Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma.
Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma.
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DOI：
10.1002/cjp2.47
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发表时间：
2016-10
影响因子：
4.1
通讯作者：
Sastre-Garau, Xavier
中科院分区：
文献类型：
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作者：
Jeannot, Emmanuelle;Becette, Veronique;Campitelli, Maura;Calmejane, Marie-Ange;Lappartient, Emmanuelle;Ruff, Evelyne;Saada, Stephanie;Holmes, Allyson;Bellet, Dominique;Sastre-Garau, Xavier
特定的人乳头瘤病毒基因型与大多数肛门生殖器癌和大部分口咽癌相关。因此，人乳头瘤病毒DNA是一种肿瘤标志物，可以在患者血液中检测到，用于临床监测。然而，关于宫颈癌患者中循环人乳头瘤病毒DNA的数据几乎没有提供临床价值，这是由于用于检测小尺寸肿瘤的测定的灵敏度不足。在这里，我们利用敏感的液滴数字PCR方法来鉴定人乳头瘤病毒相关癌患者中的循环人乳头瘤病毒DNA。回顾性分析了2002年至2013年期间诊断时采集的70份血清标本，这些血清标本来自人乳头瘤病毒-16或人乳头瘤病毒-18相关癌患者，其中47例来自宫颈，15例来自肛管，8例来自口咽。作为阴性对照，还分析了来自患有人乳头瘤病毒16相关的高度宫颈上皮内瘤变的女性的18份血清样本。血清样本储存在-80 °C（27例）或-20 °C（43例）。从200 μl血清或血浆中分离DNA，并使用人乳头瘤病毒-16 E7和人乳头瘤病毒-18 E7特异性引物进行液滴数字PCR。在61/70（87%）的癌症患者血清样本中检测到循环人乳头瘤病毒DNA，而在宫颈上皮内瘤变患者血清中未检测到。当仅使用储存在-80 °C的血清时，阳性率增加到93%。重要的是，该系列中的两名微浸润癌患者呈阳性。定量评估显示，宫颈癌患者的循环病毒DNA水平与临床分期和肿瘤大小有关，范围为55 ± 85拷贝/ml（I期）至1774 ± 3676拷贝/ml（IV期）。循环人乳头瘤病毒DNA存在于人乳头瘤病毒相关浸润性癌症患者中，甚至在亚临床阶段，其水平与肿瘤动力学相关。液滴数字PCR是一种很有前途的人乳头瘤病毒DNA定量检测方法。在人乳头瘤病毒相关高度宫颈上皮内瘤变患者中未发现阳性。
Specific human papillomavirus genotypes are associated with most ano‐genital carcinomas and a large subset of oro‐pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus‐associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus‐16 or human papillomavirus‐18‐associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro‐pharynx. As negative controls, 18 serum samples from women with human papillomavirus‐16‐associated high‐grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at −80°C (27 cases) or at −20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus‐16 E7 and human papillomavirus‐18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at −80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus‐associated invasive cancers even at sub‐clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus‐associated high grade cervical intraepithelial neoplasia.