ORIGINAL ARTICLE
Significance of mammalian target of rapamycin in patients with locally advanced stage IV head and neck squamous cell carcinoma receiving induction chemotherapy with docetaxel, cisplatin, and fluorouracil Shau-Hsuan Li, MD,1,7 Wei-Che Lin, MD, PhD,2,7 Tai-Lin Huang, MD,1,7 Chang-Han Chen, PhD,3,7 Tai-Jan Chiu, MD,1,7 Fu-Min Fang, MD, PhD,4,7 Wan-Ting Huang, MD,5 Cheng-Ming Hsu, FACS, MD,6,7 Sheng-Dean Luo, MD, FACS,6,7 Chi-Chih Lai, MD,6,7 Yan-Ye Su, MD,6,7 Hui-Ching Chuang, MD, PhD, FACS,6,7 Chih-Yen Chien, MD, FACS6,7* 1
Department of Hematology–Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 2Department of Diagnostic Radiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 3Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 4Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 5Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 6Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 7Kaohsiung Chang Gung Head and Neck Oncology Group, Cancer Center, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.
Accepted 22 April 2015 Published online 18 July 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.24111
ABSTRACT: Background. This study evaluated the significance of mammalian target of rapamycin (mTOR) activation on the prognosis of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) receiving induction chemotherapy with docetaxel, cisplatin, and fluorouracil (TPF). Methods. Immunohistochemistry (IHC) for phosphorylated-mTOR and phosphorylated-p70 ribosomal S6 protein kinase (p70S6K) examined in 107 patients with locally advanced HNSCC receiving TPF was correlated with treatment outcome. The effect of mTOR inhibition on HNSCC cell lines was investigated in vitro and in vivo. Results. Phosphorylated-mTOR expression was independently significantly associated with response to TPF, progression-free survival (PFS),
and overall survival (OS). In cell lines and xenograft models, mTOR inhibitor, everolimus, enhanced the effect of docetaxel. Conclusion. In patients with locally advanced HNSCC treated with TPF, phosphorylated-mTOR expression was independently associated with prognosis. In vitro and in vivo, concomitant inhibition of mTOR enhanced the effect of docetaxel. Our findings suggest the potential of mTOR as a C 2015 Wiley Periodicals, therapeutic target for locally advanced HNSCC. V Inc. Head Neck 38: E844–E852, 2016
INTRODUCTION
for patients with locally advanced HNSCC to improve survival and for organ preservation.3–6 In a Meta-Analysis of Chemotherapy in Head and Neck Cancer, the addition of induction chemotherapy using cisplatin and 5-fluorouracil to local treatment was associated with an improvement in overall survival (OS) and distant failures.4 Recently, randomized trials5,7 revealed induction chemotherapy regimens with docetaxel, cisplatin, and fluorouracil (TPF) significantly improved progression-free survival (PFS) and OS, compared to the conventional regimen of cisplatin and 5-fluorouracil. Several studies8 have shown that patients whose disease responded to neoadjuvant chemotherapy had a better survival rate in comparison with those who received ineffective chemotherapy. After induction chemotherapy with TPF, clinical tumor response is found in 60% to 70% of patients.5,7 However, there is still a portion of patients who do not respond well to induction chemotherapy with TPF. In addition, there are substantial differences in the response to induction chemotherapy and survival between patients with the same clinical stage. Therefore, it is principal to identify patients who are unlikely to respond to induction
Head and neck squamous cell carcinoma (HNSCC) is one of the 10 most frequent cancers worldwide, with an estimate of over 500,000 new cases being diagnosed annually.1 In Taiwan, HNSCC is the fourth most common cancer in men,2 and more than half of the patients present with locally advanced disease, technically unresectable, or with low surgical curability. Although patients with locally advanced HNSCC receive surgery and/or radiotherapy, 40% to 60% of patients eventually develop locoregional and distant recurrences.3 In an attempt to improve treatment outcome, combining chemotherapy with surgery and radiotherapy has been explored. Currently, induction chemotherapy is one of the standard treatment modalities
*Corresponding author: C.-Y. Chien, Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, 123 Ta-Pei Road, Niaosung District, Kaohsiung, Taiwan. E-mail: [email protected] Contract grant sponsor: This work was supported by grants from Chang Gung Memorial Hospital (CMRPG8D0681 and CMRPG8B1263)
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KEY WORDS: head and neck cancer, squamous cell carcinoma, mammalian target of rapamycin (mTOR), induction chemotherapy, docetaxel
PHOSPHORYLATED
