Maintenance of head and neck tumor gene expression profiles upon lymph node metastasis

Spread of cancer and development of solid metastases at distant sites is the main cause of cancer-related deaths. To understand and treat metastases, it is important to determine at which stages the most pivotal steps for development of metastases occur. In head and neck squamous cell carcinoma (HNSCC), metastasis nearly always occurs first in local lymph nodes before development of distant metastasis. Here, we have investigated gene expression patterns in HNSCC lymph node metastases using DNA microarrays. Several types of analyses show that the gene expression patterns in lymph node metastases are most similar to the corresponding primary tumors from which they arose, as long as samples contain sufficient proportions of tumor cells. Strikingly, gene expression patterns of metastatic primary HNSCC are largely maintained upon spread to the lymph node. Only a single gene, metastasis-associated gene 1 (MTA1), was found to show consistently changed expression between a large number of matched primary tumor-lymph node metastasis pairs. The maintained expression pattern includes the predictive signature for HNSCC lymph node metastasis. These results underscore the importance of the primary tumor gene expression profile for development and treatment of metastasis. The findings also agree with the concept that disseminated cancer cells alter the surrounding tissue into a metastatic environment that resembles the primary tumor microenvironment.     

The role of heparanase in lymph node metastatic dissemination: dynamic contrast-enhanced MRI of Eb lymphoma in mice

Heparanase expression has been linked to increased tumor invasion, metastasis, and angiogenesis and with poor prognosis. The aim of the study was to monitor the effect of heparanase expression on lymph node metastasis, in heparanase-overexpressing subcutaneous Eb mouse T-lymphoma tumors, and their draining lymph node. Dynamic contrast-enhanced magnetic resonance imaging (MRI) using biotin-BSA-GdDTPA-FAM/ROX was applied for analysis of blood volume, vascular permeability, and interstitial convection, and for detection of very early stages of such metastatic dissemination. Eb tumors increased extravasation, interstitial convection, and lymphatic drain of the contrast material. Interstitial flow directions were mapped by showing radial outflow interrupted in some tumors by directional flow toward the popliteal lymph node. Heparanase expression significantly increased contrast enhancement of the popliteal lymph node but not of the primary tumor. Changes in MR contrast enhancement preceded the formation of pathologically detectable metastases, and were detectable when only a few enhanced green fluorescent protein (EGFP)-expressing Eb cells were found near and within the nodes. These results demonstrate very early, heparanase-dependent vascular changes in lymph nodes that were visible by MRI following administration of biotin-BSA-GdDTPA-FAM/ROX, and can be used for studying the initial stages of lymph node infiltration.     

肝素酶基因表达与肺癌转移活性的初步研究

目的：探讨肝素酶基因表达与肺癌转移的关系。方法：应用肝素酶基因引物、逆转录聚合酶链反应技术（RT－PCR）检测肺巨细胞癌株和56例原发性肺癌及正常肺组织中肝素酶基因表达,结果：低转移和高转移的肺巨细胞癌株均为肝素酶基因表达阳性;已有淋巴转移的肺癌阳性率为80％（20／25）,而无淋巴结转移的肺癌阳性率为6．5％（2／31）,两组间差异有显著性（P＜0．001）。提示伴有淋巴结转移的病例肝素酶基因表达阳性率明显高于不伴转移者。肝素酶基因表达多见于鳞癌（P＜0．05）和低分化癌（P＜0．01）,亦提示肝素酶基因表达可能与组织学分型及分化程度有关。结论：初步结果显示肝素酶基因表达可能为肺癌细胞获得转移活性的可靠标志之一,其检测可用于辅助判断肺癌患者预后。     

