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Prognostic significance of hypoxia-inducible factor-1α expression in advanced pharyngeal cancer without human papillomavirus infection

Published online by Cambridge University Press:  10 June 2021

S Agena
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
H Hirakawa
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
T Ikegami
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
H Kinjyo
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
N Kise
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
H Maeda
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
J Uezato
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
S Kondo
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
A Kiyuna
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
Y Yamashita
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
N Hasegawa
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
M Suzuki*
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
A Ganaha
Affiliation:
Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Department of Otolaryngology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
*
Author for correspondence: Dr Mikio Suzuki, Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nakagami-gun, Okinawa903-0215, Japan E-mail: suzuki@med.u-ryukyu.ac.jp Fax: +81 895 1428
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Abstract

Objective

This study aimed to clarify the association between both hypoxia-inducible factor-1α and glucose transporter type-1 expression and survival outcome in advanced pharyngeal cancer without human papillomavirus infection.

Method

Twenty-five oropharyngeal and 55 hypopharyngeal cancer patients without human papillomavirus infection were enrolled. All patients had stage III–IV lesions and underwent concurrent chemoradiotherapy or surgery. Hypoxia-inducible factor-1α and glucose transporter type-1 expression were investigated in primary lesions by immunohistochemistry.

Results

There were 41 and 39 cases with low and high hypoxia-inducible factor-1α expression, and 28 and 52 cases with low and high glucose transporter type-1 expression, respectively. There was no significant correlation between hypoxia-inducible factor-1α and glucose transporter type-1 expression. In univariate analysis, nodal metastasis, clinical stage and high hypoxia-inducible factor-1α expression, but not glucose transporter type-1 expression, predicted significantly worse prognosis. In multivariate analysis, hypoxia-inducible factor-1α overexpression was significantly correlated with poor overall survival, disease-specific survival and recurrence-free survival.

Conclusion

High hypoxia-inducible factor-1α expression was an independent risk factor for poor prognosis for advanced human papillomavirus-unrelated pharyngeal cancer.

Type
Main Articles
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
Copyright © The Author(s), 2021. Published by Cambridge University Press

Introduction

The head and neck regions are closely associated with quality of life and social activity, so their functional preservation is important in the treatment of head and neck cancer as well as for fair prognosis. For organ preservation, concurrent chemoradiotherapy has been successfully introduced for the treatment of head and neck cancer.Reference Brizel, Albers, Fisher, Scher, Richtsmeier and Hars1,Reference Forastiere, Goepfert, Maor, Pajak, Weber and Morrison2 However, patients sometimes have severe early and late adverse toxic reactions such as mucositis, disturbance of salivary secretion, dysphagia, laryngeal necrosis and mandibular osteomyelitis.Reference Trotti, Bellm, Epstein, Frame, Fuchs and Gwede3Reference Takehana, Kodaira, Tachibana, Kimura, Shimizu and Makita5 These adverse events decrease the treatment completion rate and increase the mortality rate. Salvage surgery is used to control tumours after concurrent chemoradiotherapy failure. However, surgical treatment after concurrent chemoradiotherapy can cause a number of complications, such as local infection and suture breakage because of scarring and decreased local blood flow.Reference Ganly, Patel, Matsuo, Singh, Kraus and Boyle6Reference Pang, Jeannon and Simo9 Thus, biomarkers for predicting the effects of concurrent chemoradiotherapy have been examined.

Because cancer cells proliferate chaotically, angiogenesis in a tumour cannot maintain cancer growth and the vascular network, leading to hypoxia. Cancer cells in a heterogeneously hypoxic environment acquire an adaptive capacity to the hypoxic environment through changes in their signalling system, in which a key molecule is hypoxia-inducible factor-1.Reference Semenza, Jiang, Leung, Passantino, Concordet and Maire10Reference Semenza and Wang13