chemotherapy to spare them the toxic effects of ineffective chemotherapy and cause a delay for other therapeutic options. A reliable marker that can correctly predict the tumor response to induction chemotherapy in patients with locally advanced HNSCC is important, and this marker may serve as a promising target in the future. The mammalian target of rapamycin (mTOR) is a 289kDa serine/threonine kinase that phosphorylates key eukaryotic translation regulators, including p70 ribosomal S6 protein kinase (p70S6K) that phosphorylates the ribosomal protein S6, and the eukaryotic translation initiation factor 4E binding protein 1, which represses the eukaryotic initiation factor 4E.9 Therefore, mTOR signaling is involved in the regulation of ribosomal protein translation, cap-dependent translation, and protein synthesis, which is necessary for cell cycle progression, cell growth, and metabolism.9 Dysregulation in mTOR signaling is frequently associated with angiogenesis, tumor growth, and metastasis.9 The mTOR, a key downstream signaling component of phosphatidylinositol 3-kinase/AKT signaling pathway,9 is an attractive target for cancer therapeutics because effective inhibitors, such as everolimus or temsirolimus, are available and exhibit potent activity against various cancers in clinical and preclinical settings.10 Several studies reported that activation of the mTOR signaling pathway is involved in multiple anticancer drug-resistance mechanisms in several types of cancer.11 Therefore, further elucidation of the mTOR activation in human cancers is important for anticancer therapy targeting the mTOR signaling pathway. However, the significance of mTOR activation in the sensitivity of induction chemotherapy with TPF and its effect on the prognosis of locally advanced HNSCC treated with induction chemotherapy with TPF remain largely undefined. Thus, we evaluated mTOR activity by immunohistochemistry (IHC) and investigated the prognostic role of mTOR activation in 107 patients with locally advanced stage IV HNSCC who were treated with induction chemotherapy with TPF. Besides, we also used HNSCC cell lines to determine whether mTOR inhibition could enhance the effect of chemotherapy in vitro and in vivo.
MATERIALS AND METHODS Patient population Patients with locally advanced stage IV HNSCC, who were treated with induction chemotherapy with TPF at Kaohsiung Chang Gung Memorial Hospital, were retrospectively reviewed. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. All patients selected for this retrospective study were required to have pretreatment specimens of biopsy for IHC. The clinical staging was determined according to the American Joint Committee on Cancer (AJCC) staging system, seventh edition. To be selected for induction chemotherapy with TPF in the present study, patients were required to have Eastern Cooperative Oncology Group performance status 0 or 1, age 18 years, adequate bone marrow function (absolute neutrophil count 1.75 3 109/L, platelet count 100 3 109/L), hepatic function (serum total bilirubin 1.5 mg/dL and serum levels of aspartate aminotransferase and alanine aminotransferase
MTOR WITH INDUCTION CHEMOTHERAPY IN
HNSCC
2.5 3 upper limit of normal), and renal function (serum creatinine 1.5 mg/dL). During this period, 107 patients with locally advanced stage IV HNSCC who were treated with induction chemotherapy with TPF were identified.
Treatment and assessment Because the dosage of TPF regimen used in those phase III clinical trials5,7,12 is, in fact, intolerable for most Asian patients and low socioeconomic status patients with severe hematological toxicity,13,14 we modified the dosage of these agents to reduce toxicity and increase tolerability. Docetaxel was administered at dosages of approximately 60 to 65 mg/m2 by intravenous infusion for 1.5 hours, cisplatin at dosages of approximately 60 to 75 mg/m2 by intravenous infusion for 4 hours, and 5fluorouracil (5-FU) at dosages of approximately 600 to 750 mg/m2 per 24 hours as a 96-hour continuous intravenous infusion, which was repeated every 3 weeks per cycle. All patients received 2 to 3 cycles of induction chemotherapy with dose-modified TPF. Prophylactic granulocyte colony-stimulating factors and antibiotics were not routinely used. Within 3 to 6 weeks after the last cycle of induction chemotherapy, radiation was delivered during a 7-week period with the use of conventional fractionation (total dose, 66–70 Gy), which was concurrent with chemotherapy of weekly cisplatin at a dosage of 40 mg/m2 by intravenous infusion. Salvage surgery was performed 6 to 12 weeks after the completion of chemoradiotherapy for patients who had residual disease after chemoradiotherapy, and was also allowed for patients without response to induction chemotherapy with resectable disease. Tumor responses were assessed by clinical evaluation and imaging studies according to the Response Evaluation Criteria in Solid Tumors guideline version 1.0. PFS was calculated from the date of the first induction of chemotherapy to the date of progression or death from any cause, whichever occurred first. OS was calculated from the date of diagnosis until death or last follow-up.
