p16 Expression in cutaneous squamous carcinomas with neck metastases: A potential pitfall in identifying unknown primaries of the head and neck

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1 ORIGINAL ARTICLE p16 Expression in cutaneous squamous carcinomas with neck metastases: A potential pitfall in identifying unknown primaries of the head and neck Beth M. Beadle, MD, PhD, 1 William N. William, Jr, MD, 2 Michael S. McLemore, MD, MPH, 3 Erich M. Sturgis, MD, MPH, 4 Michelle D. Williams, MD 3 * 1 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, 2 Department of Thoracic and Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, 3 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, 4 Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas. Accepted 12 September 2012 Published online 29 October 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed ABSTRACT: Background. Human papillomavirus (HPV) positivity (þ) has been used to identify oropharyngeal squamous carcinomas (SCCs) presenting as unknown primaries in the neck. p16 overexpression correlates with HPVþ in the oropharynx; however, the use of p16 alone as a surrogate marker of oropharyngeal HPVþ tumors has not been validated. Methods. We immunohistochemically analyzed p16 expression in surgically resected aggressive cutaneous head and neck SCC primaries and their nodal metastases from 24 patients to determine the potential overlap of p16 expression outside of the oropharynx. Results. Five of 24 primary tumors (20.8%) and 3 lymph node metastases (12.5%) in levels II, III, and V, and the periparotid region diffusely expressed p16. HPV (high-risk types by in situ hybridization) was negative. Conclusions. p16 expression is relatively common in lymph nodepositive cutaneous head and neck SCCs; thus, p16 expression as an independent biomarker and mechanism to determine the oropharyngeal source of an unknown primary is not advised. VC 2012 Wiley Periodicals, Inc. Head Neck 35: , 2013 KEY WORDS: p16, unknown primary, cutaneous squamous carcinoma, neck metastases INTRODUCTION Human papillomavirus (HPV) is well established as a risk factor for squamous carcinoma (SCC) of the oropharynx, and patients with HPV-positive (HPVþ) SCC have shown dramatically improved survival over patients with HPVnegative (HPV-) tumors. 1 4 Testing oropharyngeal primary tumors for HPV status, specifically high-risk type 16, has thus become routine practice. Direct testing for HPV is predominantly performed by in situ hybridization or polymerase chain reaction (PCR) methods. However, PCR is overly sensitive and does not allow for morphologic assessment, whereas in situ hybridization allows visualization of probe hybridization within the nucleus of the tumor cells but is more expensive to perform and less sensitive than PCR. 5 Thus, a controversy remains as to the methodology that affords the best balance of sensitivity and specificity for practical routine clinical practice. As the understanding of HPV-associated oropharyngeal cancer has progressed, p16 overexpression has been consistently documented (95%) in such tumors and has been suggested as a surrogate marker for HPV-associated tumors. 3,6 However, less emphasized is a subgroup of SCCs with high p16 expression unassociated with HPV. 7 *Corresponding author: M. D. Williams, Department of Pathology, Unit 085, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX mdwillia@mdanderson.org Ehab Y. Hanna, MD, was recused from consideration of this manuscript. p16 is a tumor-suppressor gene involved in cell cycle regulation. Through gene mutations and deletions, alterations in p16 protein expression occur in a wide variety of cancers, including head and neck aerodigestive tract SCCs. 8,9 Elevated p16 expression is theorized to be related to HPV E7 protein expression-induced interference with the Rb protein causing its inactivation, and secondarily upregulation of p16 expression by compensatory feedback pathways. 3 p16 expression is easily assayed by immunohistochemical analysis with commercially available antibodies at a relatively lower cost than that of HPV in situ hybridization testing. Moreover, studies of oropharyngeal SCCs that overexpressed p16 but were HPV- by in situ hybridization (p16þ) have shown that these tumors behavior correlate most closely with that of HPVþ tumors. 