Evaluation of the Xpert® HPV assay in the detection of Human Papillomavirus in formalin-fixed paraffin-embedded oropharyngeal carcinomas

Oral Oncology 72 (2017) 117–122

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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology

Evaluation of the XpertÒ HPV assay in the detection of Human Papillomavirus in formalin-fixed paraffin-embedded oropharyngeal carcinomas Maria Gabriella Donà a,1, Francesca Rollo b,1, Barbara Pichi c, Giuseppe Spriano c, Raul Pellini c, Renato Covello b, Edoardo Pescarmona b, Giulia Fabbri b, Manuela Scalfari b, Tarik Gheit d, Maria Benevolo b,⇑ a

Sexually Transmitted Infection (STI) Unit, San Gallicano Dermatologic Institute, IRCCS, Via Elio Chianesi 53, 00144 Rome, Italy Pathology Department, Regina Elena National Cancer Institute, IRCCS, Via Elio Chianesi 53, 00144 Rome, Italy c Otolaryngology Head Neck Surgery Department, Regina Elena National Cancer Institute, IRCCS, Via Elio Chianesi 53, 00144 Rome, Italy d Infections and Cancer Biology Group, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, France b

a r t i c l e

i n f o

Article history: Received 20 February 2017 Received in revised form 26 May 2017 Accepted 14 July 2017

Keywords: Orophrayngeal neoplasms Head and neck cancer Human Papillomavirus FFPE p16ink4a HPV DNA tests

a b s t r a c t Objectives: The increasing incidence of HPV-related Oropharyngeal Squamous Cell Carcinoma (OPSCC) and the improved survival of HPV-positive OPSCC highlight the need for effective tools in evaluating HPV status on formalin-fixed paraffin-embedded (FFPE) cancers. To date, there is no agreement regarding the most appropriate method for HPV testing on FFPE materials. We aimed to investigate the performance of the XpertÒ HPV assay (Cepheid) on crude lysates from OPSCC FFPE tissues. Materials and methods: Crude lysates were obtained by proteinase K digestion of FFPE tissues that had already been analyzed by the INNO-LiPA HPV assay and p16ink4a immunostaining. Results: 159 FFPE OPSCCs were evaluated. All the samples provided valid results with the Xpert, whereas three samples (1.8%) were invalid using the INNO-LiPA. Among the remaining 156 cases, 65 (41.7%) were concordantly positive and 87 (55.8%) concordantly negative (raw agreement 0.97, 95% CI: 0.93–0.99; Cohen K 0.95, 95% CI: 0.90–0.99). Type-specific data for the cases that were positive by both methods were completely concordant. Three samples were HPV16-positive with Xpert but negative with INNOLiPA, while one OPSCC tested negative with Xpert and positive with INNO-LiPA. A very good agreement was observed between the Xpert and the p16 results, which was slightly higher than that for INNO-LiPA (Cohen K 0.87 vs. 0.85). Conclusion: The Xpert HPV assay appears to be a very good method for HPV detection and genotyping on FFPE OPSCCs, and requires no prior purification of nucleic acids. This assay showed a very good agreement with INNO-LiPA and p16 findings. Ó 2017 Elsevier Ltd. All rights reserved.

Introduction Over the last few decades, it has become clear that Human Papillomavirus (HPV) does not only cause ano-genital cancers, but also represents the etiological agent of a subset of Head and Neck Squamous Cell Carcinomas (HNSCC) [1]. The majority of the HPV-driven HNSCC are oropharyngeal squamous cell carcinomas (OPSCC) which arise at the tonsils or base of the tongue. Recently, a significant increase in the incidence of HPV-related OPSCC was observed in the US [2] and several European countries [3,4]. HPV represents a strong independent prognostic factor for OPSCCs, ⇑ Corresponding author. 1

E-mail address: [email protected] (M. Benevolo). These authors equally contributed to the work.

http://dx.doi.org/10.1016/j.oraloncology.2017.07.016 1368-8375/Ó 2017 Elsevier Ltd. All rights reserved.

