Human papillomavirus infection and p16 ...

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Human papillomavirus infection and p16 overexpression in oropharyngeal squamous cell carcinoma: a case series from 2010 to 2014 Maria Gabriella Donà*,1, Giuseppe Spriano2, Barbara Pichi2, Francesca Rollo3, Valentina Laquintana3, Renato Covello3, Raul Pellini2, Massimo Giuliani1, Edoardo Pescarmona3 & Maria Benevolo3 Aim: Human papillomavirus (HPV) associated oropharyngeal squamous cell carcinomas (OPSCC) show better prognosis and response to therapy. We evaluated HPV-DNA prevalence, p16 overexpression and HPV-attributable fraction in recent consecutive OPSCCs. Materials & methods: 140 OPSCCs were analyzed for HPV-DNA using the INNO-LiPA assay and p16 overexpression using CINtec® Histology kit. Results: HPV-DNA prevalence was 40.7%, and was significantly higher in basaloid and nonkeratinizing SCCs, but similar in tonsillar and base of tongue SCCs. Moreover, 98.0% of the HPV-positive OPSCCs overexpressed p16, compared with 12.3% of the HPV-negative cases (p < 0.0001). Based on HPV-DNA and p16 double positivity, 39.8% of the cases were HPV related. Conclusion: The high estimate of the HPV-attributable fraction suggests that a substantial proportion of OPSCC patients may be managed less intensively. In recent years, the etiologic role of human papillomavirus (HPV) in the development of a substantial fraction of head and neck squamous cell carcinomas (HNSCC) has been clearly established. In particular, a large body of experimental and epidemiologic data indicates that HPV infection plays a major role in the etiology of oropharyngeal cancer (OPSCC). Indeed, the International Agency for Research on Cancer (IARC) has recognized genotype HPV16 as a cause of oropharyngeal cancer [1] . Importantly, HPV-negative and -positive OPSCCs show distinct epidemiologic profiles, especially regarding age-at-onset, association with alcohol consumption, tobacco use and sexual behavior [2] . They also differ according to pathological features (e.g., tumor size and nodal involvement). Notably, patients with HPV-positive cancers display a significantly improved prognosis and response to therapy compared with those with HPV-negative tumors [3] . Recently, increasing incidence trends for HPV-positive OPSCC have been reported in many countries [4–7] , so this phenomenon has been defined as a virus-related cancer epidemic [8] . Owing to the abovementioned reasons, interest in the proportion of OPSCCs attributable to HPV has greatly increased, as well as in effective tools that can be used to evaluate the HPV status in patients with head and neck cancers [9] . Most of the available data on HPV prevalence in OPSCCs derive from studies conducted in the USA and northern Europe. Conversely, there are little data available concerning southern Europe and only a few of these studies, with small sample sizes, have been conducted in Italy [10–14] . There is now consolidated evidence that HPV-DNA detection alone is insufficient for the identification of HPV-related HNSCCs. Although the detection of viral transcripts is considered as the gold

KEYWORDS

s head and neck neoplasms s human papillomavirus s oropharyngeal neoplasms s p16INK4 protein s prevalence

Sexually Transmitted Infection (STI) Unit, San Gallicano Dermatologic Institute, IRCCS, Via Elio Chianesi 53, 00144 Rome, Italy Otolaryngology Head Neck Surgery Department, Regina Elena National Cancer Institute, IRCCS, Via Elio Chianesi 53, 00144 Rome, Italy 3 Pathology Department, Regina Elena National Cancer Institute, IRCCS, Via Elio Chianesi 53, 00144, Rome, Italy *Author for correspondence: Tel.: +39 06 5266 5393; Fax: +39 06 5266 6254; [email protected] 1 2

