Relationships of cervical lymph node metastasis to histopathological malignancy grade, tumor angiogenesis, and lymphatic invasion in tongue cancer

Cervical lymph node (CLN) metastasis from oral cancer correlates with poor prognosis. Therefore, accurate assessment of CLN status is crucial in treatment planning. However, there are few reports focusing on CLN metastasis from tongue cancer. Further, the growth and progress of the tumor are known to be profoundly related to histological malignancy, tumor angiogenesis, and lymphangiogenesis. Thus, this study aimed to identify predictive factors for CLN metastasis in tongue squamous cell carcinoma (SCC). Initial biopsy specimens obtained from 30 patients with tongue SCC were examined to evaluate histological malignancy according to Anneroth’s classification. In addition, blood vessel density, lymph vessel density, and lymphatic invasion in the tumor were evaluated immunohistochemically using CD31, CD34, D2-40, and AE1/AE3, and then the relationships of CLN metastasis to these parameters were investigated. Histological malignancy grade, blood vessel density, and lymphatic invasion were significantly related to CLN metastasis (P < 0.05), but there was no relationship between lymph vessel density and CLN metastasis. However, double immunostaining showed that lymphatic invasion by tumor cells was significantly related to CLN metastasis. The results indicate that Anneroth’s histological malignancy grade of 16 or more, tumor blood vessel density of more than 37, and the presence of lymphatic invasion by tumor cells can be predictive factors for CLN metastases in tongue SCC.     

舌鳞状细胞癌淋巴管密度与淋巴转移的关系

目的 探讨舌鳞癌组织中淋巴管密度与鳞癌侵袭和转移的关系.方法 选择62例舌鳞癌标本,采用免疫组化SP法检测血管内皮生长因子受体-3(VEGFR-3)和血管内皮生长因子-D(VEGF-D)的表达.结果 62例舌鳞癌标本中有56例标本的VEGFR-3呈阳性表达,但功能性淋巴管主要分布在癌组织周围,癌间质中未见明显的成形淋巴管.舌鳞癌组织中淋巴管密度与舌鳞癌患者年龄、性别无关,而与舌鳞癌病理分级、临床分期和有无淋巴结状况密切相关(P＜0.05).VEGF-D在癌细胞胞质呈阳性表达,且随着VEGF-D表达增强,癌组织周围淋巴管密度亦明显增加.结论 VEGF-D和VEGFR-3途径可促进鳞癌淋巴管新生和淋巴转移.     

Regional difference in intratumoral lymphangiogenesis of oral squamous cell carcinomas evaluated by immunohistochemistry using D2-40 and podoplanin antibody: an analysis in comparison with angiogenesis

BACKGROUND: Whether tumor cells induce lymphangiogenesis intratumorally or permeate pre-existing lymphatic vessels in the peritumoral area still remains unclear. In this study, we investigated in detail the intratumoral lymphangiogenesis of oral squamous cell carcinomas (SCC) in comparison with tumor angiogenesis. METHODS: Immunohistochemistry with D2-40, podoplanin antibody, and CD34 antibody were used to evaluate the lymphatic vessel density (LVD) and blood microvessel density (MVD). Vascular endothelial growth factor (VEGF) and VEGF-C expressions of oral SCC were also assessed by immunohistochemistry. RESULTS: LVD significantly increased in the superficial area of tumor tissue compared with normal mucosa, whereas it decreased in the deep area of intratumoral tissue near the invasion front, in sharp contrast to MVD, which significantly increased throughout tumor tissue. Consistent with the decreased intratumoral LVD and increased intratumoral MVD, VEGF-C expression of tumor cells was down-regulated in the deep area of tumor tissue, while VEGF expression of tumor cells was up-regulated throughout the tumor tissue. CONCLUSIONS: Lymphangiogenesis in oral SCC varies depending on the region within the tumor tissue. It is not induced in the genuine tumor stroma near the invasion front, probably due to the down-regulation of VEGF-C expression of tumor cells, which is different from VEGF-mediated induction of intratumoral angiogenesis.     