Immunohistochemistry IHC staining was performed using an immunoperoxidase technique. Staining was performed on slides (4 lm) of formalin-fixed, paraffin-embedded tissue sections with primary antibodies against phospho-mTOR (Ser2448, clone 49F9, 1:50; Cell Signaling Technology, Boston, MA), phospho-p70S6K (Thr389, Clone 1A5, 1:75; Cell Signaling Technology), and p16INK4 (BD Biosciences, San Jose, CA). Briefly, after deparaffinization and rehydration, the retrieval of the antigen was performed by treating the slides in 10 mmol/L citrate buffer (pH 6.0) in a hot water bath (958C) for 20 minutes. Endogenous peroxidase activity was blocked for 15 minutes in 0.3% hydrogen peroxide. After blocking with 1% goat serum for 1 hour at room temperature, the sections were incubated with primary antibodies for at least 18 hours at 48C overnight. Immunodetection was performed using the LSAB2 kit (Dako, Carpinteria, CA) followed by 3-30 diaminobenzidine for color development and hematoxylin for counterstaining. Incubation without the primary antibody was used as a negative control. Normal gastric gland15 was used as a positive control. The staining HEAD & NECK—DOI 10.1002/HED
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assessment was independently carried out by 2 pathologists (S.L.W. and W.T.H.) without any information about clinicopathological features or prognosis. The fraction of tumor cells with phosphorylated-mTOR (p-mTOR) and pp70S6K expressions (0% to 100%) were recorded, and the average value of results from 2 pathologists was calculated in each patient. Then, positive p-mTOR and pp70S6K expressions were defined as the presence of at least staining in 10% of tumor cells.15,16 Positive p16 expression was defined as strong nuclear staining in 50% or more of the tumor cells.17,18
Cell culture and methyl thiazolyl-tetrazolium assay Two HNSCC cell lines, SAS (tongue squamous cell carcinoma), and OECM1 (gingival squamous cell carcinoma), were used in this study. SAS cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured in Dulbecco modified Eagle’s medium supplemented with 10% fetal calf serum. OECM1 cells were purchased from the Bioresource Collection and Research Center and cultured in the Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal calf serum. Everolimus (RAD001) was purchased from Sigma–Aldrich (St. Louis, MO). To test the effects of cell proliferation of chemotherapy alone and in combination with everolimus on cell proliferation, cells were plated into 96-well, flat-bottomed plates at 3 3 103 cells per 100 lL per well in Dulbecco modified Eagle’s medium containing 10% FBS. After overnight incubation, triplicate wells were treated with chemotherapy alone, everolimus alone, or chemotherapy in combination with everolimus for 48 hours. The relative percentages of metabolically active cells compared with untreated controls were then determined based on mitochondrial conversion of 3-(4.5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide to formazine. In brief, after incubation, 10 lL of methyl thiazolyl-tetrazolium (Sigma) solution (5 mg/mL) was added to each well for 3 hours, and then the medium was replaced with 150 lL of dimethyl sulfoxide per well. Results were assessed in a 96-well format plate reader by measuring the absorbance at a wavelength of 540 nm using a Titertek Multiscan (Thermo, Finland).
