Cancer Epidemiology 39 (2015) 37–41
Contents lists available at ScienceDirect
Cancer Epidemiology The International Journal of Cancer Epidemiology, Detection, and Prevention journal homepage: www.cancerepidemiology.net
Prevalence of human papillomavirus in laryngeal and hypopharyngeal squamous cell carcinomas in northern Spain Juan P. Rodrigo a,*, Mario A. Hermsen a, Manuel F. Fresno c, Ruud H. Brakenhoff d, Fabian Garcı´a-Velasco a, Peter J.F. Snijders b, Danie¨lle A.M. Heideman b, Juana M. Garcı´a-Pedrero a a
Department of Otolaryngology, IUOPA, Hospital Universitario Central de Asturias, University of Oviedo, Oviedo, Spain Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands c Department of Pathology, Hospital Universitario Central de Asturias, University of Oviedo, Oviedo, Spain d Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 October 2014 Received in revised form 10 November 2014 Accepted 13 November 2014 Available online 29 November 2014
Background: Recent studies support a role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (SCCs); however, the significance of HPV in non-oropharyngeal head and neck cancers is uncertain. The aim of this study was to determine the prevalence of HPV in a large cohort of laryngeal and hypopharyngeal SCCs in northern Spain. Materials and methods: Clinical records and paraffin-embedded tumor specimens of 124 consecutive patients surgically treated for laryngeal (62 cases) and hypopharyngeal (62 cases) SCCs between 2002 and 2007 were retrieved. All cases were histologically evaluated, and presence of HPV was assessed by p16-immunohistochemistry followed by GP5+/6+-PCR-based DNA detection. Samples positive in both assays were subjected to HPV genotyping and HPV E6 transcript analysis. Results: Seventeen cases (14%) were positive for p16 immunostaining, of which 2 (1 larynx, 1 hypopharynx, 1.6% of total series) were found positive for HPV DNA by subsequent GP5+6+-PCR. Both SCCs contained HPV type 16 and showed HPV16 E6 mRNA expression. Conclusions: HPV is only occasionally involved in laryngeal and hypopharyngeal SCC patients in northern Spain. ß 2014 Elsevier Ltd. All rights reserved.
Keywords: Larynx Hypopharynx Squamous cell carcinoma Human papillomavirus Immunohistochemistry PCR In situ hybridization
1. Introduction Most head and neck cancers are squamous cell carcinomas (SCCs), which are related with tobacco smoking and excessive alcohol consumption. Human papillomavirus (HPV) infection, particularly HPV type 16 (HPV16), has been acknowledged as an etiological factor in a subset of head and neck SCCs [1]. Head and neck SCCs caused by HPV mainly comprise oropharyngeal SCCs, such as tonsillar cancer, with unique clinical and pathological features and prognostic significance [2,3]. In tumors of other anatomical sites of the upper aerodigestive tract (oral cavity, larynx, hypopharynx), HPV presence has been reported in several studies, but controversy exists regarding its role in carcinogenesis.
* Corresponding author at: Department of Otorrinolaringologı´a, Hospital Universitario Central de Asturias, 33011 Oviedo, Spain. Tel.: +34 985 107956; fax: +34 985 108015. E-mail address: [email protected] (J.P. Rodrigo). http://dx.doi.org/10.1016/j.canep.2014.11.003 1877-7821/ß 2014 Elsevier Ltd. All rights reserved.
Thus, the clinical significance of HPV infection in non-oropharyngeal head and neck SCCs remains uncertain. Although recently overshadowed by its involvement in oropharyngeal carcinogenesis, the classical location of HPV infection in the upper aerodigestive tract has been the larynx, with laryngeal papillomatosis caused by low-risk HPV types 6 and 11. The reported prevalence of HPV DNA in laryngeal carcinomas ranges widely (from 0% to 58%) being approximately 25% in metaanalysis [3–6]. Similarly, few studies have addressed the relationship of HPV with hypopharyngeal carcinomas, and also a wide variation (from 3% to 74%) in HPV prevalence has been reported in these cancers [3–5,7]. There are various possible reasons to explain this disparity, such as geographical differences, prevalence of smoking and drinking in the studied populations, anatomical subsite of the tumor, sample selection, time period of the study and/or distinct methods employed for HPV detection. The presence of HPV DNA in a tumor does not necessarily indicate that the virus is biologically active to drive and contribute to tumor development or progression. Viral DNA may well be a ‘bystander’ since HPV DNA
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J.P. Rodrigo et al. / Cancer Epidemiology 39 (2015) 37–41
