The Pilot Study On The Relationship Between Linearly Patterned Programmed Cell Necrosis And Vasculogenic Mimicry And Its Regulation In Human Melanoma

Objective:To identify if LPPCN(linearly Patterned Programmed cell necrosis) and VM (Vasculogenic Mimicry) existing in human melanoma and to study the distribution of them by morphological observation and indicate the clinical and pathological significance in melanoma.To investigate the expression of VEGF and its receptors Flt-1 and Flk-1 in surrounding tumor cells of LPPCN-positive group and VM-positive group in 70 melanoma cases,and study the interactions between VEGF and invasion and metastasis-associated proteins,to investigate their roles in tumor novel vessel construction and their clinical significance.Methods:1,Seventy melanoma cases undergoing surgery in Tumor Hospital affiliated of Tianjin Medical University from 1996 to 2007,which meet the requirements of experiments (undergoing surgery with definite pathologic diagnosis and integrated follow-up document),were collected in this study.The information of samples including gender,age, location,tumor size, date of operation,recurrence and metastasis were collected and analyzed the relationship between the clinicopathologic character of patients and prognostic.2,HE staining and CD31/PAS double staining was performed on the slides to confirm the existence of LPPCN and VM.Besides,to count the VM,LPPCN,MVD and analyze the relation among them in melanoma.Based on these,to explore the correlation between the existence of LPPCN,VM and clinicopathologic parameter clinical prognosis in the patients of melanoma.3,Immunohistochemical method was used to detect the expression of VEGF and its receptors Flt-1 and Flk-1 in 1～3 layers,4～6 layers and 7～9 layers of melanoma cells surrounding LPPCN and VM in LPPCN-positive group and VM-positive group in 70 cases.At the same time, to detect the expression of invasion and metastasis-associated proteins Twist1,MMP-2,MMP-9 in LPPCN-positive group and LPPCN-negative group,VM-positive group and VM-negative group,and in melanoma cells surrounding LPPCN.Furthermore,to analysis the relevance between the expression of VEGF and invasion and metastasis-associated proteins, to investigate their roles in the formation of VM.Results:1,LPPCN and VM exists in melanomaHE shows 38 cases existed LPPCN in 70 cases melanoma. The tumor cells undergoing LPPCN were a cluster of spindle cells with condensed cytoplasm and dark nuclei which were distributed in patterns of lines and networks in the HE staining. CD31/PAS double staining shows 33 cases existed VM. VM was tumor cells formed channel with CD31-negative,there was PAS-positive material separate tumor cells and red cells. Near these structures, there were no hemorrhage,necrosis and inflammatory cells infiltration.2,The relation between LPPCN,VM and clinicopathologic data in melanomaThe melanoma was prone to generate VM and LPPCN when its volume exceed 20cm3 (P<0.05).The occurrence of VM and LPPCN was not correlated with gender,age, location,recurrence and metastasis (P>0.05). Kaplan-Meier survive plot shows the survive time of VM-positive group was shorter than VM-negative group, the survive time of LPPCN-positive group was shorter than LPPCN-negative group (P<0.05)3,The expression of VEGF and its receptors Flt-1 and Flk-1 in surrounding LPPCN and VM tumor cellsThe expression of VEGF and Flk-1 in 1-3 layers surrounding LPPCN was higher than those in 4-61ayers and 7-91ayers(P<0.05).The expression of Flt-1 had no statistical significance(P>0.05).The expression of VEGF,Flt-1 and Flk-1 surrounding VM also had no statistical significance (P>0.05).Kaplan-Meier survive plot showed that the survival time of VEGF-positive surrounding LPPCN group was shorter than VEGF-negative group (P<0.05).The positive rate of VM in VEGF-positive group was higher than that in VEGF-negative group (P<0.05)4,The expression of Twistl,MMP-2,MMP-9 in melanoma and the relation between VEGF and themThe expression of Twistl in LPPCN-positive group was higher than LPPCN-negative group (P<0.05).The expression of Twistl in VM-positive group was higher than VM-negative group (P<0.05).The expression of MMP-2 and MMP-9 in VM-positive group was higher than VM-negative group (P<0.05).The expression of MMP-2 in LPPCN-positive group was higher than LPPCN-negative group (P<0.05).The expression of MMP-9 in LPPCN-positive group and in LPPCN-negative group had no statistical significance (P>0.05)There is a highly positive correlation between VEGF and MMP-2(r=0.334, P=0.024).There is a positive correlation between VEGF and MMP-9(r=0.118, P=0.037).There is a highly positive correlation between VEGF and Twist1 (r=0.526,P=0.003).Conclusion:1,VM and LPPCN existed in melanoma and the patients with VM or/and LPPCN has worse clinical prognosis than those without VM and LPPCN.2,LPPCN are closely related with the formation of VM and endothelium-dependent vessels in melanoma.Some tumor cells undergoing LPPCN can establish a special foundation for VM and endothelium-dependent vessels.3, VEGF and Flk-1 can play critical roles in the early stage of formation of VM in melanoma and the expression of VEGF and its receptor Flk-1 are associated with clinical prognosis of the patients with melanoma.4^ The proteins of MMP-2,MMP-9 and Twistl expressed by melanoma play a important role in the formation of VM.And them cooperate with VEGF in the formation of VM.