5,10,11 These observations have led to the belief that p16 testing is preferable to and more sensitive than direct HPV analysis by in situ hybridization for determining HPV status. As a result of these findings, and because p16 immunohistochemical analysis has become more readily available, there is an emerging trend toward foregoing direct HPV testing of tumors and instead testing only for p16 expression and considering p16-positive tumors to be HPVþ. This creates a potential pitfall as p16 expression in SCC is not exclusively associated with HPV, as emphasized by Schache et al 5 who showed that p16 overcalls HPV status and is suboptimal with a specificity of only 82% compared to PCR-based HPV status. With the recent understanding about the increasing incidence of HPV-associated oropharyngeal SCC, HPV and HEAD & NECK DOI /HED NOVEMBER

2 BEADLE ET AL. p16 testing of unknown primary carcinoma metastatic to a cervical lymph node is commonly used to facilitate identification of the site of origin of the primary tumor For patients who present with lymph nodes positive for SCC in the neck, standard practice is to evaluate for a primary site using detailed imaging, an examination under anesthesia, and directed biopsies. If these methods do not identify a primary tumor, pathologic assessment of lymph node specimens may provide further information. The presence of HPV favors an oropharyngeal primary and, thus, more targeted locoregional treatment. 3,15 However, the utility of p16 expression as an independent single marker to identify an HPVþ oropharyngeal carcinoma primary that presents as unknown primary SCC metastatic to neck lymph nodes remains unclear. p16 expression in lymph node metastases may reflect an oropharyngeal primary site; however, the rate of p16 expression in other primary head and neck SCCs that commonly present with lymph node metastases is not well established. Namely, cutaneous SCCs also may present with metastatic disease in neck lymph nodes. p16 positivity has been reported in some cutaneous SCCs from various sites; however, the rate of p16 positivity in cutaneous SCCs from the head and neck region specifically is unknown. Our study thus sought to evaluate patients with lymph node-positive cutaneous SCC from the head and neck region to determine the rates of p16 expression and hence understand the potential benefits and risks of using p16 alone to identify the primary tumor in patients with metastatic adenopathy. We immunohistochemically analyzed p16 expression in surgically resected aggressive cutaneous head and neck SCC primaries and their nodal metastases from 24 patients to determine the potential overlap of p16 expression outside of the oropharynx. MATERIALS AND METHODS After institutional review board approval of our study, we searched The University of Texas MD Anderson Cancer Center Pathology Database for patients treated with surgical resection for aggressive cutaneous head and neck SCCs (defined as primary SCCs with concurrent lymph node metastases) in the period from 2002 to Our search identified 24 patients with available paired primary and metastatic formalin-fixed paraffin-embedded tumor samples. For eligible patients, hematoxylin and eosin pathology slides were reviewed verifying the diagnosis of SCC, the pathologic features of each tumor, the level of lymph nodes involved by SCC, and to select blocks for immunohistochemical and in situ hybridization studies. Corresponding patient records were reviewed for clinical outcome and follow-up. Immunohistochemical analysis, p16 For this study, p16 immunohistochemical evaluation (p16 ink4a [mouse anti-human clone E6H4],1:3 (of predilute), CINtec 9517, MTM Laboratories, Westborough, MA) was performed in a standard manner on 4-micronthick unstained whole paraffin tissue sections using the automated BOND MAX IHC stainer by Vision Biosystems (Norwell, MA) and standard procedures. In brief, after tumor samples were deparaffinized using Bond Dewax Solution, washed, and alcohol rinsed, antigen retrieval was achieved by steaming the slides in 10 mm citrate buffer (ph 6.0) for 10 minutes. The primary antibody was applied for 15 minutes, followed by blocking of endogenous peroxidases using (3%) hydrogen peroxide. A polymer enhancer was used before adding the secondary anti-mouse antibody for 8 minutes. Slides were developed using DAB reagent (Dako), counterstained with hematoxylin, dehydrated, and mounted under a coverslip. Tumor cells were evaluated for p16 cytoplasmic and nuclear staining using the following scale: 0 ¼ <5%, 1þ ¼5% to 33%, 2þ ¼33% to 66%, and 3þ ¼>66% tumor cell staining for p16. 