乙酰肝素酶基因表达与胃癌临床病理特点的关系

目的探讨乙酰肝素酶基因(HPAmRNA)在胃癌中的表达及其与临床病理因素之间的相互关系。方法选用43例胃癌组织和10例癌旁正常胃组织,用逆转录聚合酶链反应(RTPCR)检测标本中HPAmRNA的表达,并结合患者临床病理指标进行分析。结果43例胃癌中,29例HPAmRNA表达阳性,其阳性率显著高于癌旁正常胃组织(P=0.013)。HPAmRNA的表达与TNM分期、有无浆膜浸润、淋巴结转移、远处转移以及与肿瘤大小有相关性(P<0.05);与患者年龄、性别、肿瘤所在部位、肿瘤的Borrmann分型、组织学类型、分化程度、腹膜转移和肝转移无相关性(P>0.05)。结论HPAmRNA阳性表达的胃癌有较高的侵袭转移性,HPA可能是胃癌侵袭转移中的一个重要酶,可能与胃癌的淋巴结转移有关。     

CCR7 and CXCR4 Expression in Primary Head and Neck Squamous Cell Carcinomas and Nodal Metastases – a Clinical and Immunohistochemical Study

Background: Squamous cell carcinomas (SCCs) are common head and neck malignancies demonstrating lymph node LN involvement. Recently chemokine receptor overxpression has been reported in many cancers. Of particular interest, CCR7 appears to be a strong mediator of LN metastases, while CXCR4 may mediate distant metastases. Any relations between their expression in primary HNSCCs and metastatic lymph nodes need to be clarified. Aims: To investigate CCR7 andCXCR4 expression in primary HNSCCs of all tumor sizes, clinical stages and histological grades, as well as involved lymph nodes, then make comparisons, also with control normal oral epithelium. Materials and Methods: The sample consisted of 60 formalin-fixed, paraffin-embedded specimens of primary HNSCCs, 77 others of metastasi-positive lymph nodes, and 10 of control normal oral epithelial tissues. Sections were conventionally stained with H&E and immunohistochemically with monoclonal anti-CCR7 and monoclonal anti-CXCR4 antibodies. Positive cells were counted under microscopic assessment in four fields (X40) per case. Results: There was no variation among primary HNSCC tumors staining positive for CCR7 and CXCR4 with tumor size of for CCR7 with lymph node involvement. However, a difference was noted between primary HNSCC tumors stained by CXCR4 with a single as compared to more numerous node involvement. CXCR4 appear to vary with the clinical stagebut no links were noted with histological grades. Staining for primary HNSCC tumors and metastatic lymph nodes correlated.     

Heparanase is overexpressed in lung cancer and correlates inversely with patient survival

BACKGROUND: Heparanase is an endo-beta-D-glucuronidase that is capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size (approximately 5-7 kDa). Heparanase activity has been detected frequently in several cell types and tissues. Heparanase activity correlates with the metastatic potential of tumor-derived cells, a correlation that has been attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix barrier. METHODS: In this study, the authors examined heparanase expression in 114 patients with lung cancer by means of immunohistochemistry and correlated clinical-pathologic data with heparanase immunostaining and cellular localization. RESULTS: Heparanase was overexpressed in 75% of the study patients. Heparanase expression was correlated with lung cancer lymph node status and metastasis classification (P = .04 and P = .01, respectively) and was correlated inversely with patient survival (P = .007). It is noteworthy that this adverse effect depended largely on the cellular localization of heparanase. Thus, whereas cytoplasmic staining of heparanase is associated with a poor prognosis, nuclear heparanase predicts a favorable outcome for patients with lung cancer. CONCLUSIONS: The current findings suggest that heparanase expression and cellular localization are decisive for lung cancer patients' prognosis, most likely because of heparanase-mediated tumor cell dissemination by blood and lymph vessels.     

