2025+CAP指南：头颈癌的高危HPV检测（更新版）

Abstract

High-risk human papillomavirus (HPV) plays a significant role in the pathogenesis of certain head and neck (HN) can-cers, especially squamous cell carcinomas (SCC) arising in the orophanyx. Distinction between HPV-independent and HPV-associated HNSCC has important implications for clinical management and patient prognosis in specific anatomic subsites of the head and neck. In 2018, the College of American Pathologists (CAP) issued 14 evidence-based guideline statements related to the testing, application, interpretation, and reporting of HPV and surrogate marker testing for HN carcinomas. Since that time, significant amounts of new data have been published which serves as the foundation for the updated 2025 CAP Guideline resulting in 16 evidence-based recommendations. These statements refine issues related to the application of high-risk HPV testing in patients with HNSCC, and they address some issues not fully covered in the 2018 CAP Guideline. This brief review discusses the changes that were made to the original guideline and provides a quick reference for the testing recommendations across head and neck sites.

Keywords HPV·Head and neck·Squamous cell carcinoma· Testing· Oropharynx· RNA ISH· Sinonasal carcinoma· p16

Introduction

High-risk human papillomavirus (HR-HPV) plays a critical role in the pathogenesis of certain head and neck squamous cell carcinomas (HNSCC), particularly those arising in the

oropharynx [1-3]. The distinction between HPV-indepen-dent and HPV-associated HNSCC carries significant impli-cations for both clinical management and patient prognosis in specific anatomic subsites of the head and neck. Beyond its prognostic value, determining the HR-HPV status of a

William C. Faquin and James S. Lewis have contributed equally as co-first authors.

\underline{ } $ $ $ } $ $Wfaquin@mgh.harvard.edu

1 Department of Pathology, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, WRN 219,Boston,MA 02114,USA

2 Department of Laboratory Medicine and Pathology, Mayo Clinic Arizona, Scottsdale, AZ, USA

<BeginOfPara>3 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA

<BeginOfPara>4 Department of Pathology, UT Southwestern Medical Center,Dallas, TX, USA

_2D Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

$^{6}$ Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

$^{7}$ Office of the Director, The Joint Pathology Center, Silver Springs, MD, USA

${ }^{8}$ Department of Otolaryngology- Head and Neck Surgery,Ohio State University Wexner Medical Center, Columbus,OH, USA

$^{9}$ Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, USA

$^{10}$ Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

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tumor has multiple applications. Notably, HR-HPV status is a component of the staging criteria for oropharyngeal squamous cell carcinoma(OPSCC) in the 8th edition of the American Joint Committee on Cancer(AJCC) staging manual[4, 5]. Furthermore, the HR-HPV status often deter-mines patient eligibility for clinical trials, particularly those investigating treatment de-intensification strategies such as radiotherapy de-escalation[3, 6, 7]. HR-HPV testing also plays a diagnostic role in cases of metastatic squamous cell carcinoma of unknown primary involving cervical lymph nodes. In such cases, detection of HR-HPV supports the oropharynx, specifically the ipsilateral palatine tonsil or base of tongue, as the likely primary site[8].

Despite its clinical importance, there is often uncertainty among pathologists and treating physicians regarding the appropriate application and methodology of HR-HPV test-ing in HN carcinomas[9-11]. To address this, the College of American Pathologists(CAP) convened an expert panel in 2013 to develop evidence-based recommendations for HR-HPV testing in head and neck(HN) carcinoma[12].This effort culminated in the publication of 14 guideline statements in 2018, providing comprehensive recommenda-tions on testing methodologies, interpretation, and reporting for HN carcinomas[12-15].

Since the publication of the 2018 guideline, substan-tial new evidence has emerged in the published literature.This has led to the development of an updated 2025 CAP guideline, which includes 16 evidence-based statements[6].These updated recommendations offer refinements to the 2018 guideline and address previously unresolved issues concerning the use of HR-HPV testing in patients with HNSCC.

Rather than covering all 16 of the recommendations from the 2025 CAP guideline on HR-HPV testing in HNSCC,this article focuses on 8 of the recommendations(#'s 2, 4,5,7,8,10,11,12) where changes or additions were made to the original 2018 guideline. We summarize the updates and provide a simple“user’s guide” or“desk reference” for applying the overall updated recommendations(Table 1).As in the original guideline and the update, the term HR-HPV testing is used here to refer to HPV-specific testing and/or the surrogate immunohistochemical marker p16.