3þ was considered diffuse p16 overexpression (see Discussion). Human papillomavirus analysis by in situ hybridization A tissue microarray was created from the formalinfixed paraffin tissue blocks consisting of two 1-mm cores from the primary SCCs and two 1-mm cores from the metastasis from the lymph nodes. A 4-micron-thick section of the tissue microarray was evaluated by in situ hybridization for HPV nucleic acids using the automated BenchMark per the manufacturer s recommended protocol. Ventana Inform HPV III Family 16 probe was used, which contains a cocktail of labeled HPV genomic probes that target the most common HPV genotypes "high-risk types" found to be associated with cancer including genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66 (Ventana Medical Systems, Tucson, AZ). Tumor cells were evaluated for positive nuclear expression with concurrent positive and negative controls reviewed. Statistical analysis Correlation of clinical and pathologic findings with the marker assessment was performed by univariate analysis using Fisher exact probability test with the Freeman Halton extension. Any p values less than.05 were considered statistically significant. Graphpad Prism 5 (Graphpad Software, La Jolla, CA) was used to assess Kaplan Meier survival curves via the log-rank (Mantel Cox) Test. RESULTS Clinical findings Aggressive cutaneous SCCs of the head and neck region with lymph node metastases occurred in 23 men and 1 woman from 27 to 85 years of age (median age, 75 years); the clinical characteristics of this cohort are outlined in Table 1. Primary tumor sites included the scalp, temple or forehead, ear, lip, and nose/cheek. Metastases in these 24 patients involved 35 separate levels in the neck and were distributed throughout levels I to V and the periparotid region with each level involved in 11.4% to 22.9% of cases. Nine patients (37.5%) had metastatic SCC involving more than 1 level (7 [29.2%] with 2 levels involved and 2 [8.3%] with 3 levels involved). Perineural involvement was common in the primary tumors, and was present in 10 of 24 patients (41.7%) HEAD & NECK DOI /HED NOVEMBER 2013

3 P16 EXPRESSION IN CUTANEOUS SQUAMOUS CARCINOMA TABLE 1. Clinical parameters of aggressive cutaneous squamous carcinoma of the head and neck region. Total (n ¼ 24) (%) p16 þ (3þ) primary (n ¼ 5) (%) p16 (0 2þ) primary (n ¼ 19) (%) p16 (3þ) LN (n ¼ 3) (%) p16 (0 2þ) LN (n ¼ 21) (%) Age Range Median Sex Male 23 (95.8) 4 (80.0) 19 (100.0) 3 (100.0) 20 (95.2) Female 1 (4.2) 1 (20.0) 0 (0.0) 0 (0.0) 1 (4.8) Primary sites Scalp 9 (37.5) 3 (60.0) 6 (31.6) 2 (66.7) 7 (33.3) Temple 3 (12.5) 2 (40.0) 1 (5.3) 0 (0.0) 3 (14.3) Ear 4 (16.7) 0 (0.0) 4 (21.1) 0 (0.0) 4 (19.0) Lip 4 (16.7) 0 (0.0) 4 (21.1) 0 (0.0) 4 (19.0) Nose/cheek 4 (16.7) 0 (0.0) 4 (21.1) 1 (33.3) 3 (14.3) SCC-positive LN levels (35)* (10) (15) (6) (29) I 7 (20.0) 1 (10.0) 6 (24.0) 0 (0.0) 7 (24.1) II 7 (20.0) 2 (20.0) 5 (20.0) 3 (50.0) 4 (13.8) III 4 (11.4) 1 (10.0) 3 (12.0) 1 (16.7) 3 (10.3) IV 4 (11.4) 1 (10.0) 3 (12.0) 0 (0.0) 4 (13.8) V 5 (14.3) 2 (20.0) 3 (12.0) 1 (16.7) 4 (13.8) Periparotid 8 (22.9) 3 (30.0) 5 (20.0) 1 (16.7) 7 (24.1) Distant metastases Absent 16 (66.7) 3 (60.0) 13 (68.4) 2 (66.7) 14 (66.7) Present 8 (33.3) 2 (40.0) 6 (31.6) 1 (33.3) 7 (33.3) Perineural invasion Absent 14 (58.3) 2 (40.0) 12 (63.2) 1 (33.3) 13 (61.9) Present 10 (41.7) 3 (60.0) 7 (36.8) 2 (66.7) 8 (38.1) Tumor grade (differentiation) Well 1 (4.2) 0 (0.0) 1 (5.3) 0 (0.0) 1 (4.8) Moderate 10 (41.7) 2 (40.0) 8 (42.1) 0 (0.0) 10 (47.6) Poor 13 (54.1) 3 (60.0) 10 (52.6) 3 (100.0) 10 (47.6) Follow-up ANED 8 (33.3) 2 (40.0) 6 (31.6) 1 (33.3) 7 (33.3) AWD 5 (20.8) 2 (40.0) 3 (15.8) 1 (33.3) 4 (19.0) DOD 11 (45.8) 1 (20.0) 10 (52.6) 1 (33.3) 10 (47.6) Abbreviations: LN, lymph node; SCC, squamous carcinoma; ANED, alive with no evidence of disease; AWD, alive with disease; DOD, dead of disease. * Seven patients with 2 levels and 2 patients with 3 levels involved. Median follow-up time for the 13 patients alive at last follow-up was 15 months (range, 5 96 