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Purpose: Definitive or postoperative chemoradiation (CRT) is curative for human papillomavirus-associated (HPV+) oropharynx cancer (OPC) but induces significant toxicity. As a deintensification strategy, we studied primary transoral surgery (TOS) and reduced postoperative radiation therapy (RT) in intermediate-risk HPV+ OPC., Methods: E3311 is a phase II randomized trial of reduced- or standard-dose postoperative RT for resected stage III-IVa (American Joint Committee on Cancer-seventh edition) HPV+ OPC, determined by pathologic parameters. Primary goals were feasibility of prospective multi-institutional study of TOS for HPV+ OPC, and oncologic efficacy (2-year progression-free survival) of TOS and adjuvant therapy in intermediate-risk patients after resection. TOS plus 50 Gy was considered promising if the lower limit of the exact 90% binomial confidence intervals exceeded 85%. Quality of life and swallowing were measured by functional assessment of cancer therapy-head and neck and MD Anderson Dysphagia Index., Results: Credentialed surgeons performed TOS for 495 patients. Eligible and treated patients were assigned as follows: arm A (low risk, n = 38) enrolled 11%, intermediate risk arms B (50 Gy, n = 100) or C (60 Gy, n = 108) randomly allocated 58%, and arm D (high risk, n = 113) enrolled 31%. With a median 35.2-month follow-up for 359 evaluable (eligible and treated) patients, 2-year progression-free survival Kaplan-Meier estimate is 96.9% (90% CI, 91.9 to 100) for arm A (observation), 94.9% (90% CI, 91.3 to 98.6]) for arm B (50 Gy), 96.0% (90% CI, 92.8 to 99.3) for arm C (60 Gy), and 90.7% (90% CI, 86.2 to 95.4) for arm D (66 Gy plus weekly cisplatin). Treatment arm distribution and oncologic outcome for ineligible or step 2 untreated patients (n = 136) mirrored the 359 evaluable patients. Exploratory comparison of functional assessment of cancer therapy-head and neck total scores between arms B and C is presented., Conclusion: Primary TOS and reduced postoperative RT result in outstanding oncologic outcome and favorable functional outcomes in intermediate-risk HPV+ OPC., Competing Interests: Robert L. FerrisThis author is a member of the Journal of Clinical Oncology Editorial Board. Journal policy recused the author from having any role in the peer review of this manuscript.Stock and Other Ownership Interests: NovasentaConsulting or Advisory Role: Merck, Pfizer, EMD Serono, Numab, Macrogenics, Aduro Biotech, Novasenta, Sanofi, Zymeworks, Bristol Myers SquibbResearch Funding: Bristol Myers Squibb, AstraZeneca/MedImmune, Merck, Tesaro, Novasenta Gregory S. WeinsteinPatents, Royalties, Other Intellectual Property: Olympus Shuli LiEmployment: TakedaStock and Other Ownership Interests: Takeda Harry QuonEmployment: Johns Hopkins HospitalLeadership: Pistevo HealthStock and Other Ownership Interests: Oncospace, Pistevo HealthHonoraria: Sanofi/RegeneronConsulting or Advisory Role: Pinnacle Biologics, Tactile MedicalResearch Funding: Toshiba, Vibrent HealthPatents, Royalties, Other Intellectual Property: Filed under Hopkins, we have several patents related to mobile informatics solutionsOther Relationship: EPIC Ranee MehraStock and Other Ownership Interests: GlaxoSmithKline (I)Consulting or Advisory Role: Bayer, Rakuten MedicalResearch Funding: AstraZeneca, Merck Christine H. ChungConsulting or Advisory Role: Bristol Myers Squibb, CUE Biopharma, Mirati Therapeutics, Sanofi/Regeneron, ExelixisResearch Funding: AstraZeneca, Bristol Myers Squibb, Lilly, Merck, Regeneron, Ignyta, Pfizer, Brooklyn ImmunoTherapeutics, Iovance BiotherapeuticsTravel, Accommodations, Expenses: AstraZeneca, Mirati Therapeutics Maura L. GillisonConsulting or Advisory Role: Bristol Myers Squibb, Merck, EMD Serono, Kura Oncology, BioNTech, Shattuck Labs, Bayer, Debiopharm Group¸ Ipsen, Gilead Sciences¸ Bicara Therapeutics, Nektar¸ Istari, LLX Solutions, OncLive, Seagen, Kura Oncology, Mirati Therapeutics, Sensei BiotherapeuticsResearch Funding: Bristol Myers Squibb, Genocea Biosciences, Cullinan Oncology, Genentech, Agenus, Kura Oncology Umamaheswar DuvvuriConsulting or Advisory Role: ACTIVARTIS BiotechResearch Funding: Kolltan Pharmaceuticals, Medrobotics Bert W. O'Malley JrPatents, Royalties, Other Intellectual Property: Receive royalties from University of Pennsylvania via a licensed technology to Olympus Inc Neil D. GrossHonoraria: Intuitive SurgicalConsulting or Advisory Role: PDS Biotechnology, Verb Surgical, Sanofi/Regeneron, Shattuck LabsResearch Funding: Regeneron, MedImmune, GenentechPatents, Royalties, Other Intellectual Property: UpToDate R. Bryan BellHonoraria: Merck, RegeneronConsulting or Advisory Role: MacrogenicsSpeakers' Bureau: Merck, RegeneronResearch Funding: Bristol Myers Squibb/Celgene Nabil F. SabaHonoraria: Merck, CUE Biopharma, BioNTech AG, GlaxoSmithKlineResearch Funding: Bristol-Myers Squibb, ExelixisTravel, Accommodations, Expenses: Merck, Pfizer, Blueprint Medicines Barbara BurtnessConsulting or Advisory Role: Merck, Debiopharm Group, CUE Biopharma, Maverick Therapeutics, Rakuten Medical, Nanobiotix, Macrogenics, ALX Oncology, IO Biotech, Ipsen, Genentech/Roche, Kura Oncology, Merck KGaA, PPD Global, ExelixisResearch Funding: Merck (Inst), Aduro Biotech (Inst), Formation Biologics (Inst), Bristol Myers (Inst), CUE Biopharma (Inst)Travel, Accommodations, Expenses: Merck, Debiopharm GroupNo other potential conflicts of interest were reported.

Early studies estimate that 5% to 10% of oropharyngeal squamous cell carcinomas overexpress p16 but are unassociated with transcriptionally-active high-risk human papillomavirus (HPV). Patients with discordant HPV testing may experience clinical outcomes that differ from traditional expectations. To document the rate of p16 and HPV mRNA positivity, characterize patients with discordant testing, and identify features that may warrant selective use of HPV-specific testing after p16 IHC, a multi-institutional, retrospective review of oropharyngeal squamous cell carcinoma patients with p16 IHC and HPV mRNA testing by reverse transcriptase polymerase chain reaction was performed. Of the 467 patients, most had T1 or T2 tumors (71%), 82% were p16 positive, and 84% were HPV mRNA positive. Overall, most tumors were nonkeratinizing (378, 81%), which was strongly associated with p16 and HPV positivity (93% and 95%, respectively). Overall, 81% of patients were double positive, 14% double negative, and 4.9% discordant (3.4% p16 negative/HPV mRNA positive and 1.5% p16 positive/HPV mRNA negative). The survival rates of these discordant patient groups fell squarely between the 2 concordant groups, although in multivariate analysis for both disease-free survival and overall survival, discordant patients were not found to have statistically significantly different outcomes. Reclassifying patients by applying HPV mRNA testing when p16 results and morphology do not match, or when p16 results are equivocal, improved prognostication slightly over p16 or HPV mRNA testing alone. Patients with discordant testing demonstrate a borderline significant trend toward survival differences from those with concordant tests. When evaluated independently, patients who were p16 negative but HPV mRNA positive had a prognosis somewhat closer to double-positive patients, while those who were p16 positive, but HPV mRNA negative had a prognosis closer to that of double-negative patients. We suggest an algorithm whereby confirmatory HPV mRNA testing is performed in patients where p16 status is not consistent with tumor morphology. This captures a majority of discordant patients and improves, albeit modestly, the prognostication., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the NIH grant R01DE026471 (X.W.). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Lower prevalence HPV infection has been previously reported in Thai population when compared with Western countries. p16 expression indicates HPV-associated oropharyngeal squamous cell carcinoma (OPSCC), but not non-OPSCC. We therefore evaluated the characteristic and association of p16 and HPV in Thai patients with HNSCC., Methods: We used immunohistochemistry and qPCR, respectively, to detect p16 and HPV DNA in archrival formalin-fixed paraffin-embedded HNSCC tissues. Patient characteristics and survival were analyzed., Results: p16 expression was detected in tumors of 72 of 662 (10.9%) patients with HNSCC and was significantly associated with higher-grade histology, advanced nodal stage, and oropharynx. p16 was expressed in 28 and 6.5% of patients with OPSCC or non-OPSCC, respectively, and HPV DNA was detected in 15.6 and 1% of patients, respectively. Using p16 as a surrogate for HPV status, sensitivities were 80 and 25% in OPSCC and non-OPSCC, respectively. Positive and negative predictive rates of OPSCC were 38 and 95%. Discordance rates between HPV and p16 were 23 and 7% in OPSCC and non-OPSCC, respectively. Overall survival (OS) were significantly longer in both p16-positive OPSCC (p = 0.049), and non-OPSCC (p = 0.003)., Conclusions: Low prevalence of p16 and HPV associated OPSCC and non-OPSCC were confirmed in Thai patients. High discordance and low positive predictive rates of p16 were observed in HPV-associated OPSCC. p16 was a significant prognostic factor for OS for patients with OPSCC or non-OPSCC. Therefore, HPV testing should be performed to assess the association of HPV with HNSCC regardless of p16 expression.