10.2217/FMB.15.55 © 2015 Future Medicine Ltd

Future Microbiol. (2015) 10(8), 1283–1291

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PRELIMINARY COMMUNICATION Donà, Spriano, Pichi et al. standard when estimating the HPV-attributable cancer fraction, overexpression of the cyclindependent kinase inhibitor p16INK4A (p16) is also regarded as a biomarker of transforming and active HPV infections [15] . In this study, we assessed HPV-DNA prevalence and genotype distribution in a recent series of well-characterized OPSCCs, also analyzed for p16INK4A (p16) overexpression. Moreover, we evaluated the HPV-attributable fraction based on the positivity for both p16 and HPV-DNA. To our knowledge, this represents the largest Italian case series of OPSCCs analyzed with an up-to-date HPV genotyping assay. Materials & methods ●●Study population

Formalin-fixed paraffin embedded (FFPE) tissues were retrieved from the archive of the Regina Elena National Cancer Institute (Italy) from consecutive cases of oropharyngeal SCCs. Inclusion criteria were as follows: an incidental diagnosis of primary untreated SCC of the oropharynx between June 2010 and November 2014 (International Classification of Diseases-10 codes for the oropharynx: base of the tongue (BOT), C01; soft palate, C05.1; tonsil, C02.4, C09.0–9; uvula, C05.2; other parts of the oropharynx, C10.0–9; Waldeyer’s ring, C14.2); confirmation of cancer in the first and last hematoxylin and eosin (H&E) stained sections (see below); sufficient material for HPV testing. The study was approved by the Ethics Committee of the Regina Elena National Cancer Institute (CE/485/12).

●●Study protocol

From each FFPE block, the following sections were obtained: 2 × 2 μm outer sections, used for H&E staining and diagnosis confirmation; 1 × 5 μm up to 3 × 5 μm inner sections, depending on the tissue size, for HPV detection and genotyping; 1 × 2 μm section for p16 staining, which was obtained after the sections for HPV testing. Strict conditions were adopted in order to avoid contamination [16] . OPSCC diagnosis was confirmed by two pathologists. ●●HPV DNA testing

DNA was isolated from the inner sections using the DNeasy Blood and Tissue Kit (Qiagen) as previously described [16] . HPV-DNA detection and genotyping were performed using the INNO-LiPA HPV Genotyping Extra kit (Fujirebio), which detects 28 genotypes. This assay includes a human DNA control (HLDDPB1 gene) to monitor sample quality and extraction efficiency. Manufacturer’s instructions were followed and 10 μl of extract was used as a template for amplification. All the hybridization steps up to color development were carried out in a Profiblot T48 instrument (Tecan). HPV carcinogenic risk was classified in accordance with IARC indications for cervical cancer [1] . Specifically, the 12 HPVs of Group I were considered as high-risk (HR) types. ●●p16 immunohistochemistry

Overexpression of p16 was investigated using the CINtec® Histology kit (Roche Diagnostics), following the manufacturer’s instructions. The

Figure 1. Representative examples of p16INK4A immunohistochemistry in HPV-positive oropharyngeal squamous cell carcinoma. (A) HPV16-positive tonsillar cancer; (B) HPV33-positive base of tongue cancer. Homogeneous and strong nuclear and cytoplasmic staining is visible. 3′,3′ diaminobenzidine (DAB) was used as a chromogenic substrate. Counterstaining with hematoxylin. 200× magnification.

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Human papillomavirus infection & p16 overexpression in OPSCC

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Table 1. Descriptive characteristics of the 140 oropharyngeal squamous cell carcinomas of the study population. Characteristic

OPSCC, n = 140

Median age, years (IQR) Sex distribution, n (%): – Men – Women Year of diagnosis, n (%): – 2010 (June–December) – 2011 – 2012 – 2013 – 2014 Cancer subsite, n (%): – Tonsil – Base of tongue – Other† Differentiation grade n (%): – G2 – G3 Basaloid type, n (%) Keratinizing, n (%)

62 (54–69) 116 (82.9) 24 (17.1) 17 (12.2) 31 (22.1) 31 (22.1) 30 (21.5) 31 (22.1) 64 (45.7) 60 (42.9) 16 (11.4) 44 (31.4) 96 (68.6) 30 (21.4) 83 (59.3)

Soft palate, epiglottic vallecula, posterior wall. IQR: Interquartile range; OPSCC: Oropharyngeal squamous cell carcinoma.