can be detected in non-tumor and even normal tissue from the upper aerodigestive tract [5,8]. The most informative test for a biologically relevant association between HPV infection and cancer is the analysis of viral E6/E7 oncogene transcripts in tumor specimen, but this is technically challenging when using low-quality mRNA from archival formalin-fixed paraffin-embedded (FFPE) samples. Accordingly, Smeets et al. [9] proposed a test algorithm to assess HPV involvement using FFPE materials, comprising of p16 (INK4A, CDKN2A) immunohistochemistry (IHC) followed by HPV-DNA GP5+/6+ PCR on the p16positive cases. This approach has recently been validated: comparison of mRNA E6/E7 RT-PCR and p16 IHC/HPV-DNA PCR on both frozen and FFPE tissue samples of 82 patients showed a concordance of 98% [12]. This test algorithm is based in the fact that all HPV-positive cases show overexpression of p16 protein. As the HPV E7 oncogene product inhibits the activity of retinoblastoma protein (pRb), p16 is upregulated via the loss of the negative feedback control of pRb expression. Thus p16 expression may serve as a surrogate marker of HPV related cellular transformation [1,9]. In contrast, p16 expression is usually absent in tobacco-related HNSCCs due to inactivation of the p16INK4A gene, although some cases do not harbor this inactivation and could show p16 overexpression. In addition, p53 IHC was also included in this study since most HPV-related cases have an intact TP53 gene and normally show negative p53 protein expression due to its rapid proteosome degradation, whereas TP53 gene mutations (very common in tobacco-related HNSCCs) enable protein stabilization, accumulation, and p53 protein detection by IHC [1]. Here, we analyzed the possible association of HPV infection with laryngeal and hypopharyngeal SCC using FFPE specimen collected in a Spanish study population (n = 124) and the above mentioned test algorithm [9]. The cases that were positive in both assays were further subjected to genotyping of GP5+/6+ PCR products and HPV E6 transcript analysis. 2. Materials and methods 2.1. Patients and tissue specimens Surgical tissue specimens from 124 consecutive patients with laryngeal (62 cases) or hypopharyngeal (62 cases) SCC who underwent surgical treatment at the Hospital Universitario Central de Asturias between 2002 and 2007 were retrospectively collected, following institutional review board guidelines. Informed consent was obtained from all patients. Representative tissue sections were obtained from archival, formalin-fixed and paraffin-embedded (FFPE) blocks and the histological diagnosis was confirmed by an experienced pathologist (MFF).
in 1 mol/L sodium thiocyanate to reduce cross-links. Subsequently, the tissue pellet was digested for 3 days in lysis buffer with high doses of proteinase K (final concentration, 2 mg/mL, freshly added twice daily). Finally, DNA extraction was done by using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). 2.3. Immunohistochemistry The TMAs were cut into 3-mm sections and dried on Flex IHC microscope slides (DakoCytomation, Glostrup, Denmark). Immunohistochemistry was performed in all 124 samples using an automatic staining workstation (Dako Autostainer, Dako Cytomation, Glostrup, Denmark) with the Envision system and diaminobenzidine chromogen as substrate. The following primary antibodies were used: anti-p53 clone DO-7 (DAKO, Glostrup, Denmark), anti-p16 clone E6H4 (Roche mtm laboratories AG, Heidelberg, Germany). P16 and p53 immunostainings were evaluated by two independent observers (MFF and JPR). P16 immunostaining was scored as negative (0), weak to moderate staining (1+: 10–75% of diffuse nuclear and cytoplasmic stained cells) or strong staining (2+: more than 75% diffuse nuclear and cytoplasmic stained cells). Scores 1 were considered as p16-positive expression (Fig. 1B). P53 immunostaining was evaluated as positive when >10% of the malignant cells showed nuclear staining (Fig. 1C). 2.4. HPV DNA detection and genotyping by GP5+/6+-PCR-EIA and luminex assay Following the algorithm of Smeets et al. [9], only those cases showing positive p16-immunostaining were subjected to high-risk HPV DNA detection and genotyping as described previously [9,12]. In short, isolated DNA was subjected to GP5+/6+-PCR with an enzyme-immuno-assay (EIA) read-out for detection of 14 high-risk HPV types (i.e. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Subsequent genotyping of EIA-positive cases was performed by bead-based array on the Luminex platform. Sample quality after DNA extraction was controlled by b-globin PCR [12]. 2.5. Detection of HPV16 E6 mRNA EIA-positive cases were subjected to detection of HPV16 E6 mRNA as previously described [9]. In short, RT-PCR with EIA readout was performed to detect the most abundant splice variant within the HPV16 E6 open reading frame, namely E6*I. For each clinical sample, RT-PCR without reverse transcriptase and b-glucuronidase RT-PCR were performed as controls.