Hypoxia-inducible factor-1 is a transcription factor composed of two subunits, hypoxia-inducible factor-1α and hypoxia-inducible factor-1β. Hypoxia-inducible factor-1β is constitutively expressed not only in cancer cells but also in normal cells, and hypoxia-inducible factor-1α levels are extensively regulated by the concentration of oxygen.Reference Kaelin and Ratcliffe12 Under normal oxygen conditions, hypoxia-inducible factor-1α is hydroxylated by a prolyl hydroxylase and is degraded through the ubiquitin pathway via binding to the von Hippel–Lindau tumour suppressor.Reference Semenza, Jiang, Leung, Passantino, Concordet and Maire10 However, under low oxygen conditions, hypoxia-inducible factor-1α is stabilised without being degraded and moves into the nucleus. In the nucleus, it subsequently binds to hypoxia-inducible factor-1β to function as a transcription factorReference Bredell, El-Kochairi, Dahlem, Ikenberg and Schumann14 that promotes the expression of many hypoxic adaptation-related factors, including glucose transport proteins such as glucose transporter type-1. Because these adaptations to a hypoxic environment influence treatment resistance,Reference Brown and Wilson11,Reference Bredell, El-Kochairi, Dahlem, Ikenberg and Schumann14Reference Brizel, Dodge, Clough and Dewhirst17 the expression levels of hypoxia-inducible factor-1α and glucose transporter type-1 could be used to predict therapeutic effect, recurrence and prognosis in advanced head and neck cancer.Reference Swartz, Pothen, Stegeman, Willems and Grolman15,Reference Zhou, Huang, Wang, Yuan, Dong and Zhang18,Reference Kunkel, Reichert, Benz, Lehr, Jeong and Wieand19

Hypoxia-inducible factor-1α is overexpressed in various types of cancer, including head and neck cancer.Reference Zhong, De Marzo, Laughner, Lim, Hilton and Zagzag20,Reference Beasley, Leek, Alam, Turley, Cox and Gatter21 In head and neck cancer, hypoxia-inducible factor-1α overexpression has been investigated extensively in patients with oral squamous cell carcinoma (SCC).Reference Zhou, Huang, Wang, Yuan, Dong and Zhang18,Reference Qian, Wenguang, Zhiyong, Yuntao and Wei22,Reference Fillies, Werkmeister, van Diest, Brandt, Joos and Buerger23 Although hypoxia-inducible factor-1α overexpression has been linked with poor prognosis in oral SCC,Reference Zhou, Huang, Wang, Yuan, Dong and Zhang18,Reference Qian, Wenguang, Zhiyong, Yuntao and Wei22 the findings of these reports have not been conclusive.Reference Jayaraj, Kumarasamy, Madurantakam Royam, Devi and Baxi24 By contrast, there are a relatively small number of reports examining the relationship between hypoxia-inducible factor-1α expression and disease prognosis in oropharyngeal or hypopharyngeal SCC. Hypoxia-inducible factor-1α is overexpressed in the vast majority of patients with oropharyngeal SCC, and its degree of expression has predictive and prognostic significance in patients undergoing radiation therapy.Reference Aebersold, Burri, Beer, Laissue, Djonov and Greiner25 However, one report demonstrated that human papillomavirus (HPV) status, and not hypoxia-inducible factor-1α expression, was a predictor of survival outcome in patients with oropharyngeal SCC.Reference Hong, Zhang, Veillard, Jahanbani, Lee and Jones26

Patients with advanced hypopharyngeal and HPV-unrelated oropharyngeal carcinoma have a poor prognosis among patients with head and neck cancer. Thus, the aim of this study was to clarify the association between both hypoxia-inducible factor-1α and glucose transporter type-1 expression and survival outcome in advanced pharyngeal cancer patients without HPV infection.

Materials and methods

Patients

Patients were diagnosed with oropharyngeal SCC or hypopharyngeal SCC by pathologic examination of biopsy samples and were treated by surgery or concurrent chemoradiotherapy with curative intent at the Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Japan, between 2006 and 2017.

Clinical tumour staging was performed according to the Union for International Cancer Control tumour–node–metastasis classification (7th edition, 2009). All patients had clinical stage III or IV disease. Because HPV-related oropharyngeal SCC has a fair survival rate, patients with HPV-related oropharyngeal SCC or hypopharyngeal SCC were excluded. The minimum follow-up period was set to six months after completion of treatment. Finally, this study enrolled 80 treatment-naive patients without distant metastasis. Human papillomavirus status was determined by polymerase chain reaction analysis of HPV DNA and immunohistochemistry of p16 protein, as reported previously.Reference Deng, Hasegawa, Aoki, Matayoshi, Kiyuna and Yamashita27 No patients in the present study had HPV DNA or p16 overexpression in the primary lesion.Reference Yamashita, Ikegami, Hirakawa, Uehara, Deng and Agena28

In order to determine clinical stage and to detect concomitant multiple primary cancers, the patients underwent physical and endoscopic examinations of the upper gastrointestinal tract, ultrasonic inspection of the neck, and computed tomography (CT) and 18F-fluorodeoxyglucose-positron emission tomography-CT (PET-CT) imaging. Patient evaluation and the decision-making processes were conducted by head and neck surgeons and radiation oncologists before treatment was initiated.