which show improved survival and response to treatment when HPV-driven [5]. The unique profile of HPV-associated OPSCCs has led to proposals of new staging systems for these tumors [6], and, most importantly, to clinical trials on de-intensified treatment protocols for patients with HPV-related OPSCC [7]. Taken together, these findings are fueling the need for effective tools to evaluate cancer HPV status. Whereas fresh tissues are rarely collected or available in routine clinical practice, formalin-fixed paraffin-embedded (FFPE) biopsies, which are used for histopathological evaluation, are easily available. A variety of methods for direct and indirect detection of HPV infection using FFPE samples are available. At present, at least two different assays allow E6/E7 mRNA detection on these specimens by in situ hybridization (ISH) [8–10]. Additionally, the immunohistochemical evaluation of p16ink4a protein (p16), which

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is considered by many authors as a reliable surrogate marker of transforming HPV infections [11–13], is also easily applicable to FFPE specimens, and is currently accepted as a standalone test for the identification of HPV-associated OPSCCs according to several major guidelines, e.g., College of American Pathologists and Cancer Care Ontario. However, p16 may be up-regulated also in response to virus-unrelated mechanisms, and may thus give false-positive results. In fact, HPV-negative but p16-positive tumors have been reported by numerous studies [14–16]. The last WHO Classification for Head and Neck Tumours underscores the importance of direct HPV testing [17]. Detection of HPV-DNA, together with p16 and/ or HPV mRNA evaluation, represents one of the most reliable diagnostic approaches for the identification of patients with HPV-driven OPSCC. To date, there is no agreement regarding the most appropriate method for the detection of HPV-DNA from high-risk (HR) types on FFPE tissues. PCR-based methods that use the SPF10 primer set, such as the INNO-LiPA HPV Genotyping assay, are currently considered to be the most suitable for the detection of HPV-DNA on FFPE tissues. In fact, the limited size of the HPV target region limits the risk of false-negative/invalid results that may arise because of the unpredictable degree of DNA fragmentation that occurs during fixation and DNA extraction. Indeed, HPV detection systems based on this primer set have been used in international surveys to assess HPV prevalence in archival cancer tissues [18–20]. Several platforms designed for HPV detection in cervical samples have been already applied to HNSCC samples [21–23]. Among these platforms, the XpertÒ HPV assay (Xpert) has received very little attention regarding its use on HNSCC samples. This test is based on a qualitative real-time PCR, which is very fast and easy to perform, and which gives a partial genotyping result. To the best of our knowledge, only one published study has investigated the performance of this assay on a very small series of FFPE HNSCC tissues, running the test on purified nucleic acids [24], whereas another investigation analyzed crude lysates, showing preliminary promising results [unpublished data, 25]. The use of crude lysates represents a unique advantage compared to the use of purified nucleic acids, since extraction and purification steps may deeply affect HPV testing, thus producing variability in the results obtained both on cytological [26] and FFPE samples [27,28]. In this context, we evaluated the performance of the Xpert on the crude lysates obtained from a large series of FFPE OPSCCs in order to investigate whether this assay has the ability to detect HPV on this type of specimen and may therefore help identify HPV-driven OPSCC cases. To this aim, we compared the Xpert results with those obtained using the INNO-LiPA system and p16 immunostaining.

types, was used, while for the samples analyzed from 2015, the INNO-LiPA HPV Genotyping Extra II (Fujirebio) was utilized. The latter is designed for the identification of 32 genotypes. Both tests are based on the SPF10 primer set for the amplification of a 65 bp fragment within the L1 region of the viral genome and are able to detect, among others, the 14 genotypes classified as HR or possibly/probably HR by the International Agency for Research on Cancer [1], i.e., HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Both tests amplify a region of the HLA-DPB1 gene as a human DNA control to monitor sample quality and extraction efficiency. All the steps of HPV amplification and genotyping are the same for both assays, and were performed following the previously described protocol [29]. Briefly, total nucleic acids were isolated from the FFPE sections using the DNeasy Blood and Tissue Kit (Qiagen, Milan, Italy) and measured by a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Monza, Italy); 10 ll of the extract was used as a template for amplification. All the hybridization steps were carried out in a Profiblot T48 instrument (Tecan, Mannedorf, Switzerland). Xpert HPV assay

