Risk Classication of Human Papillomavirus-related Oropharyngeal Squamous Cell Carcinoma in Japanese Patients

Purpose The validity of the risk classication according to Ang for human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) remains to be studied in patients treated by methods other than chemoradiotherapy and in Japanese patients. In this study, the validity of Ang's risk classication was studied in Japanese patients treated using various methods, including surgery. Material and Method Between 2010 and 2018, 122 patients with HPV-related OPSCC stages III and IV according to the TNM classication 7th edition (TNM-7) were treated curatively at a single institution in Japan. The median age was 62.7 years. Sixty-seven patients (54.9%) were classied as stage I according to the TNM 8th edition (TNM-8). Over 50% of the patients underwent surgery with or without adjuvant therapy. The inuence of multiple factors on survival was determined. Results Age, amount of smoking, secondary cancer, and N-stage according to the TNM-7 signicantly inuenced survival. Ang's risk classication was also predictive of prognosis, but if 30 pack-years (PYs) instead of 10 PYs is employed to dichotomize the amount of smoking, the new risk classication can signicantly better predict prognosis. According to the new risk classication, favorable and unfavorable risk patients showed 5-year progression-free survival, disease-specic survival, and overall survival rates of 72.7% and 35.9%, 94.6% and 76.2%, and 92.6% and 62.7%, respectively. Conclusions Even in patients treated by methods other than chemoradiotherapy and in Japanese patients, the combination of the amount of smoking and neck node status is useful in prognosis prediction.


Background
The incidence of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in developed countries [1]. In Japan, a similar increase in HPV-related OPSCC has been observed [2]. In contrast to HPV-unrelated OPSCC, HPV-related OPSCC has been reported to have a favorable prognosis [3][4][5][6]. Although tobacco smoking is one of the major causes and prognostic factors of HPV-unrelated OPSCC, the prognostic impact of tobacco smoking remains controversial in HPV-related OPSCC [7][8][9][10]. A milestone study by Ang et al. revealed the impact of tobacco smoking on overall survival (OS) and progression-free survival (PFS) in HPV-related OPSCC, and the amount of smoking expressed by pack-years (PYs) ≤10 or PY >10 was used as one of the criteria for risk classi cation along with the extent of neck lymph node involvement [3]. The risk classi cation according to Ang was derived from HPV-related OPSCC patients treated exclusively with chemoradiation therapy according to the RTOG 0129 protocol [3]. However, it has not yet been reported whether the risk classi cation is also valid in HPVrelated OPSCC patients treated with other modalities such as surgery, and a validity analysis has not been performed in Japanese patients with HPV-related OPSCC.
In this retrospective study, the validity of Ang's risk classi cation was veri ed in patients with HPV-related OPSCC treated with various modalities in a single institution in Japan. Additionally, we studied whether a modi cation of Ang's risk classi cation can improve the predictive power with respect to prognosis.

Patients
This single center retrospective study was approved by the Institutional Review Board (No. 2017-091 and 2018-179). Because of the retrospective nature of this study, informed consent was waived.
Between 2010 and 2018, 240 patients with non-metastatic OPSCC with a known p16 status were treated at a single institution in Japan; of these, 143 (59.6%) had p16-positive OPSCC and 97 (40.4%) had p16negative OPSCC. To diagnose HPV-related OPSCC, p16 immunohistochemical staining (IHC) was used as a surrogate in this study. An expert head and neck pathologist (YM) diagnosed p16 positivity if nuclear and cytoplasmic staining was apparent in >75% of tumor cells. Two of the 143 patients with HPV-related OPSCC were treated palliatively because of poor performance status and were excluded from this study, and the remaining 141 patients were treated with curative intent. Because the study by Ang et al. analyzed only OPSCC patients in stages more advanced than T1-2N0M0 in the TNM 7th edition (TNM-7) [3], 19 patients with T1-T2N0M0 (TNM-7) were excluded from further analysis. The remaining 122 patients with p16-positive OPSCC in stages III and IV in TNM-7 were analyzed in this study. The clinical characteristics of these 122 patients are presented in Table 1. More than 80% of patients were men and the median age was 62.7 years (range, 35-83 years). Approximately 72% of all patients were current or former smokers. The amount of tobacco smoking is expressed in PYs with a median of 25.3 PY among smokers. Synchronous or metachronous secondary cancers were found in 35 patients (28.7%). Alcohol intake was not considered in this study because of the lack of detailed information on the type of alcohol consumed. Staging was performed with physical examinations, laryngopharyngeal endoscopy, computed tomography (CT), and magnetic resonance imaging (MRI). Positron emission tomography (PET)-CT or PET-MRI was performed in selected patients. More than 70% of the patients had a primary lesion in the tonsil, which was followed by base of the tongue primary in terms of frequency. Stage classi cation was performed according to the TNM 8th edition (TNM-8) and N classi cation (N-7) of the TNM-7. Sixty-seven patients were classi ed as stage I by the TNM-8.