In a retrospective review, we identified 332 patients with 338 pathologically diagnosed primary oropharyngeal carcinomas (OPC) between January 2013 and March 2020 with known p16/HPV status from a tumor registry at Northwestern Memorial Hospital. The tumors predominantly involved the palatine tonsil (51 %) and the base of the tongue/lingual tonsil (38 %). The most common type of cancer was non-keratinizing squamous cell carcinoma (60 %), and the majority of primaries were p16 positive/HPV-mediated (86 %). A cohort of p16 positive/HPV mediated OPC (27/283, 9.5 %) presented with aggressive clinical behavior, including multiple distant metastases at unusual sites. Tumor size >2 cm and the presence of tumor anaplasia/multinucleation were significantly associated with an increased rate of distant metastases in p16 positive/HPV mediated cases, both in unadjusted and adjusted analyses (all P < 0.05). Of the 332 individuals in the overall cohort, 38 individuals died due to their disease within the observed follow-up time. Among the 283 patients with p16 positive/HPV mediated tumors, survival was estimated at 97 % (95 % CI 95 %, 100 %) at 1 year, 95 % (95 % CI 92 %, 98 %) at 2 years, and 80 % (95 % CI 72 %, 89 %) at 5 years. The presence of tumor anaplasia/multinucleation and distant metastasis were both significantly associated with poorer disease-specific survival in p16 positive/HPV mediated cases (both P < 0.05), with the survival effect of tumor anaplasia/multinucleation likely mediated in part through its association with distant metastasis. For p16 positive/HPV-mediated OPC, age, smoking status, tumor status, and lymph node status were not significantly associated with disease-specific survival in our study., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: A remarkably better prognosis is associated with oropharyngeal squamous cell carcinomas (OPSCC) driven by human papillomaviruses (HPV) compared with HPV-negative OPSCC. Consequently, de-escalation of standard treatment has been suggested. Due to modest specificity rates, debates are ongoing, whether p16 INK4a , a surrogate marker for HPV-driven OPSCC, is sufficient to correctly identify those tumours and avoid substantial HPV misattribution and thus undertreatment of patients by de-escalation. Robust data estimating the proportion of potentially undertreated patients are missing., Methods: We assessed a large-scale cohort of consecutively included OPSCC diagnosed between 2000 and 2017 for HPV-DNA, HPV genotypes, p16 INK4a expression and multiple tumour- and patient-related risk factors, and investigated their impact on patients' survival in comprehensive uni- and multivariate analyses., Results: Aetiological relevance of HPV (p16 INK4a - and high-risk HPV-DNA-positivity) was detected in 27.1% (n = 192) of OPSCC, with HPV 16 being the most abundant HPV type (94.6%). In 5.5% patients (n = 39), p16 INK4a overexpression but no HPV-DNA was detected. Principal component and survival analyses revealed that 60.6% of these p16 INK4a -positive OPSCC lacking HPV-DNA did not resemble HPV 16 -driven but HPV-negative OPSCC regarding risk-factor profile and overall survival. Notably, this group represented 10.6% of all p16 INK4a -overexpressing OPSCC., Conclusions: p16 INK4a as a single marker appears insufficient to indicate OPSCC patients suitable for treatment de-escalation.

Background: Incidence of oropharyngeal squamous cell carcinoma (OPSCC) is rising rapidly in many western countries due to Human papillomavirus (HPV) and tobacco smoking, with a considerable overlap. Immunotherapy directed at the PD1/PD-L1 axis have shown promise in head and neck cancer and other cancer types. PD-L1 expression may indicate a poorer prognosis, and at the same time indicate a possible benefit of anti-PD-L1 immunotherapeutic agents. The primary aim of this study was to establish the prognostic effect of PD-L1 expression after primary curative radiotherapy alone. Material and methods: A cohort of 303 OPSCC patients treated with primary, curative intended radiotherapy was established. PD-L1 expression was evaluated by immunohistochemistry on formalin fixed, paraffin embedded tissue sections. PD-L1 positivity was defined as a Combined Positive Score (CPS) ≥1, indicating staining of either tumor cells, lymphocytes or macrophages. Results: Median follow-up was 5.3 years. With 199 deaths, there was no difference in overall survival between patients with PD-L1+ and PD-L1- tumors (adjusted hazard ratio [aHR] and 95% confidence interval [CI]: 1.0 [0.71-1.4]). Also, locoregional failure was similar between the two groups (aHR 1.1 [CI: 0.68 - 1.7]). Tumors were PD-L1+ in 76% of cases, significantly more among HPV p16+ tumors (82% vs. 70%, p  = .01). Interestingly, higher prevalence of PD-L1+ expression was seen in HPV p16+ patients with <10 pack-years of tobacco-smoking (93%) compared to HPV p16+ smokers (76%) or HPV p16-negative patients (70%) ( p  = .003). Conclusion: PD-L1 expression had no prognostic significance in OPSCC patients treated with primary radiotherapy alone. A substantial proportion of OPSCC tumors show PD-L1 overexpression, especially in HPV p16+ tumors in patients with little or no smoking history.

Purpose: A 15-gene hypoxia classifier has been developed and validated as a predictive factor for patients with head and neck squamous cell carcinoma treated with radiotherapy and nimorazole. This paper aimed to investigate the role of this hypoxia classifier as a prognostic factor for patients with oropharyngeal cancer (OPC) treated with accelerated chemoradiotherapy., Methods: P16 and 15-gene hypoxia classifier status, categorising tumours as more or less hypoxic, were determined for 136 OPC patients. Locoregional recurrence rate (LRR) and overall survival (OS) were estimated with cumulative incidence function and Kaplan-Meier method, respectively, stratified according to p16 and hypoxia status., Results: P16-positive patients (34.6%) had significantly better LRR and OS than p16-negative patients. The 5‑year LRR of patients with more hypoxic OPC was similar to those with less hypoxic OPC in the overall patient population (27.3% versus 25.1%; p = 0.98; HR = 1.01 [CI95% 0.47;2.17]) and in the p16-negative OPC (36.4% versus 30.1%; p = 0.70; HR = 1.17 [CI95% 0.53;2.56]). No significant OS differences could be observed in neither p16-negative nor p16-positive subgroup with a 5-year OS for p16-negative more hypoxic OPC of 44.2% versus 49.0% in the less hypoxic OPC (p = 0.92; HR 0.97 [CI95% 0.51;1.84])., Conclusion: No significant outcome differences were observed between more or less hypoxic tumours, as determined by the 15-gene hypoxia classifier. These results suggest that the 15-gene hypoxia classifier may not have prognostic value in an OPC patient cohort treated with accelerated chemoradiotherapy.

The field of head and neck pathology was just developing 50 years ago but has certainly come a long way in a relatively short time. Thousands of developments in diagnostic criteria, tumor classification, malignancy staging, immunohistochemistry application, and molecular testing have been made during this time, with an exponential increase in literature on the topics over the past few decades: There were 3506 articles published on head and neck topics in the decade between 1969 and 1978 (PubMed source), with a staggering 89266 manuscripts published in the most recent decade. It is daunting and impossible to narrow the more than 162000 publications in this field and suggest only a few topics of significance. However, the breakthrough in this anatomic discipline has been achieved in 3 major sites: oropharyngeal carcinoma, salivary gland neoplasms, and sinonasal tract tumors. This review will highlight selected topics in these anatomic sites in which the most profound changes in diagnosis have occurred, focusing on the information that helps to guide daily routine practice of surgical pathology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: Limited information is available regarding the precise differences in the tumour immune microenvironment (TIM) of patients with human papilloma virus (HPV)-associated and non-HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Here, we retrospectively reviewed 137 patients with OPSCC treated with a definitive treatment to identify molecular relationships in the TIM., Materials and Methods: We used immunohistochemical analysis to assess p16 status, programmed death ligand 1 (PD-L1) level, and/or CD8 + tumour-infiltrating lymphocyte (TIL) density, followed by prognostic evaluation of these immune-related parameters., Results: Multivariate analyses demonstrated that PD-L1 level on immune cells but not on tumour cells or CD8 + TIL density was a significant predictive factor of disease-free survival (DFS) and overall survival (OS). Additionally, subgroup analyses demonstrated that patients positive for p16 and PD-L1 expression on immune cells had favourable DFS and OS, whereas patients negative for p16 and PD-L1 expression on immune cells showed worse DFS and OS., Conclusions: We demonstrated that PD-L1 expression on immune cells but not tumour cells might represent a useful prognostic biomarker in patients with OPSCC receiving a definitive treatment. We propose that a co-assessment of p16 and PD-L1 expression on immune cells would have greater prognostic potential compared with evaluation of each factor alone in patients with OPSCC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Oral cavity squamous cell carcinoma (OCC) and oropharyngeal squamous cell carcinoma (OPC) are among the most common cancers worldwide and are associated with high mortality and morbidity. The purpose of this study is to identify potential biomarkers to distinguish OCC/OPC from normal controls and to distinguish OCC patients with and without nodal metastasis. We tested saliva samples from 101 OCC, 58 OPC, and 35 normal controls using four analytical platforms (NMR, targeted aqueous by LC-MS/MS, global aqueous and global lipidomics by LC-Q-TOF). Samples from OCC and normal controls were divided into discovery and validation sets. Using linear regression adjusting for age, sex, race and experimental batches, we found the levels of two metabolites (glycine and proline) to be significantly different between OCC and controls (FDR < 0.1 for both discovery and validation sets) but did not find any appreciable differences in metabolite levels between OPC and controls or between OCC with and without nodal metastasis. Four metabolites, including glycine, proline, citrulline, and ornithine were associated with early stage OCC in both discovery and validation sets. Further study is warranted to confirm these results in the development of salivary metabolites as diagnostic markers., Competing Interests: The authors have declared that no competing interests exist.