months) with 8 patients (33.3%) alive with no evidence of disease 5 to 39 months after resection, 4 with distant metastases, and 1 with local recurrence 13 to 96 months after resection. Eleven patients were deceased (45.8%); 5 died from their SCC 7 to 20 months after resection. The crude distant metastatic rate in this cohort was 8 of 24 patients (33.3%). p16 immunohistochemical evaluation Paired primary and lymph node metastases samples were analyzed for p16 expression by immunohistochemical analysis in all 24 patients. p16 cytoplasmic and nuclear staining for p16 was present in 8 primary tumors and varied from focal staining, 1þ in 2 tumors and 2þ in 1 tumor, to diffuse staining (3þ) in 5 tumors (Figure 1A). The remaining 16 primary tumors were negative for p16 expression. Lymph nodes metastases showed similar p16 expression, with 2 metastases showing focal staining (1þ) and 3 diffusely positive (3þ) SCC lymph node metastases (Figure 1B). The remaining 19 cases were negative with no p16 expression. The discordant primary and lymph node metastases included 3 diffusely positive primary tumors (3þ), with 2 corresponding lymph node metastases showing focal p16 staining and 1 with no p16 staining. One primary tumor with focal (1þ) staining had a lymph node metastasis with diffusely positive (3þ) p16 staining (Figure 1C and D). Human papillomavirus analysis by in situ hybridization All primary and metastatic cutaneous SCCs in this cohort were analyzed for high-risk types of HPV by in situ hybridization and were negative for hybridization (HPV), regardless of p16 expression status. p16 expression and clinical characteristics The relationship between clinical characteristics and p16 expression are shown in Table 1 and include the features of primary tumors as well as lymph node metastases with diffuse (3þ) p16-positive expression. Primary and metastatic SCCs that were p16 negative/focal expression showed shorter median overall survival (28 months vs HEAD & NECK DOI /HED NOVEMBER

4 BEADLE ET AL. FIGURE 1. Aggressive cutaneous head and neck squamous carcinomas (SCCs) analyzed for p16 by immunohistochemistry. A and B, Primary cutaneous SCC and corresponding lymph node metastasis, respectively, with overexpression of p16. C and D, A discordant primary cutaneous SCC (C) with focal (1þ, <33% staining) p16 expression and the corresponding lymph node metastasis with strong diffuse (3þ) p16 overexpression (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 51 months) than that for those that were p16 positive (p ¼.40). No correlation was found between p16 expression and either primary tumor site or level of lymph node metastases. DISCUSSION Our findings show that p16 is expressed in approximately one third of aggressive primary cutaneous SCCs arising in the head and region, of which 75% have strong/diffuse p16 expression, and p16 is strongly expressed in 12.5% of cutaneous SCC lymph node metastases. The quality of p16 expression diffuse and strong seen in a subset of SCCs of cutaneous origin, was similar although less frequently seen than in HPVassociated oropharyngeal carcinomas. These findings suggest that in isolation, p16 expression in a lymph node metastasis is not a robust indicator of an oropharyngeal primary tumor. Patients with cutaneous SCC of the head and neck often present with metastatic adenopathy, and a notable subset of patients may demonstrate p16 expression in lymph node specimens, similar to findings in patients with an oropharyngeal carcinoma primary. Additionally, cutaneous SCCs with metastatic adenopathy sometimes present with a previously ablated primary, a regressed/clinically absent primary, or an innocuous/ subtle primary tumor. Our finding that p16 is overexpressed in a subset of aggressive, lymph node-positive cutaneous SCCs of the head and neck is consistent with prior reports including the Hodges study of moderate to strong p16 expression in 30% of invasive SCCs Küsters Vandevelde et al 18 demonstrated p16 expression in approximately 45% of primary as well as metastatic cutaneous SCCs arising in various sites of the body, predominantly in the absence of HPV of cutaneous serotypes. Cutaneous serotypes of HPV are not associated with mucosal sites, their viral proteins do not bind Rb or p53, as seen with the mucosal types of HPV, and would not account for p16 overexpression. 