Treatment protocol

Patients with T3 or T4 hypopharyngeal SCC were usually treated with one cycle of induction chemotherapy for organ preservation and prevention of distant metastasis. The basic regimen of induction chemotherapy was 1 or 2 cycles of a combination of 5-fluorouracil (600 mg/m2 on days 1–5), nedaplatin (60 mg/m2 on day 2) and docetaxel (60 mg/m2 on day 2).

The therapeutic response was evaluated using the four categories of the Response Evaluation Criteria in Solid Tumours (version 1.1) guidelines: complete response, partial response, stable disease and progressive disease. The initial CT or magnetic resonance imaging scans were used as reference images. The response to induction chemotherapy was classified into the response (complete response or partial response) and no response (stable disease or progressive disease) groups. Those cases for whom a partial response or complete response to induction chemotherapy was achieved underwent concurrent chemoradiotherapy as organ preservation treatment, and cases with stable disease or progressive disease were recommended to undergo total pharyngo-laryngectomy.

As a general rule, we performed post-operative radiotherapy (RT; 60 Gy) with a triweekly infusion of 80 mg/m2 cisplatin 3 times within 6 weeks of surgery if the patients had the following pathological high-risk factors for recurrence: lymph node metastasis with extracapsular extension or a positive or close surgical margin (tumour located less than 5 mm from the surgical margin). Hypopharyngeal SCC patients with concurrent chemoradiotherapy underwent definitive RT (either a total of 50.4 Gy with 1.8 Gy per day, or a total of 50 Gy with 2 Gy per day, 5 times per week) that was administered to the primary site and whole neck including the bilateral neck lymph nodes. The primary site and metastatic lymph nodes were subsequently treated with boost doses of a further 16.2 or 20 Gy in 9 or 10 fractions, respectively. Thus, the cumulative dose to the gross primary tumour and metastatic neck lymph nodes was 66.6 Gy or 70 Gy (once daily fraction).

In patients with oropharyngeal SCC, the primary treatment was concurrent chemoradiotherapy. The patients received platinum-based chemotherapy (nedaplatin and 5-fluorouracil, given twice with a 4-week interval) combined with 66.6 Gy RT. The radiological response of the primary lesion was determined at 39.6 Gy irradiation in all patients by CT, according to the revised Response Evaluation Criteria in Solid Tumours guidelines (version 1.1). If the primary lesion showed a partial response, concurrent chemoradiotherapy was continued as per the protocol. When the primary tumour failed to show a partial response regardless of the neck lymph node response, the patients underwent curative surgery for the primary lesion combined with neck dissection. Patients with N2 and N3 lesions underwent neck dissection at 2–3 months after concurrent chemoradiotherapy.Reference Hasegawa, Maeda, Deng, Kiyuna, Ganaha and Yamashita29

Immunohistochemical study

For hypoxia-inducible factor-1α and glucose transporter type-1 immunohistochemistry, 4 μm thick sections from paraffin-embedded block samples were deparaffinised in xylene and hydrated in a graded series of alcohol. Epitope retrieval was achieved by heating at 100°C for 10 minutes in 1 mM ethylene diamine triacetic acid buffer (pH 8.0) for hypoxia-inducible factor-1α immunohistochemistry or in 10 mM citrate buffer (pH 6.0) for glucose transporter type-1 immunohistochemistry.

Endogenous peroxidase activity was quenched by incubating the sections in 0.3 per cent hydrogen peroxide in methanol for 20 minutes at room temperature. A SAB-PO Kit (Nichirei Bioscience, Tokyo, Japan) was used to detect immunoreactivity to hypoxia-inducible factor-1α and glucose transporter type-1, according to the manufacturer's protocol.

After blocking non-specific reactions by incubation in 10 per cent goat serum, the sections were incubated with primary antibodies for 1 hour at room temperature. A mouse monoclonal anti-hypoxia inducible factor-1α antibody (H1α67-immunoprecipitation assay grade; Abcam, Tokyo, Japan) was diluted with Protein Block Serum-Free (Dako; Agilent Technologies, Santa Clara, USA) at 1:100 for hypoxia-inducible factor-1α immunostaining, and a rabbit monoclonal anti-glucose transporter type-1 antibody (ab115730; Abcam, Tokyo, Japan) was diluted with Protein Block Serum-Free at 1:250 for glucose transporter type-1 immunostaining. Subsequently, a biotin-labelled secondary antibody and peroxidase-labelled streptavidin were applied. Immunolabelling was visualised by incubation in 3–3′-diaminobenzidine, and stained slides were counterstained with haematoxylin.

