Given the growing clinical significance of human papillomavirus status in oropharyngeal squamous cell carcinoma, the College of American Pathologists established a set of evidence-based recommendations for high-risk human papillomavirus testing for publication in a guideline.
To evaluate the impact of the recommendations on human papillomavirus ancillary test ordering habits by comparing compliance before and after the guideline was published.
We retrospectively reviewed head and neck squamous cell carcinoma biopsy or resection specimens from outside institutions during a 2.5-year period around guideline publication to determine whether human papillomavirus testing was performed in accordance with the guideline.
Human papillomavirus testing deviated from the guideline in 45 of 107 cases (42.1%) before and 93 of 258 cases (36.0%) after its publication (P = .29). This included 6 of 26 cases of oropharyngeal squamous cell carcinoma (23.1%) before and 5 of 55 cases (9.1%) after (P = .16), with 5 of 5 (100.0%) after due to not performing p16 immunohistochemistry. This also included 30 of 68 cases of nonoropharyngeal carcinoma (44.1%) before and 69 of 163 (42.3%) after the guideline was published (P = .88), with 29 of 30 (96.7%) before and 67 of 69 (97.1%) after due to unnecessary use of p16 immunohistochemistry. Nodal metastasis testing deviated in 9 of 13 cases (69.2%) before and 19 of 40 cases (47.5%) after (P = .21) with marked variability in testing, including 3 of 9 (33.3%) before and 8 of 19 (42.1%) after, for not confirming certain p16 immunohistochemistry–positive tumors with human papillomavirus–specific testing.
Pathologists continue to deviate from the testing guideline significantly in everyday practice. Further education and discussion about the appropriate handling of head and neck cancer specimens may be needed.
High-risk human papillomavirus (HR-HPV)–related oropharyngeal squamous cell carcinoma (OPSCC) is an increasingly common subset of head and neck squamous cell carcinoma (HNSCC).1–4 Histologically, HR-HPV–positive OPSCCs typically exhibit nonkeratinizing morphology, defined as a tumor with limited or no maturing squamous differentiation5,6 that often grows in ribbon-like nests with pushing borders and has cells with high nuclear to cytoplasmic ratios.7,8 These tumors are also anatomically confined to the lymphoid-containing subsites of the oropharynx, specifically the palatine tonsils and base of tongue.9 Patients with HR-HPV–positive OPSCC have a much better prognosis and better treatment-related outcomes than their non-HPV, predominantly tobacco- and alcohol-related, counterparts.2,10–13 As such, this entity is now integrated into the American Joint Committee on Cancer staging manual14,15 as a separate staging system for HR-HPV OPSCC, and the HPV status increasingly determines specific treatment strategies in routine clinical practice.16,17
Until recently, the testing practices used to determine HR-HPV status in HNSCC biopsies and resections were widely variable. Given the increasing clinical significance of HPV status in OPSCC, the College of American Pathologists (CAP) identified 11-person expert and 9-person advisory panels in 2014 to establish a set of evidence-based recommendations for HR-HPV testing in HNSCC.9 The published guideline, released online by the Archives of Pathology & Laboratory Medicine on December 18, 2017, and then in print in May 2018, aimed to standardize HR-HPV testing. The guideline had several goals including to encourage pathologists to perform necessary testing and avoid unnecessary testing, to increase consistency across practice settings, to provide standardized wording for results, and to serve as a baseline from which future improvements could be made.
The guideline provided an algorithmic approach to HR-HPV testing in HNSCC specimens, based on anatomic location and specific clinical information (see CAP Guideline for the complete recommendations).9 It comprised many statements including several of the most pertinent summarized here:
Pathologists should perform HR-HPV testing for all patients with newly diagnosed OPSCC on the primary biopsy or resection or on a cervical nodal metastasis biopsy or dissection specimen in a patient with a clinically apparent oropharyngeal primary (Strong Recommendation).
HR-HPV testing on OPSCCs should be performed by using p16 immunohistochemistry (IHC) as a surrogate marker (Recommendation).
Pathologists should not routinely perform HR-HPV testing for patients with nonoropharyngeal primary tumors of the head and neck (Recommendation).
Pathologists should routinely perform HR-HPV testing for patients with metastatic SCC of unknown primary in a cervical upper- or mid-jugular chain lymph node (Recommendation).