Nude mouse xenograft model Six-week-old male athymic nude mice were used, and all animal experiments conformed to the protocols approved by the Experimental Animal Committee of Chang Gung Memorial Hospital. SAS cells (2 3 106 cells per implantation; 6 mice per group) were implanted subcutaneously on the dorsal gluteal region. Measurements of tumor volume with calipers began 1 week after implantation and were performed twice a week. Tumor volume was calculated from the following formula: tumor volume (mm3) 5 length 3 width2/2, where length and width were the longer and shorter dimensions of the tumor, respectively. When the xenograft tumors reached an average size of 100 to 200 mm3, the mice were divided into 4 groups; each with 6 mice and an even distribution of tumor sizes, and treated as follows: group 1: intraperitoneal phosphate-buffered saline once a week; group 2: intraperitoneal docetaxel 20 mg/kg once a week; group 3: E846
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TABLE 1. Characteristics of 107 patients with locally advanced stage IV head and neck squamous cell carcinoma receiving induction chemotherapy with docetaxel, cisplatin, and 5-fluorouracil. Characteristics
Age,y Median Mean Range Sex Male Female Primary tumor site Hypopharynx Larynx Oropharynx Oral cavity Clinical T classification T1 T2 T3 T4a T4b Clinical N classification N0 N1 N2a N2b N2c N3 Clinical AJCC Stage IVA IVB p16 expression Negative Positive p-mTOR expression Low expression Overexpression p-p70S6K expression Low expression Overexpression Smoking Absent Present Alcohol Absent Present Response to induction chemotherapy CR PR Stable disease Progressive disease
53 52 29–72 101 6 8 (8%) 11 (10%) 39 (36%) 49 (46%) 2 (2%) 11 (10%) 6 (6%) 34 (32%) 54 (50%) 22 (21%) 11 (10%) 1 (1%) 36 (34%) 29 (27%) 8 (7%) 49 (46%) 58 (54%) 103 (96%) 4 (4%) 61 (57%) 46 (43%) 63 (59%) 44 (41%) 10 (9%) 97 (91%) 19 (18%) 88 (82%) 7 (7%) 63 (59%) 25 (23%) 12 (11%)
Abbreviations: AJCC, American Joint Committee on Cancer; p-mTOR, phosphorylated mammalian target of rapamycin; p-p70S6K, phosphorylated p70 ribosomal S6 protein kinase; CR, complete response; PR, partial response.
everolimus 5 mg/kg by oral gavage twice a week; and group 4: intraperitoneal docetaxel 20 mg/kg once a week plus everolimus 5 mg/kg by oral gavage twice a week.
Statistical analysis For patient data, statistical analysis was performed using an SPSS 17 software package (IBM, Armonk, NY).
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FIGURE 1. Immunohistochemical staining of phosphorylatedmammalian target of rapamycin (p-mTOR) in head and neck squamous cell carcinoma. (A) Representative example of low p-mTOR expression. (B) Representative example of p-mTOR overexpression. (C) Representative example of low phosphorylated p70 ribosomal S6 protein kinase (p-p70S6K) expression. (D) Representative example of pp70S6K overexpression. Original magnification 3200.
The chi-square test or Fisher’s exact test was used to compare data between the 2 groups. Multivariate analysis of the response of induction chemotherapy was performed by logistic regression, and all variables were entered into the model. For survival analysis, the Kaplan–Meier method was used for univariate analysis, and the difference between survival curves was tested by a log-rank test. In a stepwise forward fashion, all parameters were, in principle, entered into the Cox regression model to analyze their relative prognostic importance. For all analyses, 2-sided tests of significance were used with p < .05 considered significant. For the cell line experiments, the t test was used for the statistical analysis. Each experiment was carried out independently at least twice, with 3 repeats each. For mice experiments, tumor volumes were compared among the phosphate-buffered saline placebo group, and everolimus group, and docetaxel group, and docetaxel plus everolimus group using the 2-way analysis of variance followed by the Bonferroni post-hoc test.
showed N0 in 22 patients (21%), N1 in 11 patients (10%), N2a in 1 patient (1%), N2b in 36 patients (34%), N2c in 29 patients (27%), and N3 in 8 patients (7%), respectively. Additional analyses according to the AJCC staging system demonstrated a stage IVA tumor for 49 patients (46%) and stage IVB for 58 patients (54%). The primary tumor site was found in the hypopharynx in 8 patients (8%), the larynx in 11 patients (10%), the oropharynx in 39 patients (36%), and the oral cavity in 49 patients (46%). The overall response rate for the 107 patients was 66%. Seven patients (7%) achieved complete response (CR); 63 patients (59%) achieved partial response (PR); 25 patients (23%) had stable disease, and 12 patients (11%) showed progressive disease. At the time of the last analysis, patients had been followed for a minimum of 24 months. The median periods of follow-up were 28.9 months (range, 24.0–42.6 months) for the 50 survivors. The median PFS and OS were 15.6 and 26.4 months, respectively. The 2-year PFS and OS were 45% and 51%, respectively.