Perivascular epithelioid cell tumors (PEComas) in the head and neck region are rare, with 26 cases described in literature. These distinct mesenchymal tumors normally express both myoid and melanocytic markers. We here report an interesting and challenging case of malignant PEComa that showed transcription factor E3 (TFE3) protein expression and rearrangement, paucity of muscle and melanocytic marker expression, and morphologically mimicked alveolar soft part sarcoma. Awareness of this morphologic pitfall and recognition of TFE3 gene-rearranged PEComa, as a distinct subtype of PEComa, is essential to avoid misdiagnosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: High-risk human papilloma virus (HPV) testing should be performed on all patients with newly diagnosed oropharyngeal HPV-associated squamous cell carcinoma (OPHPVSCC), and p16 immunostaining can be used as a surrogate marker. Although in surgical pathology specimens p16 staining in > 70% of the tumor cells is considered a positive result, the interpretation in fine-needle aspiration (FNA) specimens has remained controversial., Study Design: FNA of neck lymph nodes and corresponding surgical specimens from 42 patients with OPHPVSCC were reviewed., Results: In FNA specimens, 38 cases (90.5%) had viable tumor cells, 32 (76.2%) had keratin debris, and 36 (85.7%) had degenerated keratinized tumor cells. Twenty-seven of 27 (100%) had positive p16 staining in > 70% of viable tumor cells, while the degenerated tumor cells were negative. Twenty of 24 (83.3%) primary OPHPVSCC exhibited focal degenerated keratinized tumor cells and/or keratin debris., Conclusions: This study showed that the majority of the OPHPVSCC metastases in lymph nodes had degenerated keratinized tumor cells and keratin debris. Many primary OPHPVSCC also demonstrated focal keratinization and/or degeneration. The degenerated tumor cells showed no immunoreactivity to p16. The same 70% cutoff used in histologic specimens should be applied in cytologic specimens, but only the viable tumor cells should be counted., (© 2018 S. Karger AG, Basel.)

Background: Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed., Methods: CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided., Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04)., Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types., (© The Author 2017. Published by Oxford University Press.)

The oral cavity and oropharynx have historically been viewed as a single anatomic compartment of the head and neck. The practice of combining the oral cavity and oropharynx has recently been revised, largely owing to the observation that human papillomavirus (HPV)-related carcinogenesis has a strong predilection for the oropharynx but not the oral cavity. The purpose of this study was to determine whether HPV is evenly distributed across squamous cell carcinomas of the oropharynx including those sites that do not harbor tonsillar tissues such as the soft palate. A search of the medical records of the Johns Hopkins Hospital identified 32 primary squamous cell carcinomas of the soft palate (n=31) and posterior pharyngeal wall (n=1). All were evaluated with p16 immunohistochemistry and high-risk HPV in situ hybridization (ISH) (29 by RNA ISH and 3 by DNA ISH). For comparison, we also reviewed the medical records to obtain the HPV status of patients who had undergone HPV testing of primary tonsillar carcinomas over the same time interval as part of their clinical care. High-risk HPV as detected by ISH was present in just 1 (3.1%) of the 32 oropharyngeal squamous cell carcinomas, including 1 of 2 p16-positive carcinomas. The difference in HPV detection rates between tonsillar and nontonsillar sites was significant (1/32, 3.1% vs. 917/997, 92%; P<0.0001). HPV is not frequently detected in squamous cell carcinomas arising from nontonsillar regions of the oropharynx. Indeed, squamous cell carcinomas of the soft palate more closely resemble those arising in the oral cavity than those arising in areas of the oropharynx harboring tonsillar tissue. This finding not only further sharpens our understanding of site-specific targeting by HPV, but may have practical implications regarding HPV testing and even the way the oral vault is oncologically compartmentalized to partition HPV-positive from HPV-negative cancers.

High-risk human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas have a more favorable prognosis than HPV-negative ones. p16 immunohistochemistry has been recommended as a prognostic test in clinical practice. Several p16 antibodies are available, and their performance has not been directly compared. We evaluated three commercially available p16 antibody clones (E6H4, JC8 and G175-405) utilizing 199 cases of oropharyngeal squamous cell carcinoma from a tissue microarray, read by three pathologists with three different cutoffs for positivity: any staining, >50% and >75%. Positive predictive values for high-risk HPV status by RNA in situ hybridization for the E6H4, JC8 and G175-405 clones were 98%, 100% and 99% at the 75% cutoff, but negative predictive values were much more variable at 86%, 69% and 56%, respectively. These improved using the 50% cutoff, becoming similar for all three antibodies. Intensity varied substantially, with 85% of E6H4, 72% of JC8 and 67% of G175-405 showing strong (3+) intensity. With Kaplan-Meier survival plots at the 75% cutoff, the E6H4 clone showed the largest differential in disease specific and overall survival between p16-positive and -negative results. Decreasing the cutoff to 50% increased correlation with HPV in situ hybridization and improved the survival differential for the JC8 and G175-405 clones without worsening of performance for the E6H4 clone. Interobserver agreement was also assessed by kappa scores and was highest for the E6H4 clone. Overall, these study results show modest but important performance differences between the three different p16 antibody clones, suggesting that the E6H4 clone performs best because of strongest staining intensity, greatest differential in outcomes between positive and negative results, lowest interobserver variability, and lowest background, nonspecific staining. The results also suggest that a 75% cutoff is very functional but that, in this patient population with high HPV incidence, 50% and any staining cutoffs may be more effective, particularly for the non-E6H4 clones.

Follicular dendritic cell sarcoma is a rare mesenchymal neoplasm that most commonly occurs in cervical lymph nodes. It has histologic and clinical overlap with the much more common p16-positive human papillomavirus (HPV)-related squamous cell carcinoma of the oropharynx, which characteristically has nonkeratinizing morphology and often presents as an isolated neck mass. Not surprisingly, follicular dendritic cell sarcomas are commonly misdiagnosed as squamous cell carcinoma. Immunohistochemistry is helpful in separating the 2 entities. Follicular dendritic cell sarcoma expresses dendritic markers such as CD21 and CD23 and is almost always cytokeratin negative. However, in many cases of HPV-related oropharyngeal carcinoma, only p16 immunohistochemistry as a prognostic and surrogate marker for HPV is performed. p16 expression in follicular dendritic cell sarcoma has not been characterized. Here, we investigate the expression of p16 in follicular dendritic cell sarcoma and correlate it with retinoblastoma protein expression. A pilot study of dendritic marker expression in HPV-related oropharyngeal squamous cell carcinoma was also performed. We found that 4 of 8 sarcomas expressed p16 with strong and diffuse staining in 2 cases. In 2 of the 4 cases, p16 expression corresponded to loss of retinoblastoma protein expression. Dendritic marker expression (CD21 and CD23) was not found in HPV-related oropharyngeal squamous cell carcinomas. As such, positive p16 immunohistochemistry cannot be used as supportive evidence for the diagnosis of squamous cell carcinoma as strong and diffuse p16 expression may also occur in follicular dendritic cell sarcoma. Cytokeratins and dendritic markers are critical in separating the two tumor types., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives We analyze the relationship between CD44, epidermal growth factor receptor (EGFR), and p16 expression in oral cavity and oropharyngeal cancers in a diverse population. We also describe whether particular patterns of staining are associated with progression-free survival and overall survival. Study Design Prospective study, single-blind to pathologist and laboratory technologist. Setting Hospital based. Subjects and Methods Immunohistochemistry, comprising gross staining and cellular expression, was performed and interpreted in a blinded fashion on 24 lip/oral cavity and 40 oropharyngeal cancer specimens collected between 2007 and 2012 from participants of a larger study. Information on overall survival and progression-free survival was obtained from medical records. Results Nineteen cases were clinically p16 positive, 16 of which were oropharyngeal. Oral cavity lesions were more likely to exhibit strong CD44 membrane staining ( P = .0002). Strong CD44 membrane and strong EGFR membrane and/or cytoplasmic staining were more common in p16-negative cancers ( P = .006). Peripheral/mixed gross p16 staining pattern was associated with worse survival than the universal staining on univariate and multivariate analyses ( P = .006, P = .030). This held true when combining gross and cellular localization for p16. For CD44, universal gross staining demonstrated poorer overall survival compared with the peripheral/mixed group ( P = .039). CD44 peripheral/mixed group alone and when combined with universal p16 demonstrated the best survival on multivariate analysis ( P = .010). Conclusion In a diverse population, systematic analysis applying p16, CD44, and EGFR gross staining and cellular localization on immunohistochemistry demonstrates distinct patterns that may have prognostic potential exceeding current methods. Larger studies are warranted to investigate these findings further.