口腔癌VEGF—C表达和血管及淋巴管生成与淋巴道转移关系的研究

目的　比较口腔正常及鳞癌组织血管、淋巴管密度与血管内皮生长因子 C (VEGF C)表达及淋巴道转移关系。方法　VEGF C免疫组化染色、血管、淋巴管酶组化染色、光镜及图像分析观察血管、淋巴管总体面数密度 (TNa)。结果　VEGF C表达阳性的淋巴管TNa明显高于阴性 (P <0 .0 1) ;血管TNa略高于阴性 (P >0 .0 5 )。鳞癌比正常组织、淋巴结转移组比无淋巴结转移组血管、淋巴管TNa均有增加 (P <0 .0 1)。随临床T分期增加 ,淋巴管TNa增加 (P <0 .0 1) ,而血管TNa与之无关 (P >0 .0 5 )。随病理分级增加 ,血管TNa(P <0 .0 1)、淋巴管TNa(P <0 .0 5 )均有增加。结论　VEGF C主要介导了癌周淋巴管生成 ,对血管生成有一定的影响 ;血管、淋巴管密度的同时增加可能与VEGF、VEGF C及其受体的协同表达有一定的关系     

Angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma of minor salivary glands

J Oral Pathol Med (2012) 41: 603-609 Background: Mucoepidermoid carcinoma is the most common malignant tumor of salivary glands. This tumor is characterized by a great variability in clinical behavior, and little is known about the pathological mechanisms involved in its variance. Angiogenesis is an important step in tumor progression and is believed to be an essential event for metastatic dissemination. Methods: We aimed to investigate angiogenesis and lymphangiogenesis in mucoepidermoid carcinoma measuring the density of neoformed and lymphatic vessels using CD105 and D2-40 antibodies, respectively, and by immunohistochemical evaluation of VEGF-A and VEGF-C proteins. It was also investigated the expression of D2-40 in neoplastic cells. Results: We studied 26 cases of mucoepidermoid carcinoma, which showed great angiogenic activity measured by neoformed vessel density. However, a low density of lymphatics was observed. VEGF-A, VEGF-C, and D2-40 were commonly detected in mucoepidermoid carcinoma, but only VEGF-A expression correlated with neoformed vessel density. Recurrence and nodal metastasis were associated with low VEGF-A expression and low neoformed vessel density, indicating that impaired angiogenesis could lead to an aggressive phenotype. Conclusions: Angiogenesis seems important in the modulation of mucoepidermoid carcinoma pathogenesis; however, none of the parameters analyzed could predict tumor behavior.     

Computer‐assisted morphometric analysis of lymphatic vessel changes in hamster tongue carcinogenesis

J Oral Pathol Med (2010) 39 : 518–524 Background: To characterize lymphangiogenesis in early‐stage hamster tongue carcinoma development, morphological features and spatial relationships of lymphatic vessels. Methods: Lymphatic vessels were examined histochemically, using 5′‐Nase‐ALPase enzyme and combined light and electron microscopy to measure lymphatic vessel area (LVA) and lymphatic vessel density (LVD). Results: In atypical hyperplastic tissues, LVA was found to be 1429.97 and LVD was found to be 39, in carcinoma in situ LVA was 2538.33 and LVD was 48, and in micro‐invasive carcinoma LVA was 5733.74 and LVD was 59. Increased lymphangiogenesis was seen in pre‐neoplastic states and in early‐stage oral squamous cell carcinoma (OSCC). Small regular lymphatic vessels predominated in atypical hyperplasia, and large, irregular lymphatic vessels in early‐stage OSCC. Lymphatic endothelial vessels were stretched and porous over large areas. Conclusions: Newly formed lymphatics and patulous intercellular junctions may be optimally suited for tumor cell metastasis through lymphatic channels in early‐ and middle‐phase carcinogenesis. Lymphatic capillary LVA and LVD became enlarged, and positively correlated, with malignancy, but show no correlation with 7,12‐dimethylbenz[a]anthracene‐induced time.     

金黄地鼠舌癌血管生成的组织学定量研究

目的：动态研究金黄地鼠舌癌发生、发展过程中血管生成的变化规律。方法：采用组织学定量方法，结果：舌部癌前阶段即有血管生成现象，从癌前病变、原位癌、浸润癌发展的不同时期，血管生成现象持续存在，并随着癌细胞的增殖、浸润更为明显，血管增生主要由周边向中心呈向心性增生，肿瘤发展至一定阶段肿瘤中心出现血管生长的停滞。结论：血管生成是舌癌生长、发展的重要先决条件。     

40例舌癌组织VEGF表达及微血管密度观察

目的:探讨血管内皮生长因子(VEGF)表达及微血管密度(MVD)与舌癌组织临床病理指标的关系.方法:40例手术切除的舌癌原发病灶蜡块标本常规切片,免疫组化染色法观察各标本VEGF表达及MVD.结果:VEGF阳性表达的舌癌组织有较高的微血管密度(p＜0.01);VEGF表达和MVD在淋巴结转移组间有显著性差异(p＜0.01)、病理分型组间有显著性差异(p＜0.05),而在原发病灶临床分级组间差异无统计学意义(p＞0.05).结论:VEGF表达和MVD与舌癌病理分型及淋巴转移关系密切,可作为预测性检测指标.     

