; however, a higher level of lymphangiogenesis has no significant impact on LNM or survival time. We suggest that tumor invasiveness, rather than lymphangiogenesis, plays an important role in LNM among BCYW. Moreover, MMP-9 and LNM were independent prognostic factors for BCYW.     

Comparative study of tumor angiogenesis and immunohistochemistry for p53, c-ErbB2, c-myc and EGFr as prognostic factors in gastric cancer

Gastric cancer (GC) continues to be a highly aggressive malignancy with poor prognosis and low survival rates. The survival of patients with GC depends mainly on the stage of the disease, with early GC having a 5 year survival of 90-100% and advanced tumors a 5 year survival of 15-25%. The role of other prognostic factors in these tumors is still under investigation. 28 gastric dysplasia, 45 Early GC and 98 Advanced Gastric Cancers were evaluated for expression of the oncogenes p53, c-ErbB2, c-myc and the EGFr in paraffin-embedded material utilizing Avidin-Biotin immunohistochemistry techniques. In 34 cases of GC microvessel density (MVD) was determined in CD34 stained sections. Statistical correlations with stage, histologic type, differentiation degree, location, size, ploidy patterns and overall survival were done. The Mantel-Cox test was performed to evaluate which factors had an independent prognostic value. Both, tumor angiogenesis and p53 protein expression were statistically associated (95% confidence intervals) with overall survival in patients with GC. p53 protein expression was also correlated with cardial location, nodal involvement and tumor stage. c-ErbB2 may recognize a group of highly aggressive well differentiated adenocarcinomas with worse prognosis. c-myc was also significantly enhanced in well differentiated tumors. EGFr showed no significant associations. Mantel-Cox was performed to compare the prognostic value of tumor stage, p53 protein expression and tumor angiogenesis. Tumor angiogenesis was the most important prognostic indicator to predict overall survival in our series. p53 expression was not independent and did not provide additional prognostic information to tumor stage. Our study suggests that angiogenesis as demonstrated by microvessel counts in CD34 stained sections is a significantly important prognostic factor for predicting survival in gastric cancer.     

Density of intranodal lymphatics and VEGF-C expression in B-cell lymphoma and reactive lymph nodes

Lymphatic vasculature in solid tumors may serve as the pathway for metastatic spread of the cancer to the regional lymph nodes and to distant organs. Controversy still exists whether tumors metastasize through existing lymphatics or through newly formed vessels (lymphangiogenesis). The role of lymphangiogenesis in lymphoma spread and proliferation is not clearly established. VEGF-C is the most potent inducer of lymphangiogenesis. LYVE-1 was shown to be a specific marker for lymphatic vessels in normal and tumor tissue. The aim of the present study was the evaluation of lymph node LYVE-1-positive lymphatic sinus density (LSD) and VEGF-C expression in patients with non-Hodgkin's lymphoma (nHL) and in reactive lymph nodes. Sixty paraffin-embedded lymph nodes from newly diagnosed patients with B-cell nHL were evaluated. Twelve lymph node biopsy specimens from adult patients with reactive lymphonodulitis were used as controls. Sections of lymph nodes were stained immunohistochemically for LYVE-1 and VEGF-C. VEGF-C expression in lymph nodes of nHL patients was low and not significantly different from that in the control (p = 0.6). Moreover, VEGF-C expression did not differ significantly between aggressive and indolent lymphomas (p = 0.53). Similarly we did not find differences in LSD in aggressive nHL and in indolent nHL (p=0.49). The mean LSD in reactive lymph nodes was higher than in nHL (p = 0.03). Only in 2 out of 12 reactive lymph nodes LYVE-1-positive vessels were absent. In all groups we demonstrated a strong positive correlation between VEGF-C and LYVE-1-expression (p = 0.0001). Higher LSD in reactive lymph nodes as compared to those of nHL patients suggests that lymphoma proliferation leads to the destruction of the existing lymphatics rather than to lymphangiogenesis within lymph nodes. NHL are not associated with increased expression of VEGF-C nor increased LYVE-1-positive lymphatic sinuses density within lymph nodes.     

Clinical significance of peritumoral lymphatic vessel density and lymphatic vessel invasion detected by D2-40 immunostaining in FIGO Ib1-IIa squamous cell cervical cancer

The clinical significance of lymphangiogenesis in cervical cancer remains controversial. Our aim was to investigate the correlation between lymphangiogenesis, lymphatic vessel invasion (LVI) and tumor metastasis, invasion and prognosis in squamous cell cervical cancer. Paraffin sections of 90 patients with FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) Ib1-IIa squamous cell cervical cancer were stained for immunohistochemistry with a D2-40 monoclonal antibody against the carcinoembryonic antigen M2A. The lymphatic vessel density (LVD) and LVI were measured, and their relationship with the clinicopathological data was analyzed. D2-40-positive lymphatic vessels were found in 75 of the 90 patients (83.3 %). All D2-40-positive vessels were located in peritumoral areas. The mean±SD of the peritumoral LVD was 10.08±4.16. The positive rate of LVI was 32.0 % (24/75). The recurrence rate of patients with LVD >10 (62.1 %, 18/29) was significantly higher than that of patients with LVD ≤10 (34.8 %, 16/46, P = 0.021). The 5-year recurrence-free survival rate of patients with LVD >10 (41.0 %) was significantly lower than that of patients with LVD ≤10 (67.0 %, P = 0.045). Univariate analysis showed that the peritumoral LVD (≤10 vs >10) was correlated with LVI (absent vs present, P = 0.016). The peritumoral LVD and LVI showed no correlation with age, FIGO stage, tumor size, tumor grade, depth of invasion, or pelvic lymph node metastasis (all: P > 0.05). Peritumoral lymphangiogenesis was correlated with the recurrence and recurrence-free survival in patients with squamous cell cervical cancer. Examination of peritumoral LVD in these patients might therefore help to estimate the risk of recurrence.