Positive hypoxia-inducible factor-1α expression was defined as having a stained nucleus and cytoplasm (Figure 1a). Positive glucose transporter type-1 expression was defined as having a stained cell membrane of tumour cells as observed for erythrocytes in the same field (Figure 1b). Sample scoring was performed by semi-quantitative microscopic analysis, considering the positive rates of cancer cells and signal intensity in 3 fields of view under ×400 magnification.

Fig. 1. Representative cases showing (a) hypoxia-inducible factor-1α and (b) glucose transporter type-1 immunohistochemistry. (a) Shows a hypopharyngeal carcinoma case. Strong hypoxia-inducible factor-1α expression was observed in nuclei, and (b) shows an oropharyngeal carcinoma case. There was strong and diffuse glucose transporter type-1 expression in cell membranes. Scale bar, 50 μm.

Considering the percentage of hypoxia-inducible factor-1α immunopositive tumour cells, the following scores were given: 1 when less than 10 per cent of cells were positive; 2 when equal to or more than 10 per cent and less than 30 per cent of cells were positive; 3 when equal to or more than 30 per cent and less than 70 per cent of cells were positive; and 4 when 70 per cent or more cells were positive. Signal intensity was scored as: negative (0), weak (1), moderate (2) and strong (3). The sum of the two scores was used to categorise hypoxia-inducible factor-1α expression as negative to weak (less than or equal to 2; hereafter, low expression) and moderate to strong (more than 3; hereafter, high expression). A representative case is shown in Figure 1a.

Considering the percentage of glucose transporter type-1 immunopositive tumour cells, the following scores were given: 1 when less than 10 per cent of cells were positive; 2 when equal to or more than 10 per cent and less than 70 per cent of cells were positive; 3 when equal to or more than 70 per cent and less than or equal to 90 per cent of cells were positive; and 4 when more than 90 per cent of cells were positive. Signal intensity was scored as negative (0), weak (1), moderate (2) and strong (3). The sum of the two scores was used to categorise glucose transporter type-1 expression as negative to weak (less than or equal to 3; hereafter, low expression) and moderate to strong (equal to or more than 4; hereafter, high expression). A representative case is shown in Figure 1b. These analyses were performed by S Agena, T Ikegami and N Hasegawa, who were blinded to the patients’ clinical information.

Survival estimation

The clinicopathological parameters and treatment outcome of each patient were recorded at scheduled intervals during the observation period. The status of each patient, including information about recurrence and metastasis, was recorded at least every four to six weeks for the first year, every two to three months from two to five years, and thereafter every six months.

Overall survival, disease-specific survival and recurrence-free survival were investigated as prognostic indicators. Survival curves were estimated according to the Kaplan–Meier method, and survival distributions were compared using the log-rank test. Final prognosis was judged in February 2017. Overall survival was defined as the time from the start of treatment to death from any cause (both related and unrelated to oropharyngeal SCC or hypopharyngeal SCC) or to February 2017. Disease-specific survival was defined as the time from the start of treatment to death related to oropharyngeal SCC or hypopharyngeal SCC or to February 2017. Disease-specific survival denotes the probability of remaining free of disease after primary treatment. Recurrence-free survival was defined as the time from the start of treatment to locoregional or distant metastasis or to February 2017. All tests were two-sided, and p-values less than 0.05 were considered statistically significant. The multivariate prognostic significance of tumour variables on overall survival, disease-specific survival and recurrence-free survival was assessed using Cox proportional hazards analysis to identify prognostic parameters. Analyses were performed using the SPSS® (version 25) statistical software. The significance level was set at p < 0.05.

This study was approved by the Institutional Review Board of the University of the Ryukyus and carried out in accordance with the 1975 Declaration of Helsinki, as revised in 2008. Informed consent was obtained from all patients before enrolment. Because the present investigation was a prognostic biomarker study of hypoxia-inducible factor-1α and glucose transporter type-1, we followed the Reporting Recommendations for Tumour Marker Prognostic Studies guideline checklist.