The accurate diagnosis of human papillomavirus (HPV) causality in oropharyngeal squamous cell carcinomas (OPSCC) is likely to influence therapeutic decisions in affected patients in the near future. We conducted a systematic review and meta-analysis to determine the diagnostic accuracy of p16 INK4a immunohistochemistry (IHC) to identify HPV-induced OPSCC. We identified all studies that performed p16 INK4a IHC (index test) and HPV E6/E7 mRNA detection using an amplification-based method (gold standard to indicate a transforming relevance of HPV) in OPSCC. Testing with one or more comparator tests (HPV DNA PCR, HPV DNA in situ hybridization (ISH) and p16 INK4a IHC/HPV DNA PCR combined testing) was an optional criterion for inclusion. Among 1,636 retrieved studies 24 fulfilled the inclusion criteria. The pooled sensitivity of p16 INK4a IHC, HPV DNA PCR, HPV DNA ISH and p16 INK4a IHC/HPV DNA PCR combined testing was 94% (95%-confidence interval (CI) 91-97%), 98% (CI 94-100%), 85% (CI 76-92%) and 93% (CI 87-97%), respectively. The pooled specificity was 83% (CI 78-88%), 84% (CI 74-92%), 88% (CI 78-96%) and 96% (CI 89-100%), respectively. p16 INK4a IHC/HPV DNA PCR combined testing was as sensitive as either p16 INK4a IHC or HPV DNA PCR alone but significantly more specific than either separate test. In conclusion, p16 INK4a IHC is highly sensitive but moderately specific to diagnose HPV-transformed OPSCC when used as a single test. Combined p16 INK4a IHC and HPV DNA PCR testing significantly enhances specificity while maintaining high sensitivity. This diagnostic test combination thus represents an attractive testing strategy for the reliable diagnosis of HPV-induced OPSCC in the clinical setting and may constitute an inclusion criterion for future therapeutic trials., (© 2016 UICC.)

Introduction and Objectives: HSP-90 is an intracellular protein that protects the cell from environmental stress situations. The overexpression of HSP-90 isoforms could serve as a mechanism of resistance to radiotherapy for tumour cells. We studied this effect in a sample of head and neck tumours., Methods: We included 87 patients diagnosed with oral cavity, oropharynx, larynx and hypopharynx tumours. We studied the expression of the HSP-90 isoforms by real-time PCR on pre-treatment biopsy samples. We analysed the relationship between HSP-90 expression levels and local relapse of the tumour with CRT decision trees., Results: The expression levels of the inducible citosolic isoform (HSP90AA) allowed the definition of 2 groups of patients with different rates of local relapse. The group with a low expression level showed a 2.9% local relapse rate, while the group with a high expression level showed a 38.2% rate. Survival curves showed differences in time to local relapse for both groups of patients. These differences did not reach statistical significance., Conclusions: Radiotherapy response was related to expression levels of HSP-90 in a sample of head and neck cancer patients. This result could prove useful in the selection of treatments for this group of patients., (Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Otorrinolaringología y Cirugía de Cabeza y Cuello. All rights reserved.)

Purpose: We conducted a retrospective evaluation of the IMCL-9815 study to examine the association of human papillomavirus (HPV) and p16 protein expression status with outcomes in patients with oropharyngeal carcinoma (OPC) receiving radiotherapy (RT) plus cetuximab or RT alone., Patients and Methods: In the IMCL-9815 study, patients were randomly allocated to receive RT plus weekly cetuximab or RT alone. A subpopulation of patients with p16-evaluable OPC was retrospectively evaluated on the basis of locoregional control (LRC), overall survival (OS), and progression-free survival (PFS). Evaluable samples from patients with p16-positive OPC were also tested for HPV DNA., Results: Tumor p16 status was evaluable in 182 patients with OPC enrolled in the IMCL-9815 study; 41% were p16 positive. When treated with RT alone or RT plus cetuximab, p16-positive patients had a longer OS than p16-negative patients (hazard ratio, 0.40; 95% CI, 0.21 to 0.74 and hazard ratio, 0.16; 95% CI, 0.07 to 0.36, respectively). The addition of cetuximab to RT increased LRC, OS, and PFS in both patients with p16-positive OPC and those with p16-negative disease. Interaction tests for LRC, OS, and PFS did not demonstrate any significant interaction between p16 status and treatment effect (P = .087, .085, and .253, respectively). Similar trends were observed when patients with p16-positive/HPV-positive OPC (n = 49) and those with p16-positive/HPV-negative OPC (n = 14) were compared., Conclusion: p16 status was strongly prognostic for patients with OPC. The data suggest that the addition of cetuximab to RT improved clinical outcomes regardless of p16 or HPV status versus RT alone., Competing Interests: Authors’ disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article., (© 2015 by American Society of Clinical Oncology.)

Human papillomavirus (HPV) infection is an indicator of good response to chemoradiotherapy in oropharyngeal squamous cell carcinoma (OPSCC), and epidermal growth factor receptor (EGFR) is a molecular-therapeutic target in head and neck squamous cell carcinoma. Here we investigated the prevalence and prognostic significance of HPV infection and EGFR alteration in OPSCC. We analyzed the presence of high-risk HPV using in situ hybridization, protein expressions of p16 and EGFR using immunohistochemistry, and the EGFR gene copy number gain using chromogenic in situ hybridization (CISH) in 105 cases of OPSCC. The biopsy specimens before chemoradiotherapy were used for these analyses. HPV infection and p16 protein overexpression were detected in 53.3% and 52.4% of the OPSCCs, and each factor was associated with better overall survival (P = .0026 and P = .0026) and nonkeratinizing histology (P = .0002 and P = .0004), respectively. EGFR gene copy number gain (high polysomy or amplification) was detected in 12.4% of the OPSCCs and was correlated with EGFR protein overexpression (P = .0667) and worse overall survival (P < .0001). HPV infection and EGFR gene copy number gain (EGFR CISH positive) were mutually exclusive. The HPV-negative/EGFR CISH-positive OPSCCs had significantly worse overall survival than did the HPV-positive/EGFR CISH-negative OPSCCs and HPV-negative/EGFR CISH-negative OPSCCs (P < .0001 and P < .0001, respectively). The EGFR CISH-negative OPSCCs had favorable prognosis irrespective of HPV infection. Our results suggest that EGFR gene copy number gain-positive tumors represent an HPV-negative, aggressive subgroup of OPSCCs. The molecular subclassification of OPSCCs based on HPV infection and EGFR status may serve as important information for appropriate therapeutic strategy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: The objective of this study was to investigate differences in dynamic contrast-enhanced MRI (DCE-MRI) parameters on the basis of the status of human papillomavirus (HPV) and epidermal growth factor receptor (EGFR) biomarkers in patients with squamous cell carcinoma (SCC) of the oral cavity and oropharynx by use of histogram analysis., Materials and Methods: A total of 22 consecutive patients with oral cavity and oropharyngeal SCC underwent DCE-MRI before receiving treatment. DCE parameter maps of the volume transfer constant (K(trans)), the flux rate constant (kep), and the extravascular extracellular volume fraction (ve) were obtained. The histogram parameters were calculated using the entire enhancing tumor volume and were compared between the patient subgroups on the basis of HPV and EGFR biomarker statuses., Results: The cumulative histogram parameters of K(trans) and kep showed lower values in the HPV-negative and EFGR-overexpression group than in the HPV-positive EGFR-negative group. These differences were statistically significant for the mean (p = 0.009), 25th, 50th, and 75th percentile values of K(trans) and for the 25th percentile value of kep when correlated with HPV status in addition to the mean K(trans) value (p = 0.047) and kep value (p = 0.004) when correlated with EGFR status. No statistically significant difference in ve was found on the basis of HPV and EGFR status., Conclusion: DCE-MRI is useful for the assessment of the tumor microenvironment associated with HPV and EGFR biomarkers before treatment of patients with oral cavity and oropharyngeal SCC.

The Ang's risk profile (based on p16, smoking and cancer stage) is a well-known prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). Whether heterogeneity in (18)F-fluorodeoxyglucose (FDG) positron emission tomographic (PET) images and epidermal growth factor receptor (EGFR) expression could provide additional information on clinical outcomes in advanced-stage OPSCC was investigated. Patients with stage III-IV OPSCC who completed primary therapy were eligible. Zone-size nonuniformity (ZSNU) extracted from pretreatment FDG PET scans was used as an index of image heterogeneity. EGFR and p16 expression were examined by immunohistochemistry. Disease-specific survival (DSS) and overall survival (OS) served as outcome measures. Kaplan-Meier estimates and Cox proportional hazards regression models were used for survival analysis. A bootstrap resampling technique was applied to investigate the stability of outcomes. Finally, a recursive partitioning analysis (RPA)-based model was constructed. A total of 113 patients were included, of which 28 were p16-positive. Multivariate analysis identified the Ang's profile, EGFR and ZSNU as independent predictors of both DSS and OS. Using RPA, the three risk factors were used to devise a prognostic scoring system that successfully predicted DSS in both p16-positive and -negative cases. The c-statistic of the prognostic index for DSS was 0.81, a value which was significantly superior to both AJCC stage (0.60) and the Ang's risk profile (0.68). In patients showing an Ang's high-risk profile (N = 77), the use of our scoring system clearly identified three distinct prognostic subgroups. It was concluded that a novel index may improve the prognostic stratification of patients with advanced-stage OPSCC., (© 2015 UICC.)