Cervical lymph nodes with metastatic SCC of unknown primary should be evaluated only with p16 IHC if located in a cervical upper- or mid-jugular chain (level II or III) lymph node; additional HPV-specific testing for p16 IHC–positive cases should be performed only if the level of the lymph node is unknown or if the tumor has keratinizing morphology (Expert Consensus Opinion).
This study determined to what degree HR-HPV test ordering practices of pathologists were consistent with the CAP guideline9 after its publication in 2017. To achieve this, we studied “referrals,” which are the slides sent for review on patients coming to our institution for treatment after a diagnosis has been rendered at an outside facility. We documented if testing was performed and, when so, what methodology was used to detect HR-HPV in these specimens. Ordering practices from before and after the guideline was released were analyzed and compared.
We assessed the impact of the CAP “Human Papillomavirus Testing in Head and Neck Squamous Cell Carcinomas” guideline on HR-HPV test ordering practices by pathologists.
A retrospective analysis of HNSCC cases from outside institutions reviewed at Vanderbilt University Medical Center (VUMC; Nashville, Tennessee) as part of our “referral” service was conducted between January 1, 2017, and December 17, 2017 (before CAP guideline online publication) and between December 18, 2017, and May 1, 2019 (after publication). Cases were selected by searching the VUMC pathology database for referrals with the term squamous cell carcinoma that were signed out by 1 of our 3 head and neck subspecialty pathologists. Cases were then manually curated and those from the nasal cavity, nasopharynx, oral cavity, oropharynx, hypopharynx, larynx, or neck lymph nodes were included (Table 1). Only pure SCC cases and established variants of SCC were included. Cytology and true consult cases were excluded as were any nonsquamous carcinomas, carcinomas with squamous differentiation that are not considered true SCC or SCC variants, and squamous dysplasia or carcinoma in situ–only specimens. The cohort of cases after the CAP guideline was published online was further divided into the following groups: December 18, 2017, to April 30, 2018 (after online publication on December 17, 2017) and May 1, 2018, to May 1, 2019 (up to 1 full year after print publication in May 2018). The cohort with procedure dates after print publication was further divided into the following subgroups, in order to track trends over time: May 1, 2018, to July 31, 2018; August 1, 2018, to December 31, 2018; and January 1, 2019, to May 1, 2019 (see Figure 1; Table 2).
The surgical pathology report issued by VUMC was reviewed for the selected cases, and the following parameters were recorded: patient age; sex; date of original procedure; type of outside practice (academic or community); biopsy site; primary or metastasis; date of review at VUMC; final diagnosis at VUMC; if ancillary studies such as p16 IHC or HPV-specific testing (HPV DNA polymerase chain reaction [PCR] or DNA or RNA in situ hybridization [ISH]) were performed at the outside institution; and if additional ancillary studies such as p16 IHC or HPV-specific testing were performed at VUMC. The original diagnostic outside pathology report was reviewed for all cases where it was available, and the following parameters were recorded: final diagnosis and if ancillary studies such as p16 IHC or HPV-specific testing were performed. The results for p16 IHC were taken directly from the pathology report, and the p16 IHC slides were not re-reviewed as part of this study. The patients' VUMC electronic medical records were reviewed for imaging reports (to confirm level of lymph node involvement) and other biopsies/resections related to the patient's HNSCC.
Cases were evaluated on the basis of whether they adhered to, or deviated from, the flow diagram for HR-HPV testing in HNSCC published in the CAP guideline (see Figure 1 of CAP guideline for the complete algorithm).9 The cases were then stratified into groups by date of procedure, anatomic site, and the way the testing (or lack of testing) deviated from the guideline. The changes in ordering habits across different chronologic groups were evaluated and the statistical significance between the groups was determined by Fisher exact test using R Studio statistical software (RStudio Team, PBC, Boston, Massachusetts; http://www.rstudio.com/).
A total of 365 specimens met criteria and were evaluated. There were 91 of 365 females (25.0%) and 274 of 365 males (75.0%). The cases included 81 of 365 oropharyngeal tumors (22.2%), 231 of 365 nonoropharyngeal tumors (63.3%; including 135 of 365 oral cavity tumors [37.0%], 13 of 365 nasal cavity and nasopharyngeal tumors [3.6%], 16 of 365 hypopharyngeal tumors [4.4%], and 67 of 365 laryngeal tumors [18.4%]), and 53 of 365 lymph node metastases (14.5%). The total number of cases before and after the guideline was published and the number of cases in each time cohort are detailed in Table 1.
