Clinical Significance Of Vasculogenic Mimicry In Tongue Squamous Cell Carcinoma

1.BackgroundOral and maxillofacial malignant tumor accounted for about0.94%of the human malignant tumor, one of the most common is squamous cell carcinoma, accounts forabout80%of oral and maxillofacial malignant tumor, the incidence of tongue squamous cell carcinoma in our country is about32.3%. Tongue squamous cell carcinoma occurred in front of the tongue where is flexible in general, and occurred in1/3edge of accounted for more than50%, and in the back,bottom and tip of the tongue in turn. Many of the most common risk factors involved in tongue squamous cell carcinoma development, such as alcohol and tobacco use, Long-term chronic mechanical stimulation and infection with the human papillomavirus (HPV) are recognized. Due to the physiological characteristics such as frequent mechanical movement of the tongue, rich lymphatic and blood supply, tumor cell metastasize by blood and lymphatic vessel easily. In order to improve the cure rate and patient’s quality of life, multidisciplinary and general treatment is the clinical treatment principle nowadays, which is conventional surgical treatment assistanted by chemotherapy and radiotherapy, but the cure rate is only10%to45%.Anti-angiogenesis therapy is another way for anticancer therapy in recent years. Tumor growth and metastasis depend on the formation of new blood vessels which is related to its invasive. In the early, tumor cell grow mainly through tissue penetration, but when the tumor diameter is more than1-2cm, it must form new blood vessels for nutrient supply. We almost think that tumor angiogenesis is a vascular endothelial dependent way. Anti-angiogenesis therapy, or treatment of tumor blood vessels, is different with traditional chemotherapy drugs. The drug action object is not tumor cells themselves, but effect by inhibiting or destruction of tumor angiogenesis at the molecular level. There are two main ways in anti-angiogenesis therapy, one is to stop the formation of new blood vessels, another is to destroy the tumor blood vessels existed in tumor tissue. Theoretically, anti-angiogenesis therapy should be effective, however, clinical studies have showed it is not satisfied. That may be related to the tumor can obtain blood supply except tumor angiogenesis.A new tumor microcirculation concept named Vasculogenic Mimicry (VM) is put forward in recent years. VM is different with classic tumor angiogenesis because it do not rely on vascular endothelial cells. Tumor cells round into pipe, which positive by PAS staining, morphologically diverse and connecting network, part of the tube cavity see red blood cells, approved showed blood flow by the angiography.These things confirm sample for vascular structure. Through the lens, transmission electron microscopy (sem) and vascular endothelial cell markers of immunohistochemical detection, all did not found that the presence of vascular endothelial cells. This is different from traditional angiogenesis of blood vessel structure, named it the VM. VM is characterized by tube cavity no vascular endothelial cells, and tumor cells by imitating the body’s blood vessels to form pipes, rounded by different thickness of PAS staining positive cell membrane matrix, and the blood flow in the pipeline. Most experts agree that tumor VM existed in have high malignant degree, rapid growth, high transfer rate, prone to metastasize blood, with no lined by endothelial cells and tumor is direct contact with blood. VM challenges traditional concepts of that endothelial dependent for blood vessels is the only way of tumor microcirculation, as well as important supplement of the theory of tumor angiogenesis. VM is the pipeline which meet the blood supply of highly invasive tumors, it is not dependent on vascular endothelial cells in a tumor microcirculation model,different from classic Angiogenesis approach. VM may explains the anti-tumor treatment of inhibiting tumor blood vessels present poor curative effect. Since VM is found, people began to recognize the endothelial dependent on tumor angiogenesis is not the only mode of microcirculation, tumor inhibition in the treatment of tumor microcirculation both VM and angiogenesis, especially attach importance to VM treatment can obtain good curative effect. The existence of VM and tumor growth, differentiation, invasion are closely related. Prognosis is worse with the VM. Some scholars put forward anti-angiogenesis and anti-vasculagenic mimicry therapy combined use of inhibiting tumor growth.Because the tongue characteristics of mechanical movement is frequent and lymphatic blood supply is rich, lymphatic metastasis easily occur in tongue squamous cell carcinoma patients. And the tumor existed VM also has the characteristic of easy happening early metastasis.VM studies specifically for tongue squamous cell carcinoma in clinical is significance of the treatment and prognosis for tongue squamous cell carcinoma. Yet domestic and foreign research has not been reported. Therefore, the VM has very important significance in the process of invasion and metastasis, evaluating the clinical and prognostic value, for effective control of the invasion and metastasis of tongue cancer postoperative for future treatment2.ObjectiveWe collected tongue squamous cell carcinoma specimens of pathology, with clinical data completed and tissue wax block intacted in