The goal of this study was to examine whether HPV genotyping on FFPE invasive cervical carcinoma specimens give comparable results with freshly frozen specimens obtained from the same patient simultaneously. Our study suggests that detection of HPV using SPF10 with LiPA technology using formalin fixed paraffin embedded tissues gives comparable results to freshly frozen specimens for HPV detection in general (Kappa index 0.26, p = 0.001), see Table 1, but is not so good for specific HPV types detection (p-value > 0.05). Some previous studies which compared the paraffin embedded tissue with other samples found high agreement [7, 8, 11]. However, these studies used exfoliated cells or cervical scrapes and not freshly frozen specimens. Besides, these were cases of preneoplastic lesions, not invasive cervical carcinoma. A study which compared FFPE tissue with cervical scrapes from women with invasive cervical carcinoma found very high HPV type specific concordance . However, the number of samples studied was small, and also the method of detection was GP5+/6+ primers.
Another interesting observation was the negative HPV results in both freshly frozen specimens and FFPE tissues. Our analysis of p16 immunostaining in samples with at least one negative result showed that all but one sample tested p16 positive, suggesting that the cases are HPV mediated and that the most probable reason for not detecting the virus is related to viral, specimen preservation or technical issues such as denatured DNA by alkali  or by formalin fixation . For specific HPV types, if one considers fresh specimens as gold standard, then discordance in HPV results from FFPE specimens could be due to a number of explanations. One is that formalin fixation could have denatured the tissue. It is known that DNA extracted from FFPE tissues are usually at low concentration and fragmented [14, 15]. Another alternative view is reduced sensitivity detection of some HPV types in paraffin specimens, especially types 42, 16, 18, 39, 56, 58, 59 and 66 [16–18]. A third possibility, which may be applicable to discordance in multiple infections is due to competition between different HPV genotypes . It is conceivable that lower discordance rates could be obtained by reducing the duration of fixation in buffered formalin.
The low detection of HPV in adenocarcinoma in both types of specimens is also of interest. A number of previous studies have also found low detection of HPV in adenocarcinoma of the cervix [9, 20, 21]. Plausible explanations are: (a) that some cervical tumours may be of endometrial origin and are therefore HPV negative ; (b) that some cervical adenocarcinomas may not be related to HPV (such as minimal deviation adenocarcinomas) [21, 23]; (c) low viral load; (d) fewer episomal copies; (e) or loss of viral genome during integration in cervical adenocarcinoma . Though some investigators found a high positive rate which they attributed to higher diagnostic accuracy and exclusion of non cervical tumours .
Our results have some implications. For areas with low resources, use of FFPE samples for HPV detection is feasible, especially HPV testing for clinical management of women with abnormal smears. In case a biopsy is taken, the paraffin embedded tissue could be referred to laboratory for HPV testing. This would make use of HPV results, whether for prognosis or detection of progression of cervical lesions like high-grade squamous intraepithelial lesion (HSIL) possible in many areas. However, there would be a need to have standard protocols so that fixation time is controlled. In some situations, the preference for fresh or paraffin tissue will depend on the amount of available tissue and the questions being addressed.
Our study had some strengths. Compared with a recent study which compared the use of cervical scrapes and tissues from women with cervical carcinoma , our sample size was big. The specimens were analyzed in two different laboratories blindly using the same HPV detection method. The SPF10 and LiPA technology are quite sensitive and specific for HPV . The study also has, however, some limitations. The piece of tissue used was divided in two, and half was used to prepare the paraffin blocks and half kept as freshly frozen tissue for HPV genotyping. Thus, they were not exactly the same tumour materials, but pieces of tissue adjacent to each other. Moreover, a small number of adenocarcinomas limits the use of statistical tests.