Results

Patient characteristics

The patients included 69 men (86 per cent) and 11 women (14 per cent) with a median age of 66 (range, 39–82) years. The median follow-up period, excluding those patients who died during this time, was 76 (range, 7–132) months. All patients were followed for at least 24 months except for 1 patient who was lost to follow up at 7 months. Of the 80 patients, there were 25 (31 per cent) with oropharyngeal SCC and 55 (69 per cent) with hypopharyngeal SCC. Clinical stage IV was observed in 66 patients (82 per cent). According to T-stage classification, there were 6, 28, 25 and 21 patients with T1, T2, T3 and T4 stages, respectively. According to N-stage classification, there were 6, 16, 52 and 6 patients with N0, N1, N2 and N3, respectively (Table 1).

Table 1. Clinical features and survival estimation

*T1 = 6; T2 = 28; T3 = 25; T4 = 21; N0 = 6; N1 = 16; **N2 = 52; N3 = 6. OS = overall survival; DSS = disease-specific survival; RFS = recurrence-free survival; CCRT = concurrent chemoradiation therapy; PND = planned neck dissection

Of the 80 patients, 30 (38 per cent) underwent surgery and 50 (62 per cent) received concurrent chemoradiotherapy as a primary treatment. Of the 30 patients in the surgery group, 24 had hypopharyngeal carcinoma.

Of the 55 hypopharyngeal SCC patients, 34 had T3 or T4 lesions. Of these 34 patients, 27 (79.4 per cent) received induction chemotherapy, and the remaining 7 did not undergo induction chemotherapy because of renal dysfunction (4 patients), previous history of irradiation to the neck (1 patient) and refusal of induction chemotherapy (2 patients).

Immunohistochemical examinations

Overall, hypoxia-inducible factor-1α expression tended to be weaker than glucose transporter type-1 expression (Figure 1a and b). For hypoxia-inducible factor-1α immunohistochemistry, 41 (51.3 per cent) of 80 samples demonstrated 0–30 per cent positive cell counts, and 59 (73.8 per cent) showed negative-to-weak staining (Table 2). Strong hypoxia-inducible factor-1α expression was observed in only 5 of 80 patients (6.3 per cent). On the other hand, 71 of 80 patients (88.8 per cent) showed weak-to-strong glucose transporter type-1 expression in more than 70 per cent of primary cancer cells, and 70 (87.5 per cent) showed moderate-to-strong staining (Table 2).

Table 2. Immunohistochemical findings

HIF-1 = hypoxia-inducible factor-1; GLUT-1 = glucose transporter type-1

High hypoxia-inducible factor-1α expression was observed in 39 cases (49 per cent), and high glucose transporter type-1 expression was found in 52 cases (65 per cent). However, there was no correlation between hypoxia-inducible factor-1α and glucose transporter type-1 expression (Table 3; p = 0.087).

Table 3. Correlation between HIF-1α and GLUT-1 expression

HIF-1 = hypoxia-inducible factor-1; GLUT-1 = glucose transporter type-1

Survival estimation

Overall survival, disease-specific survival and recurrence-free survival in relation to the clinical features, and immunoexpression of hypoxia-inducible factor-1α and glucose transporter type-1 are shown in Tables 1 and 4, respectively. Node classification and clinical stage classification showed a significant difference in univariate analysis of overall survival, disease-specific survival and recurrence-free survival. However, the other clinical features including T-stage classification and primary sites did not reach significance (Table 1).

Table 4. Immunoexpression and survival estimation

OS = overall survival; DSS = disease-specific survival; RFS = recurrence-free survival; CI = confidence interval; HIF-1 = hypoxia-inducible factor-1; GLUT-1 = glucose transporter type-1

For immunohistochemical analysis, patients with high hypoxia-inducible factor-1α expression had significantly worse overall survival, disease-specific survival and recurrence-free survival than those with low hypoxia-inducible factor-1α expression (Table 4 and Figure 2). However, glucose transporter type-1 expression had no significant impact on survival (Table 4 and Figure 3). In multivariate analysis, there was a significant difference in overall survival, disease-specific survival and recurrence-free survival between patients with low and high hypoxia-inducible factor-1α expression (Table 5). Node category and clinical stage classification did not reach significance.