Materials and methods

The Xpert HPV assay (Cepheid, Inc, Sunnyvale, CA), is a qualitative test which detects the 14 HR-HPV types. This test is based on a real-time PCR that targets a sequence of 80–150 bp in the E6/E7 region, depending on the genotype. The test result is given with a concurrent partial genotyping for HPV16 as a single result; for HPV 18, 45 as a pooled result; collectively for ‘‘other HR-HPVs”. These include three HPV groups, each detected by a different probe: HPV 31, 33, 35, 52, 58; HPV 51, 59; HPV 39, 56, 66, 68. The results can be also visualized separately for each of these groups. In addition, the assay incorporates an internal control which detects a human gene in single copy in order to monitor sample quality. Depending on the size of the lesion, 1–3  5 lm FFPE sections were obtained. The utmost care was taken during the cutting protocol in order to avoid contamination [30]. A last section was stained by hematoxylin-eosin and reviewed by two certified pathologists to confirm the diagnosis. Sections were deparaffinized with 1 ml of xylene at 56 °C for 5 min. The xylene was removed by washing with 1 ml of absolute ethanol. Tissue lysis was obtained by overnight incubation at 56 °C in 180 ml of ATL buffer with 20 ml of proteinase K (Qiagen), followed by 1 h incubation at 90 °C. The crude lysates were measured by NanoDrop 2000 Spectrophotometer. 900 ml of nuclease-free water was added to 100 ml of the crude lysate and the sample thus obtained (total volume = 1 ml) was loaded into the Xpert HPV cartridge and analyzed following the manufacturer’s instructions.

Study samples

HPV E6/E7 mRNA analysis

A retrospective series of 159 FFPE primary OPSCC cases, diagnosed between 2010 and 2016 (2010: n = 15, 2011: n = 27, 2012: n = 26, 2013: n = 21, 2014: n = 29, 2015: n = 23, 2016: n = 18), were retrieved from the archives of the Pathology department of the Regina Elena National Cancer Institute of Rome, Italy. All the cases that had already been characterized for the presence of HPV-DNA by INNO-LiPA and for the immunohistochemical expression of p16 were selected. The study was approved by the Ethics Committee of the Regina Elena National Cancer Institute.

Total RNA was purified from 3  5 lm sections using the PureLink FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. RT-PCR and cDNA amplification were performed using HPV16 or HPV51 specific primers [31], as described elsewhere [14].

INNO-LiPA HPV genotyping Extra and Extra II For the samples collected up to 2014, the INNO-LiPA HPV Genotyping Extra (Fujirebio, Pomezia, Italy), which detects 28 HPV

p16 immunohistochemistry Immunohistochemistry for p16 was performed using the CINtecÒ Histology Kit (Roche Diagnostics, Milan, Italy) following the manufacturer’s instructions. Positive controls consisted of HPV16-positive tonsillar squamous carcinomas with strong p16 expression. Immunostaining was independently examined by two investigators blinded to the HPV findings. Only diffuse

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(75% tumor cells stained) and at least moderate intensity (+2/3) staining patterns were considered as positive, as previously described [32,33].

Table 1 HPV results for the 159 cases tested with the INNO-LiPA and Xpert assay. HPV test n (%)

Statistical analysis The raw agreement and Cohen kappa were calculated to measure the agreement between the Xpert and INNO-LiPA results, both overall (HPV negative vs. positive regardless of the specific genotype/s identified) and for HPV type-specific positivity. The raw agreement and Cohen kappa were also used to assess the agreement between the HPV test results and p16 findings. All statistical analyses were performed using the SPSS statistical package (version 17.0; SPSS Inc, Chicago, Ill). Results Study samples A total of 159 OPSCC cases were retrieved for the study. Most of the cases were tonsillar carcinomas (45.9%), and almost half (49.0%) of the tumors had a nonkeratinizing morphology. Xpert HPV and INNO-LiPA results The results obtained with the two HPV testing methods are shown in Table 1. Using the INNO-LiPA, 3/159 samples (1.8%) gave an invalid result (no band was observed for the housekeeping gene). The absorbance at 260 nm of the nucleic acid extract for these three samples was 0.45, 0.35 and 1.09, whereas the mean absorbance for all samples was 1.31 (range 0.04–7.01). 66/159 specimens (41.5%) tested positive and had a single infection, caused by HPV16 in the majority of cases (86.4%). All the genotypes detected by the INNO-LiPA were among those also detectable by the Xpert. No invalid results were observed with Xpert, independently of the absorbance at 260 nm of the crude lysate, which ranged between 0.08 and 21.02 (mean: 2.99). With this method, 68/159 samples (42.7%) tested HPV-positive and HPV16 was by far the most common genotype detected (88.2%). The three samples that were found invalid using the INNO-LiPA tested negative with the Xpert. The analysis on the 156 cases that gave a valid result with both tests is shown in Table 2: 65 cases (41.7%) were concordantly positive while 87 (55.8%) were concordantly negative, with a very high raw agreement (0.97, 95% CI: 0.93–0.99) and Cohen K (0.95, 95% CI: 0.90–0.99). Three samples (1.9%) were positive for HPV16 with Xpert but negative with INNO-LiPA; two of these were also positive for p16 staining. Only 1 OPSCC (0.6%) tested negative with Xpert and positive with INNO-LiPA, which detected HPV51. However, this sample was p16-negative. These four discordant cases were analyzed for the presence of HPV mRNA. This was detected in the two samples that were Xpert HPV16-positive/INNO-LiPA negative/p16positive, while the Xpert HPV16-positive/INNO-LiPA negative/ p16-negative sample was negative for HPV mRNA (data not shown). The Xpert negative/INNO-LiPA HPV51-positive/p16negative OPSCC was negative for the specific mRNA (data not shown). Although results for both p16 and HPV mRNA were only available for these four cases, it is worth noting that they were perfectly concordant. The HPV genotyping results for the 65 cases that tested positive with both assays are shown in Table 3. Type-specific data were completely concordant (Cohen K = 1). In fact, the Xpert identified HPV16 in all the 57 samples that were also HPV16-positive with