Expression of heparanase in renal cell carcinomas: implications for tumor invasion and prognosis

PURPOSE: Heparanase activity has been detected in many malignant tumors, showing a correlation with the metastatic potential. The present study was undertaken to investigate the expression of heparanase and its prognostic significance in renal cell carcinomas (RCC). EXPERIMENTAL DESIGN: Nineteen RCCs and 6 nonneoplastic renal tissues were analyzed for heparanase mRNA expression by real-time PCR. Heparanase protein expression was semiquantitatively investigated by immunohistochemistry in 70 RCCs. Involvement of heparanase in the invasiveness of RCC cell lines, 786-O and Caki-2 cells, was examined by down-regulating the gene expression with small interfering RNA (siRNA) using the Matrigel invasion assay. RESULTS: The expression level of heparanase mRNA was significantly higher in clear cell RCCs than in papillary RCCs, chromophobe RCCs, and nonneoplastic renal tissues. Heparanase was predominantly immunolocalized to cell surface and cytoplasm of clear cell RCCs and mean expression levels of heparanase were significantly higher in clear cell RCCs than in papillary and chromophobe RCCs. The protein expression levels were positively correlated with primary tumor stage, distant metastasis, and histologic grade. Targeting of heparanase mRNA expression in 786-O and Caki-2 cells with siRNA down-regulated the mRNA expression and inhibited the Matrigel invasion by these cells, whereas nonsilencing siRNA showed no effect. Multivariate Cox analysis revealed that elevated heparanase expression was a significant and an independent predictor of disease-specific survival (odds ratio, 8.814; P = 0.019). CONCLUSIONS: These data suggest that heparanase plays an important role in invasion and metastasis and silencing of the gene might be a potential therapeutic target in clear cell RCCs.     

Staging of head and neck squamous cell carcinoma using the MET oncogene product as marker of tumor cells in lymph node metastases

In head and neck squamous cell carcinomas (HNSCC), metastasis to cervical lymph nodes is a major determinant of patient outcome. To detect metastases, we used the MET oncogene as marker, which encodes the receptor for hepatocyte growth factor/scatter factor, mediating epithelial cell motility and invasiveness. The MET gene is expressed in epithelia and over-expressed in carcinomas of specific histotypes, but not in lymphatic tissue. A total of 151 lymph nodes from 20 squamous cell carcinomas were studied with both in-depth histology and end-point and real-time quantitative RT-PCR. MET-encoded sequences were found in 61 of 151 nodes (40%), of which 24 (16%) were found metastatic by in-depth histopathology. Parallel routine histopathologic analysis of 654 lymph nodes from the same cases identified 36 metastases (5%). Real-time quantitative RT-PCR was used to measure MET gene-specific mRNA in normal tissues, primary tumors and lymphatic metastases and showed a 2-8-fold increased expression in tumor cells which metastasize. RT-PCR for 3 cytokeratins expressed in HNSCC (K4, K10 and K13) proved to be less sensitive in detecting occult lymphatic metastases. Western blot analysis demonstrated the presence of the full-size MET receptor in primary tumors and lymph node metastases; immunohistochemistry showed receptor localization in tumor cells. Altogether, these data demonstrate that the MET gene product is a valuable marker with which to detect occult tumor cells in lymph nodes, thanks to its high expression in metastatic cells. After RT-PCR analysis we were able to attribute a more advanced stage to 10 out of 20 HNSCC cases, including 5 cases classified as tumor-free after routine histopathology.     

Expression of heparanase by primary breast tumors promotes bone resorption in the absence of detectable bone metastases