Overview of updates in the 2025 CAP Guideline for HPV Testing in Head and Neck Carcinomas[6](https://www.c ap.org/protocols-and-guidelines/cap-guidelines/current-ca p-guidelines/human-papillomavirus-testing-in-head-and-ne ck-carcinomas):

Recommendation 2. For oropharyngeal tissue specimens(i.e. non-cytology), including regional lymph nodes with metastatic SCC and clinical findings consistent with an oro-pharyngeal primary, pathologists should perform HR-HPV testing by surrogate marker p16 IHC. In certain scenarios

HPV-specific testing should be performed:(1) in geographic regions with a low prevalence of HR-HPV-associated OPSCC;(2) when p16 IHC is equivocal(50%-70% stain-ing or when staining is extensive but weak);(3) when there is a discrepancy between p16 IHC and morphology;(4)for large, multisite tumors overlapping the oropharynx;(5) when specimens are from a non-tonsillar, non-base of tongue oropharyngeal site; and(6) when required by clini-cal trials. Additional HPV-specific testing may be done at the discretion of the pathologist and/or treating clinician.

This recommendation outlines specific scenarios when surrogate marker p16 IHC testing alone is not sufficient,and HPV-specific testing(e.g. PCR-based testing, RNA-ISH testing etc.) is needed. For an OPSCC with classic characteristics, p16 IHC remains the optimal option for HR-HPV testing. However, for selected scenarios as outlined in the recommendation above and discussed in detail in the CAP Guideline, HPV-specific testing should be performed,unless not possible in the practice or locale. In addition,there is evidence that having results on the tumor for both p16 IHC and HR-HPV(i.e. HPV-specific) can provide bet-ter prognostic information than having results for just p16 IHC alone, particularly in lower attributable fraction geo-graphic regions[16, 17].

Recommendation 4. Pathologists should routinely per-form HR-HPV testing on sinonasal SCC.

This is a new recommendation based upon data in the literature showing that patients with HPV-associated sino-nasal SCC have an improved prognosis relative those with HPV-independent tumors(Fig. 1)[18-21]. The propor-tion of HPV-associated sinonasal SCCs has increased over time, so that now approximately 26% of sinonasal SCCs are associated with HR-HPV[21, 22]. Patients with HR-HPV-associated sinonasal SCCs had better outcomes than HPV-independent ones when treated with surgery plus radiotherapy[23, 24]. The prognostic impact is supported by population-based registry level data(Fig. 2)[22, 23].While indeed compelling, given the relative rarity of sino-nasal SCC and the lower fraction that are HPV-associated,single institution studies to date are inherently small. Higher quality evidence from clinical trials is still needed to further validate this population level signal and to address the util-ity of HPV-status for treatment decisions in patients with sinonasal SCC akin to those with oropharyngeal SCC.

Recommendation 5. When testing a sinonasal SCC speci-men for HR-HPV, pathologists should test directly for tran-scriptionally active HR-HPV(e.g. RNA ISH); positivity for the surrogate marker p16 IHC may be used to screen tumors for confirmatory HPV-specific testing.

While p16 IHC can be used as a screening tool for HPV-associated sinonasal SCCs, HPV-specific testing must be performed for confirmation of HPV status. This is due in

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Table 1 Quick reference guide-CAP HPV testing in head and neck carcinomas

|Site|Routine testing? Test type|Confirma- tory HPV- specific testing?|||| |---|---|---|---|---|---| |Oropharynx|Yes|p16||Scenario Dependent#|hrHPV RNA ISH or hrHPV DNA PCR| |Sinonasal Tract|Yes|p16*|Yes||hrHPV RNA ISH or hrHPV DNA PCR| |Cervical Lymph Node (Unknown Primary)|Yes|p16|Yes||hrHPV RNA ISH or hrHPV DNA PCR| |FNA of Cervical LN Met (Unknown Primary)|Yes|HPV- Specific Testing|Yes||hrHPV RNA ISH or hrHPV DNA PCR or Liquid-based testing| |Nasopharynx|No (Selective)|N/A|N/A|N/A|| |Oral Cavity|No|N/A|N/A|N/A|| |Larynx|No|N/A|N/A|N/A|| |Hypopharynx|No|N/A|N/A|N/A|| |Ear/Temporal Bone|No|N/A|N/A|N/A|| |Cervical Esophagus|No|N/A|N/A|N/A|| |Distant Metastases|No (Selective)^|p16|Yes|hrHPV RNA ISH or hrHPV DNA PCR||