HPV result

INNO-LiPA

Xpert

Negative Positive

90 (56.7) 66 (41.5) 57 (86.4) 1 (1.5) 8 (12.1)b 3 (1.8) 159 (100)

91 (57.3) 68 (42.7) 60 (88.2) 1 (1.5)a 7 (10.3)c 0 (0.0) 159 (100)

HPV16 HPV18 Other HR-HPVs Invalid Total a b c

Positive for the probe that detects HPV 18 and 45 as a pool. 2 HPV33-positive; 4 HPV35-positive; 1 HPV51-positive; 1 HPV58-positive. Cases positive for the probe that detects HPV 31, 33, 35, 52, 58 as a pool.

Table 2 Comparison of INNO-LiPA and Xpert for the 156 cases with a valid result with both tests. Xpert

Positive Negative

INNO-LiPA n (%) Positive

Negative

65 (41.7) 1 (0.6)b

3 (1.9)a 87 (55.8)

Raw agreement = 0.97 (95% CI: 0.93–0.99). Cohen K = 0.95 (95% CI: 0.90–0.99). a All cases were positive for HPV16 and two of them were also p16-positive. b HPV51-positive, p16-negative.

INNO-LiPA. All the remaining samples gave a compatible result, i.e., they tested positive with the Xpert probe that detects the genotypes identified by INNO-LiPA, although the Xpert cannot provide a result for the individual genotypes except for HPV16. Comparison of HPV-DNA status by Xpert and INNO-LiPA and p16 staining All the FFPE samples analyzed in this study had been stained for p16. As shown in Table 4, almost all the cases that tested positive with INNO-LiPA were also p16-positive (65/66, 98.5%). Similarly, 67/68 (98.5%) of the Xpert-positive cases displayed p16 staining. Compared to the 9 (9.8%) Xpert-negative and p16-positive OPSCCs, a slightly higher fraction of cases were INNO-LiPA negative and p16-positive (11, 12.2%). Thus, the concordance with p16 staining was slightly higher for the Xpert (Cohen K = 0.87, 95% CI: 0.80– 0.94) than for the INNO-LiPA (Cohen K = 0.85, 95% CI: 0.76–0.93). Discussion The Xpert HPV assay is a new method for HPV testing in cervical cytology that, in around 1 h, detects and partially genotypes the 14 HPV types responsible for cervical cancer development, some of which are also involved in HNSCC etiology. This test fulfills the Meijer’s criteria for use in primary cervical cancer screening programs [34]. Based on these guidelines, the accuracy indicators of a candidate HR-HPV test should show a non-inferiority compared to the clinically validated reference HC2 test on samples from a population-based screening cohort. Indeed, in a screening population, the Xpert showed a clinical performance comparable to that of HC2 [35] and other well-established HPV assays [36]. Several HPV testing methods designed for cervical specimens, such as cobas 4800 and HC2, have been also applied to HNSCC tissues for HPV DNA detection [21–23]. In this study, we analyzed the performance of the Xpert on 159 FFPE samples from OPSCC patients, and compared the results with those obtained with the well-characterized INNO-LiPA HPV assay and with the

