Ambretti S, Venturoli S, Mirasoli M, La Placa M, Bonvicini F, Cricca M, Zerbini M, Roda A, Musiani M
Section of Microbiology, Department of Clinical and Experimental Medicine, University of Bologna, Bologna, Italy.
Br J Dermatol. 2007 Jan;156(1):38-44. doi: 10.1111/j.1365-2133.2006.07541.x.
The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high-risk (HR) HPV genotypes in primary melanoma by PCR.
To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells.
We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis.
The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60-80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity.
The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.
绝大多数旨在检测皮肤癌中人乳头瘤病毒（HPV）DNA的研究都使用了灵敏的聚合酶链反应（PCR）方法，但PCR技术尽管灵敏度高，却不适用于确定：（i）HPV的存在是否仅与少数携带该病毒的细胞有关；（ii）HPV的存在是否源于肿瘤表面污染；（iii）HPV的存在是否局限于癌细胞中，而非肿瘤活检样本中存在的正常角质形成细胞中。在最近的一项研究中，我们通过PCR在原发性黑色素瘤中发现了黏膜高危（HR）HPV基因型。
在原发性黑色素瘤样本的同一切片中定位黏膜HR-HPV核酸和肿瘤黑素细胞标志物，以了解HPV与黑色素瘤细胞之间的关系。
我们开发了一种非常灵敏的方法，该方法在同一切片中依次结合了用于检测HPV核酸（16型和18型）的酶放大荧光原位杂交（ISH）和用于检测肿瘤黑素细胞标志物HMB-45的化学发光免疫组织化学（IHC）方法。分别记录荧光ISH和化学发光IHC的数字图像，赋予不同颜色，并使用特定的图像分析软件进行合并。
荧光ISH和化学发光IHC相结合，在黑色素瘤活检样本的同一切片中显示HPV核酸与黑色素瘤标志物有明显的共定位（范围在60%-80%），具有很强的特异性和灵敏度。
黏膜HR-HPV核酸与HMB-45黑素细胞标志物的强烈共定位强调病毒核酸特异性存在于黑色素瘤细胞中，并支持HPV在恶性黑色素瘤中可能发挥的积极作用。