Heparanase is an enzyme that cleaves heparan sulfate and through this activity promotes tumor growth, angiogenesis, invasion, and metastasis in several tumor types. In human breast cancer patients, heparanase expression is associated with sentinel lymph node metastases. However, the precise role of heparanase in the malignant progression of breast cancer is unknown. To examine this, a variant of MDA-MB-231 cells was transfected with the cDNA for human heparanase (HPSE cells) or with vector alone as a control (NEO cells). Transfection produced a 6-fold increase in heparanase activity in HPSE cells relative to NEO cells. When injected into the mammary fat pads of severe combined immunodeficient mice, the tumors formed by HPSE cells initially grow significantly faster than the tumors formed by NEO cells. The rapid growth is due in part to increased angiogenesis, as microvessel densities are substantially elevated in primary HPSE tumors compared with NEO tumors. Although metastases to bones are not detected, surprisingly vigorous bone resorption is stimulated in animals bearing tumors formed by the HPSE cells. These animals have high serum levels of the C-telopeptide derived from type I collagen as well as significant elevation of the active form of tartrate-resistant acid phosphatase (TRAP)-5b. In contrast, in animals having a high tumor burden of Neo cells, the serum levels of C-telopeptide and TRAP-5b never increase above the levels found before tumor injection. Consistent with these findings, histologic analysis for TRAP-expressing cells reveals extensive osteoclastogenesis in animals harboring HPSE tumors. In vitro osteoclastogenesis assays show that the osteoclastogenic activity of HPSE cell conditioned medium is significantly enhanced beyond that of NEO conditioned medium. This confirms that a soluble factor or factors that stimulate osteoclastogenesis are specifically produced when heparanase expression is elevated. These factors exert a distal effect resulting in resorption of bone and the accompanying enrichment of the bone microenvironment with growth-promoting factors that may nurture the growth of metastatic tumor cells. This novel role for heparanase as a promoter of osteolysis before tumor metastasis suggests that therapies designed to block heparanase function may disrupt the early progression of bone-homing tumors.     

Reduced KAI1 expression in pancreatic cancer is associated with lymph node and distant metastases

KAI1 belongs to a structurally distinct family of membrane glycoproteins, which function via cell-cell and cell-extracellular matrix interactions, thereby potentially influencing the ability of cancer cells to invade tissues and to metastasize into lymph nodes and distant organs. In the present study, we examined KAI1 expression in lymph node and liver metastases in comparison with primary pancreatic cancer to evaluate its influence on metastasis. KAI1 mRNA analysis was performed by Northern blot analysis and in situ hybridization. In addition, the respective protein was studied by immunostaining. Fourteen primary pancreatic cancer samples in which no lymph node metastases were present and 25 primary pancreatic cancer samples in which lymph node metastases were present at the time of tumor resection were included. In 20 of these cases, primary pancreatic cancer tissues and corresponding lymph node metastases from the same patient were studied. Furthermore, 11 liver metastases were available for KAI1 analysis. Increased steady-state levels of KAI1 mRNA were found in 33/39 (85%) primary pancreatic cancers in comparison with normal controls. Statistical analysis of KAI1 mRNA levels and clinical parameters of the patients revealed that KAI1 mRNA levels were significantly higher in non-metastasized tumors compared with tumors in which lymph node or distant metastases were present. In lymph node metastases KAI1 mRNA expression was lower than in the corresponding primary tumors: In 14 of 20 lymph node metastases no KAI1 mRNA expression and in 6 of 20 lymph node metastases only weak KAI1 mRNA levels were present in some cancer cells. Cancer cells of distant metastases were devoid of or exhibited low KAI1 mRNA levels compared with those of primary pancreatic cancers. A similar pattern was observed by immunostaining. These data support the hypothesis that KAI1 gene expression might influence the metastatic ability of pancreatic cancer cells in vivo. Reduction of KAI1 appears to promote cancer cell spread in lymph nodes and distant organs. Int. J. Cancer (Pred. Oncol.) 79:349–355, 1998. © 1998 Wiley-Liss, Inc.     

乙酰肝素酶在乳腺癌侵袭转移中的作用及调控机制*

乙酰肝素酶（Heparanase ,HPSE）是目前发现的哺乳动物细胞中唯一能降解细胞外基质和基底膜中硫酸肝素蛋白多糖侧链—硫酸乙酰肝素的内源性糖苷酶,是目前抗肿瘤转移的理想靶点。HPSE在乳腺癌中常有高表达,并与肿瘤大小和淋巴结转移等密切相关。体外研究表明,乳腺癌细胞中HPSE启动子活性增高,转染HPSE的乳腺癌细胞在体内成瘤后,其肿瘤体积、重量、微血管密度以及癌细胞存活时间均明显高于阴性对照组。应用反义核酸技术或RNA干扰技术封闭或沉默HPSE基因表达后,乳腺癌细胞黏附和侵袭能力显著降低,表明HPSE在乳腺癌侵袭转移中发挥极其重要的作用。HPSE在乳腺癌侵袭转移中受多种机制调控,主要有:雌激素与其受体结合后作用于HPSE基因特定区域,从而提高HPSE转录活性,增强其基因和蛋白的表达;HPSE可使破骨细胞刺激因子产生增加,从而导致骨质溶解破坏,为乳腺癌骨转移奠定基础;HPSE诱导循环淋巴细胞产生刺激因子,从而促进乳腺癌的侵袭转移。此外,HPSE基因还受p53基因、ETS 基因、EGR 1 基因、PI-88因子、COX-2 等的调控。本文就HPSE在乳腺癌中的表达状况、在侵袭转移中的作用及调控机制进行综述。      