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Table 3 Comparison of the HPV genotyping results for the 65 cases positive with both the INNO-LiPA and Xpert assay. n (%) INNO-LiPA

Total

Xpert

HPV16

HPV18

Other HR-HPVa

HPV16 HPV 18/45 Other HR-HPVb Total

57 (100) 0 (0.0) 0 (0.0) 57 (100)

0 1 0 1

0 0 7 7

(0.0) (100) (0.0) (100)

(0.0) (0.0) (100) (100)

57 (100) 1 (100) 7 (100)

Raw agreement = 1 (95% CI: 0.93–1). Cohen K = 1 (95% CI: 1–1). a HPV 33, 35, 58. b HPV 31, 33, 35, 52, 58 as a pool.

Table 4 Comparison of p16 positivity and HPV status detected with the INNO-LiPA and Xpert assay for the samples with a valid HPV test result. p16 staining n (%) Positive

Negative

Total

INNO-LiPA (N = 156) Positive Negative Total

65 (98.5) 11 (12.2) 76 (48.7)

1 (1.5)a 79 (87.8) 80 (51.3)

66 (100) 90 (100) 156 (100)

Xpert (N = 159) Positive Negative Total

67 (98.5) 9 (9.8) 76 (47.8)

1 (1.5)b 82 (90.2) 83 (52.2)

68 (100) 91 (100) 159 (100)

Inno-LiPA vs. p16: raw agreement 0.92 (95% CI: 0.87–0.96); Cohen K 0.85 (95% CI: 0.76–0.93). Xpert vs. p16: raw agreement 0.94 (95% CI: 0.88–0.97); Cohen K 0.87 (95% CI: 0.80– 0.94). a HPV51-positive and Xpert negative. b HPV16-positive and Inno-LiPA negative.

immunohistochemical expression of p16, which were available for all the cases. To the best of our knowledge, only one study has been published, which has investigated the performance of the Xpert on FFPE tissues of HNSCC [24]. However, this previous investigation analyzed 74 OPSCC cases, and had p16 assessment on only 50 of them. Moreover, differently from Guerendiain and collaborators, who carried out the Xpert on nucleic acids purified from the tissue sections, in the present study, the Xpert was performed on crude lysates. Importantly, we observed no invalid results. Together with the very good agreement with the INNO-LiPA results (see below), this indicates that HPV detection on FFPE samples with the Xpert does not require prior purification of nucleic acids. This is an important point because this step is time-consuming, increases the costs of HPV-DNA testing, and inevitably introduces a source of variability, as shown by previous studies [26,28]. Therefore, the protocol used in this study is less time consuming, costsaving, and stable, since the variability associated with DNA purification is avoided. Additionally, while the other PCR-based HPV testing methods require that purification, amplification and detection steps are each performed in a different room, all these steps are carried out in a ready-to-use disposable Xpert cartridge, thus limiting the laboratory space required and minimizing the contamination risk. Moreover, because of its simplicity and ease of use, Xpert does not require highly skilled technicians. In our investigation, we tested 68 samples which were older than 5 years and all of them provided a valid Xpert result, whereas 6.7% of cases analyzed by Guerendiain and collaborators were found to be invalid even if using purified nucleic acids [24], and the only previous study that used crude lysates from 10 to 15-year-old FFPE specimens produced an invalid result in 17.6% of the cases [unpublished data, 25]. This may due to the fact that