Background: We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation., Methods: Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results., Results: A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America., Conclusions: HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To perform a prospective, multi-institutional, phase 2 study of a substantial decrease in concurrent chemoradiation therapy (CRT) intensity as primary treatment for favorable-risk, human papillomavirus-associated oropharyngeal squamous cell carcinoma., Methods and Materials: The major inclusion criteria were: (1) T0 to T3, N0 to N2c, M0; (2) human papillomavirus or p16 positive; and (3) minimal/remote smoking history. Treatment was limited to 60 Gy intensity modulated radiation therapy with concurrent weekly intravenous cisplatinum (30 mg/m(2)). The primary study endpoint was pathologic complete response (pCR) rate based on required biopsy of the primary site and dissection of pretreatment positive lymph node regions, regardless of radiographic response. Power computations were performed for the null hypothesis that the pCR rate is 87% and n=40, resulting in a type 1 error of 14.2%. Secondary endpoint measures included physician-reported toxicity (Common Toxicity Terminology for Adverse Events, CTCAE), patient-reported symptoms (PRO-CTCAE), and modified barium swallow studies., Results: The study population was 43 patients. The pCR rate was 86% (37 of 43). The incidence of CTCAE grade 3/4 toxicity and PRO-CTCAE severe/very severe symptoms was as follows: mucositis 34%/45%, general pain 5%/48%, nausea 18%/52%, vomiting 5%/34%, dysphagia 39%/55%, and xerostomia 2%/75%. Grade 3/4 hematologic toxicities were 11%. Thirty-nine percent of patients required a feeding tube for a median of 15 weeks (range, 5-22 weeks). There were no significant differences in modified barium swallow studies before and after CRT., Conclusions: The pCR rate with decreased intensity of therapy with 60 Gy of IMRT and weekly low-dose cisplatinum is very high in favorable-risk oropharyngeal squamous cell carcinoma, with evidence of decreased toxicity compared with standard therapies. ClinicalTrials.gov ID: NCT01530997., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although human papillomavirus (HPV)-related oropharyngeal carcinomas (HPV-OPCs) are generally regarded as "poorly differentiated," they actually maintain a close resemblance to the lymphoepithelium of the tonsillar crypts from which they arise: they are basaloid, exhibit minimal keratinization, and are often permeated by lymphocytes. In rare cases, the presence of cilia in a primary HPV-OPC and their persistence in lymph node metastasis can confound the distinction between a benign and malignant process. Three cases of ciliated HPV-OPCs were identified from the archives of The Johns Hopkins Head and Neck Pathology consultation service. HPV status was determined using p16 immunohistochemistry and high-risk HPV in situ hybridization. All 3 patients presented with a cystic lymph node metastasis without a known primary carcinoma. One metastasis was originally diagnosed as a branchial cleft cyst only to regionally recur 7 years later. In 2 cases, a primary HPV-OPC was found in the tonsil. The carcinomas exhibited both nonkeratinizing squamous epithelium and cystic/microcystic spaces lined by ciliated columnar cells. Both the squamous and ciliated cells were HPV positive. This report draws attention to a novel variant of HPV-related head and neck cancer that exhibits ciliated columnar cells. This variant challenges prevailing notions that: (1) HPV-OPCs are uniformly poorly differentiated cancers; (2) cilia are an infallible feature of benignancy; and (3) presence of cilia is a reliable criterion for establishing branchial cleft origin when dealing with cystic lesions of the lateral neck.

Objective: Complement C4d-containing fragments have been proposed as diagnostic markers for lung cancer. The purpose of this study was to evaluate the presence of C4d in oropharyngeal (OPSCC) and oral (OSCC) squamous cell carcinomas., Subjects and Methods: C4d staining was analyzed by immunohistochemistry in 244 OPSCC surgical specimens. C4d levels were quantified by ELISA in resting saliva samples from 48 patients with oral leukoplakia and 62 with OSCC. Plasma samples from 21 patients with leukoplakia and 30 with oral carcinoma were also studied., Results: C4d staining in OPSCC specimens was associated with nodal invasion (P = 0.001), histopathologic grade (P = 0.014), disease stage (P = 0.040), and focal-adhesion kinase expression (P < 0.001). No association was found between C4d and prognosis. Saliva C4d levels were higher in patients with oral cancer than in subjects with leukoplakia (0.07 ± 0.07 vs 0.04 ± 0.03 μg ml(-1) , P = 0.003). The area under the ROC curve was 0.63 (95%CI: 0.55-0.71). Salivary C4d levels in stage IV patients were higher than in patients with earlier stages (P = 0.028) and correlated with tumor size (P = 0.045). Plasma C4d levels also correlated with salivary C4d levels (P = 0.041), but differences between patients with oral cancer and subjects with leukoplakia were not significant (1.26 ± 0.59 vs 1.09 ± 0.39 μg ml(-1) , P = 0.232)., Conclusion: C4d-containing fragments are detected in oral primary tumors and are increased in saliva from patients with OSCC., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We assessed a population-based cohort of patients diagnosed with oropharyngeal squamous cell carcinoma in Southeast Scotland over 13 months. p16 and human papilloma virus (HPV) expression were determined, and correlated with stage, treatment, smoking and alcohol history, and disease outcomes. Retrospective analysis was performed on 60 patients. p16 immunohistochemistry and HPV genotyping were performed on formalin-fixed paraffin-embedded tissues. HPV infection (as defined by p16 positivity and/or HPV PCR positivity) was identified in 57% of samples, while dual positives were detected in 45% of cases. HPV16 was most prevalent of the HPV types and was associated with 90% of positive samples. Cause-specific 1-year and 2-year survivals were 82.5% and 78.2%, respectively. The p16-positive and HPV-positive groups demonstrated significantly increased cause-specific survival in comparison with their negative counterparts., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Human papillomavirus-associated oropharyngeal squamous cell carcinoma is now recognised as a subtype of head and neck cancer with distinct clinical, molecular and histological characteristics. The majority of these carcinomas are of non-keratinising squamous type but there is a growing number of histomorphologic variants of this disease. Here we describe the clinical, histomorphologic and immunophenotypic features of two cases of human papillomavirus-associated oropharyngeal squamous cell carcinoma demonstrating a clearly delineated biphasic differentiated and undifferentiated phenotype.

Purpose: Oral squamous cell carcinoma (OSCC) is a remarkable health problem worldwide, but its pathogenesis remains unknown. The aim of this study was to compare fat composition and secretory phospholipase-A2 (sPLA2) activity between the malignant and adjacent normal squamous tissues in patients with OSCC., Methods: Paired samples of malignant squamous and adjacent normal-appearing tissues were collected from 27 patients with OSCC. The fatty acid composition in the obtained tissues was determined by gas liquid chromatography. Tissue enzyme activities of sPLA2 were measured using the standard assay with Diheptanoyl Thio-Phosphatidylcholine as substrate., Results: In the OSCC tissue, the level of stearic acid (18:0) and activity of sPLA2 were higher (P < 0.001), and the levels of oleic acid (18:1n-9) and linoleic acid (18:2n-6) were lower than that in the adjacent normal-appearing squamous tissue (P < 0.001). The activity of sPLA2 in OSCC was strongly negatively correlated with the amount of 18:2n-6 (r = -0.41, P < 0.001). Negative significant associations were observed between the OSCC invasion and tissue levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHE)., Conclusion: The changes in the fatty acid composition and sPLA2 activity may be regarded as indicators of altered lipid metabolism occurring in vivo during squamous cell carcinogenesis.

Background: Residual neck disease after radiotherapy in advanced oropharyngeal squamous cell carcinoma (OPSCC) is associated with increased mortality, and some patients may benefit from post-radiotherapy neck dissection (PRND). The aim of the present study was to assess the value of magnetic resonance imaging (MRI) and other clinical characteristics in selecting patients for PRND., Materials and Methods: Retrospective cohort study. Consecutive patients with N+ OPSCC were included. Medical records, pathology reports and imaging reports were reviewed. Pre- and post-therapeutic imaging was re-evaluated., Results: A total of 100 consecutive patients from a three-year period were included. Neck response was evaluated with MRI two months after treatment. Sixty patients were suspicious for residual neck disease, and were offered surgery; seven of these patients had histologic evidence of carcinoma. Cumulative neck failure after three years was 14% (8.4-24%), and did not differ significantly among patients with positive compared to negative MRI (radiologist's initial description; p = 0.47, log-rank test). Applying neck failure as gold standard, sensitivity and specificity of MRI was 69% and 41%, respectively; positive and negative predictive value was 15% and 90%. Patients with p16 + disease had significantly larger lymph nodes after treatment, and imaging based on lymph node size resulted in many false positives. Analysis of receiver operating characteristic curves in 191 individual lymph nodes showed that a short axis ≥ 10 mm should be classified as suspicious. Furthermore, T-stage and p16-status were associated with increased risk of neck recurrence. Salvage was successful in four patients with early detected nodal recurrence., Conclusion: These results suggest that lymph node size, T-stage and p16 status could be used in selecting patients for PRND in OPSCC. Yet, early anatomical imaging may be inappropriate for evaluating neck response in patients with p16 + disease as enlarged lymph nodes often do not indicate residual neck disease.