our hospital. We organized the wax block of biopsy followed by HE staining and immunohistochemical staining, through optical microscope microscopic observation to know whether there was the VM in patients with tongue cancer. And We divided experimental data into VM positive group and negative group,combined with patient age, gender, lymph node metastasis, tumor differentiation degree. Through statistical methods, we analysed the relationship of the patients’age, gender, lymph metastasis, tumor differentiation degree between two group. We provided theoretical basis on guiding the clinical treatment of patients with tongue squamous cell carcinoma.3. Study subjects and methods3.1Study SubjectsWe collected surgically resected42specimens with clinical data completed and tissue wax block intacted of tongue squamous cell carcinoma in Zhujiang Hospital, Southern Medical University from2003to2011, aged40to81(61.2±9.8);31male and11female. Lymph node metastasis:14cases of lymph node metastasis,28cases without lymph node metastasis. Differentiation degree:20cases of high differentiation,16cases of Moderate differentiation,6cases of low differentiation. All cases are confirmed by postoperative pathology for tongue squamous cell carcinoma with completed clinical and pathologic data, and All patients have been followed up, including outpatient follow-up, hospital to review, telephone follow-up.3.2ReagentUsing mouse anti human monoclonal antibody CD34(clone number:QBEnd/10, Leica,7ml working liquid); Ready-to-use rapid immunohistochemical MaxVisionTM detection kits (KIT-5030110ml, fuzhou new biological technology Co LTD); PAS staining kits (BA4080A, zhuhai biological technology Co LTD).3.3Preparation of specimenWe detect the presence of VM using HE staining and double staining of CD34 and PAS in surgically resected42specimens with clinical data completed and tissue wax block intacted of tongue squamous cell carcinoma in Zhujiang Hospital, Southern Medical University from2003to2011.(1) HE staining:the tongue squamous cell carcinoma specimens of paraffin slice dewaxing, after HE dyeing, neutral rubber sealing piece, were observed under optical microscope lens.(2) CD34/PAS double staining:the tongue squamous cell carcinoma specimens of paraffin slice dewaxing, CD34immunohistochemical staining, control color development situation under microscope lens, after vascular endothelial cells coloring, termination of chromogenic reaction, then PAS staining, observed under optical microscope lens.3.4Statistical method and statistical softwareExperimental data were analyzed by using SPSS13.0. with four case table X2test and two independent samples nonparametric tests(rank-sum test), analysis the relationship between two group, p<0.05said differences with a statistical significance. We divided the collecting experimental data into VM positive group and negative group, we use statistical analysis software SPSS13.0with four case table X2test, and two independent samples nonparametric tests (rank-sum test), analysis the relationship of the patients’age, gender, lymph metastasis, tumor differentiation degree in two group.4.Results4.1VM exsit in tongue squamous cell carcinomaIn this experiment, We found the proportion of the VM was42.86%(18/42), by HE staining and CD34/PAS double staining. In HE staining, tumor cells arranged along the pipe, round into pipeline, containing red blood cells. In CD34/PAS double staining, tumor cells round into pipe, which around by PAS staining positive and CD34staining negative membrane, containing red blood cells, confirmed its not endothelial cells. In VM duct surrounding the pleomorphism cells, abundant cytoplasm, granular, nuclear hyperchromatism and nodules, lobulated, such as a variety of forms, nuclear membrane thickness, nuclear membrane appears cave, creases, nuclear membrane is serrated, chromatin edge, obvious nucleoli, appear large nucleoli, abnormal nuclear fission, nuclear mass ratio increases, confirmed to the cancer cells.4.2Statistical significance of VM presence in tongue squamous cell carcinomaIn this experiment, we detected the VM in18tongue squamous cell carcinoma pathological specimens, including4cases of high differentiation,9cases of Moderate differentiation,5cases of low differentiation. Compared age, gender between VM positive group and negative group respectively, there was no statistically significant difference. Lymph node metastasis rate with the VM positive group was50.00%, and the VM negative group was20.83%, checked by four case table X2test, P=0.049, the difference was statistically significant. We think there is different about lymph node metastasis rate in two groups, the lymph node metastasis rate in the VM positive group patients is higher than the VM negative groups. Compared tumor differentiation degree between VM positive group and negative group, using rank-sum test, p=0.002, the difference was statistically significant. We think there is different about tumor differentiation degree in two groups, confirmed that tumor differentiation degree in VM positive group is lower than VM negative group.5.Conclusions1. VM can be observed by CD34/PAS double staining in tongue squamous cell carcinoma.2. The higher malignant degree of tongue squamous cell carcinoma has stronger ability to form VM.3. Tongue squamous cell carcinoma with VM was detected showed lower differentiation degree and higher lymph node metastasis rate.