舌鳞癌淋巴管生成与颈淋巴结转移的关系

目的：研究舌鳞癌淋巴管生成及淋巴管密度／淋巴管相对面积（LVD／LVA）与颈淋巴结转移的关系，为术前准确判断颈淋巴结状况提供参考。方法：口腔颌面外科接受舌癌切除及颈淋巴清扫术的标本63例：对HE染色阴性的淋巴结，应用细胞角蛋白CK（AE1／AE3）标记检测微转移，以免疫组织化学检测结果判断淋巴结转移；以LYVE—1作为淋巴管内皮标志物，研究舌癌淋巴管生成参数——淋巴管密度（LVD）和淋巴管面积（LVA）与颈淋巴结转移及其他临床病理变量（年龄、性别、T分类、病理分级、浸润方式）之间的关系。应用SPSS10．0统计软件包对所得数据进行Mann-whitney U检验。结果：应用细胞角蛋白CK（AE1／AE3）标记免疫组化法检测微转移，提高了淋巴结转移的检出率；舌癌实质内未见到LYVE—1^＋的脉管结构，LYVE—1^＋脉管多位于癌周，癌周LVD、LVA与颈淋巴结转移及其他临床病理变量均无关。结论：舌癌癌周淋巴管密度（LVD）、淋巴管面积（LVA）与颈淋巴结转移及其他临床病理变量均无关，不宜作为术前评估淋巴结状况的指标。     

口腔鳞癌脉管侵袭的研究

对４４例口腔鳞癌组织切片用抗血型抗原ＡＢＨ抗体作免疫组化染色，以研究癌组织对脉管的侵袭。结果表明，该方法为脉管内皮细胞提供了清晰的染色，显示脉管侵袭的阳性率为４０．９％，显著高于以Ｈ·Ｅ染色显示的阳性率（２０．５％）。口腔鳞癌组织侵袭脉管与其分化程度、生长方式及淋巴结转移有密切关系。作者提示口腔鳞癌脉管侵袭的研究对判断预后有重要意义     

High interstitial fluid pressure in rat tongue cancer is related to increased lymph vessel area, tumor size, invasiveness and decreased body weight

Background:  Interstitial fluid pressure (IFP) in most tumors is high, and this high pressure has been correlated with poor prognosis. Measurements of IFP in normal tongue and in tongue cancer are lacking. Recent research suggests the existence of a relationship between increased peritumoral lymph vessels (PTLV) and survival, and a correlation of increased lymphatic vessel density with an unfavorable prognosis has been reported.
Materials and methods:  In the present study, tongue squamous cell carcinoma (SCC) was induced by adding the carcinogen 4-nitroquinoline oxide in drinking water for 19 weeks. The IFP was measured by micropuncture and immunohistochemistry was used to visualize lymph vessels.
Results:  In normal tongue, IFP averaged 3.1 ± 0.3 mmHg. The IFP, both in the tumor (29.1 ± 2.9 mmHg) and 0.5 cm anterior to it (15.4 ± 2.1 mmHg) was consistently increased ( P  <   0.005) with values ranging from 10 to 40 mmHg. The highest IFP values were measured in rats with large tumors ( P  <   0.05) and low body weight ( P  <   0.001), suggesting that IFP increases with cancer progression. Lymphatic vessel area (%), as determined with the lymphatic specific marker LYVE-1 antibody, was significantly increased in the peritumoral area when compared to intratumoral and control mucosa ( P  <   0.05). There was a significant positive correlation between IFP, PTLV area, tumor size and invasiveness.
Conclusions:  Our data show that IFP is increased in tongue cancer. Corresponding changes in PTLV area, invasiveness, tumor area and IFP suggest that the increased pressure is caused by defective lymph drainage and solid stress generated by tumor cells growing in a low compliant environment.     