RESULTS
Phosphorylated-mammalian target of rapamycin and phosphorylated p70 ribosomal S6 protein kinase expressions in patients with locally advanced stage IV head and neck squamous cell carcinoma
Patient characteristics A total of 107 patients were collected in the study with a median age of 53 years (range, 29–72 years). The characteristics of 107 patients with locally advanced stage IV HNSCC receiving induction chemotherapy with TPF are summarized in Table 1. Among them, 101 were men and 6 were women. The analyses of T classification revealed T1 in 2 patients (2%), T2 in 11 patients (10%), T3 in 6 patients (6%), T4a in 34 patients (32%), and T4b in 54 patients (51%). Further analyses of N classification
Among the 107 patients, 61 patients (57%) showed “low expression” for p-mTOR expression and the other showed “overexpression” (Figure 1). Low expression and overexpression for p-p70S6K were observed in 63 patients (59%) and 44 patients (41%), respectively. The expression of p-mTOR or p-p70S6K was not associated with any clinicopathologic parameters, including age, sex, HEAD & NECK—DOI 10.1002/HED
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TABLE 2. Associations between phosphorylated mammalian target of rapamycin and phosphorylated p70 ribosomal S6 protein kinase expressions and clinicopathologic parameters in 107 patients with locally advanced stage IV head and neck squamous cell carcinoma receiving induction chemotherapy with docetaxel, cisplatin, and 5-fluorouracil. p-mTOR expression Parameters
Age, y
Significance of mammalian target of rapamycin in patients with locally advanced stage IV head and neck squamous cell carcinoma receiving induction chemotherapy with docetaxel, cisplatin, and fluorouracil Shau-Hsuan Li, MD,1,7 Wei-Che Lin, MD, PhD,2,7 Tai-Lin Huang, MD,1,7 Chang-Han Chen, PhD,3,7 Tai-Jan Chiu, MD,1,7 Fu-Min Fang, MD, PhD,4,7 Wan-Ting Huang, MD,5 Cheng-Ming Hsu, FACS, MD,6,7 Sheng-Dean Luo, MD, FACS,6,7 Chi-Chih Lai, MD,6,7 Yan-Ye Su, MD,6,7 Hui-Ching Chuang, MD, PhD, FACS,6,7 Chih-Yen Chien, MD, FACS6,7* 1
Department of Hematology–Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 2Department of Diagnostic Radiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 3Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 4Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 5Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 6Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 7Kaohsiung Chang Gung Head and Neck Oncology Group, Cancer Center, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan.
Accepted 22 April 2015 Published online 18 July 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.24111
ABSTRACT: Background. This study evaluated the significance of mammalian target of rapamycin (mTOR) activation on the prognosis of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) receiving induction chemotherapy with docetaxel, cisplatin, and fluorouracil (TPF). Methods. Immunohistochemistry (IHC) for phosphorylated-mTOR and phosphorylated-p70 ribosomal S6 protein kinase (p70S6K) examined in 107 patients with locally advanced HNSCC receiving TPF was correlated with treatment outcome. The effect of mTOR inhibition on HNSCC cell lines was investigated in vitro and in vivo. Results. Phosphorylated-mTOR expression was independently significantly associated with response to TPF, progression-free survival (PFS),
and overall survival (OS). In cell lines and xenograft models, mTOR inhibitor, everolimus, enhanced the effect of docetaxel. Conclusion. In patients with locally advanced HNSCC treated with TPF, phosphorylated-mTOR expression was independently associated with prognosis. In vitro and in vivo, concomitant inhibition of mTOR enhanced the effect of docetaxel. Our findings suggest the potential of mTOR as a C 2015 Wiley Periodicals, therapeutic target for locally advanced HNSCC. V Inc. Head Neck 38: E844–E852, 2016
INTRODUCTION
for patients with locally advanced HNSCC to improve survival and for organ preservation.3–6 In a Meta-Analysis of Chemotherapy in Head and Neck Cancer, the addition of induction chemotherapy using cisplatin and 5-fluorouracil to local treatment was associated with an improvement in overall survival (OS) and distant failures.4 Recently, randomized trials5,7 revealed induction chemotherapy regimens with docetaxel, cisplatin, and fluorouracil (TPF) significantly improved progression-free survival (PFS) and OS, compared to the conventional regimen of cisplatin and 5-fluorouracil. Several studies8 have shown that patients whose disease responded to neoadjuvant chemotherapy had a better survival rate in comparison with those who received ineffective chemotherapy. After induction chemotherapy with TPF, clinical tumor response is found in 60% to 70% of patients.5,7 However, there is still a portion of patients who do not respond well to induction chemotherapy with TPF. In addition, there are substantial differences in the response to induction chemotherapy and survival between patients with the same clinical stage. Therefore, it is principal to identify patients who are unlikely to respond to induction