Treatment
Regarding treatment, 65 (53.3%) patients underwent surgery with or without adjuvant radiation or chemoradiotherapy. Primary resection and neck dissection were performed in 62 patients, of whom seven with base of the tongue primary underwent bilateral neck dissection. In the remaining three patients, neck lymph node metastasis was managed by neck dissection, while the primary site was irradiated de nitively. Thirty-eight of the 65 surgically treated patients were irradiated postoperatively, of which 15 patients underwent chemotherapy concurrently with postoperative radiotherapy.
The remaining 57 patients were managed with de nitive radiation therapy with (47 patients) or without (10 patients) concurrent chemotherapy. Neoadjuvant chemotherapy was administered to 11 of the 47 patients treated with concurrent chemoradiotherapy. The neoadjuvant chemotherapy regimen was DCF (docetaxel 75 mg/m 2 on day 1, cis-platinum 75 mg/m 2 on day1, 5-FU 750 mg/m 2 from day 1 to day 5, repeated at 21-day intervals) in nine patients, and CF (cis-platinum 100 mg/m 2 on day 1 and 5-FU 1000 mg/m 2 from day 1 to 5, repeated at 21-day intervals) in two patients.
In total, 62 patients underwent concurrent chemotherapy with de nitive or postoperative irradiation. In 47 patients, triweekly cis-platinum (80 mg/m 2 ) was administered, while cetuximab was administered to 15 patients with a priming dose of 400 mg/m 2 and an ensuing weekly dose of 250 mg/m 2 because of poor renal function.
De nitive and postoperative radiation therapies using 6 MV X-rays from accelerators (Varian, Palo Alto, CA, USA) were performed with intensity-modulated radiation therapy in 93 out of 95 patients. In the de nitive radiation, the gross tumor volume was irradiated with doses between 60 and 70 Gy in conventional fractionation. Prophylactic neck regions were irradiated up to 54 Gy. Postoperative irradiation to the bilateral necks was delivered in doses between 50 and 60 Gy in a conventional fractionation in cases with positive operative margins, multiple lymph node involvement, and/or extranodal invasion, with a boost dose of 6-10 Gy at the sites of extranodal invasion or positive margins. Doses of <60 Gy were applied in only two patients who were irradiated postoperatively.

Statistical analysis
Progression-free survival (PFS), disease-speci c survival (DSS), and overall survival (OS) were calculated using the Kaplan-Meier method, assuming the date of treatment initiation as day 0. For the calculation of PFS, recurrence and death from any cause were considered as events. For the calculation of DSS, death by tumor was considered as an event, and patients who died without developing recurrence and patients who were alive at the nal follow-up were censored. The difference between survival curves was tested using the log-rank test. Multivariate analysis (MVA) using Cox proportional hazard regression models was performed with PFS, DSS, and OS as endpoints. Variables with p-values of <0.1 in the univariate analyses (UVA) were included in the MVA. All analyses were performed using SPSS ver. 26. The median follow-up period for all patients was 52 months.
MVA was performed using the variables with a p-value of <0.1 in the UVA (

Discussion
This study aimed to verify the validity of Ang's risk classi cation in patients with HPV-related OPSCC treated with various modalities in a single institution in Japan.