Background: The Wnt/β-catenin signaling pathway has been noted to be upregulated in head and neck cancers, including oropharyngeal squamous cell carcinoma (OSCC). This study compared the efficacy of β-catenin immunohistochemistry (IHC), p16 IHC and automated human papillomavirus (HPV) in situ hybridization (ISH) in OSCC., Methods: Sixty-eight OSCCs (48 surgical specimens and 20 fine-needle aspirations) were evaluated. Nuclear staining only of β-catenin was assessed as 0-3+ intensity (relative to controls of benign squamous mucosa). p16 was interpreted as positive if 70% of tumor cells showed brown nuclear and cytoplasmic staining. HPV ISH was interpreted as positive if a minimum of one tumor cell showed brown punctate dot-like nuclear positivity. p16 IHC and HPV ISH were then correlated with β-catenin staining. HPV ISH was used as the gold standard., Results: Twenty-five of 48 surgical specimens (52.1%) and 11 of 20 cell blocks (55%) stained positively for β-catenin, making a total of 36 of 68 (52.9%) staining positively for β-catenin, as compared to 61.7% positive for p16 IHC and 70.6% positive by automated HPV ISH, the gold standard method for OSCC diagnosis. x03C7;2 analysis revealed no significant correlation between β-catenin and HPV ISH (p > 0.05) and demonstrated a strong correlation between p16 and HPV ISH (p < 0.05)., Conclusion: β-Catenin IHC is not a sensitive or specific marker of HPV and is unlikely to be a useful adjunct to p16 IHC or HPV ISH in the setting of advanced OSCC. However, as this study focused on samples of advanced OSCC, β-catenin IHC may still find some use in the diagnosis of early-stage OSCC., (© 2016 S. Karger AG, Basel.)

In oral and oropharyngeal squamous cell carcinoma (OCSCC and OPSCC) exist an association between clinical and histopathological parameters with cell proliferation, basal lamina, connective tissue degradation and surrounding stroma markers. We evaluated these associations in Chilean patients. A convenience sample of 37 cases of OCSCC (n=16) and OPSCC (n=21) was analyzed clinically (TNM, clinical stage) and histologically (WHO grade of differentiation, pattern of tumor invasion). We assessed the expression of p53, Ki67, HOXA1, HOXB7, type IV collagen (ColIV) and carcinoma-associated fibroblast (α-SMA-positive cells). Additionally we conducted a univariate/bivariate analysis to assess the relationship of these variables with survival rates. Males were mostly affected (56.2% OCSCC, 76.2% OPSCC). Patients were mainly diagnosed at III/IV clinical stages (68.8% OCSCC, 90.5% OPSCC) with a predominantly infiltrative pattern invasion (62.9% OCSCC, 57.1% OPSCC). Significant association between regional lymph nodes (N) and clinical stage with OCSCC-HOXB7 expression (Chi-Square test P < 0.05) was observed. In OPSCC a statistically significant association exists between p53, Ki67 with gender (Chi-Square test P < 0.05). In OCSCC and OPSCC was statistically significant association between ki67 with HOXA1, HOXB7, and between these last two antigens (Pearson's Correlation test P < 0.05). Furthermore OPSCC-p53 showed significant correlation when it was compared with α-SMA (Kendall's Tau-c test P < 0.05). Only OCSCC-pattern invasion and OPSCC-primary tumor (T) pattern resulted associated with survival at the end of the follow up period (Chi-Square Likelihood Ratio, P < 0.05). Clinical, histological and immunohistochemical features are similar to seen in other countries. Cancer proliferation markers were associated strongly from each other. Our sample highlights prognostic value of T and pattern of invasion, but the conclusions may be limited and should be considered with caution (small sample). Many cases were diagnosed in the advanced stages of the disease, which suggests that the diagnosis of OCSCC and OPSCC is made late.

It is well established that nonkeratinizing squamous cell carcinoma (SCC) of the oropharynx is causally related to transcriptionally active human papillomavirus (HPV) and has better survival as compared with carcinomas with a keratinizing phenotype (KSCC). Although the great majority of KSCCs are unrelated to HPV, transcriptionally active HPV is detected in a minority of oropharyngeal cases. To date, it has not been established whether the HPV status in KSCC also confers a survival advantage as it does in HPV-related nonkeratinizing SCC. This study compares clinical outcomes between patients with HPV-positive versus HPV-negative oropharyngeal KSCC. Among a total of 54 cases, 7 (13%) were diffusely and strongly positive for p16. HPV E6/E7 RNA was positive in 5 of the 6 (83%) p16-positive cases that were tested and in only 1 of the 47 (2%) p16-negative cases. Only 1 of the 7 (14%) p16-positive patients developed disease recurrence and died in the follow-up period. Kaplan-Meier survival analysis showed significantly better overall and disease-specific survival in the p16-positive than in the p16-negative patients (P=0.01 and 0.046, respectively). These data, although with relatively small patient numbers, suggest that HPV-related SCC in the oropharynx is associated with highly favorable outcomes, regardless of the keratinizing or nonkeratinizing phenotype.

Background: Human papillomavirus (HPV) is now recognized to be very important in the pathogenesis of oropharyngeal squamous cell carcinoma (OPSCC). It is not clear yet whether the physical status of HPV in OPSCC is similar to what is found in cervical cancer., Study Design: We performed genome-wide mate pair next generation sequencing from 20 OPSCCs patients, thirteen of which were positive for HPV16 to determine the HPV physical status and its relationship to HPV oncogene E6 and E7 expression., Results: This high throughput approach detected HPV integration events and also determined the bridged HPV coverage in each sample. Two of the HPV16-positive OPSCCs had HPV integration and one of the HPV16-negative OPSCCs had an HPV26 integration. We mapped the site of integration in the HPV genome in all integration events and the integrations were located at E1, E5, E6 and L2 region respectively. One HPV positive OPSCC had two integration events but also had a very high bridged HPV coverage, while the other two just had HPV integrated into the human genome., Conclusion: Our results are thus different from what is routinely observed in cervical cancer where HPV is almost always integrated into the human genome with loss of episomal HPV sequences. Thus more investigation should be carried out to study how episomal HPV alone can contribute to the development of most OPSCCs., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Backgrounds: A p16 protein is known to be overexpressed in human papillomavirus-positive head and neck squamous cell carcinoma specimens. p53 is a tumor suppressor protein detectable by immunohistochemistry in carcinogen-associated head and neck squamous cell carcinoma as a result of gene mutations. The purpose of this study is to investigate the prognostic impact of p16 and p53 expression in oropharyngeal squamous cell carcinomas., Methods: We retrospectively examined the relationship between prognosis, and p16 and p53 expression levels of oropharyngeal squamous cell carcinoma specimens in 53 patients using immunohistochemistry., Results: Overall, 55% of patients were p16 positive and 45% p16 negative, while 28% were p53 positive and 72% p53 negative. The p16 status showed an inverse relationship with the p53 status. A survival analysis by p16 status, p53 status, Union for International Cancer Control stage and main treatment modality demonstrated that only p16 status was related to better prognosis in terms of overall survival and disease-specific survival (3-year overall survival, 87 vs. 62%, P = 0.02; 3-year disease-specific survival, 90 vs. 62%, P = 0.02). To evaluate the practical prognostic factors in oropharyngeal squamous cell carcinoma patients, we classified patients as either p16-positive or p53-negative oropharyngeal squamous cell carcinomas, representing human papillomavirus-related oropharyngeal squamous cell carcinoma with wild-type p53 or the remaining patients with p16-negative or p53-positive OPSCCs, respectively. The former group showed survival advantages in terms of overall survival and disease-specific survival by log-tank test compared with the latter group (3-year overall survival, 96 vs. 58%, P = 0.005; 3-year disease-specific survival, 96 vs. 63%, P = 0.02)., Conclusions: A group of patients who were p16 positive/p53 negative had better prognoses in terms of overall survival and disease-specific survival than that who were p16-positive alone.

Sarcomatoid carcinoma (SC) is a variant of head and neck squamous cell carcinoma characterized by a prominent and sometimes exclusive spindle cell component. Distinction from a sarcoma or reactive stroma can be problematic, particularly in cases in which the conventional component is not obvious. The value of immunohistochemistry is limited because of the loss of cytokeratin expression in a sizable percentage of cases. Staining for p63 can enhance detection of epithelial differentiation, but its usefulness is offset by expression in various soft tissue proliferations. Staining for p40--a squamous-specific isoform of p63--could potentially improve diagnostic accuracy. Immunohistochemistry for pancytokeratin, p63, and p40 was performed on 37 head and neck SCs, 201 soft tissue neoplasms, and 40 reactive stromal proliferations. The SCs were also stained for p16 in the event that some of the tumors were human papillomavirus (HPV) related. HPV in situ hybridization was performed on p16-positive cases. Twenty-three of 37 (62%) SCs were positive for pancytokeratin, 23 of 37 (62%) were positive for p63, and 20 of 37 (54%) were positive for p40. Compared with p63, p40 staining was less likely to be observed in soft tissue tumors (5% vs. 30%) and reactive stromal proliferations (0% vs. 30%). HPV16 was detected in 3 of 10 (30%) SCs of the oropharynx but in none of the nonoropharyngeal SCs. p40 staining does not improve the sensitivity for diagnosing SC, but it does diminish the risk of misdiagnosing a sarcoma or reactive stroma as SC. The presence of a sarcomatoid variant of HPV-related oropharyngeal cancer points to HPV testing as a useful diagnostic tool for atypical spindle cell proliferations of the oropharynx.