Head and neck squamous cell carcinoma (HNSCC) is one of the 10 most frequent cancers worldwide, with an estimate of over 500,000 new cases being diagnosed annually.1 In Taiwan, HNSCC is the fourth most common cancer in men,2 and more than half of the patients present with locally advanced disease, technically unresectable, or with low surgical curability. Although patients with locally advanced HNSCC receive surgery and/or radiotherapy, 40% to 60% of patients eventually develop locoregional and distant recurrences.3 In an attempt to improve treatment outcome, combining chemotherapy with surgery and radiotherapy has been explored. Currently, induction chemotherapy is one of the standard treatment modalities
*Corresponding author: C.-Y. Chien, Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, 123 Ta-Pei Road, Niaosung District, Kaohsiung, Taiwan. E-mail: [email protected] Contract grant sponsor: This work was supported by grants from Chang Gung Memorial Hospital (CMRPG8D0681 and CMRPG8B1263)
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KEY WORDS: head and neck cancer, squamous cell carcinoma, mammalian target of rapamycin (mTOR), induction chemotherapy, docetaxel
PHOSPHORYLATED
chemotherapy to spare them the toxic effects of ineffective chemotherapy and cause a delay for other therapeutic options. A reliable marker that can correctly predict the tumor response to induction chemotherapy in patients with locally advanced HNSCC is important, and this marker may serve as a promising target in the future. The mammalian target of rapamycin (mTOR) is a 289kDa serine/threonine kinase that phosphorylates key eukaryotic translation regulators, including p70 ribosomal S6 protein kinase (p70S6K) that phosphorylates the ribosomal protein S6, and the eukaryotic translation initiation factor 4E binding protein 1, which represses the eukaryotic initiation factor 4E.9 Therefore, mTOR signaling is involved in the regulation of ribosomal protein translation, cap-dependent translation, and protein synthesis, which is necessary for cell cycle progression, cell growth, and metabolism.9 Dysregulation in mTOR signaling is frequently associated with angiogenesis, tumor growth, and metastasis.9 The mTOR, a key downstream signaling component of phosphatidylinositol 3-kinase/AKT signaling pathway,9 is an attractive target for cancer therapeutics because effective inhibitors, such as everolimus or temsirolimus, are available and exhibit potent activity against various cancers in clinical and preclinical settings.10 Several studies reported that activation of the mTOR signaling pathway is involved in multiple anticancer drug-resistance mechanisms in several types of cancer.11 Therefore, further elucidation of the mTOR activation in human cancers is important for anticancer therapy targeting the mTOR signaling pathway. However, the significance of mTOR activation in the sensitivity of induction chemotherapy with TPF and its effect on the prognosis of locally advanced HNSCC treated with induction chemotherapy with TPF remain largely undefined. Thus, we evaluated mTOR activity by immunohistochemistry (IHC) and investigated the prognostic role of mTOR activation in 107 patients with locally advanced stage IV HNSCC who were treated with induction chemotherapy with TPF. Besides, we also used HNSCC cell lines to determine whether mTOR inhibition could enhance the effect of chemotherapy in vitro and in vivo.
MATERIALS AND METHODS Patient population Patients with locally advanced stage IV HNSCC, who were treated with induction chemotherapy with TPF at Kaohsiung Chang Gung Memorial Hospital, were retrospectively reviewed. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. All patients selected for this retrospective study were required to have pretreatment specimens of biopsy for IHC. The clinical staging was determined according to the American Joint Committee on Cancer (AJCC) staging system, seventh edition. To be selected for induction chemotherapy with TPF in the present study, patients were required to have Eastern Cooperative Oncology Group performance status 0 or 1, age 18 years, adequate bone marrow function (absolute neutrophil count 1.75 3 109/L, platelet count 100 3 109/L), hepatic function (serum total bilirubin 1.5 mg/dL and serum levels of aspartate aminotransferase and alanine aminotransferase
MTOR WITH INDUCTION CHEMOTHERAPY IN
HNSCC
2.5 3 upper limit of normal), and renal function (serum creatinine 1.5 mg/dL). During this period, 107 patients with locally advanced stage IV HNSCC who were treated with induction chemotherapy with TPF were identified.
