The Correlation And Clinical Significance Between Linearly Patterned Programmed Cell Necrosis And Tumor Microvascular Architecture

Objective1.To investigate the morphological characteristics and the regulation involving the distribution of linearly patterned programmed cell necrosis in human malignant melanoma and to indicate the clinical and pathological significance of LPPCN through analyzing the correlations between linearly patterned programmed cell necrosis and clinical and histopathologic parameters of patients with human malignant melanoma.2. The expressions of regulators related with angiogenesis are tested to elucidate that the correlations between linearly patterned programmed cell necrosis and the expressions of the regulators related with angiogenesis in malignant melanoma. The computer softwares are used to analyze whether there is similarity between the distribution and tendency of linearly patterned programmed cell necrosis and that of tumor novel vessel construction. Finally, to disclose the role and significance of linearly patterned programmed cell necrosis in tumor novel vessel construction.3. To examine the expressions of apoptosis- and autophage-related proteins to demonstrate the important role of these proteins in the process of formation of linearly patterned programmed cell necrosis. The aim is to define the molecule mechanisms involving the formation of linearly patterned programmed cell necrosis in human malignant melanoma.Methods1. The method of histopathologic examination was used to identify the morphological characteristics and the regulation of the distribution and pattern of linearly patterned programmed cell necrosis in human malignant melanoma. Meanwhile, Many of general features and conventional necrosis of malignant melanoma were examined.2. Immunohistochemical staining for HMB45, Ki67, activators of angiogenesis, apoptosis- and autophage-related proteins were performed to observe the difference of the expressions of these proteins between the positive group of linearly patterned programmed cell necrosis and that of the negative group. PAS-CD31 double staining was used to observe the structure and distribution of vasculogenic mimicry and tumor novel vessel construction. To analyze the roles of these parameters in the process of linearly patterned programmed cell necrosis.3. Real time quantitation PCR were performed to detect the mRNA expressions of activators of angiogenesis and autophagy-related protiens in malignant melanoma tissues. To observe the difference of the expressions of these proteins between the positive group of linearly patterned programmed cell necrosis and that of the negative group.4. Tissue chip technique was utilized in making tmour successive sections for CD31 staining. Next, the images were obtained by computer image acquisition system.5. To analyze the vector of the two-dimensional images and to construct the three-dimensional images to demonstrate the similarity between the distribution and tendency of linearly patterned programmed cell necrosis and that of tumor novel vessel construction.Results1. The results of etailed histopathological examinations showed that linearly patterned programmed cell necrosis was identified generally present in some highly aggressive tumor tissues of patients with malignant melanoma, the positive rate of it is 54.29% in seventy samples.2. The presence of linearly patterned programmed cell necrosis has close positive correlation with the following parameters in malignant melanoma:e.g. ulceration(rs=0.350, p=0.003), Breslow's thickness(rs=0.287, p=0.016), histological subtype(rs=0.261, p=0.029), distant metastasis(rs=o.326, p=0.006), vasculogenic mimicry(rs=0.529, p=0.000), mitotic rate(rs=0.668, p=0.000), Ki67 rate (rs=0.703, p=0.000).And it had the negative correlations with melanin(rs=-0.375,p=0.001). The presence of linearly patterned programmed cell necrosis has not shown any association with gender, age, lymph node metastasis, AJCC stage, cellular phenotype and nucleolus (p>0.05).3. The result of Kaplan-Meier and Log test survival analysis showed that the total survival time and the metastasis-free survival time for patients with the presence of linearly patterned programmed cell necrosis were significantly shorter than that for patients without linearly patterned programmed cell necrosis(p=0.005). But in Cox Regression analyzing the difference lost the statistic significant (p>0.05).4. The data of analyzing the two-dimensional images shown that there is a similar trend in vectors of linearly patterned programmed cell necrosis and tumor novel vessel construction in the same district inside tumor. The three-dimensional re-conduction according the two-dimensional images demonstrated that the distribution and the pattern of linearly patterned programmed cell necrosis and tumor novel vessel construction existed similarity.5. The results of immunohistochemistry and real time PCR showed that the expressions of HIF-la, FLK-1, VEGF in both proteins and mRNA levels were higher in the positive group of linearly patterned programmed cell necrosis than that of in the negative group in malignant melanoma, the difference was signifficent(p<0.05).6. The data showed that the expressions of Beclin-1, Atg4B, LC-3, Cyto C, AIF in both proteins and mRNA levels were signifficently higher in the positive group of linearly patterned programmed cell necrosis than that of in the negative group in malignant melanoma by immunohistochemistry and real time PCR methods (p<0.05).Conclusion1. Linearly patterned programmed cell necrosis is a general phenomenon in more aggressive human malignant melanoma.2. Linearly patterned programmed cell necrosis associates with the proliferation, invasion, metastasis of the tumor, and the presence of linearly patterned programmed cell necrosis indicates the poor outcome of the patients with malignant melanoma.3. Linearly patterned programmed cell necrosis is a early event in the process of tumor novel vessel construction. Linearly patterned programmed cell necrosis can provide the spacial base for the later.4. Linearly patterned programmed cell necrosis maybe the accommodation response to the hypoxia under the tumor microenvirenment, and the apoptosis- and autophage-related proteins maybe play critical roles in the mechanism of linearly patterned programmed cell necrosis.