Pratesi Chiara, Bortolin Maria Teresa, D'Andrea Monica, Vaccher Emanuela, Barzan Luigi, Bidoli Ettore, Tedeschi Rosamaria, Zanussi Stefania, de Paoli Paolo
Microbiology Unit, Centro di Riferiferimento Oncologico, via Pedemontana Occidentale, 12, 33081, Aviano, PN, Italy.
J Clin Virol. 2003 Oct;28(2):155-64. doi: 10.1016/s1386-6532(02)00276-7.
Nasopharyngeal carcinoma (NPC) is frequently associated with Epstein-Barr virus (EBV), but little is known about the EBV DNA prevalence on peripheral blood in Western Countries, where the tumour is not endemic and its incidence is low.
To set up and evaluate an internally controlled qualitative polymerase chain reaction (PCR) followed by quantitative competitive PCR for the detection of EBV DNA in clinical specimens. To investigate whether EBV DNA load in peripheral blood was a consistent feature of Italian NPC patients.
A PCR assay based on latent membrane protein 2A (LMP2A) sequence amplification was chosen. Best assay conditions, sensitivity and reproducibility were determined. Sixty-four sera and 63 plasma from an Italian cohort of 39 NPC patients were analyzed. Samples from 5 patients followed up after radiotherapy were also assayed. Qualitative and quantitative beta-globin amplification was performed in parallel in order to provide an independent control for amplification competence of DNA and to investigate whether EBV DNA levels could be due to intracellular EBV viral genomes from cells lysed during plasma/serum collection.
Twenty-five patients had undifferentiated carcinoma (UC) and 14 squamous cell carcinoma (SCC). EBV DNA has been quantified in 58 and 9% of the UC and SCC cases, respectively. No statistically significative differences were observed between the EBV DNA levels (469 vs 750 copies/ml, P=0.16) and prevalence (64 vs 57%, chi2(1)=0.22, P=0.64) in plasma and serum samples. Increased EBV viremia was found in patients with considerable extension of the primary tumour (172 vs 2250 copies/ml, low vs high tumour burden). Three UC subjects, which had detectable pre-treatment EBV DNA levels, became negative after radiotherapy. Clinical examination revealed that all had complete tumour regression.
These PCR procedures allow an accurate and reproducible estimation of plasma/serum EBV DNA load in NPC patients living in non endemic areas, being strictly associated with UC WHO III and with tumour severity.
鼻咽癌（NPC）常与爱泼斯坦-巴尔病毒（EBV）相关，但在该肿瘤并非地方病且发病率较低的西方国家，外周血中EBV DNA的流行情况鲜为人知。
建立并评估一种内部对照的定性聚合酶链反应（PCR），随后进行定量竞争性PCR以检测临床标本中的EBV DNA。研究外周血中EBV DNA载量是否为意大利NPC患者的一个一致特征。
选择一种基于潜伏膜蛋白2A（LMP2A）序列扩增的PCR检测方法。确定最佳检测条件、灵敏度和可重复性。分析了来自意大利一组39例NPC患者的64份血清和63份血浆。还检测了5例放疗后随访患者的样本。同时进行定性和定量β-珠蛋白扩增，以便为DNA扩增能力提供独立对照，并研究EBV DNA水平是否可能归因于血浆/血清采集过程中裂解细胞的细胞内EBV病毒基因组。
25例患者为未分化癌（UC），14例为鳞状细胞癌（SCC）。UC和SCC病例中分别有58%和9%的患者EBV DNA得到定量。血浆和血清样本中的EBV DNA水平（469对750拷贝/毫升，P = 0.16）和流行率（64对57%，χ2(1)=0.22，P = 0.64）之间未观察到统计学显著差异。在原发肿瘤广泛扩展的患者中发现EBV病毒血症增加（172对2250拷贝/毫升，低肿瘤负荷对高肿瘤负荷）。3例治疗前可检测到EBV DNA水平的UC患者放疗后变为阴性。临床检查显示所有患者肿瘤均完全消退。
这些PCR方法能够准确且可重复地估计生活在非流行地区的NPC患者血浆/血清中的EBV DNA载量，且与UC WHO III级和肿瘤严重程度密切相关。