2.2. Tissue microarray (TMA) construction and DNA extraction 2.6. HPV DNA detection by in situ hibridization (ISH) Five morphologically representative areas were selected from each individual tumor paraffin block: two for DNA isolation and three for the construction of a TMA. To avoid cross-contamination, two punches of 2 mm diameter were taken first, using a new, sterile punch (Kai Europe GmbH, Solingen, Germany) for every tissue block, and stored in Eppendorf tubes at room temperature prior to DNA extraction. Subsequently, three 1 mm cylinders were taken to construct TMA blocks, as described previously [10]. A total of 5 TMAs were created, containing three tissue cores of each of the 124 laryngeal and hypopharyngeal carcinomas. In addition, each TMA included three cores of normal epithelium (pharynx) as an internal negative control and three cores of a HPV-positive cervix carcinoma as a positive control (Fig. 1A). Special care was taken to obtain high-quality DNA from the FFPE tissues by applying an elaborate extraction protocol [11], which includes thorough deparaffinization with xylene, methanol washings to remove all traces of the xylene, and a 24-h incubation
In an attempt to further confirm the results and avoid falsenegative cases, ISH with biotinylated HPV DNA probes considered to react with HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 (Y1443, DakoCytomation, Glostrup, Denmark) was performed on all 124 laryngeal and hypopharyngeal carcinomas using 3 mm formalin-fixed, paraffin-embedded tissue sections of the TMAs, according to the manufacturer’s instructions. The results were evaluated by two independent observers (MFF and JPR). Focal DAB staining in the tumor nuclei indicated the presence of HPV. 3. Results 3.1. Patient characteristics All patients had a single primary tumor and received no treatment prior to surgery. No patient had distant metastases at
J.P. Rodrigo et al. / Cancer Epidemiology 39 (2015) 37–41
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Fig. 1. Images of in situ hybridization (ISH), p16 and p53 expression in a cervix uterine carcinoma used as control (A); the HPV-positive laryngeal carcinoma, showing negative ISH, positive p16, and positive p53 expression (B); and the HPV-positive hypopharyngeal carcinoma, showing negative ISH, positive p16, and negative p53 expression (C). Magnification 10.
the time of diagnosis. Only 5 patients were female, and the mean age was 60 years (range 36–86 years). All but five patients were habitual tobacco smokers, 71 moderate (1–50 pack-years) and 48 heavy (>50 pack-years), and 107 were habitual alcohol drinkers. The stage of the tumors was determined according to the TNM system of the International Union Against Cancer (7th Edition): 12 tumors were stage I, 3 stage II, 23 stage III, and 86 stage IV. The series included 34 well, 55 moderately and 35 poorly differentiated tumors, determined according to the degree of differentiation of the tumor (Broders’ classification). None of the cases showed basaloid differentiation. Sixty-six (53%) of 124 patients received postoperative radiotherapy. An overview of all clinicopathological data is given in Table 1. 3.2. HPV prevalence in laryngeal and hypopharyngeal SCCs Seventeen of 124 cases (14%) were p16-immunopositive, nine with a 1+ score (7 laryngeal and 2 hypopharyngeal), and eight with a 2+ score (7 laryngeal and 1 hypopharyngeal). Among the p16immunopositive cases, two with a 2+ score were positive by GP5+/ 6+ PCR (1.6% of total series). Both contained HPV type 16 by genotyping of the GP5+/6+ PCR products, and were positive for HPV16 E6 mRNA by RT-PCR. The HPV-positive tumors were from male patients, with ages of 85 and 80 years and tumors located in the larynx (pT3N0 well differentiated carcinoma, Fig. 1B; case 1) and the hypopharynx (pT3N3 poorly differentiated carcinoma, Fig. 1C; case 2),
respectively. Both patients were heavy smokers (>50 pack-years) and alcohol drinkers, and remained free of locoregional recurrence during follow-up, although patient 2 developed distant metastasis in the lungs after 5 months. Case 1 was immunopositive for p53, Table 1 Clinico-pathological data of the studied patients. Total cases (%) Localization Larynx Hypoharynx
62 (50) 62 (50)
T classification T1 T2 T3 T4
17 18 44 45
N classification N0 N1–3
40 (32) 84 (68)
Stage I–II III IV
15 (12) 23 (19) 86 (69)
Histological grade Well differentiated Moderately differentiated Poorly differentiated
34 (27) 55 (45) 35 (28)
(14) (15) (35) (36)
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J.P. Rodrigo et al. / Cancer Epidemiology 39 (2015) 37–41
and case 2 was p53-immunonegative. HPV ISH was negative in both cases, as it was in all remaining 122 SCCs as well. 4. Discussion Reported proportions of HPV-positive laryngeal and hypopharyngeal SCCs vary largely in the literature, ranging from 0% up to even 75% [4–8,13–18]. Apart from differences in geographical distribution and case selection, also the applied HPV detection test plays a role in this variability. Using a validated test algorithm, we found an overall 1.6% of HPV-positive cases (1/62 laryngeal and 1/62 hypopharyngeal SCCs), diagnosed between the years 2002 and 2007 in Asturias, northern Spain. E6 viral oncogene expression supported the presence of transcriptionally active HPV16 in both cases. The 1.6% prevalence of HPV is among the lowest reported thus far in the literature for laryngeal and hypopharyngeal carcinomas, but it is in agreement with a recent large study from two highincidence regions of head and neck cancer (Latin America and Central Europe) [7], and with other recent series [13–16,18] that used reliable detection techniques. In a previous study carried out at our Hospital performed in patients treated between 1991 and 1993, none of the 13 laryngeal tumors and 2/15 (13%) of hypopharyngeal tumors analyzed showed the presence of HPV16 DNA [19]. The low numbers evaluated and the lack of surrogate markers at the time to confirm the liability of the findings made an accurate estimation difficult. Our current findings reflect the best analytical approach, as the different analyses proved to be concordant: a small proportion of the tumors showed the p16-positive/p53-negative pattern typical of HPV infection (i.e. 3 cases (2.5%), data not shown), and none of the cases were positive by ISH analysis. Of interest, apart from the anatomical site, the two HPVpositive cases also deviate for a subset of parameters from HPVrelated oropharyngeal or oral cancer patients: both were over 80 years of age, heavy smokers, neither showed a basaloid differentiation pattern, the hypopharyngeal case developed early distant metastasis, and the laryngeal HPV-positive case was also p53positive, which are characteristics opposite to those described for HPV-related HNSCCs [1]. Nonetheless, E6 transcript expression supports the idea of an active role of the virus in these cancers, despite the fact that HPV ISH failed to confirm the presence of HPV DNA in these cases. This, however, may reflect the lower sensitivity of this technique compared to PCR. Altogether, the results of the current study suggest that HPV does not play a significant role in the development of laryngeal and hypopharyngeal squamous cell carcinomas in northern Spain. One of the possible explanations for the low proportion may lie in a lower exposure to HPV, either due to a lesser presence of HPV in the general population in the north of Spain or to different sexual behavior. In fact, in a previous study we have also found a very low proportion (3.2%) of HPV-related oropharyngeal squamous cell carcinomas in our region [20]. The high prevalence of tobacco smoking and alcohol drinking in our region could also influence this low proportion of HPV-related HNSCCs. We were not able to evaluate possible associations with prognosis due to the imbalance in HPV-positive (n = 2) and HPV-negative (n = 122) cases. In conclusion, the proportion of laryngeal and hypopharyngeal squamous cell carcinomas attributable to HPV infection seems to be very low, and almost negligible, in northern Spain. Conflict of interest The authors declare that they have no potential conflicts of interests.