乙酰肝素酶mRNA表达和血管生成与胃癌发展的关系

目的　探讨乙酰肝素酶mRNA表达状况和血管生成与胃癌发展的关系。方法　应用原位杂交技术 ,检测 5 2例胃癌组织中乙酰肝素酶mRNA的表达情况 ,并用免疫组化法对全部标本用CD34抗体进行肿瘤血管内皮染色 ,计数肿瘤微血管密度 (MVD) ,分析乙酰肝素酶mRNA与MVD及其与胃癌组织分化程度、浸润深度、淋巴结转移和器官转移的关系。结果　胃癌组织乙酰肝素酶mRNA阳性表达 2 5例 (4 8.1% ) ,MVD平均值为 73.2± 2 2 .8;阴性表达 2 7例 (5 1.9% ) ,MVD平均值为 4 4 .8±11.9,两者之间差异有显著性 (P <0 .0 0 1)。乙酰肝素酶mRNA表达和MVD与胃癌组织浸润深度、淋巴结转移有关 (P <0 .0 0 5 )。结论　乙酰肝素酶与胃癌的血管生成密切相关 ,对胃癌的生长和浸润转移有促进作用 ,乙酰肝素酶可作为反映胃癌生物学行为的客观指标。     

Clinical significance of heparanase activity in primary resected non-small cell lung cancer

Clinicopathological significances of heparanase activity in non-small cell lung cancer (NSCLC) were investigated by analyzing 76 resected specimens of NSCLC. Heparanase activities in NSCLC were significantly higher than non-cancerous lung tissues (P < 0.0001). The heparanase activities of NSCLC were significantly higher in larger diameter tumors (P = 0.0141) or with metastasis to ipsilateral mediastinal lymph nodes (P = 0.0004). The activities of heparanase in primary tumors were increased significantly according to the pathological stage of the progression of the disease (P =0.0009). Among the clinicopathological parameters, histological cell type and evidence of ipsilateral lymph node metastasis showed a significant association with elevated heparanase activities, whereas age, degree of differentiation and tumor diameter did not. Kaplan-Meier curves for overall and disease-free survival demonstrated a significant difference between patients with elevated and non-elevated heparanase activity by log-rank test (P = 0.0145 and 0.0002, respectively). Multivariate analysis showed heparanase activity was an independent factor to influence disease-free survival in our study. These results suggest that heparanase activity could be used as a prognostic indicator for postoperative patients with NSCLC and heparanase might be a promising molecular target for treatment of NSCLC.     

Efficient engraftment of human primary breast cancer transplants in nonconditioned NOD/Scid mice

We describe a new human tumor xenotransplant animal model that is highly efficient for engraftment, does not need host conditioning and is suitable for in vivo studies of human tumors. Pieces of 61 freshly operated primary breast tumors were implanted into 172 irradiated and 228 nonconditioned NOD/Scid mice. A high mortality was observed in irradiated but not in nonconditioned recipients. More than 90% of analyzed implanted breast cancer specimens engrafted in the NOD/Scid mice irrespective of pretreatment. The tumors were vascularized within 3 days of implantation and maintained original histomorphology as well as expression patterns of tumor markers (cytokeratin and MUC1) and cytokines (tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4) and IL-10) released by adjacent stromal cells. A majority of tumors grew slowly, locally infiltrating host tissue, whereas some grew aggressively, developing large, fatal tumor masses and metastases within regional lymph nodes. Tumor progression in mice correlated with stage, grade, proliferation index and hormone receptor status of primary tumors. The reproducible growth behavior and preservation of characteristic features suggest that this new xenotransplant model is relevant and can be recommended for testing new anticancer therapies.     

