HPV Infection and Anemia Status Stratify the Survival of Early T2 Laryngeal Squamous Cell Carcinoma *,†,‡Hongzhi Wang, *,†Zichen Zhang, *,†,‡Rui Sun, §Hui Lin, kLong Gong, kMinjie Fang, and *,†,‡Wei-Han Hu, *yzxGuangzhou and kShenzhen, Guangdong
Summary: Objective. This study was designed to investigate the potentially prognostic indicators of early laryngeal squamous cell carcinomas (LSCCs), including human papillomavirus (HPV) status. Methods. A total of 336 patients with T2N0–1M0 LSCC were included in this study. Clinical data were collected from archival documents, and HPV infection and p16INK4A expression were detected. Results. A total of 32/318 cases of high-risk HPV infection and 10/336 cases of p16INK4A overexpression were found. Three hundred eighteen tumors were classified into a three-class model according to HPV infection and p16INK4A expression: class I, HPV+/p16+; class II, HPV+/p16; and class III, HPV/p16. Class III had a trend of decreased overall survival (OS) (P ¼ 0.076) and a markedly low relapse-free survival (RFS) (P ¼ 0.022) compared with class I and class II. HPV-positive cases (class I plus class II) had a significantly longer OS (P ¼ 0.038) and RFS (P ¼ 0.006). In multivariate analysis, HPV-positive (P ¼ 0.020), nonanemia (P ¼ 0.011), and N0 stage (P ¼ 0.005) were significant predictors for high RFS. But only HPV-positive (P ¼ 0.047) and nonanemia (P < 0.001) were significant predictors for superior OS. Conclusion. A trend of discrete survival among HPV+/p16+, HPV+/p16, and HPV/p16 classes was found in early LSCCs. We suggest that HPVinfection and hemoglobin level are the potential factors that can stratify outcome of early LSCCs. Key Words: Laryngeal SCC–Human papillomavirus–p16INK4A–Hemoglobin–N1 stage.
INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world; the laryngeal cancers significantly contribute to this statistic, with an age-standardized incidence of 3.5–5.5 per 100 000 and a mortality of 2.1–2.4 per 100 000 men worldwide.1 Tobacco and alcohol exposure have traditionally been responsible for a large proportion of laryngeal cancers.2 In recent years, human papillomavirus (HPV) infection has gained attention for its function in the carcinogenesis of HNSCC. HPV is found in 25.9% of all HNSCCs worldwide and 24.0% of laryngeal carcinomas.3 HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) has been recognized as an extremely heterogeneous group of tumors for its distinct clinical and pathologic features.4 In OPSCC, HPV RNA detection has been considered as the gold standard for meaningful HPV infection.5 p16INK4A can be upregulated indirectly by HPV oncoprotein E7, and its overexpression serves as an alternative indicator of HPV-related tumorigenesis.6 p16INK4A is a reliable biomarker of transcriptionally active HPV, and the combination with HPV DNA test offers a valuable alternative to RNA analysis in OPSCC.7 Recent study also found the biological evidence for a causal role of HPV in a small fraction (5%) of laryngeal squamous cell carcinoma (LSCC).8 Previous clinical trial reported that HPVAccepted for publication August 25, 2014. From the *Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong Province, China; yState Key Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou, Guangdong Province, China; zCollaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong Province, China; xGuangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong Province, China; and the kShenzhen People’s Hospital, The Second Clinical Medical College, Jinan University, Shenzhen, Guangdong Province, China. Address correspondence and reprint requests to Wei-Han Hu, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, 510060, China. E-mail: [email protected] or [email protected] Journal of Voice, Vol. 29, No. 3, pp. 356-362 0892-1997/$36.00 Ó 2015 The Voice Foundation http://dx.doi.org/10.1016/j.jvoice.2014.08.016
associated p16INK4A expression was a major factor for treatment response and survival in patients with head and neck cancer, in which LSCCs made up 30% of patients.9 However, there is still no large-scale investigation regarding HPVassociated survival in laryngeal carcinoma. Hemoglobin level is a traditional prognostic factor in HNSCC treated with radiation.10–12 Hemoglobin is the oxygen-carrying molecule, which was considered to be correlated with the oxygenation status of tumors. However, the correlation between the oxygenation status and hemoglobin level is not linear.13 Was the increased survival from hemoglobin completely owing to the enhanced sensitivity to radiotherapy? There were rarely studies regarding the prognostic significance of hemoglobin in patients without definitive radiotherapy. Clinical investigation regarding LSCCs is prone to focus on advanced disease. Early