\# Scenarios in the oropharynx requiring confirmatory HPV-Specific Testing:

(1) p16 immunostaining is equivocal(50-70% staining of moderate to strong intensity or diffuse weak staining)

(2) In low overall HPV-attributable geographic regions(50% or less)

(3) Discrepancy between p16 immunostaining and morphology(p16 positive keratinizing SCC or p16 negative nonkeratinizing SCC)

(4) Large, multisite tumors that overlap the oropharynx

(5) Biopsy specimens labeled as a non-tonsillar, non-base of tongue site(soft palate, uvula, tonsillar pillar,lateral wall, or posterior wall)

(6) Required for a clinical trial

*Sinonasal SCC specimens can be tested directly for transcriptionally-active, HR-HPV by RNA in situ hybridization without performing p16 immunohistochemistry

${}^{A}$ In lung metastases, HR-HPV testing can be used to distinguish primary lung SCC from metastatic HPV-associated SCC

part to the lower fraction of HPV-associated cases of carci-noma in the sinonasal cavity compared to the oropharynx,and thus there is an increased possibility of a false positive p16 IHC result, if p16 were used alone[6]. As detailed in Recommendation 12 and in the discussion below, HR-HPV(RNA ISH) testing is optimal, but HPV DNA PCR may be used as the HPV-specific test in p16 IHC-positive speci-mens. HPV DNA PCR should not be used as a standalone HR-HPV test in these patients due to the potential for a false positive result.

<BeginOfPara>Recommendation 7. For tissue specimens(i.e., non-cytology) from patients presenting with metastatic SCC of unknown primary in a cervical lymph node, pathologists should perform HPV-specific testing or surrogate marker p16 IHC, followed by HPV-specific testing for p16-positive

<EndOfPara>tumors. An explanatory note on the significance of a posi-tive HPV result is recommended.

In contrast to the prior CAP recommendation from 2018,the updated CAP Guideline recommends HPV-specific test-ing for all patients with metastatic SCC of unknown pri-mary.It also no longer limits the testing recommendation to levels II and III, but rather now applies to all cervical lymph node levels. HPV-specific testing can be performed either as a stand-alone test or in conjunction with p16 IHC which can serve as a preliminary screening test. The reason for this change in the CAP Guideline is to avoid a false positive (or a false negative) result which could lead to less-than-optimal management. The explanatory note for cases with a positive HPV result should indicate that the likely primary site of origin is the ipsilateral oropharynx [6].

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Fig.1 HPV-associated sinonasal squamous cell carcinoma. A The tumor consists of ribbons of nonkeratinizing squamous cell carcinoma with areas of obvious nuclear pleomorphism and multinucleation(H&E).B p16 IHC shows strong and diffuse nuclear and cytoplasmic reactivity(immunohisto-chemical stain). C High-risk HPV RNA in situ hybridization shows extensive granular,brown positive signal in the tumor cells(RNAISH)

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HPV Associated Sinonasal SCC Overall Survival

|Study|logHR|SE|||Estimate [95% CI]| |---|---|---|---|---|---| ||||||| |Studies with Univariable Analysis|||||| |Alos 2009|-1.61|0.72|||0.20[0.05, 0.81]| |Bishop 2013|-0.54|0.41|||0.58[0.26,1.29]| |Oliver 2020|-0.48|0.17|||0.62[0.45,0.86]| |Svajdler 2020|-0.34|0.65|||0.71[0.20,2.52]| |Amanian 2024|-0.87|0.07|||0.42[0.37,0.48]| |Random-effects(UV)|||||0.49[0.37,0.65]| |Fixed-effects(UV)|||||0.45[0.40,0.50]| |UV heterogeneity: $T^{2}$ = 0.04, $x^{2}$ = 6.98(df= 4, p= 0.137), F= 43.0%)|||||| |Effect p-value(2-test): p< 0.001|||||| |Studies with Multivariable Analysis|||||| |Alos 2009|-1.77|0.74|||0.17[0.04,0.72]| |Bishop 2013|-0.18|0.45|||0.85[0.35,2.07]| |Oliver 2020|-0.8|0.24|||0.45[0.28,0.72]| |Amanian 2024|-0.51|0.07|||0.60[0.52,0.69]| |Random-effects(MV)|||||0.57[0.46,0.70]| |Fixed-effects(MV)|||||0.58[0.51,0.67]| |MV heterogeneity: $T^{2}$ = 0.01, $x^{2}$ = 4.79(df= 3, p= 0.188), if $11.2\%$|||||| |Effect p-value(2-test): p< 0.001|||||| ||||||| ||||||| |||||||