Human papillomavirus-related (p16-positive) oropharyngeal squamous cell carcinoma patients develop recurrent disease, mostly distant metastasis, in approximately 10% of cases, and the remaining patients, despite cure, can have major morbidity from treatment. Identifying patients with aggressive versus indolent tumors is critical. Hematoxylin and eosin-stained slides of a microarray cohort of p16-positive oropharyngeal squamous cell carcinoma cases were digitally scanned. A novel cluster cell graph was constructed using the nuclei as vertices to characterize and measure spatial distribution and cell clustering. A series of topological features defined on each node of the subgraph were analyzed, and a random forest decision tree classifier was developed. The classifier (QuHbIC) was validated over 25 runs of 3-fold cross-validation using case subsets for independent training and testing. Nineteen (11.9%) of the 160 patients on the array developed recurrence. QuHbIC correctly predicted outcomes in 140 patients (87.5% accuracy). There were 23 positive patients, of whom 11 developed recurrence (47.8% positive predictive value), and 137 negative patients, of whom only 8 developed recurrence (94.2% negative predictive value). The best other predictive features were stage T4 (18 patients; 83.1% accuracy) and N3 nodal disease (10 patients; 88.6% accuracy). QuHbIC-positive patients had poorer overall, disease-free, and disease-specific survival (P<0.001 for each). In multivariate analysis, QuHbIC-positive patients still showed significantly poorer disease-free and disease-specific survival, independent of all other variables. In summary, using just tiny hematoxylin and eosin punches, a computer-aided histomorphometric classifier (QuHbIC) can strongly predict recurrence risk. With prospective validation, this testing may be useful to stratify patients into different treatment groups.

Background: Human papillomavirus (HPV) positivity (+) has been used to identify oropharyngeal squamous carcinomas (SCCs) presenting as unknown primaries in the neck. p16 overexpression correlates with HPV+ in the oropharynx; however, the use of p16 alone as a surrogate marker of oropharyngeal HPV+ 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 node-positive 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., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Carcinoma of unknown primary (CUP) of the neck are heterogeneous tumors in their clinical and biological characteristics, and a preoperative prognostic marker is desirable to optimize staging and therapy and to improve outcome and survival. For CUP syndrome, no optimized diagnostic and treatment strategy or biomarker have yet been determined., Methods: Forty-seven patients presenting with CUP syndrome were analyzed after thorough standard diagnostic staging procedures. All patients were surgically treated with tonsillectomy, neck dissection of the diseased neck, as well as adjuvant chemoradiation. The tissue of lymph node metastases (and, if found, of the primary tumor) was analyzed regarding expression of p16, epidermal growth factor receptor (EGFR), and presence of human papillomavirus (HPV) DNA., Results: In 39% of all cases (20 of 47), the primary cancer was found during diagnostic workup. If HPV DNA was detected in the neck lymph node metastasis, the primary cancer was significantly more frequently found in the oropharynx (p = .002). Patients with a p16-positive tumor had a significantly higher 5-year overall survival (OS; 33% vs 69%; p = .045, disease-free survival [DSF] 77% vs 89%; p = not significant [NS]). Patients with p16-positive neck metastasis and no detectable primary cancer had a better prognosis. Expression of EGFR in this series did not have a significant effect on prognosis., Conclusion: In patients presenting with CUP syndrome, p16 immunohistochemistry can serve to locate the primary cancer in the oropharynx. It is a positive prognostic indicator in patients with those heterogeneous cancers., (Copyright © 2013 Wiley Periodicals, Inc.)

Purpose: To explore whether tumor biomarkers and pre-treatment factors correlate with treatment outcome in patients with oropharyngeal squamous cell carcinoma (SCC)., Methods: Fifty-seven consecutive patients diagnosed with oropharyngeal SCC were treated using intensity modulated radiotherapy (IMRT). Thirty-four (60%) patients were treated with definitive chemoradiotherapy to a median total dose of 70 Gy and 23 (40%) were treated with postoperative RT to a median total dose of 66 Gy. Concurrent platinum- based chemotherapy was used in 51 patients (90%) and cetuximab in 3 (5%) patients., Results: Forty-four (77%) cases were positive and 13 (23%) were negative for p16 expression. Eighty-eight percent of non-smokers, 87% of smokers in their remote past and 56% of active smokers were diagnosed with p16-positive cancer. After 22 months median follow up, 51 (89%) patients were alive. Forty-five (77%) patients were without evidence of disease at their last follow up, 82% of the patients with p16-positive tumors vs 58% of those with p16-negative cancer, respectively (p= 0.04). Locoregional disease-free survival was 82% for the entire cohort, 91% for patients treated postoperatively and 76% for patients treated with definitive chemoradiotherapy. Five (9%) patients developed distant metastases, and 3 (5%) developed new malignancies. One third of the patients with pre-RT hemoglobin level of ≤ 11 g/dL experienced persistent/recurrent disease; 80% of patients with hemoglobin ≤ 11 g/dL were smokers and 42% had p16-negative tumors., Conclusions: Smoking, p16 expression and pre-RT anemia are interrelated and influence outcome in oropharyngeal cancer patients and should be evaluated as stratifying variables in further clinical trials.

Viral oncogene RNA expression is regarded as reliable biomarker to identify oropharyngeal squamous cell carcinomas (OPSCC) with active HPV16 involvement. This study addressed whether the expression profile of the cellular proteins p16(INK4a), pRb, Cyclin D1 and p53 provide surrogate marker combinations that identify OPSCC with active HPV16 in situations where only formalin-fixed biopsies are available. Protein expression was analyzed by immunohistochemistry on tissue microarrays created from 188 OPSCC of which the HPV16 DNA and RNA status had been established previously from snap-frozen biopsies. Associations of single markers and of marker combinations with HPV16 DNA, viral RNA expression patterns and overall survival as primary end point were evaluated by statistical analysis. Most tumors with active HPV16 involvement (RNA(+) group; n = 40) showed a specific protein pattern, that is, high p16(INK4a) (80%), low pRb (85%), low Cyclin D1 (95%) and normal p53 (73%). This pattern was significantly different from the pattern observed in HPV DNA-negative tumors (HPV(-) group) and in HPV16 DNA-positive tumors lacking viral RNA expression patterns (RNA(-) group). The combination of high p16(INK4a) and low pRb levels was distinctly superior to p16(INK4a) alone; it was strongly associated with RNA(+) tumors (OR 41.4, 95%CI 10.7-162.5), with improved survival (HR 0.37, 95%CI 0.2-0.8) and had best predictive values (78% sensitivity, 93% specificity, 78% PPV, 93% NPV). In conclusion, if only formalin-fixed biopsy material is available, the marker combination high p16(INK4a) /low pRb is well suited to identify OPSCC with biologically active HPV16 which represent a distinct OPSCC entity with improved prognosis., (Copyright © 2013 UICC.)

Aim: EGFR and HPV-associated p16 are among the most investigated biomarkers in head and neck cancer. The aim was to investigate the correlation and interaction between these two markers and to evaluate their potential prognostic significance when combined., Materials and Methods: 336 Oropharyngeal carcinomas treated with primary radiotherapy (66-68 Gy, 2fx/day, 10-12 Gy/week) and with known EGFR/p16-status estimated semiquantitatively by immunohistochemistry were included in the study. Data were evaluated by EGFR-expression (high/low) and p16-status (positive/negative) consequently dividing tumours into four groups by combination of the biomarkers. Patient/tumour characteristics and complete 5-year follow-up were available., Results: Low EGFR-expression was significantly more common in p16-positive tumours compared to p16-negative, p < 0.0001. p16 positivity showed a strong prognostic impact (p < 0.0001, HR = 0.22 [0.13-0.38]), whereas EGFR was a weak prognostic marker when local control was used as endpoint (p = 0.03, HR = 0.53 [0.29-0.94]). Combination of EGFR/p16 did not add significant information to p16 alone and by multivariable analysis only p16 showed significant prognostic information for all evaluated endpoints., Conclusions: Both EGFR and p16 bear prognostic information in oropharyngeal cancer, although p16 is, by far, the strongest prognostic factor. The markers seem to be correlated and this might have influence when evaluating the effect of EGFR inhibition in oropharyngeal tumours., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Backgrounds: Large cell neuroendocrine carcinoma (LCNEC) is well-known as a lung cancer subtype. This study assessed the prevalence of head and neck mucosal LCNEC (M-LCNEC)., Methods: M-LCNEC was studied clinically, histologically and immunohistochemically., Results: Of 814 surgically resected cases of mucosal head and neck carcinoma, only eight cases (0.98%; all men, mean age 64.6 years) were rediagnosed as M-LCNEC. They occurred in the oropharynx (n=3), larynx (n=4) and hypopharynx (n=1). Seven of the cases had regional lymph node metastases and four resulted in death. Histologically, M-LCNEC had a sheet-like trabacular organoid growth pattern of relatively large basaloid cells in which central necrosis, rosette formation, peripheral palisading and high mitotic figures were evident. M-LCNEC was immunopositive for two or three neuroendocrine markers (CD56, chromogranin-A and synaptophysin). All cases showed high proliferative activity., Conclusion: M-LCNEC in the head and neck regions is a distinct histopathological entity whose positivity for neuroendocrine markers makes its diagnosis important. As about half of the patients died of the disease, M-LCNEC has a relatively poor prognosis.