stage LSCCs are frequently neglected because of the good prognosis. Except for traditionally adverse features, were patients of early LSCCs really a homogeneous collective? Prognostic factors for early stage LSCCs have not been well investigated, particularly when HPV status was taken into account. In the present study, early LSCCs without traditional adverse features were enrolled. We aimed to investigate the prognostic indicators from the clinicopathological features, including HPV status and pretreatment hemoglobin level, to provide basis for individual management for early LSCCs. PATIENTS AND METHODS Patients and tissues We selected patients with T2N0–1M0 LSCCs consecutively treated at the Sun Yat-sen University Cancer Center from 1995 to 2009. Formalin-fixed paraffin-embedded specimens were obtained from 356 patients who received definitive surgery and were preoperatively and postoperatively evaluated without adverse features. Adverse features, including extracapsular nodal spread, positive margin, perineural invasion, and
Hongzhi Wang, et al
HPV and Anemia Stratify Survival of Early LSCC
vascular embolism, were confirmed by enhanced computed tomography/magnetic resonance imaging and/or pathology. Twenty patients were eliminated because of other malignant tumor history, induced or adjuvant chemotherapy, or no detailed surgical information. This retrospective study was approved by the Institutional Review Board and Human Ethics Committee. Treatments All patients received definitive surgery, including 23 cases of total laryngectomy, 294 cases of hemilaryngectomy, and 19 cases of CO2 laser endoscopic resection, and selective or definitive neck dissection. All patients were restaged after operation as T2N0–1M0 according to the seventh edition of the American Joint Committee on Cancer system. A total of 100 patients received postoperative radiotherapy according to institutional guidelines. There was no induction, concurrent, or adjuvant chemotherapy in this set. And we defined a pretreatment hemoglobin level of 135 g/L or less for men and 125 g/L or less for women as anemia. HPV DNA detection and genotyping Two or three 10-mm-thick sections of paraffin curls were prepared for DNA isolation. Genomic DNA was purified with the QIAamp DNA FFPE Tissue Kit (Qiagen Ltd, Hilden, Germany). HPV DNA detection and typing were performed using the Human Papillomavirus Genotying Kit (Beijing GP Medical Technologies Ltd, Beijing, China). The test was based on polymerase chain reaction amplification, which was performed with HPV L1 open reading frame consensus primers MY11 and MY09. The human b-actin gene was used as an internal control, and amplification without the HPV DNA template was used as a negative control. The HPV genotyping was performed on a W2600 system, a method based on surface plasmon resonance.14 The surface plasmon resonance chip was immobilized with 24 types of HPV-specific oligonucleotide probes targeting the L1 region of the HPV genome, which could distinguish 15 high-risk (HR) HPV subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68) and nine low-risk HPV subtypes (HPV 6, 11, 40, 42, 43, 44, 54, 70, and 81). p16INK4A immunohistochemistry Paraffin sections were cut to 5 mm, heated at 60 C for 2 hours and deparaffinized in xylene. Antigen retrieval was performed using disodium ethylene diamine tetra-acetic acid (pH 8.0) in high-pressure steam for 5 minutes. Sections were incubated with antibody clone JC8 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) in a dilution of 1:100. Specific reactions were detected using Polink-1 horseradish peroxidase Mouse for DAB Bulk Kit (GBI Labs, Mukilteo, WA), and the slides were counterstained with hematoxylin. Sections of p16INK4A-positive cervical carcinoma were used as positive controls. p16INK4A staining evaluation was performed independently by two pathologists blinded to the clinicopathological outcomes of the patients. Tumors with strong and diffuse nuclear and cytoplasmic staining in more than 70% of tumor cells were classified as p16INK4A positive. In this study, concomitant HPV DNA-positive and p16INK4A-positive patients were
357
defined as HPV transcriptionally active cases, and others were non-HPV cases. Statistical analysis The end points used were overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) since definitive surgery. Statistical analysis was performed using SPSS 16.0 software package for Windows. The chisquare test, Fisher exact test, or the Mann-Whitney test was used to evaluate the different distributions of HPV and anemia status based on clinicopathological characteristics. The statistical values of the end points were evaluated by the Kaplan-Meier analysis and compared using the log-rank test. Multivariate survival analyses were performed using the Cox proportional hazards regression model. With a two-sided test, P value of