chemical damage typically increases with the age of the tissue. Compared to the samples analyzed in the previous investigation, those tested in the present study were more recent, being 7year-old at the most. The performance of Xpert on older specimens should be investigated to better establish whether this method is suitable for epidemiological retrospective studies. Our findings may also indicate that the FFPE specimens of the present study were well preserved, with limited DNA fragmentation and cross-linking. In fact, the choice of appropriate conditions in the pre-analytical phase plays a central role in guaranteeing the quality of FFPE tissues and their use for future studies; type of fixative, pH and fixation time are among the variables that may influence the quality of these specimens. While on the one hand the lack of invalid results may be due to the fact that our samples were mostly recent and were handled properly in the pre-analytical phase, on the other hand it may also suggest that Xpert is particularly resistant to inhibition, since its performance was not affected by the use of a total cell lysate, which contain a considerable quantity of PCR inhibitors. This resistance might be due to the reagents used for the amplification, e.g., DNA polymerase and/or additives. Interestingly, the performance of Xpert was very good despite the wide variation in the quantity of nucleic acids present in the lysates, as inferred by the measures of absorbance at 260 nm. This suggests that, on the one hand, this method can be applied even when very small tissue blocks are available, and that, on the other hand, it is not inhibited by a great quantity of DNA, which is usually accompanied by a larger presence of template-associated inhibitors. We found a very good agreement between Xpert and INNO-LiPA (Cohen K = 0.95). Importantly, this very good agreement was not only observed for the HPV status but also for the genotyping results. In fact, Xpert identified HPV16 in all the samples that tested HPV16-positive with INNO-LiPA. Although Xpert does not specifically genotype all 14 detectable HR-HPVs, HPV16 typing is given as a single result, and this represents an important feature of the test since HPV16 has been shown to be responsible for the majority of the HPV-driven OPSCCs [20]. Indeed, both in our series as well as in Guerendiain’s samples, HPV16 was the most represented genotype [24]. Interestingly, the three samples that were invalid with INNOLiPA gave a valid result with Xpert; all of them tested negative and showed no p16 staining. Among the samples with a valid result with both tests, only four discordant cases were observed. The Xpert detected HPV16 in three specimens that were negative with INNO-LiPA. Two of these cases were positive both for p16 and HPV mRNA and thus represent true HPV-driven OPSCCs that the INNO-LiPA failed to reveal. The third case was negative for p16 and also for HPV mRNA and thus represents a case in which Xpert detected an HPV infection not really involved in the etiology of the tumor. In fact, in order to establish the cases associated with

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HPV infection, a multi-step algorithm should be applied that is based both on HPV-DNA testing to reveal the presence of the virus, and detection of viral activity, directly, by means of an E6/E7 mRNA test, and/or indirectly, by p16 expression analysis. It is widely accepted that the HPV-attributable OPSCCs are those that display simultaneous positivity for HPV-DNA and p16 and/or E6/E7 mRNA. Only one case was negative with Xpert and positive with INNO-LiPA. This OPSCC harboured HPV51, which is included among the types detectable with Xpert, but this sample was p16-negative as well as HPV mRNA-negative. Based on the above-mentioned criteria to establish the HPV-driven cancers, this case cannot be considered as etiologically associated to HPV infection. Therefore, Xpert, even though it did not reveal the presence of HPV51, did not miss an HPV-associated OPSCC. Previous studies that applied HR-HPV detection methods to HNSCC evidenced a variable performance for these tests. Compared to p16 immunostaining, cobas 4800 showed a higher sensitivity (100% vs. 93%) but a lower specificity (91% vs. 97%) in HPV detection [23]. Another study that applied HC2 on FFPE HNSCC found a fair agreement between this test and p16 staining (K = 0.62) [22]. Differently, we found a very good agreement between the Xpert results and p16 findings, and this was slightly higher than that observed for the INNO-LiPA (Cohen K 0.87 vs. 0.85). These results are consistent with those of Guerendiain et al., who found a 90% agreement between Xpert and p16 findings [24]. The very high rate of p16 positivity among the Xpert-positive cases suggests that this method detects HPV-DNA when HPV is not only present but also actively transcribed. This finding may support the use of this assay to establish the real fraction of HPV-associated tumors, although studies on other case series are needed, as well as investigations on the clinical outcome of OPSCCs that tested HPV-positive with Xpert. We have to acknowledge a few limitations in the use of the Xpert and, in general, of HPV DNA detection methods. Compared to RNA ISH which allows the direct visualization of transcriptionally active HPV within the tumor, PCR-based methods do not offer such advantage, may be susceptible to contamination and may lead to misdiagnosis because of the high sensitivity and inability to distinguish active and transforming infections. While in cervical cancer screening a high sensitivity is required in order to detect the majority of the high-grade lesions, in OPSCC diagnostic workup specificity is privileged. In fact, false-positive cases that may improperly undergo de-intensified treatment protocols must be avoided. Therefore, HPV-DNA detection methods cannot be used as a standalone diagnostic test to identify HPV-driven OPSCC because of their poor accuracy, and should be combined with other markers, such as p16 and/or HPV mRNA.