†

antibody included in this kit is a monoclonal antibody (clone E6H4) specific for human p16INK4A protein. Immunostaining was independently examined by two investigators blinded to the HPV findings. Similar to the criteria used by Prigge and collaborators, only diffuse (>10% tumor cells stained) and clonal (at least five contiguous positive cells) staining patterns were considered as positive, regardless of staining intensity [17] (Figure 1) .

included in the study. The median age of the patients at diagnosis was 62 years (IQR: 54–69). Descriptive characteristics of the study population are reported in Table 1. Most of the OPSCCs were diagnosed at the tonsils (45.7%) and base of tongue (42.9%). Cancer cases were predominantly keratinizing (59.3%) and poorly differentiated (68.6%). Only a reduced proportion was basaloid (21.4%). ●●HPV-DNA prevalence

●●Statistical analyses

Descriptive statistics were used to describe the basic features of the study population. Samples containing any of the 28 HPV types that were detectable using the INNO-LiPA were considered positive for HPV. Samples containing any of the 12 HPVs of Group I (see above) were considered positive for HR types. Differences between percentages were tested using Pearson chi-square. A p-value < 0.05 was considered as statistically significant. Statistical analyses were conducted using the SPSS+ package v.17.0. Results ●●Study population Based on the inclusion criteria previously described, 140 consecutive OPSCC cases were


Overall, HPV-DNA was detected in 57/140 cases (40.7%). HPV prevalence in tonsillar (46.9%) and BOT SCC (43.3%) was similar (p = 0.69) (Table 2) . HPV-DNA was detected in a significantly higher proportion of cases from these two subsites compared with other oropharyngeal subsites (45.2 vs 6.2%, p = 0.002). HPV prevalence was significantly higher in basaloid (60.0 vs 35.4%, p = 0.01) and nonkeratinizing (52.6 vs 32.5%, p = 0.017) cancer types. Moderately and poorly differentiated SCCs showed a similar rate of HPV positivity (40.9 vs 40.6%, p = 0.56). No significant difference in HPV prevalence between genders was observed (45.8 in women vs 39.6% in men, p = 0.58; data not shown). The median age for patients with HPV-negative and HPV-positive OPSCC was the same (62 years).

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PRELIMINARY COMMUNICATION Donà, Spriano, Pichi et al. Table 2. Overall HPV prevalence and stratified by cancer subsite and histopathologic characteristics. Characteristic

HPV-DNA positive n/N (%)

All OPSCCs

57/140 (40.7)

Cancer subsite

Tonsils Base of tongue Other†

30/64 (46.9) 26/60 (43.3) 1/16 (6.2)

Histopathologic characteristics

Basaloid type: – Yes – No Keratinizing: – Yes – No Grading: – G2 – G3

18/30 (60.0) 39/110 (35.4) 27/83 (32.5) 30/57 (52.6) 18/44 (40.9) 39/96 (40.6)

Soft palate, epiglottic vallecula, posterior wall. OPSCC: Oropharyngeal squamous cell carcinoma.

†

Only HR types were found. Overall, HPV16 was by far the most frequent type (50/140, 35.7%), and accounted for the majority of the HPV-positive OPSCCs, being present in 50/57 cases (87.7%), followed by HPV-33 and -35 (two cases each, 3.6%), HPV-18, -51 and -58 (1 case each, 1.7%) (Table 3) . HPV-16 prevalence did not differ significantly between women and men (72.7 vs 91.3%, p = 0.12, data not shown). No multiple infections were found. ●●p16 overexpression

Analysis of p16 overexpression was performed on 123 OPSCCs, which included 50 HPV-positive cases (43 with HPV16, one with HPV18, two with HPV33, two with HPV35, one with HPV51 and one with HPV58). Overall, 58/123 cases (47.1%) were positive for p16 (Table 4) . Positivity for p16 did not vary significantly when comparing tonsillar and BOT SCCs (50.0 vs 55.5%, p = 0.69). The proportion of tumors overexpressing p16 was significantly higher among HPV-positive than HPV-negative OPSCCs (98.0 vs 12.3%, p < 0.0001). This was also observed for tonsillar and BOT SCCs separately. In fact, among HPV-positive tonsillar SCCs, 96.0% displayed p16 staining, which is a significantly higher proportion compared with that of HPV-negative cases (10.3%, p < 0.0001). Similarly, all HPVpositive BOT SCCs were positive for p16, while 20.0% of the HPV-negative cases displayed p16 overexpression (p < 0.0001).