Fig. 2. Kaplan–Meier curves showing: (a) overall survival, (b) disease-specific survival and (c) recurrence-free survival in 80 pharyngeal carcinoma patients without human papillomavirus infection. (a) Shows overall survival in relation to hypoxia-inducible factor (HIF)-1α expression. Patients with high hypoxia-inducible factor-1α expression had worse overall survival than those with low hypoxia-inducible factor-1α expression (p = 0.033). (b) Shows disease-specific survival in relation to hypoxia-inducible factor-1α expression. Patients with high hypoxia-inducible factor-1α expression had worse disease-specific survival than those with low hypoxia-inducible factor-1α expression (p = 0.033). (c) Shows recurrence-free survival in relation to hypoxia-inducible factor-1α expression. Patients with high hypoxia-inducible factor-1α expression had worse recurrence-free survival than those with low hypoxia-inducible factor-1α expression (p = 0.015). low ex. = low expression; high ex. = high expression

Fig. 3. Kaplan–Meier curves of (a) overall survival, (b) disease-specific survival and (c) recurrence-free survival in 80 pharyngeal carcinoma patients without human papillomavirus infection. (a) Shows overall survival in relation to glucose transporter type (Glut)-1 expression. There was no significant difference in overall survival between patients with high and low glucose transporter type-1 expression. (b) Shows disease-specific survival in relation to glucose transporter type-1 expression. There was no significant difference in disease-specific survival between patients with high and low glucose transporter type-1 expression. (c) Shows recurrence-free survival in relation to glucose transporter type-1 expression. There was no significant difference in recurrence-free survival between patients with high and low glucose transporter type-1 expression. low ex. = low expression; high ex. = high expression

Table 5. Multivariate analysis of survival data

*N0 = 6, N1 = 16; N2 = 52, N3 = 6. OS = overall survival; DSS = disease-specific survival; RFS = recurrence-free survival; CI = confidence interval; HIF-1 = hypoxia-inducible factor-1

There were no significant differences in clinical characteristics between low and high hypoxia-inducible factor-1α expression (Table 6). However, the high hypoxia-inducible factor-1α expression group contained a relatively large number of patients with oropharyngeal SCC (p = 0.066). Figure 4 shows the difference in overall survival between low and high hypoxia-inducible factor-1α expression in oropharyngeal SCC and hypopharyngeal SCC patients. Hypopharyngeal SCC patients with high hypoxia-inducible factor-1α expression had worse overall survival than those with low hypoxia-inducible factor-1α expression (Figure 4b; p = 0.026). Oropharyngeal SCC patients also showed the same tendency for overall survival, despite the small number of samples (Figure 4a; p = 0.114).

Fig. 4. Kaplan–Meier curves of overall survival in patients with (a) oropharyngeal carcinoma and (b) hypopharyngeal carcinoma. (a) Shows oropharyngeal cancer patients with high hypoxia-inducible factor (HIF)-1α expression tended to have worse overall survival than those with low hypoxia-inducible factor-1α expression, but the difference did not reach significance (p = 0.114). (b) Shows hypopharyngeal cancer patients with high hypoxia-inducible factor-1α expression had worse overall survival than those with low hypoxia-inducible factor-1α expression (p = 0.026). low ex. = low expression; high ex. = high expression

Table 6. Clinical characteristics of HIF-1α-positive cases

*T1 = 6, T2 = 28; T3 = 25, T4 = 21; N0 = 6, N1 = 16; **N2 = 52, N3 = 6. HIF-1 = hypoxia-inducible factor-1; CCRT = concurrent chemoradiation therapy; PND = planned neck dissection

Discussion

In this study, high hypoxia-inducible factor-1α expression was found to be an independent risk factor for poor prognosis in patients with advanced oropharyngeal SCC or hypopharyngeal SCC. This is the first report of hypoxia-inducible factor-1α expression focusing on advanced pharyngeal cancer without HPV infection.

A meta-analysis of 1474 oral cancers demonstrated that hypoxia-inducible factor-1α was associated with tumour size, clinical stage and lymph node metastasis, and high hypoxia-inducible factor-1α expression was an indicator for worse survival outcome.Reference Zhou, Huang, Wang, Yuan, Dong and Zhang18 In subgroup analysis, this phenomenon was observed exclusively in Asian patients. According to another systematic review of hypoxia-inducible factor expression in head and neck cancer, hypoxia-inducible factor-1α overexpression was also significantly associated with poor prognosis in Asian patients, but not in European patients.Reference Hong, Lui, Hashiguchi, Hui and Chan30 Regarding tumour location in head and neck cancer, oral carcinoma, nasopharyngeal carcinoma and oropharyngeal carcinoma, but not laryngeal carcinoma, showed an association between hypoxia-inducible factor-1α overexpression and worse overall survival. A previous report on the association between hypoxia-inducible factor-1α overexpression and the survival rate in hypopharyngeal SCC demonstrated no clear relationship between hypoxia-inducible factor-1α and locoregional control and disease-specific survival.Reference Bernstein, Andrews, Slevin, West and Homer31

