| Nasopharyngeal carcinoma(NPC)is rare with an incidence of1per100000in western countries. In southern China, however, NPC is endemic, and the incidence is up to25per100000, making it the main cause of death in head and neck tumors. Due to the cryptic onset, NPC is usually presented with a loco-regionally advanced stage at diagnosis. Therefore, critical importance lies in developing more sensitive and specific test for the screening and early diagnosis. On another aspect, although much progress has been made over the past few years in local control and overall survival (OS), especially with an impressive5-year OS of more than90%in early stage disease, attributed to the improvement of radiological examination and multi-disciplinary treatment modality, distant metastasis remains a major problem with advanced tumor, thus calling for more effective and personalized therapy.Epstein-Barr virus (EBV) is correlated with the etiology and pathogenesis of NPC in endemic regions, and based on this, serologic markers such as VCA IgA and EA IgA was widely adopted for screening and diagnosis. However, these markers usually remain elevated even after disease remission is achieved. In contrast, quantification of cell-free plasma EBV DNA (pEBV DNA) using a real-time PCR technique is regarded more valuable as a NPC-related biomarker in diagnosis, pre-treatment evaluation and prediction of long-term survival. In term of this, a current obstacle is the great heterogeneity of mass quantitative data of pEBV DNA coming from different research centers, due to the use of diversified quantitative method. Meanwhile, it still remains unclear how tumor-derived EBV DNA enters the blood circulation, usually resulting in obscurity when combined with clinical practice, more evidence is needed for a better explanation.Under this circumstance, this study was designed to explore a better choice of standards in quantitative detection, and to identify the most contributive factors that influence pre-treatment pEBV DNA concentration, hence postulate a probable route of distant metastasis. It also discussed the possibility of using post-treatment pEBV DNA as an evaluation approach for early therapeutic effect. With these concerns, we compared the quantitative results using two different standards, an EBV-DNA segment-contained recombinant plasmid, and the genome DNA of Namalwa cell, a human-derived lymphoma cell line. After evaluating the quantitative accuracy and stability, we chose a better standard for the following detection in enrolled patients. Overall clinical features and radiological characteristics regarding tumor invasion and metastasis were collected, and a multi-variate linear regression model was fitted to identify the most significant factors that influence the release of EBV DNA and determine primitive pEBV DNA concentration. In addition, the change of pEBV DNA throughout different treatment modalities were recorded and compared with radiological alternations. Preceding that, we identified the measurement WHO and Response Evaluation Criteria in Solid Tumors (RECIST) that better reflects tumor burden between, based on three-dimensional gross tumor volume (GTV). Results were found as following:1. Compared with recombinant plasmid DNA, Namalwa genome DNA shows a better reasonable range of concentration that covers most plasma sample, also it retains reproducible quantitative results with repeated detections. Therefore, it’s more suitable as the standard DNA in RT-PCR.2. Pre-treatment pEBV DNA concentration is correlated with T, N staging, invasion of specific structures by primary tumor, and the metastatic characteristics of regional lymph nodes, but overlapping effect exists within these factors. With a multi-variate linear regression model, tumor volume of metastatic lymph nodes was identified to be the most contributive parameter that determines pEBV DNA concentration. Invasion of skull base and metastatic lymph nodes in lower neck are also significant factors.3. After effective treatment with different modalities, pEBV DNA concentration dramatically declined with tumor regression, while persistently high DNA load eventually correlates treatment failure and tumor progression in some patients. WHO criteria dominates RECIST1.1in reflecting tumor burden, thus is used for tumor response evaluation in this study. Compared with radiological measurement, post-treatment pEBV DNA concentration seems more sensitive in reflecting tumor response, and the persistent positive pEBV DNA may indicate potential tumor residue, especially in lymph nodes.Conclusions:Namalwa genome DNA should be used as standard DNA in RT-PCR, rather than plasmid DNA; Pre-treatment pEBV DNA concentration is mainly determined by larger metastatic lymph nodes, invasion of skull base, and metastases to lower neck (including supraclavicular ones), which are probably correlated with increased tumor apoptosis and more vulnerable blood circulation; Post-treatment pEBV DNA could hopefully serve as a new approach in evaluating preliminary tumor response to treatments. |
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