Lieveld, Marusya, Padalko, Elizaveta, Praet, Marleen, Broeck, Davy Vanden, Zappacosta, Roberta, Lattanzio, Giuseppe, Viola, Patrizia, Ianieri, Manuel Maria, Gatta, Daniela Maria Pia, and Rosini, Sandra
Dear editor Zappacosta et al1 recently published a case report concerning a human papillomavirus (HPV)-positive invasive cervical cancer in a 79-year-old women who had a history of normal Pap smears. In this article, Anyplex II HPV28 (Seegene) is used for HPV genotyping of formalin-fixed paraffin embedded (FFPE) tissue, liquid based cytology (LBC) specimens and urine samples. It is suggested that HPV53 is present exclusively in the cervical cancer cells, lymph node metastases, and atypical urinary cells of one single case while the surrounding CIN2+ tissue revealed ten different HPV strains. Unfortunately, the HPV genotype results for lymph nodes and urinary cells are not presented while these results underline the potential role of HPV53 in oncogenesis. Moreover, it is generally accepted that one lesion is caused by one HPV infection, detection of multiple HPV types thus indicates the presence of multiple infections,2 suggesting that this patient may have several lesions. In Figure 3 in Zappacosta et al1 two melting peaks (-d(RFU)/dt) for HPV53 are displayed, one detected by probe A in plasmid DNA (referred to as a positive internal control), and one detected by probe B in cervical cancer cells. However, Anyplex II HPV28 only uses one target specific primer/catcher/pitcher set for each HPV type. The internal positive control used in Anyplex II HPV28 is human β-globin and not plasmid DNA as indicated by Zappacosta et al.1 It is included to monitor whether the sample is adequate for analysis and to exclude polymerase chain reaction (PCR)-inhibition which may lead to false negative results. The test also includes external positive controls which consist of HPV type-specific plasmids, in order to validate whether the prepared master mix accurately detects the indicated HPV types. Nevertheless, the authors could opt to use synthesized plasmid HPV53 DNA as an extra internal positive control by adding it to the sample but this could not be derived from the article. In either case, melting curve analysis of the positive control will give a melting profile consisting of melting curves specific for each HPV type included in the positive control (personal communication Seegene). Because the shape of the melting curve and the position of the peak is a signature for each HPV type, infection with a particular HPV type will give rise to a particular melting curve. Therefore, it is expected that HPV53 infection in cervical cancer cells (right melting curve in Figure 3, Zappacosta et al1) will give the exact same melting profile as HPV53 observed in the positive control (left melting curve in Figure 3, Zappacosta et al1). This is not the case, as it can be deduced from Figure 3, Zappacosta et al. The melting peak of HPV53 in the positive control differs approximately 8°C from the HPV type detected in cervical cancer cells. Modified Figure 3 Real-time PCR assay performed on tissues (invasive cervical cancer, CIN2+ lesion adjacent to invasive neoplasia, metastatic lymph nodes, non-metastatic lymph nodes, and thyroid), and on liquid-based urinary samples. The Anyplex II HPV28 is based on melting curve analysis using the TOCE™ technology. In contrast to conventional High Resolution Melt (HRM) analysis, the TOCE™ technology enables identification of multiple targets (in this case, HPV types) simultaneously in a single channel by controlling the catcher melting temperature (catcher-Tm). The catcher is an artificial fluorescently labeled template; it is designed in such a way that the tagging portion of “the pitcher”, which previously recognized and hybridized with the target sequence, hybridizes with the catcher. Because each HPV-specific pitcher will hybridize with a catcher of a distinctive length causing a temperature shift during the melting out, different HPV types will be detected in one single channel using one fluorophore. Hereupon, catchers are labeled with different fluorophores (FAM, HEX, Cal Red 610, Quasar 670, Quasar 705) which are detected using different channels allowing multiplexing of 28 different HPV genotypes. Anyplex II HPV28 detects and differentiates 19 high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) and 9 low-risk HPV genotypes (6, 11, 40, 42, 43, 44, 54, 61, 70). The kit consists of two independent panels: Panel A with 14 high-risks (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and Panel B with 5 high-risks (26, 53, 69, 73, 82) and 9 low-risks HPV.3–5 Zappacosta et al described in their article that urine samples were analyzed using Hybrid capture II HPV test (HC2 HPV test).1 To our knowledge, HC2 HPV test has not yet been optimized for urine samples.6,7 In-house optimization of urine samples for HC2 HPV could have been performed, but this is not specified in the article. It is also unclear why the authors carried out the HC2 HPV test since the results generated by Anyplex II HPV28 and HC2 are not compared with each other in the Results section. Moreover, Anyplex II HPV28 detects all of the genotypes included in HC2, which makes the potential complementary use of HC2 redundant. To prevent sample-to-sample contamination, the microtome blade and working space were cleaned with DNA Away™ (Thermo Fischer Scientific) after sectioning of each FFPE test sample. However, it is unclear whether a negative control was included in the experiments in order to exclude cross-contamination. Validation for cross-contamination could be performed beforehand, but this is not reported in the article. A negative control (a tissue that is shown negative for the tested HPV types) should be cut between the test samples ideally after a positive specimen to verify absence of cross-contamination. In conclusion, based on the observation that the melting curve of the HPV type detected in the cervical cancer cells does not correspond with the melting curve for HPV53 plasmid DNA, it can be deduced that another HPV type might be involved in the cervical cancer cells. Hence, it cannot be concluded that HPV53 is solely responsible in oncogenesis in this case as previously suggested by Zappacosta et al.1