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S Paytubi & MA Pavón (May 2022). The Ampfire HPV testing for HPV detection and genotyping in formalin-fixed paraffin-embedded oropharyngeal cancer samples. www.HPVWorld.com, 197

High-risk human papillomavirus (HPV) is aetiologically linked to a group of oropharyngeal squamous cell carcinomas (OPSCCs), whose incidence rates are worldwide increasing and show better clinical outcome and response to treatment. Identifying molecular diagnostic tests able to categorise those cancers etiologically associated with HPV is a must for an accurate diagnosis of patients with oropharyngeal cancer. The identification of HPV-related tumours in the clinical setting has been mainly based on the detection of p16^INK4a overexpression determined by immunohistochemistry. However, the combination of p16^INK4a overexpression and HPV-DNA testing increases diagnostic accuracy and has a better prognostic value.¹

All commercial HPV-DNA detection tests have been developed mainly to achieve optimal performance for cervical cancer screening, although they have not been validated for cancer diagnosis using formalin-fixed, paraffin-embedded (FFPE) biopsies. HPV-DNA detection platforms are usually expensive, require well-trained staff and have been designed for a high-throughput workflow, hampering its implementation for OPSCC diagnosis. FFPE biopsies are one of the most widely performed methods for clinical sample preservation, and are frequently the only available samples for molecular testing. Those types of samples require specific processing before PCR amplification, a process that increases handling and costs, but is also crucial for HPV test accuracy. For all these reasons, it is essential to validate in FFPE specimens the cervical cancer screening HPV-DNA tests prior to their implementation for the diagnosis of OPSCCs.

Over the last two years, we have witnessed the mushrooming of publications on the Ampfire HPV assays. One of the platforms developed by Atila Biosystems Company is the Ampfire High-Risk HPV Real Time Fluorescent Detection with HPV16/18 Genotyping assay (from now onwards Ampfire Multiplex HR-HPV). The test allows multiplexed real time fluorescent detection of 15 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68 ). In addition, the test performs individual genotyping of HPV16 and HPV18 in a single assay tube by means of a 60ºC isothermal reaction during 74 min,which represents an advantage of the test, since HPV16 is by far the most common type found amongst HPV-driven OPSCCs (prevalence over the 80%).² The presence and integrity of DNA in all samples is verified by β-globin gene amplification as an internal control. Additionally, the amplicons detected by the Ampfire assay are smaller (100 bp E1 and/or L1) than those amplicons detected by other tests. The kit also provides positive and negative controls to ensure the results are trustworthy. The whole process, from DNA to detection is shown in Figure 1.

In our study, we have compared two commercial HPV tests in a routine diagnostic setting by analysing the performance of the AmpFire Multiplex HR-HPV assay on 160 DNA samples extracted from FFPE primary tumour specimens from patients with OPSCC, and compared the results with those obtained with the HPV Linear Array assay. The latest is one of the most widely used methods for testing and genotyping of FFPE samples.³ With a 99.4% concordance and a very good agreement according to Cohen’s Kappa coefficient index, the commercially available Ampfire Multiplex HR-HPV test showed a similar performance to Linear Array on the detection and genotyping of HPV on FFPE OPSCC samples (Table 1).⁴






Moreover, a concordance of 100% between Ampfire Multiplex HR-HPV assay and Linear Array was observed for the presence of types HPV16 (38/38) and 18 (0/0). In terms of “other HR-HPV non-HPV16/18” concordance, the agreement between both assays was 87.5% (7/8), being HPV45 the only discordant type not detected by the Ampfire Multiplex HR-HPV test in one sample.

Although in the present study extracted DNA was used and showed a good performance, the Ampfire Multiplex HR-HPV test, contrarily to other assays, has the capacity to be used with or without DNA extraction. This is an important point because the DNA extraction step is time-consuming, increases the costs of HPV-DNA testing, could increase the rate of invalid samples and inevitably introduces a source of variability.

The Ampfire Multiplex HR-HPV test relies on real-time isothermal PCR, which is an easier and faster procedure compared to the Linear Array technique which involves two separate time steps, PCR amplification and detection. Using the Ampfire Multiplex HR-HPV assay as the initial screen could significantly decrease the turnaround time, manual labor and expenses associated with current testing strategies.

In our experience, the Ampfire Multiplex HR-HPV test appears to be a trustable HPV detection method that could be used combined with p16INK4a immunohistochemistry in clinical laboratories with good performance in routine OPSCC FFPE samples, thus meeting the latest recommendations for HPV testing in OPSCC.


DISCLOSURE
The authors declare nothing to disclose.


References
1. Mena M et al. Double positivity for HPV-DNA/p16INK4a is the biomarker with strongest diagnostic accuracy and prognostic value for human papillomavirus related oropharyngeal cancer patients. Oral Oncol 2018; 78: 137–144. Available from: https://pubmed.ncbi.nlm.nih.gov/29496041/
2. Castellsagué X et al. HPV Involvement in Head and Neck Cancers : Comprehensive Assessment of Biomarkers in 3680 Patients. J Natl Cancer Inst 2016, 108: 1–12. Available from: https://pubmed.ncbi.nlm.nih.gov/26823521/
3. Kocjan B et al. Detection of alpha human papillomaviruses in archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens. J. Clin. Virol 2016; 76: S88–S97. Available from: https://pubmed.ncbi.nlm.nih.gov/26514313/
4. Paytubi S et al. The isothermal amplification Ampfire assay for HPV detection and genotyping in formalin-fixed paraffin-embedded oropharyngeal cancer samples. Journal of Molecular Diagnostics 2021; 24(1):79-87. Available from: https://pubmed.ncbi.nlm.nih.gov/34801703/