Laser capture microdissection of cervical human papillomavirus infections: copy number of the virus in cancerous and normal tissue and heterogeneous DNA methylation.
Laser capture microdissection of cervical human papillomavirus infections: copy number of the virus in cancerous and normal tissue and heterogeneous DNA methylation.
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DOI：
10.1016/j.virol.2009.05.006
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发表时间：
2009-08-01
期刊：
影响因子：
3.7
通讯作者：
Bernard, Hans-Ulrich
中科院分区：
文献类型：
--
作者：
Kalantari, Mina;Garcia-Carranca, Alejandro;Dalia Morales-Vazquez, Claudia;Zuna, Rosemary;Perez Montiel, Delia;Calleja-Macias, Itzel E.;Johansson, Bo;Andersson, Sonia;Bernard, Hans-Ulrich
人乳头瘤病毒（HPV）在宫颈癌发生过程中的致病性研究通常依赖于匀浆组织或培养细胞的研究。这种方法不能检测受感染组织内的分子异质性。需要了解特定组织学背景下的分子特性。我们询问存档宫颈肿瘤的激光捕获显微切割 (LCM) 结合实时聚合酶链反应和亚硫酸氢盐测序是否能够 (i) 对福尔马林固定细胞小簇进行灵敏的 DNA 诊断，(ii) 肿瘤细胞和正常细胞中 HPV DNA 的定量，以及 (iii) 分析 HPV DNA 甲基化（肿瘤进展的标志物）。我们分析了 26 个含有 HPV-16 或 18 的肿瘤。我们从 LCM 解剖的 100 至 2000 个细胞的薄片中制备 DNA，并分析了对应于 9 至 70 个细胞的等分试样。我们在每个肿瘤细胞中分别检测到 9 至 630 个 HPV-16 基因组拷贝和 1 至 111 个 HPV-18 基因组拷贝。在 26 个样本中的 17 个样本中，HPV DNA 存在于远离肿瘤边缘的组织学正常细胞中，但浓度比肿瘤中低得多，这表明 HPV 可以低水平感染而不会引起致病性变化。 HPV DNA 的甲基化是病毒整合到细胞 DNA 中的生物标志物，由于灵敏度有限，只能在少数样本中进行测量，并表明癌细胞和正常细胞小簇中的异质甲基化模式。 LCM 对于研究宫颈 HPV 感染的分子参数（如拷贝数、潜伏期和表观遗传学）非常有用。
Research on the pathogenicity of human papillomaviruses (HPVs) during cervical carcinogenesis often relies on the study of homogenized tissue or cultured cells. This approach does not detect molecular heterogeneities within the infected tissue. It is desirable to understand molecular properties in specific histological contexts. We asked whether Laser Capture Microdissection (LCM) of archival cervical tumors in combination with real-time polymerase chain reaction and bisulfite sequencing permits (i) sensitive DNA diagnosis of small clusters of formalin fixed cells, (ii) quantification of HPV DNA in neoplastic and normal cells, and (iii) analysis of HPV DNA methylation, a marker of tumor progression. We analyzed 26 tumors containing HPV-16 or 18. We prepared DNA from LCM dissected thin sections of 100 to 2000 cells, and analyzed aliquots corresponding to between nine and 70 cells. We detected nine to 630 HPV-16 genome copies and one to 111 HPV-18 genome copies per tumor cell, respectively. In 17 of the 26 samples, HPV DNA existed in histologically normal cells distant from the margins of the tumors, but at much lower concentrations than in the tumor, suggesting that HPVs can infect at low levels without pathogenic changes. Methylation of HPV DNA, a biomarker of integration of the virus into cellular DNA, could be measured only in few samples due to limited sensitivity, and indicated heterogeneous methylation patterns in small clusters of cancerous and normal cells. LCM is powerful to study molecular parameters of cervical HPV infections like copy number, latency and epigenetics.
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