Authorship contribution statement The authors declare that they have contributed to the manuscript as follows: J.P. Rodrigo: study design, data acquisition, data analysis and interpretation, statistical analysis, manuscript preparation. M.A. Hermsen: study design, data analysis, quality control of data and algorithms, manuscript preparation. M.F. Fresno: data acquisition, data analysis and interpretation, manuscript review. R.H. Brakenhoff: study design, quality control of data and algorithms, manuscript editing. F. Garcı´a-Velasco: data acquisition, manuscript review. P.J.F. Snijders: quality control of data and algorithms, data analysis and interpretation, manuscript editing. D.A.M. Heideman: data acquisition, data analysis and interpretation, manuscript editing. J.M. Garcı´a-Pedrero: study design, quality control of data and algorithms, manuscript preparation. Acknowledgments This study was supported by grants from the Plan Nacional de I+D+I 2008–2011 Instituto de Salud Carlos III (PI11-929 and EMER07-048) and Plan Nacional de I+D+I 2013–2016 ISCIII (CP13/ 00013 and PI13/00259), RD12/0036/0015 of Red Tema´tica de Investigacio´n Cooperativa en Ca´ncer (RTICC), Spain, and the FEDER Funding Program from the European Union. The authors thank Aitana Vallina, Laura Alonso-Dura´n, and Sira Potes for the tissue microarray preparation, the immunohistochemistry and the DNA extractions, and Debby Boon for excellent laboratory assistance.
References [1] Marur S, D‘Souza G, Westra WH, Forastiere AA. HPV-associated head and neck cancer: a virus-related cancer epidemic. Lancet Oncol 2010;11:781–9. [2] Fakhry C, Westra WH, Li S, Cmelak A, Ridge JA, Pinto H, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst 2008;100:261–9. [3] Ragin CCR, Taioli E. Survival of squamous cell carcinoma of the head and neck in relation to human papillomavirus infection: review and meta-analysis. Int J Cancer 2007;121:1813–20. [4] Kreimer AR, Cifford GM, Boyle P, Franceschi S. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review. Cancer Epidemiol Biomark Prev 2005;14:467–75. [5] Isayeva T, Li Y, Maswahu D, Brandwein-Gensler M. Human papillomavirus in non-oropharyngeal head and neck cancers: a systematic literature review. Head Neck Pathol 2012;6:S104–20. [6] Torrente MC, Rodrigo JP, Haigentz Jr M, Dikkers FG, Rinaldo A, Takes RP, et al. Human papillomavirus infections in laryngeal cancer. Head Neck 2011;33:581–6. [7] Ribeiro KB, Levi JE, Pawlita M, Koifman S, Matos E, Eluf-Neto J, et al. Low human papillomavirus prevalence in head and neck cancer: results from two large case– control studies in high-incidence regions. Int J Epidemiol 2011;40:489–502. [8] Duray A, Descamps G, Arafa M, Decaestecker C, Remmelink M, Sirtaine N, et al. High incidence of high-risk HPV in benign and malignant lesion of the larynx. Int J Oncol 2011;39:51–9. [9] 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. [10] Mene´ndez ST, Rodrigo JP, Alvarez-Teijeiro S, Villaronga MA´, Allonca E, Vallina A, et al. Role of HERG1 potassium channel in both malignant transformation and disease progression in head and neck carcinomas. Mod Pathol 2012;25:1069–78. [11] Lo´pez F, Llorente JL, Oviedo CM, Vivanco B, Marcos CA´, Garcı´a-Incla´n C, et al. Gene amplification and protein overexpression of EGFR and ERBB2 in sinonasal squamous cell carcinoma. Cancer 2012;118:1818–26. [12] Rietbergen MM, Leemans CR, Bloemena E, Heideman DA, Braakhuis BJ, Hesselink AT, et al. Increasing prevalence rates of HPV attributable oropharyngeal squamous cell carcinomas in the Netherlands as assessed by a validated test algorithm. Int J Cancer 2013;132:1565–71. [13] Chernock RD, Wang X, Gao G, Lewis Jr JS, Zhang Q, Thorstad WL, et al. Detection and significance of human papillomavirus, CDKN2A (p16) and CDKN1A (p21) expression in squamous cell carcinoma of the larynx. Mod Pathol 2013;26:223–31. [14] Maier M, Kraft K, Steinestel K, Schramm A, Lorenz KJ, Tisch M, et al. Human papillomavirus in squamous cell cancer of the head and neck. A study at the Ulm Military Hospital, Germany. HNO 2013;61:593–601.
