Study On Factors Associated With Pelvic Lymph Node Metastasis In The Patients With Early-stage Squamous Cell Carcinoma Of The Uterine Cervix

Globally, cervical cancer is the second most common cause of cancer death in women, with an estimated 510, 000 newly diagnosed cervical cancer cases and 288, 000 deaths in 2006. In developing countries, cervical cancer is often the most common cancer in women. Lymph node metastasis is one of the most important biological behaviors of cervical cancer including early-stage patients. Lymph node status is the most important prognostic factor in cervical cancer. However, up to date, there was no good method to evaluate pelvic lymph node status before surgery.Previous studies have demonstrated that several factors, including clinical stage, tumor size, serum squamous cell carcinoma antigen levels, histologic type, grade, depth of invasion and vascular invasion, may influence pelvic lymph node metastasis in patients with early-stage squamous cell carcinoma of the uterine cervix. However, it is very difficult to predict the status of pelvic lymph node metastasis before operation according to these clinicopathological features. The sentinel lymph node (SN) is the first node draining the lymphatic flow from a primary tumor, and represents the status of lymphatic spread. SN detection (SND) in cervical cancer is a feasible and highly accurate method toestimate nodal status. However, low detection rate, false negativity, and exigent histological assessment limited the further implications of SND in clinic. Before the standard application of the SN technique as a diagnostic tool, the results of large multicentre trials must be critically evaluated; it must be proved that SND has no negative impact on survival but reduces morbidity.Imaging technique is the major method to evaluate lymph node status. However, the sensitivities of CT and MRI are not so good, because they can not distinguish metastasis from necrosis and can not detect the lymph node less than 1 cm. Positron emission tomography (PET) imaging with the radiolabeled glucose analogue 2-(fluorine-18)-fluoro-2-deoxy-D-glucose (F—18 FDG) has been widely used to visualize primary and metastatic tumors. Although the sensitivity and specificity of lymph metastasis diagnosed by FDG-PET were both higher than by CT or MRI, some smaller metastatic lymph node might be neglected by FDG-PET.Tumor metastasis is a complex and multifactorial process which involved with oncogenes, tumor suppression genes and tumor metastasis associated genes. The difficulty in metastasis study is largely due to the complex nature of this multi-step process. Microarray is a new powerful tool for studying the molecular basis of interactions on a scale that is impossible using conventional analysis. This technique makes it possible to examine the expression of thousands of genes simultaneously. In cancer research, it will provide high-throughput and valuable insights into differences in an individual's tumor as compared with constitutional DNA, mRNA expression, and protein expression and activity. Across individuals, comparisons could provide tissue-specific disease signatures that provide diagnosis based on hundreds of informative genes. The resulting product should be a wealth of tumor-associated and tumor-specific biomarkers, which may help in cancer etiology, diagnosis,and therapy and ultimately lead to "molecular oncology" of cancers.Over the past decade, microarray technology has become a powerful tool to provide a genome-wide view of genetic or epigenetic changes associated with tumor metastasis. For example, in one report, genetic profiles of subcutaneously grown metastatic human and mouse melanoma cell lines were compared to profiles derived from subcutaneously grown, non-metastatic lines. This analysis revealed that almost all metastatic genes continued to be overexpressed in metastatic cells grown subcutaneously. Altogether, 32 genes and expressed sequence tags (ESTs) whose expression patterns positively correlated with the metastastic potential of tumor cells could be identified. In another report, an established human breast cancer cell line was used to select for subclones that specifically metastasize to bone. Gene expression profiling revealed that overexpression of interleukin-11 (IL 11), connective tissue growth factor (CTGF), the chemokine receptor CXCR4, fibroblast growth factor 5 (FGF5), the multifunctional adhesion factor and metastasisrelated protein osteopontin, and the matrix metalloprotease MMP1 correlated with bone metastasis.However, rare similar study of cervical cancer was carried out. Uniquely, Lyng et al have used microarrays to identify gene expressions associated with metastatic phenotypes of locally advanced cervical cancers. Thirty-one genes that differed in expression between the node positive and negative tumors were identified. Expressions of eight of these genes (MRPL11, CKS2, PDK2, MPS23, MSN, TBX3, KLF3, LSM3) correlated with progression free survival in univariate analysis and were therefore more strongly associated with metastatic phenotypes than the others.The goal in this study is to investigate the mechanisms of lymph node metastasis in early-stage cervical cancer. Firstly, to find the clinical high risk factors of lymph node metastasis of cervical cancer, therelationship between lymph node metastasis and clinicopathological features, serum squamous cell carcinoma antigen levels, and coagulation indexes of patients with stage ⅠB～ⅡA squamous cell carcinoma of cervix was analysed. Secondly, to identify gene expressions associated with metastatic phenotypes of early-stage cervical carcinomas, we investigated the difference of gene expression profile in cervical cancer patients with and without diagnosed lymph node metastases by use of cDNA microarray techniques. And the gene expression changes were validated at the RNA and protein levels by reverse transcription PCR and western blot analysis, respectively.Part 1 clinicopathological features influencing pelvic lymph node metastasis in patients with early-stage squamous cell carcinoma of the uterine cervixObjective: To evaluate clinical and pathologic findings predicting pelvic lymph node metastasis in patients with early-stage squamous cell carcinoma of the uterine cervix.Materials and Methods: 135 patients with stage ⅠB—ⅡA cervical squamous cell carcinoma in Women's Hospital, School of Medicine, Zhejiang University, were retrospectively studied from February 2004 to January 2007. The relationship between pelvic lymph node metastasis and age, clinical stage, tumor size, grade of differentiation, depth of muscular invasion, lymphatic vascular space invasion, pretreatment level of serum squamous cell carcinoma antigen, pretreatment plasma level of fibrinogen, pretreatment levels of hemoglobulin and platelet were evaluated by the univariate and multivariate analysis.Results: Totally, 3996 lymph nodes were dissected in 135 patients, with an average of 29.6 lymph nodes in each patient (range: 20-47). 