In the present study, all patients were Japanese (i.e. Asians) with hypopharyngeal SCC or oropharyngeal SCC, and a clear association was seen between hypoxia-inducible factor-1α expression and overall survival, disease-specific survival and recurrence-free survival. Although only a small number of reports have examined hypoxia-inducible factor-1 expression in patients with oropharyngeal SCC or hypopharyngeal SCC, the findings of the present study are in line with those of previous reports. Given that racial differences in hypoxia-inducible factor-1 expression have been observed between Asian and European countries,Reference Zhou, Huang, Wang, Yuan, Dong and Zhang18,Reference Hong, Lui, Hashiguchi, Hui and Chan30 further studies in different ethnic groups are needed to confirm our observations.

Because this study focused on advanced pharyngeal cancer, clinical tumour, node and stage classifications were not found to be significant prognostic factors in multivariate analysis. Oropharyngeal carcinomaReference Silva, Slevin, Sloan, Valentine, Cresswell and Ryder32 and head and neck cancerReference Koukourakis, Sivridis, Simopoulos and Turley33 with radiotherapy showed worse local control and survival rate when high hypoxia-inducible factor-1α expression was observed.Reference Silva, Slevin, Sloan, Valentine, Cresswell and Ryder32 On the other hand, hypoxia-inducible factor-1α expression in surgically treated patients with head and neck SCC (31 oral cavity, 23 oropharynx, 16 larynx and 9 hypopharynx) was associated with improved disease-free survival and overall survival,Reference Beasley, Leek, Alam, Turley, Cox and Gatter21 while the results of the present study were similar to those of previous reports.Reference Silva, Slevin, Sloan, Valentine, Cresswell and Ryder32,Reference Koukourakis, Sivridis, Simopoulos and Turley33

Elevated hypoxia-inducible factor-1α protein levels have been shown to be associated with increased hypoxic radiation resistance in FaDu human pharyngeal carcinoma cell line in vitro.Reference Vordermark, Kraft, Katzer, Bolling, Willner and Flentje34 The discrepancies between these contradictory results may reflect different treatment modalities, and hypoxia-inducible factor-1α-overexpressing head and neck cancer might be treated by surgery, and not by radiation-based therapy. Further study is needed to clarify an appropriate treatment modality for hypoxia-inducible factor-1α-overexpressing in head and neck cancer.

  • Hypoxia-inducible factor-1α is overexpressed in various types of cancer, including head and neck cancer

  • Few reports have examined the relationship between hypoxia-inducible factor-1α expression and disease prognosis in pharyngeal squamous cell carcinoma (SCC)

  • No study has reported the association between hypoxia-inducible factor-1α and prognosis in advanced pharyngeal cancer without human papillomavirus (HPV) infection

  • High hypoxia-inducible factor-1α expression was an independent risk factor for poor prognosis in advanced HPV-unrelated oropharyngeal or hypopharyngeal SCC

  • High glucose transporter type-1 expression was not an independent factor for poor prognosis in advanced HPV-unrelated oropharyngeal or hypopharyngeal SCC

Glucose transporter type-1 is a cell membrane transport protein that determines glucose uptake and is abnormally expressed in head and neck cancer.Reference Kunkel, Reichert, Benz, Lehr, Jeong and Wieand19 Although glucose transporter type-1 shows only weak expression in normal mucosal lesions, it is strongly expressed in dysplasia and SCC.Reference Angadi and Angadi35 Glucose transporter type-1 expression is considered to be a prognostic marker in head and neck cancer.Reference Chung, Jeong, Park, Jang, Son and Choi36,Reference Hanamoto, Tatsumi, Takenaka, Hamasaki, Yasui and Nakahara37,Reference Pak, Cheon, Nam, Kim, Kang and Chung38 However, in the present study, glucose transporter type-1 expression did not demonstrate a clear correlation with disease prognosis. We found that glucose transporter type-1 expression was much stronger than hypoxia-inducible factor-1α expression (Table 2). Because PET-CT studies have indicated that glucose uptake is highly increased in head and neck cancer,Reference Kunkel, Reichert, Benz, Lehr, Jeong and Wieand19,Reference Kuwabara, Toriihara, Yuasa-Nakagawa, Toda, Tateishi and Yoshimura39,Reference Zhao, Yang, Zhou, Dong, Bao and Yao40 the immunohistochemical approach used in the present study might not detect differences in glucose uptake ability in advanced pharyngeal carcinoma. In addition, although a significant correlation between glucose transporter type-1 and hypoxia-inducible factor-1α expression in head and neck cancer has been reported,Reference Zhao, Yang, Zhou, Dong, Bao and Yao40 we found no such association. Other methods, such as metabolic tumour volume in PET-CT, might be more appropriate than glucose transporter type-1 immunohistochemistry for evaluating glucose metabolism.