舌鳞状细胞癌浸润方式与癌淋巴管生成的特点及临床意义

目的探讨不同浸润方式舌鳞状细胞癌淋巴管生成的特点及临床意义。方法 参照Anneroth等描述的肿瘤浸润方式对41例舌鳞状细胞癌进行分型,应用免疫组化法,以D2-40标记淋巴管,计数淋巴管密度（lymphatic vessel density,LVD）。结果不同浸润方式的舌鳞状细胞癌D2-40的表达不同,浸润方式为Ⅲ、Ⅳ型的舌鳞状细胞癌,淋巴管密度（LVD）显著高于Ⅰ、Ⅱ型（P〈0.05）,且临床易出现淋巴结转移。结论Ⅲ、Ⅳ型浸润方式的舌鳞状细胞癌恶性程度高,可能具有较强的诱导淋巴管生成的能力。     

口腔鳞癌中微淋巴管密度的检测及其临床意义

目的:探讨淋巴管生成在口腔鳞癌中的作用.方法:对14 例正常口腔黏膜组织、 40 例口腔鳞癌组织标本,应用免疫双重组织化学染色区分血管和淋巴管,人工记数微淋巴管密度(MLVD),分析其与颈淋巴结转移的关系.结果:40 例口腔鳞癌组织中MLVD显著高于14 例正常口腔黏膜组织(P<0.01),口腔鳞癌组织中MLVD与肿瘤的TNM分期及其淋巴结转移密切相关(P<0.01).结论:淋巴管生成可能参与了口腔鳞癌的生长、浸润及转移,提示测定口腔鳞癌中MLVD对评估其淋巴结转移和预后判断可能具有重要意义.     

舌癌不同浸润方式ki67表达的组织学研究

目的:探讨舌癌不同浸润方式ki67表达的差异及临床意义。方法:按Anneroth等介绍的方法将43例病理资料完整的舌癌标本进行浸润方式分型,免疫组化SP法检测ki67的表达情况。结果:ki67的表达与肿瘤的浸润方式有关。Ⅲ、Ⅳ型浸润方式的舌癌癌细胞ki67表达高于Ⅰ、Ⅱ型(P<0.05),且临床上易发生颈淋巴结转移。结论:浸润方式可作为舌癌恶性程度的有效指标之一,且分型越高其ki67表达越强,有助于判断舌癌的淋巴结转移情况,为临床治疗方案设计提供依据。     

野黄芩苷对鼠舌癌移植瘤新生脉管及弹性和胶原纤维的影响

目的:检测野黄芩苷对人舌鳞癌SAS细胞裸鼠移植瘤中的新生脉管及弹性和胶原纤维的表达和分布的影响,及对肿瘤生长的作用.方法:不同浓度的野黄芩苷、生理盐水及顺铂处理肿瘤,观察移植瘤体积的变化.结果:应用免疫组化观察CD105和Prox-1标记的血管和淋巴管的表达,Masson和醛品红染色检测弹性和胶原纤维的分布.野黄芩苷治...     

口腔鳞状细胞癌中血管内皮生长因子-C的表达及其与血管、淋巴管生成、淋巴结转移的关系

目的探讨血管内皮生长因子-C（VEGF-C）在口腔鳞状细胞癌（OSCC）组织中的表达情况及其与血管、淋巴管生成、淋巴结转移之间的关系。方法调查拥有完整临床病理资料的67例口腔鳞癌患者的手术切除标本,采用SP免疫组化技术检测VEGF-C的表达情况并分析其与微血管密度（MVD）、淋巴管密度（LVD）及其他临床病理指标的关系。结果晚期病例、淋巴结转移阳性病例的VEGF-C表达明显升高（P值分别为0.015和<0.001）,而VEGF-C表达与患者性别、肿瘤部位、肿瘤分化程度无关（P>0.05）。VEGF-C高表达组的LVD明显高于VEGF-C低表达组（P=0.001）,但两组间MVD无统计学差异（P=0.125）。此外,淋巴结转移阳性组的LVD明显高于淋巴结转移阴性组（P=0.026）。结论VEGF-C可能主要通过参与诱导口腔鳞癌淋巴管生成促进淋巴结转移。     

A cervical lymph node metastatic model of human tongue carcinoma: Serial and orthotopic transplantation of histologically intact patient specimens in nude mice.

PURPOSE: A lymph node metastatic model of human tongue carcinoma using orthotopic and serial transplantation was established in nude mice to study the invasive and metastatic properties of human tongue cancer. Materials and Methods: Lymph node metastatic specimens of human tongue carcinoma were transplanted into nude mice orthotopically. Tumors dissected from the metastatic lymph nodes of the nude mice were serially transplanted into tongues of disease-free nude mice at 4-week intervals. RESULTS: All mice developed aggressive and diffuse well-differentiated squamous cell carcinoma at tongue recipient sites. Tumor cells invaded to lymphatic vessels. In addition, increased cervical lymph node metastasis was noted in the first (3 of 14, or 21%), second (4 of 11, or 36%), third (6 of 10, or 60%), or fourth (11 of 14, or 79%) transplantation. In mice, 2 of 14 lung metastases were found in the fourth round of transplantation. CONCLUSION: After surgical specimens of the lymph node metastasis for human tongue cancer were transplanted into the tongue of nude mice, the clinical characteristics of human tongue carcinoma, especially invasion and metastasis, were observed. This metastatic model involving orthotopic and serial transplantation should be useful for studies on the mechanisms, treatment, and prevention of human carcinoma of tongue.     