J.P. Rodrigo et al. / Cancer Epidemiology 39 (2015) 37–41 [15] Halec G, Holzinger D, Schmitt M, Flechtenmacher C, Dyckhoff G, Lloveras B, et al. Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma. Br J Cancer 2013;109:172–83. [16] Lee SY, Cho NH, Choi EC, Kim WS, Kim SH. Is human papillomavirus a causative factor of glottic cancer? J Voice 2011;25:770–4. [17] Ernoux-Neufcoeur P, Arafa M, Decaestecker C, Duray A, Remmelink M, Leroy X, et al. Combined analysis of HPV DNA, p16, p21 and p53 to predict prognosis in patients with stage IV hypopharyngeal carcinoma. J Cancer Res Clin Oncol 2011;137:173–81.
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[18] Wilson DD, Rahimi AS, Saylor DK, Stelow EB, Jameson MJ, Shonka DC, et al. p16 not a prognostic marker for hypopharyngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2012;138:556–61. [19] Alvarez Alvarez I, Sanchez Lazo P, Ramos Gonzalez S, Rodrigo JP, Nunez Batalla F, Suarez C. Using polymerase chain reaction to human papillomavirus in oral and pharyngolaryngeal carcinomas. Am J Otolaryngol 1997;18:375–81. [20] Rodrigo JP, Heideman DA, Garcı´a-Pedrero JM, Fresno MF, Brakenhoff RH, Dı´az Molina JP, et al. Time trends in the prevalence of HPV in oropharyngeal squamous cell carcinomas in northern Spain (1990–2009). Int J Cancer 2014;134:487–92.
Contents lists available at ScienceDirect
Cancer Epidemiology The International Journal of Cancer Epidemiology, Detection, and Prevention journal homepage: www.cancerepidemiology.net
Prevalence of human papillomavirus in laryngeal and hypopharyngeal squamous cell carcinomas in northern Spain Juan P. Rodrigo a,*, Mario A. Hermsen a, Manuel F. Fresno c, Ruud H. Brakenhoff d, Fabian Garcı´a-Velasco a, Peter J.F. Snijders b, Danie¨lle A.M. Heideman b, Juana M. Garcı´a-Pedrero a a
Department of Otolaryngology, IUOPA, Hospital Universitario Central de Asturias, University of Oviedo, Oviedo, Spain Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands c Department of Pathology, Hospital Universitario Central de Asturias, University of Oviedo, Oviedo, Spain d Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 October 2014 Received in revised form 10 November 2014 Accepted 13 November 2014 Available online 29 November 2014
Background: Recent studies support a role for human papillomavirus (HPV) in oropharyngeal squamous cell carcinomas (SCCs); however, the significance of HPV in non-oropharyngeal head and neck cancers is uncertain. The aim of this study was to determine the prevalence of HPV in a large cohort of laryngeal and hypopharyngeal SCCs in northern Spain. Materials and methods: Clinical records and paraffin-embedded tumor specimens of 124 consecutive patients surgically treated for laryngeal (62 cases) and hypopharyngeal (62 cases) SCCs between 2002 and 2007 were retrieved. All cases were histologically evaluated, and presence of HPV was assessed by p16-immunohistochemistry followed by GP5+/6+-PCR-based DNA detection. Samples positive in both assays were subjected to HPV genotyping and HPV E6 transcript analysis. Results: Seventeen cases (14%) were positive for p16 immunostaining, of which 2 (1 larynx, 1 hypopharynx, 1.6% of total series) were found positive for HPV DNA by subsequent GP5+6+-PCR. Both SCCs contained HPV type 16 and showed HPV16 E6 mRNA expression. Conclusions: HPV is only occasionally involved in laryngeal and hypopharyngeal SCC patients in northern Spain. ß 2014 Elsevier Ltd. All rights reserved.
Keywords: Larynx Hypopharynx Squamous cell carcinoma Human papillomavirus Immunohistochemistry PCR In situ hybridization
1. Introduction Most head and neck cancers are squamous cell carcinomas (SCCs), which are related with tobacco smoking and excessive alcohol consumption. Human papillomavirus (HPV) infection, particularly HPV type 16 (HPV16), has been acknowledged as an etiological factor in a subset of head and neck SCCs [1]. Head and neck SCCs caused by HPV mainly comprise oropharyngeal SCCs, such as tonsillar cancer, with unique clinical and pathological features and prognostic significance [2,3]. In tumors of other anatomical sites of the upper aerodigestive tract (oral cavity, larynx, hypopharynx), HPV presence has been reported in several studies, but controversy exists regarding its role in carcinogenesis.