Overall, HR-HPV testing deviated from the guideline for 45 of 107 patients (42.1%) before and 93 of 258 (36.0%) after the guideline was released (P = .29). Non–guideline-conforming testing decreased appreciably over time from 48.1% (39 of 81) in the December 2017 to April 2018 cohort (immediately after online publication) to 26.0% (13 of 50) in the May to July 2018 cohort (immediately after print publication) (P = .02) and remained lower at approximately 32% after this time (Table 2). This included an appreciable decrease in the use of HPV-specific testing when considered to not be routinely indicated by the CAP guideline across all anatomic sites: 17.8% (8 of 45) from before the guideline to 7.5% (7 of 93) after the guideline was released (P = .08).
For OPSCCs, 6 of 26 cases (23.1%) before and 5 of 55 cases (9.1%) after the guideline was released had HR-HPV testing practices that deviated from the guideline (P = .16) (Table 2). The majority of non–guideline-conforming testing (4 of 6, 66.7%) performed before guideline publication consisted of HPV RNA-ISH or HPV DNA PCR that was ordered instead of/in addition to p16 IHC when considered not routinely indicated per CAP guideline recommendations. Conversely, 100% of the cases (n = 5) with non–guideline-conforming testing after the guideline were due to a failure to order p16 IHC in an oropharyngeal primary tumor.
For non-OPSCCs, including those from the oral cavity, nasal cavity/nasopharynx, hypopharynx, and larynx, 30 of 68 cases (44.1%) before and 69 of 163 (42.3%) after the guideline was released had HR-HPV testing performed against the published recommendations (P = .88) (Table 2). Across non-OPSCC primaries, testing for HR-HPV using IHC, ISH, or PCR was performed most often in the hypopharynx (2 of 3 cases [66.7%] before; 7 of 13 cases [53.8%] after) followed by the oral cavity (19 of 42 cases [45.2%] before; 43 of 93 cases [46.2%] after), the larynx (9 of 23 cases [39.1%] before; 14 of 44 cases [31.8%] after), and lastly nasal cavity/nasopharynx (0 of 0 [0.0%] cases before; 5 of 13 [38.5%] cases after) (Supplemental Table 1, see supplemental digital content at https://meridian.allenpress.com/aplm in the September 2021 table of contents). In all non-OPSCC cases, the most frequently ordered HR-HPV ancillary test was p16 IHC, accounting for 29 of 30 cases (96.7%) before and 67 of 69 cases (97.1%) after the guideline was published. The oral cavity was the most common non-OPSCC site to deviate from guideline recommendations owing to the frequency of cases, with 18 of 19 of the deviation cases (94.7%) from before and 42 of 43 of the deviation cases (97.6%) from after publication due to unnecessary use of p16 IHC. Despite the similar rates of nonconforming testing between the 2 periods, there was a shift toward adherence to guideline recommendations with 62.5% (15 of 24) of cases with non–guideline-conforming testing performed in the December 2017 to April 2018 cohort, and only 36.8% (7 of 19) in the January to May 2019 cohort (P = .13) (Table 2).
Lastly, cervical lymph node metastasis HR-HPV evaluation deviated from the guideline in 9 of 13 cases (69.2%) before and 19 of 40 cases (47.5%) after the publication of the guideline (P = .21) (Figure 2, A through C; Table 2), with marked variability in the types of testing performed. Of the non–guideline-conforming testing, reasons for deviation included not ordering p16 IHC and/or HPV-specific testing when recommended in 9 of 9 cases (100%) before and 17 of 19 cases (89.5%) after or, alternatively, ordering p16 IHC and/or HPV-specific testing when not routinely indicated in no cases before but 2 of 19 cases (10.5%) after the guideline (Table 2). The specific scenarios included not ordering p16 IHC for any lymph node metastasis at a level II or level III, unknown location, or from a patient with a known oropharyngeal primary; ordering HPV-specific testing instead of p16 IHC on lymph node metastases from level II or level III sites with nonkeratinizing morphology or from a patient with a known oropharyngeal lesion; failing to confirm HPV status of p16 IHC–positive lymph node metastases when the lymph node level was unknown, the tumor had keratinizing morphology, or the primary tumor was of overlapping primary sites (ie, oral cavity and oropharynx); or ordering p16 IHC when the patient had a known nonoropharyngeal primary tumor. While the case numbers in this cohort were too low to determine any statistically significant changes across periods, a trend toward better use of ancillary testing was appreciated with 63.6% (7 of 11) of cases in the December 2017 to April 2018 cohort having non–guideline-conforming testing performed, while only 28.6% (2 of 7) of cases in the January to May 2019 cohort deviated from the guideline (P = .33).