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Finally, considering these 123 cancer cases that were tested both for HPV-DNA and p16 overexpression, we observed that 49 (39.8%) were positive for both biomarkers (data not shown). Discussion The indisputable role of HPV in the etiology of OPSCC has fuelled the research on the fraction of cases related to this infection, especially in view of its significance regarding prognosis and response to therapy. A large body of data is available from the USA and several European countries, while only few studies, also with limited case series, have been conducted in Italy. To our knowledge, this represents the first significant sized study on HPV-DNA prevalence and genotyping in OPSCCs in Italy, carried out using an up-to-date genotyping assay. We observed an overall HPV-DNA prevalence of 40.7%, which is in line with the most recent meta-analysis which estimated a pooled worldwide prevalence of 45.8% for 3946 OPSCC cases and a pooled prevalence of 41.4% for Europe [18] . Our findings are similar to those of other European countries, such as Germany and Sweden [18] and to some Italian studies [11,12] . However, they substantially differ from the overall prevalence of 24.2% estimated for southern Europe [18] and from other Italian investigations that evidenced a much lower prevalence [14,19] . This large variation may be


Human papillomavirus infection & p16 overexpression in OPSCC


Table 3. HPV type-specific prevalence among the 57 oropharyngeal squamous cell carcinomas positive for HPV-DNA. HPV genotype

HPV-positive n (%)

16 18 33 35 51 58 All

50 (87.7) 1 (1.7) 2 (3.6) 2 (3.6) 1 (1.7) 1 (1.7) 57 (100.0)

explained by the relative contribution of the cancer subsites, the characteristics of the study population, such as percent of males, patient age and period of diagnosis. Noteworthy, our study population included cases diagnosed in the last 5 years, and this may account for the higher HPV prevalence compared with other Italian studies which analyzed cases diagnosed in less recent time periods. Moreover, the diversity of HPV testing methods also contributes to such variation. The Italian studies either utilized assays that do not provide type-specific information [12,13] , or limited the analysis to HPV 16 and 18 [10] , and, differently from the present report, none of them used the INNO-LiPA assay. This PCR-based method, which allows us to detect 28 HPV genotypes, is among the most valid assays for HPV detection and genotyping on FFPE samples because of the very limited size of the amplicon [20,21] . Importantly, the INNO-LiPA HPV Genotyping Extra has been shown to give the highest rate of HPV positivity compared with other six methods when testing FFPE biopsies from oral and oropharyngeal squamous cell carcinomas [22] . Finally, the INNO-LiPA employs SPF-10 broad-spectrum primers, used in international surveys to assess HPV prevalence in cancers other than cervical carcinoma [23,24] . Our study confirms that HPV prevalence in tonsillar and BOT SCCs is significantly higher

than in other OPSCCs [18] . Additionally, we detected no significant difference in HPV-DNA prevalence between tonsillar and BOT cancers, a fact also evidenced in the meta-analysis (pooled prevalence being 53.9% for tonsillar and 47.8% for BOT SCC) [18] and other European studies [25] . Conversely, a retrospective study on over 4000 HNSCCs from all over the world reported a much higher prevalence for tonsillar (47.0%) compared with BOT SCCs (18.5%) [Castellsaguè X, Unpublished Data] . A similar finding has been reported in another international multicenter study [19] . Our study confirms that HPV-positive OPSCCs are predominantly basaloid and nonkeratinizing, as already evidenced in other studies [26,27] . Although our case series lacked G1 tumors, we did not observe any association between HPV positivity and differentiation grade. Similar to other Italian studies [10–12] and global data that show that OPSCC is much more common in men than women [28–30] , the OPSCCs of our study population mostly occurred in men (82.9%). Regarding the positivity for HPV-DNA according to gender, we evidenced a higher prevalence among women than men, although this difference was not significant and our female study sample had a limited size. Our findings are similar to pooled European data that showed a male-to-female ratio of HPV