* Corresponding author at: Department of Otorrinolaringologı´a, Hospital Universitario Central de Asturias, 33011 Oviedo, Spain. Tel.: +34 985 107956; fax: +34 985 108015. E-mail address: [email protected] (J.P. Rodrigo). http://dx.doi.org/10.1016/j.canep.2014.11.003 1877-7821/ß 2014 Elsevier Ltd. All rights reserved.
Thus, the clinical significance of HPV infection in non-oropharyngeal head and neck SCCs remains uncertain. Although recently overshadowed by its involvement in oropharyngeal carcinogenesis, the classical location of HPV infection in the upper aerodigestive tract has been the larynx, with laryngeal papillomatosis caused by low-risk HPV types 6 and 11. The reported prevalence of HPV DNA in laryngeal carcinomas ranges widely (from 0% to 58%) being approximately 25% in metaanalysis [3–6]. Similarly, few studies have addressed the relationship of HPV with hypopharyngeal carcinomas, and also a wide variation (from 3% to 74%) in HPV prevalence has been reported in these cancers [3–5,7]. There are various possible reasons to explain this disparity, such as geographical differences, prevalence of smoking and drinking in the studied populations, anatomical subsite of the tumor, sample selection, time period of the study and/or distinct methods employed for HPV detection. The presence of HPV DNA in a tumor does not necessarily indicate that the virus is biologically active to drive and contribute to tumor development or progression. Viral DNA may well be a ‘bystander’ since HPV DNA
38
J.P. Rodrigo et al. / Cancer Epidemiology 39 (2015) 37–41
can be detected in non-tumor and even normal tissue from the upper aerodigestive tract [5,8]. The most informative test for a biologically relevant association between HPV infection and cancer is the analysis of viral E6/E7 oncogene transcripts in tumor specimen, but this is technically challenging when using low-quality mRNA from archival formalin-fixed paraffin-embedded (FFPE) samples. Accordingly, Smeets et al. [9] proposed a test algorithm to assess HPV involvement using FFPE materials, comprising of p16 (INK4A, CDKN2A) immunohistochemistry (IHC) followed by HPV-DNA GP5+/6+ PCR on the p16positive cases. This approach has recently been validated: comparison of mRNA E6/E7 RT-PCR and p16 IHC/HPV-DNA PCR on both frozen and FFPE tissue samples of 82 patients showed a concordance of 98% [12]. This test algorithm is based in the fact that all HPV-positive cases show overexpression of p16 protein. As the HPV E7 oncogene product inhibits the activity of retinoblastoma protein (pRb), p16 is upregulated via the loss of the negative feedback control of pRb expression. Thus p16 expression may serve as a surrogate marker of HPV related cellular transformation [1,9]. In contrast, p16 expression is usually absent in tobacco-related HNSCCs due to inactivation of the p16INK4A gene, although some cases do not harbor this inactivation and could show p16 overexpression. In addition, p53 IHC was also included in this study since most HPV-related cases have an intact TP53 gene and normally show negative p53 protein expression due to its rapid proteosome degradation, whereas TP53 gene mutations (very common in tobacco-related HNSCCs) enable protein stabilization, accumulation, and p53 protein detection by IHC [1]. Here, we analyzed the possible association of HPV infection with laryngeal and hypopharyngeal SCC using FFPE specimen collected in a Spanish study population (n = 124) and the above mentioned test algorithm [9]. The cases that were positive in both assays were further subjected to genotyping of GP5+/6+ PCR products and HPV E6 transcript analysis. 2. Materials and methods 2.1. Patients and tissue specimens Surgical tissue specimens from 124 consecutive patients with laryngeal (62 cases) or hypopharyngeal (62 cases) SCC who underwent surgical treatment at the Hospital Universitario Central de Asturias between 2002 and 2007 were retrospectively collected, following institutional review board guidelines. Informed consent was obtained from all patients. Representative tissue sections were obtained from archival, formalin-fixed and paraffin-embedded (FFPE) blocks and the histological diagnosis was confirmed by an experienced pathologist (MFF).
