鼻咽癌患者血浆游离EBV DNA定量检测及其临床意义

发布时间：2018-05-29 08:20

本文选题：鼻咽癌 + 血浆游离EBV　；参考：《复旦大学》2013年博士论文

【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。
[Abstract]:The incidence of nasopharyngeal carcinoma ( NPC ) in China , South - East Asia and the Mediterranean has been significantly improved in China , South - East Asia and the Mediterranean region . In recent years , with the development of imaging and radiotherapy technology and the development of multi - disciplinary integrated therapy , the overall survival rate of nasopharyngeal carcinoma is more than 90 % .

In the etiology and pathogenesis of nasopharyngeal carcinoma ( NPC ) , Epstein - Barr virus ( EBV ) has special status , and its associated serological markers such as VCAIgA and EAIgA are widely used in clinical diagnosis and screening , but it is not timely to reflect the tumor situation in vivo .

Based on the above background , this experiment is divided into two parts : the optimization of the quantitative method of plasma free EBVDNA , the main influencing factors of the pre - treatment concentration and the possible relationship between the possible blood feeding mechanism and distant metastasis .

1 . The concentration range of the Namalwa DNA standard is more reasonable relative to the standard - quality grain DNA , which can cover almost all of the samples to be tested , not too high , and has a higher retest stability , and is therefore more suitable for use as an absolute quantification of plasma free EBV DNA .

2 . The plasma free EBV DNA concentration and clinical T , N stage , primary tumor invasion range and lymph node metastasis were correlated with each other .

3 . In different treatment modes , the plasma free EBV DNA of most patients decreased significantly with the tumor regression . In individual patients , EBV DNA at the end of treatment indicated the progression of tumor control and post - treatment . The WHO two - dimensional radial line assay was superior to the one - dimensional method of RECIST1.1 in reflecting the actual load of tumor . As a means of imaging assessment of recent efficacy , compared with the changes in plasma free EBV DNA , it was found that the latter was more sensitive to tumor response , and its persistence was indicative of tumor residual , especially lymph node residue .

In conclusion , it is concluded that the genomic DNA of Namalwa cell is more suitable as the standard for the quantification of free EBV DNA in nasopharyngeal carcinoma .
Large volume of metastatic lymph nodes , skull base bone involvement , and lymph node metastasis on the lower neck and clavian bone resulted in increased tumor apoptosis and increased risk of blood feeding , thus affecting the plasma free EBV DNA concentration before treatment ;
The change of plasma free EBV DNA concentration after treatment is expected to be a new auxiliary standard , which can be applied to the evaluation of the recent curative effect of anti - tumor therapy .
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.63

【共引文献】