Brousset P, Butet V, Chittal S, Selves J, Delsol G
Anatomical Pathology Laboratory, Centre Hospitalier Universitaire Purpan, Toulouse, France.
Lab Invest. 1992 Oct;67(4):457-64.
The detection of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) may be of diagnostic importance, particularly in cases from nonendemic areas. For cellular localization of viral genomes, cold in situ hybridization methods for the demonstration of EBV-associated NPC remain difficult and relatively insensitive for routinely processed tissues.
The aim of the present study was to assess the importance of tissue processing and the hybridization targets to improve the sensitivity of the cold in situ hybridization method. In situ hybridization was performed in six cases of NPC using three biotinylated EBV cDNA probes (BamHI W/IR1, BamHI Y/EBNA2, XhoI/latent membrane protein) and two cocktails of EBER and BHLF1 oligonucleotides labeled with fluorescein isothiocyanate on routinely fixed and paraffin embedded sections. In two cases, in situ hybridization was also performed on specially processed (ModAMeX) sections. Immunohistochemistry was used to detect EBV-induced antigens using monoclonal antibodies against latent membrane protein, EBNA2 and ZEBRA (BZLF1).
All cases showed EBV nucleic acids regardless of the tissue preparation with the three cDNA probes and on routinely processed sections with EBER oligonucleotides. By using cDNA probes, the best EBV DNA signal was obtained with BamHI W without heating of slides in tissue sections processed by ModAMeX, which probably gives rise to large amounts of single stranded DNAs. All cases positive with cDNA probes were found to be positive with EBER oligonucleotides and negative with BHLF1. However, on routinely processed paraffin sections, the signals with EBER oligonucleotides were stronger than with BamHI W cDNA probe. Dual labeling with in situ hybridization and immunohistochemistry showed that the hybridization signals were restricted to malignant epithelial cells. Latent membrane protein expression was detectable in four of six EBV nucleic acid-positive cases on both ModAMeX and routinely processed sections. The anti-EBNA2 and anti-ZEBRA antibodies were found to be negative on the two cases processed by ModAMeX.
Cold in situ hybridization, in particular with EBER oligonucleotides, appears to be more reliable than immunohistochemistry with anti-latent membrane protein antibody to detect EBV in NPC in routine pathology. These findings confirm a distinctive phenotype (latent membrane protein +/-, EBNA2-, ZEBRA-) of EBV-positive NPC. The negative staining for BHLF1 oligonucleotides further supports the viral latency.
在鼻咽癌（NPC）中检测爱泼斯坦-巴尔病毒（EBV）可能具有诊断意义，尤其是在非流行地区的病例中。对于病毒基因组的细胞定位，用于显示EBV相关鼻咽癌的冷原位杂交方法对于常规处理的组织来说仍然困难且相对不敏感。
本研究的目的是评估组织处理和杂交靶点对提高冷原位杂交方法敏感性的重要性。使用三种生物素化的EBV cDNA探针（BamHI W/IR1、BamHI Y/EBNA2、XhoI/潜伏膜蛋白）以及两种用异硫氰酸荧光素标记的EBER和BHLF1寡核苷酸混合物，在六例NPC的常规固定和石蜡包埋切片上进行原位杂交。在两例中，还在经过特殊处理（ModAMeX）的切片上进行了原位杂交。使用针对潜伏膜蛋白、EBNA2和ZEBRA（BZLF1）的单克隆抗体通过免疫组织化学检测EBV诱导的抗原。
无论使用三种cDNA探针进行何种组织制备，以及在使用EBER寡核苷酸的常规处理切片上，所有病例均显示EBV核酸。通过使用cDNA探针，在经过ModAMeX处理的组织切片中，不加热载玻片的情况下，使用BamHI W可获得最佳的EBV DNA信号，这可能会产生大量单链DNA。所有用cDNA探针检测为阳性的病例在用EBER寡核苷酸检测时也为阳性，而用BHLF1检测时为阴性。然而，在常规处理的石蜡切片上，EBER寡核苷酸产生的信号比BamHI W cDNA探针更强。原位杂交和免疫组织化学双重标记显示杂交信号局限于恶性上皮细胞。在ModAMeX处理的切片和常规处理的切片上，六例EBV核酸阳性病例中有四例可检测到潜伏膜蛋白表达。在经过ModAMeX处理的两例病例中，抗EBNA2和抗ZEBRA抗体呈阴性。
在常规病理学中，冷原位杂交，尤其是使用EBER寡核苷酸，在检测NPC中的EBV方面似乎比使用抗潜伏膜蛋白抗体的免疫组织化学更可靠。这些发现证实了EBV阳性NPC的独特表型（潜伏膜蛋白 +/ - ，EBNA-，ZEBRA-）。BHLF1寡核苷酸的阴性染色进一步支持了病毒潜伏期。