in 1 mol/L sodium thiocyanate to reduce cross-links. Subsequently, the tissue pellet was digested for 3 days in lysis buffer with high doses of proteinase K (final concentration, 2 mg/mL, freshly added twice daily). Finally, DNA extraction was done by using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). 2.3. Immunohistochemistry The TMAs were cut into 3-mm sections and dried on Flex IHC microscope slides (DakoCytomation, Glostrup, Denmark). Immunohistochemistry was performed in all 124 samples using an automatic staining workstation (Dako Autostainer, Dako Cytomation, Glostrup, Denmark) with the Envision system and diaminobenzidine chromogen as substrate. The following primary antibodies were used: anti-p53 clone DO-7 (DAKO, Glostrup, Denmark), anti-p16 clone E6H4 (Roche mtm laboratories AG, Heidelberg, Germany). P16 and p53 immunostainings were evaluated by two independent observers (MFF and JPR). P16 immunostaining was scored as negative (0), weak to moderate staining (1+: 10–75% of diffuse nuclear and cytoplasmic stained cells) or strong staining (2+: more than 75% diffuse nuclear and cytoplasmic stained cells). Scores 1 were considered as p16-positive expression (Fig. 1B). P53 immunostaining was evaluated as positive when >10% of the malignant cells showed nuclear staining (Fig. 1C). 2.4. HPV DNA detection and genotyping by GP5+/6+-PCR-EIA and luminex assay Following the algorithm of Smeets et al. [9], only those cases showing positive p16-immunostaining were subjected to high-risk HPV DNA detection and genotyping as described previously [9,12]. In short, isolated DNA was subjected to GP5+/6+-PCR with an enzyme-immuno-assay (EIA) read-out for detection of 14 high-risk HPV types (i.e. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Subsequent genotyping of EIA-positive cases was performed by bead-based array on the Luminex platform. Sample quality after DNA extraction was controlled by b-globin PCR [12]. 2.5. Detection of HPV16 E6 mRNA EIA-positive cases were subjected to detection of HPV16 E6 mRNA as previously described [9]. In short, RT-PCR with EIA readout was performed to detect the most abundant splice variant within the HPV16 E6 open reading frame, namely E6*I. For each clinical sample, RT-PCR without reverse transcriptase and b-glucuronidase RT-PCR were performed as controls.
2.2. Tissue microarray (TMA) construction and DNA extraction 2.6. HPV DNA detection by in situ hibridization (ISH) Five morphologically representative areas were selected from each individual tumor paraffin block: two for DNA isolation and three for the construction of a TMA. To avoid cross-contamination, two punches of 2 mm diameter were taken first, using a new, sterile punch (Kai Europe GmbH, Solingen, Germany) for every tissue block, and stored in Eppendorf tubes at room temperature prior to DNA extraction. Subsequently, three 1 mm cylinders were taken to construct TMA blocks, as described previously [10]. A total of 5 TMAs were created, containing three tissue cores of each of the 124 laryngeal and hypopharyngeal carcinomas. In addition, each TMA included three cores of normal epithelium (pharynx) as an internal negative control and three cores of a HPV-positive cervix carcinoma as a positive control (Fig. 1A). Special care was taken to obtain high-quality DNA from the FFPE tissues by applying an elaborate extraction protocol [11], which includes thorough deparaffinization with xylene, methanol washings to remove all traces of the xylene, and a 24-h incubation
In an attempt to further confirm the results and avoid falsenegative cases, ISH with biotinylated HPV DNA probes considered to react with HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 (Y1443, DakoCytomation, Glostrup, Denmark) was performed on all 124 laryngeal and hypopharyngeal carcinomas using 3 mm formalin-fixed, paraffin-embedded tissue sections of the TMAs, according to the manufacturer’s instructions. The results were evaluated by two independent observers (MFF and JPR). Focal DAB staining in the tumor nuclei indicated the presence of HPV. 3. Results 3.1. Patient characteristics All patients had a single primary tumor and received no treatment prior to surgery. No patient had distant metastases at
J.P. Rodrigo et al. / Cancer Epidemiology 39 (2015) 37–41
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Fig. 1. Images of in situ hybridization (ISH), p16 and p53 expression in a cervix uterine carcinoma used as control (A); the HPV-positive laryngeal carcinoma, showing negative ISH, positive p16, and positive p53 expression (B); and the HPV-positive hypopharyngeal carcinoma, showing negative ISH, positive p16, and negative p53 expression (C). Magnification 10.
the time of diagnosis. Only 5 patients were female, and the mean age was 60 years (range 36–86 years). All but five patients were habitual tobacco smokers, 71 moderate (1–50 pack-years) and 48 heavy (>50 pack-years), and 107 were habitual alcohol drinkers. The stage of the tumors was determined according to the TNM system of the International Union Against Cancer (7th Edition): 12 tumors were stage I, 3 stage II, 23 stage III, and 86 stage IV. The series included 34 well, 55 moderately and 35 poorly differentiated tumors, determined according to the degree of differentiation of the tumor (Broders’ classification). None of the cases showed basaloid differentiation. Sixty-six (53%) of 124 patients received postoperative radiotherapy. An overview of all clinicopathological data is given in Table 1. 3.2. HPV prevalence in laryngeal and hypopharyngeal SCCs Seventeen of 124 cases (14%) were p16-immunopositive, nine with a 1+ score (7 laryngeal and 2 hypopharyngeal), and eight with a 2+ score (7 laryngeal and 1 hypopharyngeal). Among the p16immunopositive cases, two with a 2+ score were positive by GP5+/ 6+ PCR (1.6% of total series). Both contained HPV type 16 by genotyping of the GP5+/6+ PCR products, and were positive for HPV16 E6 mRNA by RT-PCR. The HPV-positive tumors were from male patients, with ages of 85 and 80 years and tumors located in the larynx (pT3N0 well differentiated carcinoma, Fig. 1B; case 1) and the hypopharynx (pT3N3 poorly differentiated carcinoma, Fig. 1C; case 2),