HGF的表达与口腔鳞癌VEGF-C表达及淋巴管生成关系的研究

目的：探讨口腔鳞状细胞癌（OSCC）中肝细胞生长因子（HGF）的表达与血管内皮生长因子-C（VEGF-C）的表达及淋巴管生成的关系。方法：纳入经病理证实的70例口腔鳞癌患者的手术切除标本,采用SP免疫组化技术检测HGF及VECF-C的表达情况并分析其与淋巴管密度（LVD）的关系,并结合临床病理参数作相关统计学分析。结果：淋巴结转移阳性病例的VEGF—C表达明显升高（P=0．001）,VEGF—C的表达与其他病理参数无相关,另外,HGF的表达与临床病理参数无相关。VEGF-C高表达组的瘤周淋巴管密度（PLD）明显高于VEGF—C低表达组（P=0．001）,HGF表达及VEGF—C低表达组与瘤内及瘤周淋巴管的密度无相关性。另外,HGF的表达与VEGF—C的表达间无相关。结论：VEGF—C的表达与口腔鳞癌淋巴结转移密切相关,可能主要通过参与诱导口腔鳞癌淋巴管生成促进淋巴结转移。     

Immunoexpression of matrix metalloproteinase-2 and matrix metalloproteinase-9 in the metastasis of squamous cell carcinoma of the human tongue

Background: Squamous cell carcinoma of the tongue has frequent lymph node metastases and poor prognosis. Matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9) are reported to degrade basement membrane, facilitating invasion and metastasis. This study determined the expression of MMP‐2/MMP‐9 in primary tongue cancer with or without cervical metastases and analysed the significance of such expression in relation to the presence or absence of metastases. Methods: Expressions of MMP‐2/MMP‐9 were detected by immunohistochemistry in 10 specimens of normal oral mucosa, 20 lymph node‐negative tongue cancers, 41 lymph node‐positive tongue cancers and their metastasized tumours in cervical lymph nodes. Results: MMP‐2/MMP‐9 expression was seldom found in normal epithelium. In lymph node‐negative tongue cancer, 45% and 40% of these primary tumours were positively stained for MMP‐2/MMP‐9, respectively. Importantly, in lymph node‐positive tongue cancer, 71% and 79% of these primary tumours were positive for MMP‐2/MMP‐9, respectively. Overexpression of MMP‐2/MMP‐9 was present in the metastatic lymph nodes. Conclusions: Our results imply that MMP‐2 and/or MMP‐9 play an important role in invasion and metastasis in tongue cancer, and that analysis of MMP expression and/or activity in primary tumours may have a predictive value for the actual or potential presence of cervical metastases.     

口腔鳞状细胞癌组织中HIF—1α，VEGF-C的表达及与淋巴管生成的关系

目的：研究口腔鳞状细胞癌（Orals quamous cell carcinoma,OSCC）组织中乏氧诱导因子-1α（Hypoxiainducible factor-1α,HIF—1α）,血管内皮生长因子-C（vascular endothelial growth factor,VEGF-C）表达水平及微淋巴管密度（LVD）,探讨其临床病理意义及在口腔鳞状细胞癌中的相互关系。方法：应用免疫组织化学SP法检测65例口腔鳞状细胞癌中HIF-1α,VEGF—C,淋巴管内皮细胞透明质酸受体-1（Lymphatic vessel endothelial hyaluronic acid receptor--1,LYVE—1）的表达并计数LVD。结果：65例OSCC中有43例HIF-1α呈（66．2％）高表达,VEGF--C有34例（52．3％）高表达。HIF-1α和VEGF—C高表达的病例中都出现高淋巴管密度。HIF-1a高表达分别与VEGF--C高表达显著相关（P〈0．05,χ2检验）,与高淋巴管密度显著相关（P〈0．01,Mann—Whitneyu—test）,与区域淋巴结转移显著相关（P〈0.01,χ2检验）而且也同UICCTNM分期相关（P〈0．05,χ2检验）。结论：OSCC中HIF-1α可能通过上调VEGF—C的表达来诱导淋巴管生成并最终导致肿瘤细胞发生区域淋巴结转移。