Conclusion In conclusion, we confirmed the data of Guerendiain’s study on a larger series of tumors which were homogeneously derived from the oropharynx, which, to date, represent the only HNSCC with a significant association with HPV infection and important biological differences compared to the HPV-negative counterpart. Most importantly, the Xpert HPV assay, a simple and rapid cartridgebased method that minimizes the risk of contamination, displayed a high resistance to inhibition and can thus be used on crude lysates from FFPE tissues. It also showed a very good agreement with the INNO-LiPA and p16 findings, and thus appears to be a reliable HPV detection method for FFPE OPSCCs and a promising tool for the assessment of the HPV-driven fraction in combination with other biomarkers such as p16 and/or HPV mRNA.

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Acknowledgments The authors would like to thank Dr. Michael Kenyon for his review of the English language, and Dr. Massimo Tommasino for providing the collaboration of his group in performing the HPV mRNA analysis. Funding This work was partly supported by the Italian Ministry of Health (GR-2011-02349732). Part of the reagents (Xpert HPV Assay) have been provided for free by Cepheid S.r.l. Role of the funding source Cepheid S.r.l. did not have any role in study design, conduction, data analysis, and in the decision of submitting data for publication. Conflict of interest statement None declared. References [1] International Agency Research Cancer. IARC monographs on the evaluation of carcinogenic risks to humans, vol. 100B. A review of human carcinogens: biological agents. Lyon, France: IARC; 2011. [2] Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, et al. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. J Clin Oncol 2011;29:4294–301. [3] Carlander AF, Gronhoj Larsen C, Jensen DH, Garnaes E, Kiss K, Andersen L, et al. Continuing rise in oropharyngeal cancer in a high HPV prevalence area: a Danish population-based study from 2011 to 2014. Eur J Cancer 2017;70:75–82. [4] Tinhofer I, Johrens K, Keilholz U, Kaufmann A, Lehmann A, Weichert W, et al. Contribution of human papilloma virus to the incidence of squamous cell carcinoma of the head and neck in a European population with high smoking prevalence. Eur J Cancer 2015;51:514–21. [5] Coordes A, Lenz K, Qian X, Lenarz M, Kaufmann AM, Albers AE. Meta-analysis of survival in patients with HNSCC discriminates risk depending on combined HPV and p16 status. Eur Arch Otorhinolaryngol 2016;273:2157–69. [6] Dahlstrom KR, Garden AS, William Jr WN, Lim MY, Sturgis EM. Proposed staging system for patients with HPV-related oropharyngeal cancer based on nasopharyngeal cancer N categories. J Clin Oncol 2016;34:1848–54. [7] Masterson L, Moualed D, Liu ZW, Howard JE, Dwivedi RC, Tysome JR, et al. Deescalation treatment protocols for human papillomavirus-associated oropharyngeal squamous cell carcinoma: a systematic review and metaanalysis of current clinical trials. Eur J Cancer 2014;50:2636–48. [8] Bishop JA, Ma XJ, Wang H, Luo Y, Illei PB, Begum S, et al. Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. Am J Surg Pathol 2012;36:1874–82. [9] Kerr DA, Arora KS, Mahadevan KK, Hornick JL, Krane JF, Rivera MN, et al. Performance of a branch chain RNA in situ hybridization assay for the detection of high-risk human papillomavirus in head and neck squamous cell carcinoma. Am J Surg Pathol 2015;39:1643–52. [10] Rooper LM, Gandhi M, Bishop JA, Westra WH. RNA in-situ hybridization is a practical and effective method for determining HPV status of oropharyngeal squamous cell carcinoma including discordant cases that are p16 positive by immunohistochemistry but HPV negative by DNA in-situ hybridization. Oral Oncol 2016;55:11–6. [11] Smeets SJ, Hesselink AT, Speel EJ, Haesevoets A, Snijders PJ, Pawlita M, et al. A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer 2007;121:2465–72. [12] Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tan PF, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 2010;363:24–35. [13] Ukpo OC, Flanagan JJ, Ma XJ, Luo Y, Thorstad WL, Lewis Jr JS. High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma. Am J Surg Pathol 2011;35:1343–50. [14] Gheit T, Anantharaman D, Holzinger D, Alemany L, Tous S, Lucas E, et al. Role of mucosal high-risk human papillomavirus types in head and neck cancers in central India. Int J Cancer 2017;141:143–51. [15] Lewis Jr JS, Thorstad WL, Chernock RD, Haughey BH, Yip JH, Zhang Q, et al. P16 positive oropharyngeal squamous cell carcinoma: an entity with a favorable prognosis regardless of tumor HPV status. Am J Surg Pathol 2010;34:1088–96.

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