Purpose: We have previously reported that metabolic tumor volume (MTV) obtained from pretreatment (18)F-fluorodeoxydeglucose positron emission tomography (FDG PET)/ computed tomography (CT) predicted outcome in patients with head-and-neck cancer (HNC). The purpose of this study was to validate these results on an independent dataset, determine whether the primary tumor or nodal MTV drives this correlation, and explore the interaction with p16(INK4a) status as a surrogate marker for human papillomavirus (HPV)., Methods and Materials: The validation dataset in this study included 83 patients with squamous cell HNC who had a FDG PET/CT scan before receiving definitive radiotherapy. MTV and maximum standardized uptake value (SUV(max)) were calculated for the primary tumor, the involved nodes, and the combination of both. The primary endpoint was to validate that MTV predicted progression-free survival and overall survival. Secondary analyses included determining the prognostic utility of primary tumor vs. nodal MTV., Results: Similarly to our prior findings, an increase in total MTV of 17 cm(3) (difference between the 75th and 25th percentiles) was associated with a 2.1-fold increase in the risk of disease progression (p = 0.0002) and a 2.0-fold increase in the risk of death (p = 0.0048). SUV(max) was not associated with either outcome. Primary tumor MTV predicted progression-free (hazard ratio [HR] = 1.94; p < 0.0001) and overall (HR = 1.57; p < 0.0001) survival, whereas nodal MTV did not. In addition, MTV predicted progression-free (HR = 4.23; p < 0.0001) and overall (HR = 3.21; p = 0.0029) survival in patients with p16(INK4a)-positive oropharyngeal cancer., Conclusions: This study validates our previous findings that MTV independently predicts outcomes in HNC. MTV should be considered as a potential risk-stratifying biomarker in future studies of HNC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tumor human papillomavirus (HPV) status is a prognostic factor for oropharyngeal cancer, but classification methods are not standardized. Here we validate the HPV classification methods used in US cooperative group trials. Tumor DNA and RNA purified from 240 paraffin-embedded oropharyngeal cancers diagnosed from 2000 to 2009 were scored as evaluable if positive for DNA and mRNA controls by quantitative polymerase chain reaction (PCR). Eighteen high-risk (HR) HPV types were detected in tumors by consensus PCR, followed by HR-HPV E6/7 oncogene expression analysis by quantitative reverse transcriptase PCR. The sensitivity (S), specificity (SP), and positive (PPV) and negative predictive values (NPV) of p16 expression detected by immunohistochemistry (IHC) and HPV16 detected by in situ hybridization (ISH) were evaluated in comparison with HR-HPV E6/7 oncogene expression. Interrater agreement among 3 pathologists was evaluated by κ statistics. Of 235 evaluable tumors, 158 (67%; 95% confidence interval, 61.2-73.3) were positive for HR-HPV E6/7 oncogene expression [HPV type 16 (92%), 18 (3%), 33 (3%), 35 (1%), or 58 (1%)]. p16 IHC had high sensitivity (S 96.8%, SP 83.8%, PPV 92.7%, and NPV 92.5%), whereas HPV16 ISH had high specificity (S 88.0%, SP 94.7%, PPV 97.2%, and NPV 78.9%) for HR-HPV oncogene expression. Interrater agreement was excellent for p16 (κ=0.95 to 0.98) and HPV16 ISH (κ=0.83 to 0.91). Receiver operating curve analysis determined the cross-product of p16 intensity score and percentage of tumor staining to optimally discriminate HR-HPV E6/7-positive and HR-HPV E6/7-negative tumors. p16 IHC and HPV16 ISH assays show excellent performance, with high sensitivity and specificity, respectively. A new validated H-score for p16 IHC assessment is proposed. Appropriate assay choice depends on clinical implications of a false-positive or false-negative test.

Oropharyngeal squamous cell carcinoma (SCC) is frequently related to high risk human papillomavirus. This tumor expresses p16, frequently has a nonkeratinizing morphology, and has improved outcomes. Despite having a good prognosis, tumors can have focal or diffuse nuclear anaplasia or multinucleation, the significance of which is unknown. From a database of 270 oropharyngeal SCCs with known histologic typing (using our established system) and p16 immunohistochemistry, all surgically resected cases (149) were reviewed. Anaplasia was defined as any × 40 field with ≥ 3 tumor nuclei with diameters ≥ 5 lymphocyte nuclei (~25 μm), and multinucleation was defined as any × 40 field with ≥ 3 tumor cells with multiple nuclei. p16 was positive in 128 cases (85.9%), 64 cases (43.0%) showed anaplasia, and 71 (47.7%) showed multinucleation. Anaplasia and multinucleation were highly related (P<0.001), and both also correlated with histologic type (P<0.001 and P=0.01, respectively), p16 status (P=0.09 and 0.03, respectively), and partially with nodal extracapsular extension. There was no correlation with any of the other variables. In univariate analysis, cases showing anaplasia or multinucleation had worse overall, disease-specific, and disease-free survival (P<0.006 for all). Higher T-stage, keratinizing histologic type, extracapsular extension, and smoking also all correlated with worse survival. In multivariate analysis, anaplasia and multinucleation both predicted worse disease-specific survival (hazard ratio 9.9, P=0.04; and hazard ratio 11.9, P=0.02, respectively) independent of the other variables. In summary, among surgically resectable oropharyngeal SCC (including among just the p16-positive cohort), tumor cell anaplasia and multinucleation independently correlated with disease recurrence and poorer survival.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is associated with favorable patient survival. Tumor HPV status at primary diagnosis is critical for proper management, and p16 immunohistochemistry (IHC) has emerged as a reliable, single, surrogate marker. It is not known, however, if small biopsy specimens are completely adequate for p16 evaluation. From a database of oropharyngeal SCC for which p16 IHC and histologic typing were already performed, all patients (32) who had available in-house primary tumor biopsy specimens and also subsequent surgical resections were analyzed. p16 IHC was performed along with histologic typing into: Type 1 keratinizing SCC, Type 2 nonkeratinizing SCC with maturation, and Type 3 nonkeratinizing SCC. Staining was graded on both biopsies and resections as follows: 0=negative; 1+ =1-25% of tumor cells positive; 2+ =26-50%; 3+ =51-75%; 4+ =76-100%. Strictly considering p16 score, perfect biopsy-resection correlation was present in 28 of 32 cases (85%), including 6/9 (67%) Type 1, 6/7 (86%) Type 2, and 16/16 (100%) Type 3 cases. Considering p16 expression binarily as 51% tumor cell staining or more (3+ or 4+) being positive and lesser amounts (0, 1+, or 2+) as being negative, there was perfect biopsy-resection correlation for all 32 cases. With p16 expression in resection specimens considered the gold standard, p16 IHC in biopsies was both 100% sensitive and specific. Our results demonstrate that p16 staining in diagnostic biopsies reliably reflects whole tumor staining results, and suggest that biopsies do not suffer from false negatives or positives.

High-grade neuroendocrine carcinoma of the head and neck is an aggressive neoplasm which rarely arises in the oropharynx. Here we report a series of 8 oropharyngeal neuroendocrine carcinomas associated with both human papillomavirus (HPV) infection and tobacco exposure. The tumor occurred predominantly in male patients (6 of 8) at a mean age of 59 years. Histologically, these cases were all classified as poorly differentiated neuroendocrine carcinoma (small cell carcinoma) with high mitotic activity [mean 53.3 mitoses per 10 HPF], necrosis, high nuclear-to-cytoplasmic ratio, and nuclear molding. One case also exhibited a moderately differentiated component, and one other case had a component of squamous cell carcinoma with basaloid features. Neuroendocrine differentiation was confirmed by immunoreactivity for synaptophysin and/or chromogranin A in all cases. P63 staining was negative, except in 1 case. Seven of the 8 cases showed strong and diffuse p16 expression, a surrogate marker for high-risk HPV infection. HPV infection was confirmed in 6 of these 7 cases by HPV in situ hybridization and/or polymerase chain reaction analysis. HPV subtypes 16, 18, and 33 were identified in 1 case each by polymerase chain reaction testing. Six of the 7 patients for whom clinical history was available presented with advanced disease (4 with regional lymph node metastases, 1 with distant metastases, and 1 with distant and locoregional metastases). Disease recurred in 5 of the 6 patients with available clinical follow-up, with 3 developing distant metastases to brain, bones, lung, pleura, adrenal glands, and pancreas. These 3 cases were all from the HPV-positive group. In summary, neuroendocrine carcinoma of the oropharynx represents a rare novel HPV-associated entity with high-grade histologic features and aggressive clinical behavior.