3,19 In situ HPV testing of high-risk "mucosal" types (types 16, 18, 33, and others, as tested for in this study) do not cross-react with cutaneous HPV serotypes and remains a practical method for HPV testing of oropharyngeal metastases. 19 HPV typing of cutaneous lesions is not routinely/clinically performed. In the skin, p16 seems to become progressively overexpressed as cutaneous lesions progress from actinic keratosis to in situ and invasive SCCs entirely unrelated to HPV The progressive increase in overexpression of p16 from premalignancy to carcinoma is thought to be related to oncogene-induced tumor cell senescence and feedback loops. 20 p16 in oropharynx squamous carcinoma Based on the wealth of literature in recent years, p16 protein expression evaluation by immunohistochemistry has become a surrogate marker for HPV-associated SCC arising in the oropharynx (including the tonsils and base of tongue). 2,3,10 The trend toward using p16 preferentially over direct HPV testing to determine a tumor s HPV status has developed secondary to: (1) the ease of immunohistochemical evaluation of p16, (2) the challenges of 1530 HEAD & NECK DOI /HED NOVEMBER 2013

5 P16 EXPRESSION IN CUTANEOUS SQUAMOUS CARCINOMA FIGURE 2. Overexpression of p16 in head and neck squamous carcinomas (SCCs) of the upper aerodigestive tract: A, oropharynx (human papillomavirus [HPV] positive), (B) oral tongue, (C) nasopharynx, and (D) supraglottic larynx. Concurrent HPV testing was negative in nonoropharyngeal locations (B, C, and D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] lower sensitivity and higher cost of HPV testing by in situ hybridization, and (3) clinical studies suggesting that patients with p16 þ/hpv- tumors arising in the oropharynx have favorable outcomes similar to those of patients with HPVþ tumors. 3,10,11 However, as noted by our current study, there are limitations to the use of p16 as an isolated surrogate marker of HPV association and for determining the site of a presumed oropharyngeal primary tumor. The use of immunohistochemical markers like p16 as potential biomarkers must be rigorously tested and standardized. The methodology used to perform the immunohistochemical test (antibody clone, dilution), how an antibody is to be interpreted by a pathologist as positive or negative, and what threshold correlates to the clinical parameter of interest all must be clearly defined. Numerous studies have reported p16 immunohistochemical analysis in SCCs; however, there has been a dearth of consistent definitions of p16 overexpression; the general consensus has been that strong and diffuse staining of p16 in the majority of tumor cells is associated with HPV positivity. 3,12,21 Although this high threshold of p16 diffuse expression will not detect approximately 5% of HPVþ tumors, which only show focal/patchy or even negative p16 expression. 3,5 Equally important is our understanding of the specificity of a marker and under what conditions that specificity is maintained. In the case of oropharyngeal SCC, the association of p16 with HPV positivity has been defined; however, the meaning of p16 expression outside the oropharyngeal region varies and may not be associated with HPV, and because of the aforementioned, the use of p16 alone as an independent biomarker for a primary tumor s site of origin is not supported for SCCs. 22 p16 in other head and neck neoplasms and tumors of unknown primary p16 overexpression is not limited to oropharyngeal SCC and has also been reported in various sites within the head and neck region (Figure 2) Harris et al 24 reported overexpression of p16 in 39% (11 of 28) of oral tongue SCCs in young adults without an association with HPV. Duray et al 23 reported 22% of laryngeal SCCs expressing p16 were negative for HPV by PCR analysis. Similarly, although 89% of nasopharyngeal carcinomas were positive for p16 expression, 32% of those tumors were negative for HPV and Epstein Barr virus by PCR. 25 In the sinonasal region, both SCCs and sinonasal undifferentiated carcinomas may overexpress p16 unrelated to HPV. 26 These findings suggest that p16 expression is not limited to oropharyngeal cancers, despite p16 s clear association with HPV positivity among oropharyngeal cancers and, moreover, that p16 expression in metastatic adenopathy cannot be considered a consistent indicator of an oropharyngeal primary. However, with the advances in imaging technology, the inability to identify a primary site of a head and neck tumor that presents as metastatic adenopathy is uncommon. Through a comprehensive evaluation, including examination under anesthesia, biopsies, and imaging, only 5% to 10% of patients who present with SCC metastases to the neck will remain without an identifiable primary tumor. 