12.7% patients (17/135) had metastasized pelvic lymph node. Tumor size,pretreatment levels of fibrinogen and platelet, depth of muscular invasion and lymphatic vascular space invasion were significantly related with lymph node metastasis (relative risk = 2.701, 1.011, 3.574, 3.165 and 3. 930, respectively). Multivariate analysis showed that pretreatment plasma level of fibrinogen and lymphatic vascular space invasion were independently related to lymph node metastasis in patients with early-stage squamous cell carcinoma of the uterine cervix. Conclusions: In early-stage cervical squamous cell carcinoma, pretreatment plasma levels of fibrinogen and lymphatic vascular space invasion were independently related to lymph node metastasis. Part 2 Gene expression profile in early-stage cervical squamous cell carcinoma with pelvic lymph node metastasisObjective: To investigated the difference of gene expression profile in cancer tissues between early-stage cervical squamous cell carcinoma patients with and without lymph node metastasis.Materials and Methods: By human genome array chip containing 38500 genes (Affymetrix U133 plus 2.0), the gene expression profile between cervical squamous cell cancer tissue with and without lymph node metastasis and normal cervical tissue were compared and analyzed.Results: A total of 322 differentially expressed genes were screened out in cervical squamous cell cancer tissue with lymph node metastasis. Among these genes, 182 were up-regulated and the other 140 were down-regulated. 23 genes were identified as markedly differentially expressed (>4 fold), among which, 14 were up-regulated and the other 9 were down-regulated in cancer tissue with lymph node metastasis. No differentially expressed gene was found between normal cervical tissue and cancer tissue without lymph node metastasis. There were 33 genes differentially expressedbetween cervical tissues of squamous cervical cancinoma with and without pelvic lymph node metastasis, identified (>2 folds). Among these, 17 genes were up-regulated and 16 genes were down-regulated in cancer tissue with lymph node metastasis. Among the up-regulate genes in cancer tissue without lymph node metastasis, 8 genes were down-regulated in cancer tissue with lymph node metastasis, and no gene was further up-regulated in cancer tissue with lymph node metastasis,. And among down-regulate genes in cancer tissue without lymph node metastasis, there were 6 genes up-regulated in cancer tissue with lymph node metastasis.Conclusions: Lymph node metastasis of cervical squamous cell cancer was a process, in which, numerous genes expression were involved. Microarray is a powerful tool to screen out metastasis associated genes, which may help in prediction of lymph node metastasis.Part 3 Expression of VSNL1, VCAM1,autotaxin,septin 11 in early-stage cervical squamous cell carcinoma and its association with pelvic lymph node metastasisObjective: To validate the association between several differentially expressed genes screened out by gene chips and pelvic lymph node metastasis in cervical squamous cancer.Methods: Fluorescent real-time quantitative RT-PCR and Western blot were employed to validate the different expression of 4 candidate genes: VCAM1, VSNL, autotaxin and septin 11 in tissues from normal cervical, and squamous cervical cancer with and without pelvic lymph node metastasis. Results: The relative expression level of VCAM1 mRNA in cervical cancer tissues was significantly higher than that in normal cervical tissues. Compared with cancer without lymph metastasis, the expression of VCAM1mRNA was further elevated in cancer with lymph metastasis (30.473 vs 828.259). The relative expression level of VSNL mRNA in cervical cancer tissues was significantly higher than that in normal cervical tissues. However, there was no significant difference between the expression of VSNL mRNA in cancer with and without lymph metastasis (731.472 vs 69.363). The relative expression level of autotaxin mRNA in cervical cancer tissues was significantly lower than that in normal cervical tissues. Compared with cancer without lymph metastasis, the expression of autotaxin mRNA was further decreased in cancer with lymph metastasis (36.944 vs 0.408). The relative expression level of septin 11 mRNA in cervical cancer tissues was significantly lower than that in normal cervical tissues. Compared with cancer without lymph metastasis, the expression of autotaxin septin 11 was further decreased in cancer with lymph metastasis (53.970 vs 0.058). The expression level of VCAM1 protein in cervical cancer tissues was significantly higher than that in normal cervical tissues (0.414 vs 0.262). Compared with cancer without lymph metastasis, the expression of VCAM1 protein was further elevated in cancer with lymph metastasis (0.414 vs 0.907). The expression level of VSNL protein in cervical cancer tissues was significantly higher than that in normal cervical tissues (0.436 vs 0.253. However, there was no significant difference between VSNL protein expression in cancer with and without lymph metastasis. The expression level of septin 11 protein in cervical cancer tissues was significantly lower than that in normal cervical tissues (0.509 vs 0.780). However, there was no significant difference between septin 11 protein expression in cancer with and without lymph metastasis.Conclusions: 1. The expression of VCAM1 was significantly up-regulated in tissues of early stage cervical squamous cell carcinoma with pelvic lymph node metastasis. And it was indicated that VCAM1 play an important role in the development of pelvic lymph node metastasis in early stage cervicalsquamous cell carcinoma. 2. The expression levels of VSNL1, autotaxin, and septin 11 in early stage cervical squamous cell carcinoma with pelvic lymph node metastasis were different from other malignancies, and the roles of these genes in the development of pelvic lymph node metastasis need further studies.