Discrimination of Cancer Stem Cell Markers ALDH1A1, BCL11B,BMI-1, and CD44 in Different Tissues of HNSCC Patients

Cancer stem cells (CSCs) are accountable for the progress of head and neck squamous cell carcinoma (HNSCC). This exploratory study evaluated the expression of molecular CSC markers in different tissues of HNSCC patients. Tissue specimens of primary tumor, lymph node metastases and macroscopically healthy mucosa of 12 consecutive HNSCC patients, that were treated with surgery and adjuvant radio(chemo)therapy upon indication, were collected. Samples were assessed for the expression of p16 as a surrogate for HPV-related disease and different molecular stem cell markers (ALDH1A1, BCL11B, BMI-1, and CD44). In the cohort, seven patients had HPV-related HNSCC; six thereof were oropharyngeal squamous cell carcinoma. While expression of BMI-1 and BCL11B was significantly lower in healthy mucosa than both tumor and lymph node metastasis, there were no differences between tumor and lymph node metastasis. In the HPV-positive sub-cohort, these differences remained significant for BMI-1. However, no significant differences in these three tissues were found for ALDH1A1 and CD44. In conclusion, this exploratory study shows that CSC markers BMI-1 and BCL11B discriminate between healthy and cancerous tissue, whereas ALDH1A1 and CD44 were expressed to a comparable extent in healthy mucosa and cancerous tissues.     

乙酰肝素酶mRNA在大肠癌中的表达及意义

目的:探讨乙酰肝素酶在大肠癌中的表达及意义.方法:应用原位杂交方法检测乙酰肝素酶mRNA在45例大肠癌组织中的定位及表达.结果:45例大肠癌组织中,乙酰肝素酶mRNA阳性表达22例(48.88%).26例大肠癌伴淋巴结转移者,其原发灶内乙酰肝素酶mRNA表达明显高于无转移组,有显著差异(P＜0.01).乙酰肝素酶mRNA表达与大肠癌组织浸润深度、淋巴结转移有关.结论:乙酰肝素酶可能在大肠癌的生长、浸润和转移中起一定的作用.     

Expression of heparanase in hepatocellular carcinoma has prognostic significance: a tissue microarray study

Heparanase plays an important role in invasion and metastasis of tumor cells. In this study, we explored the expression and clinicopathological significance of heparanase protein in hepatocellular carcinogenesis to investigate their roles in invasion and the relationship between biological behavior and prognosis of hepatocellular carcinoma (HCC) in tissue microarrays (TMAs). Heparanase expression was examined by immunohistochemistry in TMAs comprising 120 cases of HCC, 48 cases of adjacent tumor liver, 62 cases of cirrhosis, and 23 cases of normal liver tissues. Statistical analyses were determined to access the correlation between heparanase expression and the clinicopathological features of HCC. The results showed a positive level of heparanase in HCC tissues that was significantly higher than that in adjacent tumor liver, cirrhosis, and normal liver tissue. Heparanase was expressed lower in clinical TNM stages I and II than in III and IV. Moreover, the expression of heparanase in cases without metastasis within 20 months was statistically lower than in those with metastasis. Furthermore, heparanase expression in groups of alpha-fetoprotein (AFP) > or = 400 microg/L, portal vein tumor emboli, multiple tumor nodes, and tumor diameter > or = 5 cm were significantly higher than those of corresponding groups, while it was not associated with patients' age, sex, histological classification, cirrhosis, or tumor capsular infiltration. In conclusion, TMA is a powerful tool for the rapid identification of molecular alterations in HCC. The overexpression of heparanase may play an important role in hepatocarcinogenesis, progression, and metastases of HCC. It could serve as a determining factor for clinical prognosis and curative effect.