Conclusion

In conclusion, high hypoxia-inducible factor-1α expression, but not high glucose transporter type-1 expression, was an independent risk factor for poor prognosis in advanced HPV-unrelated oropharyngeal SCC and hypopharyngeal SCC patients who underwent concurrent chemoradiotherapy as a primary treatment. Racial differences in the association of hypoxia-inducible factor-1α expression with survival outcome between Asian and European countries and treatment modality in cases with high hypoxia-inducible factor-1α expression should be examined to facilitate the design of better treatment protocols.

Acknowledgements

This study was supported by and conducted in cooperation with the Ryukyu Society for the Promotion of Oto-Rhino-Laryngology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by Japan Society for the Promotion of Science Kakenhi (to M Suzuki, grant number: JP17K11390).

Competing interests

None declared

Footnotes

Dr M Suzuki takes responsibility for the integrity of the content of the paper

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Figure 0

Fig. 1. Representative cases showing (a) hypoxia-inducible factor-1α and (b) glucose transporter type-1 immunohistochemistry. (a) Shows a hypopharyngeal carcinoma case. Strong hypoxia-inducible factor-1α expression was observed in nuclei, and (b) shows an oropharyngeal carcinoma case. There was strong and diffuse glucose transporter type-1 expression in cell membranes. Scale bar, 50 μm.

Figure 1

Table 1. Clinical features and survival estimation

Figure 2

Table 2. Immunohistochemical findings

Figure 3

Table 3. Correlation between HIF-1α and GLUT-1 expression

Figure 4

Table 4. Immunoexpression and survival estimation

Figure 5

Fig. 2. Kaplan–Meier curves showing: (a) overall survival, (b) disease-specific survival and (c) recurrence-free survival in 80 pharyngeal carcinoma patients without human papillomavirus infection. (a) Shows overall survival in relation to hypoxia-inducible factor (HIF)-1α expression. Patients with high hypoxia-inducible factor-1α expression had worse overall survival than those with low hypoxia-inducible factor-1α expression (p = 0.033). (b) Shows disease-specific survival in relation to hypoxia-inducible factor-1α expression. Patients with high hypoxia-inducible factor-1α expression had worse disease-specific survival than those with low hypoxia-inducible factor-1α expression (p = 0.033). (c) Shows recurrence-free survival in relation to hypoxia-inducible factor-1α expression. Patients with high hypoxia-inducible factor-1α expression had worse recurrence-free survival than those with low hypoxia-inducible factor-1α expression (p = 0.015). low ex. = low expression; high ex. = high expression

Figure 6

Fig. 3. Kaplan–Meier curves of (a) overall survival, (b) disease-specific survival and (c) recurrence-free survival in 80 pharyngeal carcinoma patients without human papillomavirus infection. (a) Shows overall survival in relation to glucose transporter type (Glut)-1 expression. There was no significant difference in overall survival between patients with high and low glucose transporter type-1 expression. (b) Shows disease-specific survival in relation to glucose transporter type-1 expression. There was no significant difference in disease-specific survival between patients with high and low glucose transporter type-1 expression. (c) Shows recurrence-free survival in relation to glucose transporter type-1 expression. There was no significant difference in recurrence-free survival between patients with high and low glucose transporter type-1 expression. low ex. = low expression; high ex. = high expression

Figure 7

Table 5. Multivariate analysis of survival data

Figure 8

Fig. 4. Kaplan–Meier curves of overall survival in patients with (a) oropharyngeal carcinoma and (b) hypopharyngeal carcinoma. (a) Shows oropharyngeal cancer patients with high hypoxia-inducible factor (HIF)-1α expression tended to have worse overall survival than those with low hypoxia-inducible factor-1α expression, but the difference did not reach significance (p = 0.114). (b) Shows hypopharyngeal cancer patients with high hypoxia-inducible factor-1α expression had worse overall survival than those with low hypoxia-inducible factor-1α expression (p = 0.026). low ex. = low expression; high ex. = high expression

Figure 9

Table 6. Clinical characteristics of HIF-1α-positive cases