The cohort was a compilation of referral cases from both academic (55 of 365 [15.1%]) and community (310 of 365 [84.9%]) practices. There was no significant difference in ordering habits between these 2 groups: non–guideline-conforming testing was performed in 10 of 23 academic (43.5%) and 35 of 84 community (41.7%) cases before (P = .62) and 16 of 32 academic (50.0%) and 77 of 226 community (34.1%) cases after (P = .11) the guideline was released.
To confirm if any HR-HPV testing was performed at the time of the original diagnosis, both the VUMC pathology report and the original diagnostic pathology report from the referring institution were reviewed. In non-OPSCC cases where HR-HPV ancillary testing was performed at the referring institution against guideline recommendation, we found that in approximately 25% (24 of 95) of cases, the reviewing VUMC pathologist did not document outside hospital ancillary testing in the VUMC pathology report. There were also 31 cases of non-OPSCC where there was no mention of HR-HPV ancillary testing performed at the referring institution in the VUMC report, and the original diagnostic pathology reports were not available for confirmatory review. Thus, there was a small cohort of patients for which we cannot assess if (or what type of) HR-HPV may have been performed. In these cases, we assumed that ancillary testing was not performed when conducting the statistical analysis. However, from the patterns of our other available data, we can also impute that of the 31 cases with no diagnostic report available for review, approximately 8 likely had ancillary testing performed at the outside institution that was not captured by our study, thus would only increase the rates of deviation from the guideline.
This study assessed the degree to which HR-HPV test ordering practices were consistent with the CAP guideline9 after its publication in 2017 and showed only a slight, statistically insignificant increase in compliance with HR-HPV ancillary testing practices since publication. A variety of reasons could account for the CAP guideline not being more widely followed amongst pathologists. This study explores those reasons, reiterates the potential harm from not following the CAP guideline, and concludes with possible action steps that could be taken to result in a more consistent adherence to the CAP guideline.9
The difficulty of new pathology guidelines being quickly and effectively adopted is a well-known obstacle when recommending new testing procedures. Old practices are ingrained and hard to modify across large numbers of people. However, in the case of adherence to the CAP guideline for HR-HPV test ordering practices for HNSCC there are some specific limiting factors, beyond generally applicable difficulties in updating testing procedures, contributing to the slow rate of adoption.
General reasons for the slow dissemination of updated guidelines relevant to pathology likely include the inherent difficulties in bringing a specialized procedure to a broad audience, limitations on community pathologists' resources, and practice inertia. Some of the general limitations are that general pathologists have a variety of responsibilities, and it is not feasible to maintain a regular review of every journal publication and updated database in a timely manner necessary to perform their job functions. Rather, things tend to “trickle down” over time. Limitations on community pathologists' resources include a lack of provided clinical information, unclear anatomic location for the specimens at the time of diagnosis, lack of in-house ancillary testing, and external pressures to perform certain testing procedures independently of guidelines or proven clinical indication. Practice inertia includes the slow incorporation of new approaches that are inconsistent with a previous practice to which pathologists have grown accustomed.