27 The possible sites of occult primary tumors leading to neck metastases include the upper aerodigestive tract, skin, and other distant sites. In patients whose diagnosis remains "unknown primary SCC" metastatic to the neck HEAD & NECK DOI /HED NOVEMBER

6 BEADLE ET AL. even after an exhaustive workup, optimal management remains controversial. Some centers favor treating only the disease-positive neck site with either surgery, radiation, or a combination of the two. 28 Other centers favor comprehensively treating both sides of the neck and all potential sources of the primary tumor in the upper aerodigestive mucosa. 29 How extensive the mucosal coverage should be and the optimal radiotherapy doses also remain subjects of debate. 30 Comprehensive radiotherapy is associated with relative morbidity because of the extent of the treatment fields, and it is widely accepted that any localizing information regarding the source of the primary tumor would be helpful in minimizing treatment and potential side effects. With the increasing body of literature linking HPV and p16 expression with oropharyngeal SCCs, testing of SCC neck metastases from unknown primary SCCs has been advocated to identify a possible primary region and to further delineate treatment fields The design of these studies, however, has focused on the detection of p16 and HPV in lymph node metastases from known oropharyngeal primaries, with fewer samples from other sites. The design of this study, whereas representing a limited sampling of cutaneous head and neck SCC with metastases, highlights the potential limitations of p16 overexpression in isolation. Although the incidence of encountering the proposed scenario of a p16þ/hpv- (in situ) metastatic SCC in a lymph node cannot be determined by this study, Desai et al 31 identified 4 of 41 metastatic SCCs in the neck to be p16þ and HPV- by PCR with confirmed primary SCCs outside of the oropharynx for a potential rate of 10.0%. Additional studies showed a 3% to 8% discordant rate of p16þ/hpv- tumors that were ultimately located outside the oropharynx However, the limited number of p16þ/hpv- cases in these studies limits a conclusion of how to triage these patients and, in combination, emphasizes the real potential of encountering this clinical scenario. CONCLUSIONS Our study evaluating head and neck cutaneous SCCs highlights the potential limitations of using p16 expression alone to identify the primary site or region of origin of SCCs diagnosed with metastatic lymphadenopathy. Biologically, p16 overexpression may be secondary to an association with HPV or secondary to other causes altering the Rb pathway. An awareness of a significant rate of p16 expression in aggressive cutaneous SCCs of the head and neck must be considered when evaluating metastatic SCCs from an unknown primary and attempting to identify a site of origin. With the above knowledge, the approach to an unknown primary SCC metastatic to the neck may include testing for p16 and HPV; however, because p16 expression may be present in lymph nodepositive cutaneous head and neck SCCs, as well as SCCs from other sites, the association of a p16þ/hpv- SCC metastasis to the neck specifically from an oropharyngeal primary cannot be inferred on the basis of p16 positivity alone, and the metastatic SCC should remain a tumor of unknown primary. p16 analysis may be sufficient to determine HPV positivity in known oropharyngeal primary tumors; however, further HPV testing by a more sensitive method such as PCR is prudent in the analysis of these discrepant cases of unknown primary cancer as needed to facilitate clinical management. REFERENCES 1. Dahlstrom KR, Adler Storthz K, Etzel CJ, et al. Human papillomavirus type 16 infection and squamous cell carcinoma of the head and neck in never-smokers: a matched pair analysis. 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