Summary: Objective. This study was designed to investigate the potentially prognostic indicators of early laryngeal squamous cell carcinomas (LSCCs), including human papillomavirus (HPV) status. Methods. A total of 336 patients with T2N0–1M0 LSCC were included in this study. Clinical data were collected from archival documents, and HPV infection and p16INK4A expression were detected. Results. A total of 32/318 cases of high-risk HPV infection and 10/336 cases of p16INK4A overexpression were found. Three hundred eighteen tumors were classified into a three-class model according to HPV infection and p16INK4A expression: class I, HPV+/p16+; class II, HPV+/p16; and class III, HPV/p16. Class III had a trend of decreased overall survival (OS) (P ¼ 0.076) and a markedly low relapse-free survival (RFS) (P ¼ 0.022) compared with class I and class II. HPV-positive cases (class I plus class II) had a significantly longer OS (P ¼ 0.038) and RFS (P ¼ 0.006). In multivariate analysis, HPV-positive (P ¼ 0.020), nonanemia (P ¼ 0.011), and N0 stage (P ¼ 0.005) were significant predictors for high RFS. But only HPV-positive (P ¼ 0.047) and nonanemia (P < 0.001) were significant predictors for superior OS. Conclusion. A trend of discrete survival among HPV+/p16+, HPV+/p16, and HPV/p16 classes was found in early LSCCs. We suggest that HPVinfection and hemoglobin level are the potential factors that can stratify outcome of early LSCCs. Key Words: Laryngeal SCC–Human papillomavirus–p16INK4A–Hemoglobin–N1 stage.
INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world; the laryngeal cancers significantly contribute to this statistic, with an age-standardized incidence of 3.5–5.5 per 100 000 and a mortality of 2.1–2.4 per 100 000 men worldwide.1 Tobacco and alcohol exposure have traditionally been responsible for a large proportion of laryngeal cancers.2 In recent years, human papillomavirus (HPV) infection has gained attention for its function in the carcinogenesis of HNSCC. HPV is found in 25.9% of all HNSCCs worldwide and 24.0% of laryngeal carcinomas.3 HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) has been recognized as an extremely heterogeneous group of tumors for its distinct clinical and pathologic features.4 In OPSCC, HPV RNA detection has been considered as the gold standard for meaningful HPV infection.5 p16INK4A can be upregulated indirectly by HPV oncoprotein E7, and its overexpression serves as an alternative indicator of HPV-related tumorigenesis.6 p16INK4A is a reliable biomarker of transcriptionally active HPV, and the combination with HPV DNA test offers a valuable alternative to RNA analysis in OPSCC.7 Recent study also found the biological evidence for a causal role of HPV in a small fraction (5%) of laryngeal squamous cell carcinoma (LSCC).8 Previous clinical trial reported that HPVAccepted for publication August 25, 2014. From the *Department of Radiation Oncology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong Province, China; yState Key Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou, Guangdong Province, China; zCollaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong Province, China; xGuangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong Province, China; and the kShenzhen People’s Hospital, The Second Clinical Medical College, Jinan University, Shenzhen, Guangdong Province, China. Address correspondence and reprint requests to Wei-Han Hu, Department of Radiation Oncology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong, 510060, China. E-mail: [email protected] or [email protected] Journal of Voice, Vol. 29, No. 3, pp. 356-362 0892-1997/$36.00 Ó 2015 The Voice Foundation http://dx.doi.org/10.1016/j.jvoice.2014.08.016
associated p16INK4A expression was a major factor for treatment response and survival in patients with head and neck cancer, in which LSCCs made up 30% of patients.9 However, there is still no large-scale investigation regarding HPVassociated survival in laryngeal carcinoma. Hemoglobin level is a traditional prognostic factor in HNSCC treated with radiation.10–12 Hemoglobin is the oxygen-carrying molecule, which was considered to be correlated with the oxygenation status of tumors. However, the correlation between the oxygenation status and hemoglobin level is not linear.13 Was the increased survival from hemoglobin completely owing to the enhanced sensitivity to radiotherapy? There were rarely studies regarding the prognostic significance of hemoglobin in patients without definitive radiotherapy. Clinical investigation regarding LSCCs is prone to focus on advanced disease. Early stage LSCCs are frequently neglected because of the good prognosis. Except for traditionally