Table 4. p16 positivity by HPV status in the 123 oropharyngeal squamous cell carcinoma cases tested for both biomarkers. HPV status Positive Negative Total

p16-positive, n/N (%) Tonsillar SCC, n = 54

Base of tongue SCC, n = 54 Other OPSCC†, n = 15

Total

24/25 (96.0) 3/29 (10.3) 27/54 (50.0)

24/24 (100.0) 6/30 (20.0) 30/54 (55.5)

49/50 (98.0) 9/73 (12.3) 58/123 (47.1)

1/1 (100.0) 0/14 (0.0) 1/15 (6.7)

Soft palate, epiglottic vallecula, posterior wall. OPSCC: Oropharyngeal squamous cell carcinoma; SCC: Squamous cell carcinoma.

†



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PRELIMINARY COMMUNICATION Donà, Spriano, Pichi et al. prevalence in OPSCC of 1.0 [30] . Conversely, in North America, Australia and some European countries HPV-positive OPSCCs are much more likely to occur in men [29] . Similar to other Italian studies [10] , no significant difference emerged in the median age of patients with HPV-positive and HPV-negative OPSCCs. However, others have evidenced that patients with HPV-positive HNSCCs are usually younger (3–5 years) than those with HPVnegative tumors [2] . HPV16 was by far the most common genotype among our HPV-positive OPSCCs (87.7%), and the other genotypes were much less frequent, in line with previous reports [18,25,31] . In particular, we found HPV 33 and 35 in 3.6% of the samples in both cases, and HPV 18, 51 and 58 in 1.7% of the samples in all three cases. Pooled data showed that HPV33 is the second most frequent genotype worldwide in OPSCCs, followed by HPV18 [18] . Interestingly, we found one positive case for HPV51, while in the pooled analysis of 2975 OPSCCs, none of the cases harbored this genotype [18] . HPV51 has been detected in oral rinses [32,33] , but rarely in HNSCC, and mainly of the oral cavity and hypopharynx [18] . However, it must be underlined that the OPSCC harboring HPV51 was the only HPV-positive cancer that did not show p16 overexpression (see below). This finding suggests lack of involvement of HPV51 in the development of this cancer, thus classifiable as an HPV-inactive tumor [34] . None of the cases analyzed harbored a multiple infection, in accordance with recent data that showed that multiple infections are very rare among HNSCCs (approximately 1%, ten-times lower than in cervical cancer) [18,31] . A subgroup of our cases was also analyzed for p16 overexpression. This is considered a reliable surrogate biomarker of transforming HPV infections, especially in cervical cancer but also in HNSCCs. We found that 98.0% of the HPV-positive cases were also positive for p16. This p16 percentage positivity is in line with other Italian [10,35] and European data [36,37] . Noteworthy, all HPV-positive cases displayed p16 overexpression irrespective of the genotype, except for the OPSCC harboring HPV51, as previously described. We also observed that 12.3% of the HPVnegative cases were positive for p16. This proportion is in line with other reports [10,38] . Although a partial misclassification of the cases