Fig.2 Sinonasal SCC. Meta-analysis with forest plots showing indi-vidual study estimates, and both random and fixed effects pooled esti-mates for studies with univariable and multivariable analysis of over-all survival in patients with sinonasal SCC. Pooled estimates for both indicate a significantly more favorable prognosis for HPV-associated

Recommendation 8. Pathologists should not routinely perform HR-HPV testing on patients with primary oral cav-ity, laryngeal, nasopharyngeal, or hypopharyngeal SCCs of the head and neck for prognostic purposes. Note: HR-HPV testing in nasopharyngeal SCCs can be used at the discre-tion of the pathologist and/or treating clinician.

Except for SCC of the sinonasal tract, there is insuffi-cient evidence in the published literature to support reflex testing for HR-HPV in non-oropharyngeal SCCs. Unlike the oropharynx, identifying HPV in primary oral cavity,laryngeal, and hypopharyngeal SCCs does not have clear and consistent implications for patient prognosis or patient management, supported by large amounts of new data since the original guideline[25-28]. HPV associated nasopharyn-geal carcinomas appear distinct clinically, but the literature is inconsistent in supporting prognostic and therapeutic val-ues given limitations in study size, consistency of compari-son groups(i.e. vs EBV or vs virally negative counterparts),and overall low attributable fractions[29]. Nonetheless,HR-HPV testing may be performed at the discretion of the pathologist or treating physician.

Recommendation 10. Pathologists should perform HR-HPV testing on head and neck FNAs of nodal SCC samples from all patients with(1) clinical findings of an oropharyn-geal or sinonasal primary or(2) metastatic SCC of unknown primary.

This recommendation is intended for those cases where HR-HPV testing has not already been performed on a patient's known primary oropharyngeal or sinonasal SCC,

$\text{sinonasal carcinomas while}x^{2}\text{test shows no significant heterogeneity}$across studies. $I^{2}$ indicates moderate variation due to heterogeneity for the univariable analyses, but low variation in the multivariable analy-ses. The reliability of the heterogeneity metrics, however, is limited by the small number of studies in both groups

as well as for all FNA cases of metastatic SCC of unknown primary site. As mentioned above in the discussion of other guideline recommendations, there is very strong evidence in the literature to support HR-HPV testing since it has impli-cations for both patient prognosis and clinical management[14,30,31]. Given the tendency of HPV-associated OPSCC to metastasize to cervical lymph nodes, FNA is often the first tissue biopsy to detect and diagnose these metastatic carcinomas[15,30].

Recommendation 11. For FNA specimens, pathologists should perform HPV-specific testing.

Note: In selected circumstances, p16 IHC can be per-formed instead of HPV-specific testing. If the result of HR-HPV testing on the FNA sample is negative, testing should be performed on tissue if it becomes available.

This is a significant change from the previous guideline.While p16 IHC has been applied to evaluate the HR-HPV status of FNA samples in cell blocks, published data consis-tently shows that p16 IHC is neither an effective screening tool nor an effective stand-alone method for determining if a metastatic SCC is HPV-positive[32-37]. Instead, HPV-specific methodologies including RNA in situ hybridization,PCR, and liquid-based tests have been shown to be more accurate and are therefore preferred(Fig. 3)[35, 37-39].

Recommendation 12. For HPV-specific testing, patholo-gists should use tests that exhibit optimal performance char-acteristics-such as RNA-ISH or DNA PCR-and have adequate coverage of non-HPV 16 high-risk types. DNA-ISH is not recommended.