Treatment and assessment Because the dosage of TPF regimen used in those phase III clinical trials5,7,12 is, in fact, intolerable for most Asian patients and low socioeconomic status patients with severe hematological toxicity,13,14 we modified the dosage of these agents to reduce toxicity and increase tolerability. Docetaxel was administered at dosages of approximately 60 to 65 mg/m2 by intravenous infusion for 1.5 hours, cisplatin at dosages of approximately 60 to 75 mg/m2 by intravenous infusion for 4 hours, and 5fluorouracil (5-FU) at dosages of approximately 600 to 750 mg/m2 per 24 hours as a 96-hour continuous intravenous infusion, which was repeated every 3 weeks per cycle. All patients received 2 to 3 cycles of induction chemotherapy with dose-modified TPF. Prophylactic granulocyte colony-stimulating factors and antibiotics were not routinely used. Within 3 to 6 weeks after the last cycle of induction chemotherapy, radiation was delivered during a 7-week period with the use of conventional fractionation (total dose, 66–70 Gy), which was concurrent with chemotherapy of weekly cisplatin at a dosage of 40 mg/m2 by intravenous infusion. Salvage surgery was performed 6 to 12 weeks after the completion of chemoradiotherapy for patients who had residual disease after chemoradiotherapy, and was also allowed for patients without response to induction chemotherapy with resectable disease. Tumor responses were assessed by clinical evaluation and imaging studies according to the Response Evaluation Criteria in Solid Tumors guideline version 1.0. PFS was calculated from the date of the first induction of chemotherapy to the date of progression or death from any cause, whichever occurred first. OS was calculated from the date of diagnosis until death or last follow-up.
Immunohistochemistry IHC staining was performed using an immunoperoxidase technique. Staining was performed on slides (4 lm) of formalin-fixed, paraffin-embedded tissue sections with primary antibodies against phospho-mTOR (Ser2448, clone 49F9, 1:50; Cell Signaling Technology, Boston, MA), phospho-p70S6K (Thr389, Clone 1A5, 1:75; Cell Signaling Technology), and p16INK4 (BD Biosciences, San Jose, CA). Briefly, after deparaffinization and rehydration, the retrieval of the antigen was performed by treating the slides in 10 mmol/L citrate buffer (pH 6.0) in a hot water bath (958C) for 20 minutes. Endogenous peroxidase activity was blocked for 15 minutes in 0.3% hydrogen peroxide. After blocking with 1% goat serum for 1 hour at room temperature, the sections were incubated with primary antibodies for at least 18 hours at 48C overnight. Immunodetection was performed using the LSAB2 kit (Dako, Carpinteria, CA) followed by 3-30 diaminobenzidine for color development and hematoxylin for counterstaining. Incubation without the primary antibody was used as a negative control. Normal gastric gland15 was used as a positive control. The staining HEAD & NECK—DOI 10.1002/HED
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assessment was independently carried out by 2 pathologists (S.L.W. and W.T.H.) without any information about clinicopathological features or prognosis. The fraction of tumor cells with phosphorylated-mTOR (p-mTOR) and pp70S6K expressions (0% to 100%) were recorded, and the average value of results from 2 pathologists was calculated in each patient. Then, positive p-mTOR and pp70S6K expressions were defined as the presence of at least staining in 10% of tumor cells.15,16 Positive p16 expression was defined as strong nuclear staining in 50% or more of the tumor cells.17,18
Cell culture and methyl thiazolyl-tetrazolium assay Two HNSCC cell lines, SAS (tongue squamous cell carcinoma), and OECM1 (gingival squamous cell carcinoma), were used in this study. SAS cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and cultured in Dulbecco modified Eagle’s medium supplemented with 10% fetal calf serum. OECM1 cells were purchased from the Bioresource Collection and Research Center and cultured in the Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal calf serum. Everolimus (RAD001) was purchased from Sigma–Aldrich (St. Louis, MO). To test the effects of cell proliferation of chemotherapy alone and in combination with everolimus on cell proliferation, cells were plated into 96-well, flat-bottomed plates at 3 3 103 cells per 100 lL per well in Dulbecco modified Eagle’s medium containing 10% FBS. After overnight incubation, triplicate wells were treated with chemotherapy alone, everolimus alone, or chemotherapy in combination with everolimus for 48 hours. The relative percentages of metabolically active cells compared with untreated controls were then determined based on mitochondrial conversion of 3-(4.5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide to formazine. In brief, after incubation, 10 lL of methyl thiazolyl-tetrazolium (Sigma) solution (5 mg/mL) was added to each well for 3 hours, and then the medium was replaced with 150 lL of dimethyl sulfoxide per well. Results were assessed in a 96-well format plate reader by measuring the absorbance at a wavelength of 540 nm using a Titertek Multiscan (Thermo, Finland).