adverse features, were patients of early LSCCs really a homogeneous collective? Prognostic factors for early stage LSCCs have not been well investigated, particularly when HPV status was taken into account. In the present study, early LSCCs without traditional adverse features were enrolled. We aimed to investigate the prognostic indicators from the clinicopathological features, including HPV status and pretreatment hemoglobin level, to provide basis for individual management for early LSCCs. PATIENTS AND METHODS Patients and tissues We selected patients with T2N0–1M0 LSCCs consecutively treated at the Sun Yat-sen University Cancer Center from 1995 to 2009. Formalin-fixed paraffin-embedded specimens were obtained from 356 patients who received definitive surgery and were preoperatively and postoperatively evaluated without adverse features. Adverse features, including extracapsular nodal spread, positive margin, perineural invasion, and
Hongzhi Wang, et al
HPV and Anemia Stratify Survival of Early LSCC
vascular embolism, were confirmed by enhanced computed tomography/magnetic resonance imaging and/or pathology. Twenty patients were eliminated because of other malignant tumor history, induced or adjuvant chemotherapy, or no detailed surgical information. This retrospective study was approved by the Institutional Review Board and Human Ethics Committee. Treatments All patients received definitive surgery, including 23 cases of total laryngectomy, 294 cases of hemilaryngectomy, and 19 cases of CO2 laser endoscopic resection, and selective or definitive neck dissection. All patients were restaged after operation as T2N0–1M0 according to the seventh edition of the American Joint Committee on Cancer system. A total of 100 patients received postoperative radiotherapy according to institutional guidelines. There was no induction, concurrent, or adjuvant chemotherapy in this set. And we defined a pretreatment hemoglobin level of 135 g/L or less for men and 125 g/L or less for women as anemia. HPV DNA detection and genotyping Two or three 10-mm-thick sections of paraffin curls were prepared for DNA isolation. Genomic DNA was purified with the QIAamp DNA FFPE Tissue Kit (Qiagen Ltd, Hilden, Germany). HPV DNA detection and typing were performed using the Human Papillomavirus Genotying Kit (Beijing GP Medical Technologies Ltd, Beijing, China). The test was based on polymerase chain reaction amplification, which was performed with HPV L1 open reading frame consensus primers MY11 and MY09. The human b-actin gene was used as an internal control, and amplification without the HPV DNA template was used as a negative control. The HPV genotyping was performed on a W2600 system, a method based on surface plasmon resonance.14 The surface plasmon resonance chip was immobilized with 24 types of HPV-specific oligonucleotide probes targeting the L1 region of the HPV genome, which could distinguish 15 high-risk (HR) HPV subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68) and nine low-risk HPV subtypes (HPV 6, 11, 40, 42, 43, 44, 54, 70, and 81). p16INK4A immunohistochemistry Paraffin sections were cut to 5 mm, heated at 60 C for 2 hours and deparaffinized in xylene. Antigen retrieval was performed using disodium ethylene diamine tetra-acetic acid (pH 8.0) in high-pressure steam for 5 minutes. Sections were incubated with antibody clone JC8 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) in a dilution of 1:100. Specific reactions were detected using Polink-1 horseradish peroxidase Mouse for DAB Bulk Kit (GBI Labs, Mukilteo, WA), and the slides were counterstained with hematoxylin. Sections of p16INK4A-positive cervical carcinoma were used as positive controls. p16INK4A staining evaluation was performed independently by two pathologists blinded to the clinicopathological outcomes of the patients. Tumors with strong and diffuse nuclear and cytoplasmic staining in more than 70% of tumor cells were classified as p16INK4A positive. In this study, concomitant HPV DNA-positive and p16INK4A-positive patients were
357
defined as HPV transcriptionally active cases, and others were non-HPV cases. Statistical analysis The end points used were overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) since definitive surgery. Statistical analysis was performed using SPSS 16.0 software package for Windows. The chisquare test, Fisher exact test, or the Mann-Whitney test was used to evaluate the different distributions of HPV and anemia status based on clinicopathological characteristics. The statistical values of the end points were evaluated by the Kaplan-Meier analysis and compared using the log-rank test. Multivariate survival analyses were performed using the Cox proportional hazards regression model. With a two-sided test, P value of