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 Fig.3 Application of HPV-specific testing to an FNA cell block. A FNA of metastatic squamous cell carcinoma of unknown primary to a level II lymph node(Papanicolaou stain); B Corresponding cell block

(H&E);C RNA-ISH for HR-HPV is positive, supporting a metastatic HPV-associated SCC(ISH)

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 Fig.3(continued)

Instead of recommending a particular HPV-specific test, the updated CAP guideline recognizes multiple HPV-specific testing options, the most common of which are RNA-ISH and HPV DNA PCR. For applications to tissue biopsies and cell blocks, the ideal test would be one that demonstrates transcriptionally-active HPV as reflected by HR-HPV E6/E7 mRNA[40, 41]. RNA ISH for HR-HPV is now readily available for use in clinical laboratories and has shown high sensitivities and specificities of approxi-mately 93% and 94%, respectively[42]. The advantage of RNA ISH over DNA PCR testing is that the latter does not distinguish transcriptionally-active HPV within tumor cells from passenger DNA or non-specific background HPV. For this reason, when evaluating tissue-based specimens, HPV DNA testing should only be utilized as the HPV-specific test in patients with a p16-positive carcinoma[6]. For RNA specimens, HPV RNA-ISH, DNA PCR, and other liquid-based DNA amplification testing modalities can be used as a standalone test since p16 IHC is no longer recommended for cytology samples.

It is important that whichever HPV-specific test is used,that it cover not only HPV subtypes 16 and 33, the most common HPV subtypes found in HNSCC, but also several of the other HR-HPV subtypes which have most often been associated with HPV-associated HN SCC such as HPV 18,26,35, and 58[6,43,44]. In contrast to RNA-ISH, DNA-ISH for HR-HPV suffers from poor sensitivity at low viral loads, difficulties in interpreting the ISH results, and lack

of information about transcriptional activity[6]. As such,DNA-ISH is no longer recommended for use as an HPV-specific test[40,41,45].

Concluding Remarks

Among the most important updates in the 2025 CAP guide-line for HPV testing in HNSCCs are several instances where HPV-specific testing should be performed either alone or in conjunction with p16 IHC. Included among these is the use of HPV-specific testing for FNAs and tissue specimens of metastatic SCC to cervical lymph nodes since p16 testing alone on a cell block is no longer considered reliable, and p16 positivity alone is not specific for an HPV-associated carcinoma. In general, allowances are made for laboratories where: resources are limited, HPV-specific testing meth-ods are not practical, or where p16 IHC testing may be the only testing option. The other important update to the CAP guideline is the inclusion of sinonasal SCC as a second head and neck cancer type where reflex HR-HPV testing is indi-cated due to implications for patient prognosis.

The CAP Guideline for HPV testing in the head and neck is assessed every 5 years, and based upon published evi-dence in the literature, updates are made to the guideline if warranted. Looking ahead, the development of liquid biopsy technologies-particularly circulating tumor HPV DNA(ctHPV DNA) assays-holds promise for advancing

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HPV-associated head and neck cancer[46-49]. Although not currently incorporated into the CAP guideline,ctHPV DNA is being explored in academic centers and clinical trials as a tool for diagnosis, post-treatment surveillance,and early detection of recurrence. As evidence continues to emerge, these approaches may complement or extend cur-rent tissue-based testing strategies[46-49].

Acknowledgements We acknowledge the significant contributions that Christina Lacchetti and the staff at the College of American Pathologists (Carol Colasacco and Tanja Kalicanin) made in the devel-opment of the 2025 CAP Guideline update.

Author Contribution W.F. and J.L. contributed equally as co-first au-thors. W.F. and J.L. wrote the initial manuscript draft and prepared Figures 1 and 3.J.L.prepared Table 1.R.S. prepared Fig. 2.W.F., J.L.,B.B., J.B., R.C., J.K., J.M., J.R., M.S., and R.S. all contributed sig-nificantly to the development and preparation of the 2025 CAP Guide-line update which forms the content upon which this review article is based. All co-authors reviewed drafts of the manuscript during its development, and provided content edits and suggestions for modi-fications.

Funding This study was not supported by any funding. The authors have disclosed that they have no significant relationships with or fi-nancial interest in any commercial companies pertaining to this article.

Data Availability Available upon request.

Code Availability Not applicable.

Declarations

Conflict of interest The authors declare that they have no conflict of interest. The identification of specific products or scientific instrumen-tation is considered an integral part of the scientific endeavor and does not constitute endorsement or implied endorsement on the part of the authors, Department of Defense, or any component agency.The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army/Navy/Air Force, Depart-ment of Defense, or U.S. Government.

Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors.

Consent to Participate For this type of study informed consent is not required.

Consent for Publication For this type of study consent for publication is not required.

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