The specific limiting factors may include a lack of subject matter expertise in head and neck pathology, a misunderstanding of the anatomic boundaries between oropharynx and adjacent structures, and confusing differences in practice guidelines between the CAP9 and subsequent American Society of Clinical Oncology (ASCO) guidelines.18
A difference between CAP and ASCO guideline recommendations for testing could prevent adherence to the CAP guideline. Even though the CAP and ASCO guidelines are mostly consistent, at the time of its publication, ASCO included qualifying statements that provided clinicians with slightly different recommendations for testing than the earlier published CAP guideline. Most of ASCO's qualifying statements18 are centered around confirming transcriptionally active HR-HPV with HPV-specific testing in lesions that are p16 IHC positive, whereas the CAP guideline recommends performing p16 IHC exclusively. These situations include oropharyngeal primary tumors of questionably HPV-driven pathogenesis or nonsquamous differentiation, and in all metastatic lesions to the neck of unknown primary location, regardless of lymph node level or nonkeratinizing morphology. The differences between the 2 guidelines may play a role in the miscommunication between pathologist and oncologist, contributing to the lack of adherence to the CAP guideline.9
The relatively recent publication of the CAP guideline relative to the time period we studied could be responsible for the overall lack of adherence we observed. This study assessed adherence to the CAP guideline–recommended testing standards for a period of only 1.5 years after its publication. This period may have been insufficient for adoption and perhaps HR-HPV testing in this group of tumors will continue to improve over time.
Before suggesting solutions to the reasons possibly contributing to the lack of adherence, this study assessed the harm that could come from not following the CAP guideline and identified several important scenarios where persistent deviation from the CAP guideline may lead to negative outcomes in clinical practice (summarized in Table 3).
Stressing the importance of evaluating all OPSCCs with some form of HR-HPV testing was a major aim of the CAP guideline and this recommendation was endorsed by ASCO. This study shows that a proportion of OPSCC patients still are not receiving the appropriate HR-HPV evaluation at the time of diagnosis. Failure to perform HR-HPV testing on an oropharyngeal tumor or a metastatic lesion of likely oropharyngeal origin (ie, nonkeratinizing and/or at level II-III) has potential diagnostic, staging, prognostic, and therapeutic implications. HPV status in OPSCC affects eligibility for certain treatment protocols, including novel clinical trials,16,17 and HPV-positive OPSCC has a significantly better prognosis than HPV-negative SCC of the head and neck regardless of clinical management.10–12,19,20 Furthermore, HPV-positive metastatic lesions of the neck have a strong likelihood of origin in the oropharynx,2,10,19,21 and failure to identify the tumor as HR-HPV driven may delay timely diagnosis of an oropharyngeal primary, particularly as oropharyngeal tumors are frequently subtle and not always clinically apparent.
Another frequently observed deviation from the CAP guideline was failure to confirm p16 IHC–positive neck metastases with HPV-specific testing when the lesion lacks the characteristic features of an oropharyngeal metastasis (ie, outside level II-III or exhibiting keratinizing morphology). In this scenario, ASCO's qualifying statement further recommends that all p16 IHC–positive neck lesions of unknown primary should be evaluated by an HPV-specific method, regardless of location or morphology. p16 IHC is an excellent surrogate marker of viral infection in the correct clinical context.22 However, because p16 is a surrogate marker, it is not always associated with HR-HPV, and non-HPV–related tumors occasionally show strong diffuse staining with p16, including 20% to 30% of aggressive head and neck cutaneous SCCs23,24 and approximately 20% of lung squamous cell carcinomas.25,26 As such, failure to confirm that p16 IHC positivity is due to transcriptionally active HR-HPV can have significant diagnostic implications for the patient.
The most frequently observed deviation from the CAP guideline in this study was performing HR-HPV testing outside of the oropharynx, particularly performing p16 IHC on oral cavity tumors. While this practice may not result in direct patient harm, the CAP expert panel, endorsed by ASCO, did not find evidence to support this practice because there is no proven prognostic or therapeutic difference in HPV status in sites outside of the oropharynx. It is possible that a positive HPV result in this context could be misleading to the patient and/or clinician as to the biologic behavior or appropriate treatment. In speaking with a variety of pathologists from other institutions who have observed the same issue for the past several years, the authors have heard numerous reports of pathologists feeling pressured by oncologists to perform HR-HPV testing in nonoropharyngeal primary tumors. Although these reports are anecdotal, if they are an accurate reflection of the testing environment, it would account for the frequency at which this testing is being performed.
While there are certain clinical scenarios where testing of an apparent nonoropharyngeal tumor may be appropriate (ie, large tumors of multiple sites overlapping the oropharynx or when evaluating for a recurrent HPV-related tumor versus a new primary), the frequency of p16 IHC evaluation outside the oropharynx observed in this study indicates that it is frequently being performed in situations where it simply is not indicated.