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as HPV-negative is hypothetically possible, the method used for HPV-DNA detection in this study is very sensitive, and particularly suitable for FFPE samples. The limited size of the amplicon makes it possible to reduce the rate of false-negative cases which might arise because of DNA degradation in this type of specimen. When HPV infection is absent, p16 overexpression might still occur because of the activation of other cellular oncogenic pathways. When we estimated the HPV-attributable fraction considering the simultaneous positivity for HPV-DNA and p16, we found that 39.8% of the OPSCCs tested positive for both markers. This fraction is similar to the pooled prevalence of 45.0% calculated in the most recent meta-analysis based on the data from five European studies, but is also similar to the estimated percentage of dual-positive OPSCCs, in other words, 39.7% [18] . This meta-analysis also evidenced that the HPV-driven fraction for OPSCCs was similar when considering either p16 (39.7%) or E6/E7 mRNA positivity (39.8%) in addition to HPV-DNA positivity. The detection of viral transcripts represents the gold standard biomarker of HPV oncogenic activity, because the presence of these transcripts indicates the HPV etiologic role in HNSCC development [39] . However, mRNA detection is particularly challenging in FFPE tissues, generally used for retrospective studies. Additionally, HPV mRNA detection requires genotype-specific assays. Differently, p16 immunohistochemistry on FFPE tissues is easy to perform, widely utilized in pathology departments and it can be employed irrespective of the genotype present in the tumor. There are a few limitations in the present study. The HPV-attributable fraction estimation was not based on mRNA detection, as just mentioned. Second, this was a single-center study, thus our findings may not reflect exactly the HPV contribution to OPSCCs in Italy. Conclusion The current study evidenced that a substantial fraction of OPSCCs is positive for HPV-DNA, confirmed that HPV16 is the dominant genotype, and that HPV positivity is associated with basaloid and nonkeratinizing OPSCC cases. It also showed a comparable HPV prevalence in tonsillar and base of tongue SCC and a slightly higher prevalence in women compared with men, although it was


Human papillomavirus infection & p16 overexpression in OPSCC not statistically significant. Finally, considering p16 overexpression in addition to HPVDNA positivity, our estimate of HPV-driven OPSCCs is particularly high. This finding may have a significant impact on the management of OPSCC patients, a substantial fraction of which may be eligible for less intensive treatments. Additionally, it indicates that a significant proportion of OPSCC cases may be prevented through the currently available HPV vaccines. A future Italian multicenter study would be essential in order to confirm our findings and to assess conclusively the HPVattributable fraction in our country. Future perspective The exact contribution of HPV infection to orophrayngeal squamous cell carcinoma is still unknown and much variability has been observed regarding the HPV-attributable fraction, depending on the case series analyzed and the biomarkers used to assess transforming HPV infections. Because of the improved prognosis of HPV-positive head and neck cancers, considerable efforts will be made to elucidate which is the most suitable and useful biomarker/algorithm to identify such tumors. At present, management of patients with HPV-positive oropharyngeal cancer is not influenced by knowledge of their HPV status. Results of ongoing and future clinical trials will


be essential to establish whether these patients may be eligible for therapy de-intensification. Finally, a considerable research effort will be devoted to clarify the natural history of oral HPV infection, mostly unknown, and to investigate the possibility of preventing oral HPV and HPV-associated head and neck cancers through the available prophylactic HPV vaccines. Acknowledgements The authors acknowledge M Kenyon for his revision of the English language.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

EXECUTIVE SUMMARY Background ●

HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) show better prognosis and response to therapy.

●

Data on HPV contribution to OPSCC are scarce for southern Europe.

Materials & methods ●

Archival tissues from 140 consecutive primary OPSCCs were analyzed for HPV-DNA and p16 overexpression.

Results ●

HPV prevalence was 40.7%.

●

Tonsillar and base of tongue SCCs displayed similar HPV prevalence.

●

HPV positivity did not significantly differ in cancers arising in men and women.

●

p16 overexpression was observed in almost all HPV-positive cases (98.0%).

●

Based on HPV-DNA and p16 double positivity, the HPV-related fraction was 39.8%.

Conclusions ●

A substantial fraction of OPSCCs is attributable to HPV infection.

●

A high proportion of OPSCC patients may be eligible for less intensive treatments.



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PRELIMINARY COMMUNICATION Donà, Spriano, Pichi et al. patients with surgically treated oropharyngeal squamous cell carcinoma. J. Clin. Oncol. 24(36), 5630–5636 (2006).

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