【作者】 周鑫；

【导师】 胡超苏；

【作者基本信息】 复旦大学 ， 临床医学（专业学位）， 2013， 博士

【摘要】 鼻咽癌(Nasopharyngeal carcinoma, NPC)发病率在西方国家约1/10万人年,但在我国、东南亚及地中海等地区却具有显著的地方性,尤其在我国华南其发病率高达25/10万人年,发病率和死亡率均居于头颈部恶性肿瘤之首。由于该肿瘤发病的隐匿性,早期症状不明显,许多患者就诊时已是中晚期,因此,发展敏感度和特异度兼具的筛查和早期诊断手段十分重要。近年来,随着影像学、放射治疗技术的进步和多学科综合治疗的发展,鼻咽癌的局控率和远期生存预后均有了大幅提高,早期肿瘤的5年生存率达90%以上。然而在晚期肿瘤中,伴随局控率的提高,远处转移却仍旧没有明显改善,寻求更有效的治疗手段及治疗方案的个体化势在必行。在流行地区鼻咽癌的病因和发病学中,EB病毒(Epstein-Barr virus,EBV)具有特殊地位,其相关的血清学指标如VCAIgA, EAIgA等被广泛用于临床诊断与筛查,但其不能及时反映体内肿瘤情况,即便在肿瘤完全缓解后,其滴度仍相当高。相较之下,荧光实时定量聚合酶链反应(Real-time polymerase chain reaction, RT-PCR)测定的血浆游离EBV DNA(Cell-free plasma EBV DNA,cfEBVDNA)被视作更具价值的肿瘤相关生物标志物,在鼻咽癌诊断与评估、长期生存预后方面具有积极意义。然而,目前相关数据来自多个研究中心,其检测方法不统一,定量结果差别极大,因此尚无法整合得到统一的量化指标;另一方面,其产生和入血的机制和环节尚不明确,如何与临床诊治有机结合,仍有待进一步证据支持。基于以上背景,本实验分为基础和临床两大部分,旨在探讨血浆游离EBVDNA定量方法的优化,治疗前浓度的主要影响因素及其可能入血机制,与远处转移的可能关系,以及将其应用于治疗近期疗效评估的合理性与可行性。首先在小样本血浆样品中,分别应用含目的片段的重组标准品质粒DNA和Namalwa人淋巴瘤细胞基因组DNA(目前最主流的两种定量参照)作为绝对定量标准品,通过平行测定对比及反复复测,评价两种定量方法的优劣,并择优进行后续大样本临床检测。此后,通过对比治疗前不同浓度血浆游离EBV DNA及其临床和影像学上的浸润和转移特征,通过多元线性模型寻找影响该指标的最显著因素,建立线性模型进行预测和归纳,进而探讨在肿瘤发生发展中,影响EBVDNA入血的关键环节。最后,观察分析放化疗前后血浆游离EBV DNA的变化情况,并和反映肿瘤负荷变化的影像学缓解评估手段进行对比,讨论其是否能够用于肿瘤早期缓解评估。为此,研究先行对比了三维肿瘤大体体积(Gross tumor volume, GTV)金标准下,一维和二维径线测定法反映鼻咽癌肿瘤负荷的能力,并从WHO标准和实体瘤疗效评价标准(Response evaluation criteria in solid tumors, RECIST)中择优作为最佳影像学评估手段。实验发现：1.相对标准品质粒DNA, Namalwa DNA标准品的浓度范围更加合理,能覆盖几乎全部待测样本,不会过高测定,并且其复测稳定性更高,因此更适合用作血浆游离EBV DNA的绝对定量。2.治疗前血浆游离EBV DNA的浓度和临床T、N分期,原发肿瘤侵犯范围和淋巴结转移特性等均相相关,但各因素间存在重叠。经多元线性模型筛选后,发现淋巴结体积为最显著的影响因素,其他因素包括颅底胃质受累,下颈部(含锁骨上)淋巴结转移等。三者分别可导致血浆游离EBV DNA升高至原先5,4.2和6.7倍3.在不同的治疗模式下,多数患者血浆游离EBV DNA随肿瘤退缩发生明显下降。而个别患者中,治疗结束时高载量EBV DNA预示肿瘤未控和治疗后进展。在反映肿瘤实际负荷上,WHO二维径线测定法优于RECIST1.1一维法。。将其作为近期疗效的影像学评估手段,与血浆游离EBV DNA变化进行对比后发现,后者在反映肿瘤缓解上更为敏感,且其持续阳性提示肿瘤残留,尤其是淋巴结残留。综上,本实验得出以下结论：Namalwa细胞基因组DNA较质粒DNA更适于作为鼻咽癌血浆游离EBV DNA定量的标准品；大体积的转移淋巴结,颅底骨质受累,以及下颈部及锁骨上淋巴结转移等致使肿瘤凋亡增加和入血风险增高,从而影响治疗前血浆游离EBV DNA浓度；治疗后血浆游离EBV DNA浓度变化有望作为新的辅助标准,应用于抗肿瘤治疗近期疗效的评估。更多还原

【Abstract】 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.更多还原