Additionally, an observed practice in this study that is not strictly a deviation from the CAP guideline was the performance of HPV-specific testing in addition to, or instead of, p16 IHC. In the guideline publication, the CAP determined that for OPSCC specimens, p16 IHC is an effective surrogate for transcriptionally active HR-HPV in the proper contexts, as it was considered to be the most practical, inexpensive, and reproducible option, performing well compared to RNA-ISH and DNA PCR when evaluating patients' survival outcomes.27–29 However, even the CAP guideline allows that “additional HPV-specific testing may be performed at the discretion of the pathologist.”9 In its endorsement publication, ASCO actually recommends confirmative HPV-specific testing in certain scenarios, particularly involving the neck. As is often the case with nuanced areas of guidance, a thorough understanding of the subject matter is necessary for it to be applied properly and although there are situations where testing for RNA-ISH and DNA PCR would be appropriate, using them as a substitute for p16 IHC is a more expensive and potentially less reliable option that could muddy the waters for treating physicians. Furthermore, it is important to reiterate that DNA ISH is not as sensitive as any of the other methods and may miss HPV positivity, as well documented in a recent study on CAP proficiency testing by Keung et al,30 where HPV-positive cell lines were used and laboratory results using DNA ISH showed decreased sensitivity relative to RNA ISH. Using DNA ISH as the only method of HPV detection is strongly discouraged.9,30
The frequency with which this study found deviations from the CAP guideline and the possible harm originally described in the CAP guideline, which was further expanded in this discussion, indicate that further steps should be undertaken to ensure that the CAP guideline recommendations are followed. This study listed a number of general and specific obstacles that could contribute to the insignificant impact the guideline has had on testing procedures since its publication in 2017. However, for the most part the solution to each of those contributing impediments is the same: more comprehensive education and awareness. Changing existing testing procedures, convincing pathologists of the benefits from updated guidelines, and alerting practitioners to the more complicated and nuanced elements of determining what tests should be run requires that there be a patient benefit or gained efficiency justifying the effort. As this study established a pattern of nonadherence to the CAP guideline and outlined the possible harm that could result from nonadherence, it will hopefully bring the appropriate attention necessary for testing procedures to change going forward.
There are several significant limitations to this retrospective study, the primary being that compliance was defined in the study as a strict adherence to the flow diagram contained in the CAP guideline. We recognize that various clinical scenarios, as well as following certain qualifying statements of the ASCO guidelines, would result in “noncompliance” according to this study while possibly representing “best practice” in a clinical sense. However, binary thresholds had to be established for testing purposes and every area where a deviation from the CAP guideline was assessed is worthy of further discussion. Second, our study numbers are still relatively small in some subgroups, which may have limited our statistical power. Third, because the original outside diagnostic pathology reports were unavailable for review in a subset of cases, there may be an underestimation of the rates of “unnecessary” ancillary testing in certain scenarios. Similarly, addendums could have been made to the original pathology reports with additional ancillary testing that the authors were not privy to at the time of this study. Fourth, the timeline after publication of the guideline (up to 1.5 years afterwards) may simply not have been long enough to really evaluate the lasting impact of the guideline over time. Lastly, the study did not allow for the evaluation of some of the guideline recommendations, including evaluation of HR-HPV testing in nonsquamous head and neck tumors (only squamous cell carcinomas were included) or the correct interpretation of p16 IHC (p16 IHC expression patterns were not documented or obtained, nor the actual slides re-reviewed as part of the study).
Overall, this study shows that HR-HPV testing in routine clinical practice still frequently deviated from the CAP guideline during the 1.5 years post publication, which may be due to both general pathology– and head and neck pathology–specific factors. This study identified several important scenarios where persistent deviation from the CAP guideline may lead to negative clinical outcomes. Additional education and awareness of the guideline are needed.
The authors have no relevant financial interest in the products or companies described in this article.
Supplemental digital content is available for this article at https://meridian.allenpress.com/aplm in the September 2021 table of contents.
Preliminary data collected for this study were presented at the United States and Canadian Academy of Pathology (USCAP) annual meeting as a poster; March 19, 2019; Washington, District of Columbia.