HPV-related OPSCC is known to have different etiologies and therapeutic responses to HPV-unrelated
OPSCC. HPV-related OPSCC is caused by infection with high-risk HPV and responds well to therapeutic interventions with a favorable prognosis, in contrast to HPV-unrelated OPSCC [3][4][5][6]. The TNM-8 separates HPV-related and HPV-unrelated OPSCC, taking into account the etiological and prognostic differences. The TNM-8 uses p16 positivity as a surrogate marker for HPV infection, similar to the methods employed in this study. Although some studies have reported better differentiation of OS according to the stages of the TNM-8 than by the stages of the TNM-7 [11][12][13][14], some reports indicate that prognosis is inadequately differentiated by the TNM-8 [15,16]. The current study also showed that the stages de ned by the TNM-8 were inadequate in differentiating PFS, OS, and DSS. The OS of patients with stage III in this study was better than that of the other reported series [11][12][13][14]. DeFelice et al. also demonstrated a favorable 5-year OS of 86.2% in patients with stage III HPV-related OPSCC and denied the utility of the TNM-8 [15].
Because of the favorable prognosis of HPV-related OPSCC, therapeutic de-escalation has been attempted [17][18][19]. However, to demonstrate the validity of de-escalated therapeutic strategies, it is vital to select patients who can be managed with less intensive therapy [17,19]. To identify appropriate patients, proper prognostic criteria are indispensable, and the TNM-8 staging seems to be inadequate to extract HPVrelated OPSCC patients with a good prognosis who would be candidates for de-escalated treatment.
The risk classi cation proposed by Ang et al. was derived from patients treated exclusively with chemoradiation therapy [3]. Studies have demonstrated the validity of Ang's risk classi cation in HPVrelated OPSCC patients managed by radiation therapy with and without chemotherapy [15,[20][21][22][23], but its validity has not been con rmed in surgically treated patients and Japanese patients. The current study demonstrated that Ang's risk classi cation is effective in differentiating the prognosis of HPV-related OPSCC in a Japanese population treated using various modalities including surgery. In this series, the amount of smoking dichotomized at 30 could better differentiate prognosis than when dichotomized at 10 PYs, as used in Ang's risk classi cation. It remains unclear whether this difference is due to the different sensitivities of the Japanese population to tobacco smoking, as ethnic differences in the effects of tobacco smoking on carcinogenesis have been reported [24,25]. However, if we replace the division at 10 PYs with 30 PYs, the new classi cation can differentiate PFS, DSS, and OS quite well, with signi cant differences observed in both the UVA and MVA.
This study demonstrated that the basic principles of Ang's risk classi cation combining smoking PYs and N-7 stage are valid for identifying Japanese HPV-related OPSCC patients with a good prognosis who are treated by various strategies including surgery. In the TNM-8, bilateral involvement of neck lymph nodes (N2c in TNM-7) is classi ed as a higher N stage [11]. However, the current series showed that unilateral multiple lymph node involvement (N2b in TNM-7) is more predictive of an unfavorable prognosis of HPV-related OPSCC, which is similar to ndings of Ang [3]. Using the new risk classi cation, a larger number of patients are classi ed into the favorable risk group than when using Ang's risk classi cation, which suggests that more patients would be candidates for the therapeutic de-escalation trial in HPV-related OPSCC.
The retrospective nature of this study limits its value. Additionally, important information regarding alcohol consumption, which was reported to be a signi cant prognosticator, is lacking in this study [26]. The proposed risk classi cation method must be validated in a prospective trial involving Japanese patients.

Conclusions
In the current series, the TNM-8 could not adequately differentiate the prognosis of patients with HPVrelated OPSCC. In contrast, the combination of smoking PYs and N stage by TNM-7, such as in Ang's risk classi cation, could better differentiate prognosis and was shown to be valid even in Japanese patients treated with surgery as well as chemoradiotherapy. However, by replacing the cutoff point of 10 PYs in Ang's risk classi cation with a cutoff point of 30 PYs, the prognosis of patients with HPV-related OPSCC could be better differentiated.

Consent for publication
All the authors agreed to publish this manuscript in BMC cancer.

Availability of data and material
The datasets used and analysed during the current study are available from the corresponding author on reasonable request after the approval of National Cancer Center review board.
Competing Interests J Itami reports grants and lecture honoraria from ITOCHU and Elekta, personal fees from Hekabio, AlphaTau, and Palette Life Science.
K Kobayashi reports no con ict of interest.
T Mori reports no con ict of interest.
Y Honma reports no con ict of interest.
Y Kubo reports no con ict of interest.
N Murakami reports no con ict of interest.
G Omura reports no con ict of interest.
K Okuma reports no con ict of interest.
K Inaba reports no con ict of interest.
K Takahashi reports no con ict of interest.
T Kashihara reports no con ict of interest.
Y Shimizu reports no con ict of interest.
A Takahashi reports no con ict of interest.
Y Nakayama reports honoraria for lectures and personal fee from AstraZeneca.
F Matsumoto reports no con ict of interest.
S Yoshimoto reports no con ict of interest.
H Igaki reports a research grant from Hekabio, honoraria for lectures from Itochu and AstraZeneca, and a personal fee from Hekabio.

Funding
This work was nancially supported in part by research grant from ITOCHU. The funding body has nothing to do with content of the study.
Author's contributions JI, KK, NM, GO, FM, and SY designed the study, TM did the p16 immunohistochemistry and diagnosis, YH reviewed chemotherapy, YK reviewed radiological examinations, KO, KI, KT, TK, YS, AT, YN collected clinical records, the results were interpreted by JI, KK, FM, SY, and HI. All the coauthors approved to submit this study to "BMC cancer".