respectively. Both patients were heavy smokers (>50 pack-years) and alcohol drinkers, and remained free of locoregional recurrence during follow-up, although patient 2 developed distant metastasis in the lungs after 5 months. Case 1 was immunopositive for p53, Table 1 Clinico-pathological data of the studied patients. Total cases (%) Localization Larynx Hypoharynx
62 (50) 62 (50)
T classification T1 T2 T3 T4
17 18 44 45
N classification N0 N1–3
40 (32) 84 (68)
Stage I–II III IV
15 (12) 23 (19) 86 (69)
Histological grade Well differentiated Moderately differentiated Poorly differentiated
34 (27) 55 (45) 35 (28)
(14) (15) (35) (36)
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and case 2 was p53-immunonegative. HPV ISH was negative in both cases, as it was in all remaining 122 SCCs as well. 4. Discussion Reported proportions of HPV-positive laryngeal and hypopharyngeal SCCs vary largely in the literature, ranging from 0% up to even 75% [4–8,13–18]. Apart from differences in geographical distribution and case selection, also the applied HPV detection test plays a role in this variability. Using a validated test algorithm, we found an overall 1.6% of HPV-positive cases (1/62 laryngeal and 1/62 hypopharyngeal SCCs), diagnosed between the years 2002 and 2007 in Asturias, northern Spain. E6 viral oncogene expression supported the presence of transcriptionally active HPV16 in both cases. The 1.6% prevalence of HPV is among the lowest reported thus far in the literature for laryngeal and hypopharyngeal carcinomas, but it is in agreement with a recent large study from two highincidence regions of head and neck cancer (Latin America and Central Europe) [7], and with other recent series [13–16,18] that used reliable detection techniques. In a previous study carried out at our Hospital performed in patients treated between 1991 and 1993, none of the 13 laryngeal tumors and 2/15 (13%) of hypopharyngeal tumors analyzed showed the presence of HPV16 DNA [19]. The low numbers evaluated and the lack of surrogate markers at the time to confirm the liability of the findings made an accurate estimation difficult. Our current findings reflect the best analytical approach, as the different analyses proved to be concordant: a small proportion of the tumors showed the p16-positive/p53-negative pattern typical of HPV infection (i.e. 3 cases (2.5%), data not shown), and none of the cases were positive by ISH analysis. Of interest, apart from the anatomical site, the two HPVpositive cases also deviate for a subset of parameters from HPVrelated oropharyngeal or oral cancer patients: both were over 80 years of age, heavy smokers, neither showed a basaloid differentiation pattern, the hypopharyngeal case developed early distant metastasis, and the laryngeal HPV-positive case was also p53positive, which are characteristics opposite to those described for HPV-related HNSCCs [1]. Nonetheless, E6 transcript expression supports the idea of an active role of the virus in these cancers, despite the fact that HPV ISH failed to confirm the presence of HPV DNA in these cases. This, however, may reflect the lower sensitivity of this technique compared to PCR. Altogether, the results of the current study suggest that HPV does not play a significant role in the development of laryngeal and hypopharyngeal squamous cell carcinomas in northern Spain. One of the possible explanations for the low proportion may lie in a lower exposure to HPV, either due to a lesser presence of HPV in the general population in the north of Spain or to different sexual behavior. In fact, in a previous study we have also found a very low proportion (3.2%) of HPV-related oropharyngeal squamous cell carcinomas in our region [20]. The high prevalence of tobacco smoking and alcohol drinking in our region could also influence this low proportion of HPV-related HNSCCs. We were not able to evaluate possible associations with prognosis due to the imbalance in HPV-positive (n = 2) and HPV-negative (n = 122) cases. In conclusion, the proportion of laryngeal and hypopharyngeal squamous cell carcinomas attributable to HPV infection seems to be very low, and almost negligible, in northern Spain. Conflict of interest The authors declare that they have no potential conflicts of interests.
Authorship contribution statement The authors declare that they have contributed to the manuscript as follows: J.P. Rodrigo: study design, data acquisition, data analysis and interpretation, statistical analysis, manuscript preparation. M.A. Hermsen: study design, data analysis, quality control of data and algorithms, manuscript preparation. M.F. Fresno: data acquisition, data analysis and interpretation, manuscript review. R.H. Brakenhoff: study design, quality control of data and algorithms, manuscript editing. F. Garcı´a-Velasco: data acquisition, manuscript review. P.J.F. Snijders: quality control of data and algorithms, data analysis and interpretation, manuscript editing. D.A.M. Heideman: data acquisition, data analysis and interpretation, manuscript editing. J.M. Garcı´a-Pedrero: study design, quality control of data and algorithms, manuscript preparation. Acknowledgments This study was supported by grants from the Plan Nacional de I+D+I 2008–2011 Instituto de Salud Carlos III (PI11-929 and EMER07-048) and Plan Nacional de I+D+I 2013–2016 ISCIII (CP13/ 00013 and PI13/00259), RD12/0036/0015 of Red Tema´tica de Investigacio´n Cooperativa en Ca´ncer (RTICC), Spain, and the FEDER Funding Program from the European Union. The authors thank Aitana Vallina, Laura Alonso-Dura´n, and Sira Potes for the tissue microarray preparation, the immunohistochemistry and the DNA extractions, and Debby Boon for excellent laboratory assistance.
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