DCE-MRI Assessment Of The Effect Of DNAzyme Targeted To EBV-encoded Latent Membrane Protein-1 On Nasopharyngeal Carcinoma In Clinical And Experimental Research

The search for new anticancer therapeatics has shifted away from traditional cytotoxic agents to mechanism-driven drugs. Nasopharyngeal carcinoma (NPC) is one of common malignant tumors in China, and DNAzyme targeted to EBV-LMP1 mRNA may be one of the targeted molecular therapeatics for NPC. Imaging biomarkers are emerging and promising tools during the development of anticancer drugs. With the use of DCE-MRI, quantitative kinetic parameters can be generated as indicators for changes of tumor microcirculation. In this study, we attempt to investigate the effect of DNAzyme targeted to LMP1 mRNA on NPC in clinical research and animal model by using DCE-MRI.Part?Objectives:To understand the quantitative kinetic parameters of DCE-MRI in NPC, and to standardize method of DCE-MRI scanning technology and data analysis, thus laying the foundation of DCE-MRI to monitor treatment effect of NPC.Methods:24 NPC patients with pathologically diagnosed as poorly differentiated squamous cell carcinoma underwent conventional MRI and DCE-MRI. The nordicICE DCE software was used to post-process the raw data and obtain Ktrans?kep?Ve parameter map. The kinetic parameter values of NPC tumor tissue and lateral pterygoid muscle were calculated.Results:There were statistically significant differences of Ktrans? kep?Ve values between NPC tumor tissue and the lateral pterygoid muscle. The Ktrans value 95% CI of NPC tumor tissue was 0.1637 (minute-1) 0.2164 (minute-1), and the lateral pterygoid muscle was 0.0745 (minute-1)?0.1020 (minute-1). There was a positive correlation between Ktrans and kep value of NPC tumor tissue, and Pearson correlation coefficient was 0.552 (P= 0.009). Negative correlation existed between Ve and kep value of NPC tumor tissue, and Pearson correlation coefficient was -0.473 (P= 0.020).Conclusion:Based on two-chamber kinetic model with arterial flow function deconvolution technology, standardized method of DCE-MRI scanning can generate quantitative kinetic parameters (Ktrans?kep?Ve). The differences of quantitative DCE-MRI kinetic parameters between NPC tumor tissue and lateral pterygoid muscle may be due to the difference of the microcirculation between the two. There are some correlation among the DCE-MRI kinetic parameters of NPC tumor tissue.Part IIObjective:To investigate the effect and vascular changes following the treatment with combination of DNAzyme targeted to EBV-LMP1 mRNA with radiation therapy in NPC using DCE-MRI.Methods:24 NPC patients with EBV-LMP1 positive expression were randomly divided into two groups:combined treatment group(radiotherapy +DNAzyme) and radiotherapy alone group (radiotherapy+saline). All patients underwent conventional MRI and DCE-MRI scans in 1 to 2 days before radiotherapy?during the radiotherapy (radiation dose of 50Gy)?at the end of radiotherapy (radiation dose of 70Gy) and 3 months after the end of radiotherapy. The Ktrans?kep?Ve values of NPC tumor tissue in different periods were obtained, and the change rates of Ktrans and tumor volume were calculated.Results:In combined therapy group, there were statistically significant differences of the Ktrans value?Kep values of tumor tissue between pre-radiotherapy and post-radiotherapy(including during the radiotherapy?at the end of radiotherapy and 3 months after the end of radiotherapy), and the latter were lower than the former. In radiotherapy alone group, there was no significant difference of the Ktrans value of tumor tissue between pre-radiotherapy and during the radiotherapy?at the end of radiotherapy, and statistically significant difference of the Ktrans value only existed between pre-radiotherapy and 3 months after the end of radiotherapy(P<0.05); There were statistically significant differences of the Kep values of tumor tissue between pre-radiotherapy and post-radiotherapy(including during the radiotherapy?at the end of radiotherapy and 3 months after the end of radiotherapy), and the latter were lower than the former. During the process of treatment, there were a similar trend of the change rate of Ktrans?the change rate of tumor volume between combined therapy group and radiotherapy alone group, and the absolute values both increased gradually. However,3 months after the end of radiotherapy, the change rate of Ktrans?the change rate of tumor volume were significantly different between the two groups (P<0.05). Conclusion:With radiotherapy alone or combined use of DNAzyme targeted to LMP1 mRNA, the Ktrans value, Kep value of NPC tumor tissue are both decreased. DCE-MRI quantitative kinetic parameters can be used to non-invasively assess the changes of tumor angiogenesis and vascular permeability during the treatment of NPC, providing timely quantitative indicators for the assessment of the efficacy. Combined with radiotherapy, the use of DNAzyme targeted to LMP1 mRNA can further promote tumor volume regression, and play a role in the decline of tumor tissue Ktrans.Part?Objectives:To evaluate the effect of DNAzyme targeted to LMP1 mRNA on vascular permeability and angiogenesis of NPC xenograft in nude mice by using DCE-MRI, and to attempt to clarify the molecular mechanism of its enhancing radiosensitivity in NPC xenograft, combined with histopathology and immunohistochemistry.Methods:The nude mice bearing NPC xenograft were randomly divided into 6 groups:DNAzyme treated group, oligonucleotide group, saline group, low dose radiation (5Gy)+DNAzyme group, high dose radiation (10Gy)+oligonucleotide group, high dose radiation (10Gy) group, and intervented respectively in different ways. All nude mice underwent routine MRI and DCE-MRI after interventions, and the Ktrans value of tumor tissue were obtained. Tumor specimens were immunostained with anti-human VEGF monoclonal antibody and CD34 antibody.Results:There was a significant difference of the Ktrans value in xenografts tumor tissue between DNAzyme group and saline group (P <0.05), the former was lower than the latter. The VEGF expression in DNAzyme treated group is "±", but that in oligonucleotide group and saline group is "+". The CD34 expression in DNAzyme treated group is "±", but that in oligonucleotide group and saline group is "+". There were no significant difference of the Ktrans value in xenografts tumor tissue among Low-dose radiotherapy (5Gy)+DNAzyme group?high dose radiation (10Gy)+oligonucleotides group.Conclusion:Combination of immunohistochemistry with Ktrans assessment can confirm that DNAzyme targeted to LMP1 mRNA could inhibit angiogenesis and decrease vascular permeability of NPC xenografts, and the pathophysiology basis is that DNAzyme targeted to LMP1 mRNA can inhibit the expression of VEGF. Ktrans can provide evidence for the mechanism of DNAzyme targeted to LMP1 mRNA enhancing radiosensitization.