Nude mouse xenograft model Six-week-old male athymic nude mice were used, and all animal experiments conformed to the protocols approved by the Experimental Animal Committee of Chang Gung Memorial Hospital. SAS cells (2 3 106 cells per implantation; 6 mice per group) were implanted subcutaneously on the dorsal gluteal region. Measurements of tumor volume with calipers began 1 week after implantation and were performed twice a week. Tumor volume was calculated from the following formula: tumor volume (mm3) 5 length 3 width2/2, where length and width were the longer and shorter dimensions of the tumor, respectively. When the xenograft tumors reached an average size of 100 to 200 mm3, the mice were divided into 4 groups; each with 6 mice and an even distribution of tumor sizes, and treated as follows: group 1: intraperitoneal phosphate-buffered saline once a week; group 2: intraperitoneal docetaxel 20 mg/kg once a week; group 3: E846
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TABLE 1. Characteristics of 107 patients with locally advanced stage IV head and neck squamous cell carcinoma receiving induction chemotherapy with docetaxel, cisplatin, and 5-fluorouracil. Characteristics
Age,y Median Mean Range Sex Male Female Primary tumor site Hypopharynx Larynx Oropharynx Oral cavity Clinical T classification T1 T2 T3 T4a T4b Clinical N classification N0 N1 N2a N2b N2c N3 Clinical AJCC Stage IVA IVB p16 expression Negative Positive p-mTOR expression Low expression Overexpression p-p70S6K expression Low expression Overexpression Smoking Absent Present Alcohol Absent Present Response to induction chemotherapy CR PR Stable disease Progressive disease
53 52 29–72 101 6 8 (8%) 11 (10%) 39 (36%) 49 (46%) 2 (2%) 11 (10%) 6 (6%) 34 (32%) 54 (50%) 22 (21%) 11 (10%) 1 (1%) 36 (34%) 29 (27%) 8 (7%) 49 (46%) 58 (54%) 103 (96%) 4 (4%) 61 (57%) 46 (43%) 63 (59%) 44 (41%) 10 (9%) 97 (91%) 19 (18%) 88 (82%) 7 (7%) 63 (59%) 25 (23%) 12 (11%)
Abbreviations: AJCC, American Joint Committee on Cancer; p-mTOR, phosphorylated mammalian target of rapamycin; p-p70S6K, phosphorylated p70 ribosomal S6 protein kinase; CR, complete response; PR, partial response.
everolimus 5 mg/kg by oral gavage twice a week; and group 4: intraperitoneal docetaxel 20 mg/kg once a week plus everolimus 5 mg/kg by oral gavage twice a week.
Statistical analysis For patient data, statistical analysis was performed using an SPSS 17 software package (IBM, Armonk, NY).
PHOSPHORYLATED
MTOR WITH INDUCTION CHEMOTHERAPY IN
HNSCC
FIGURE 1. Immunohistochemical staining of phosphorylatedmammalian target of rapamycin (p-mTOR) in head and neck squamous cell carcinoma. (A) Representative example of low p-mTOR expression. (B) Representative example of p-mTOR overexpression. (C) Representative example of low phosphorylated p70 ribosomal S6 protein kinase (p-p70S6K) expression. (D) Representative example of pp70S6K overexpression. Original magnification 3200.
The chi-square test or Fisher’s exact test was used to compare data between the 2 groups. Multivariate analysis of the response of induction chemotherapy was performed by logistic regression, and all variables were entered into the model. For survival analysis, the Kaplan–Meier method was used for univariate analysis, and the difference between survival curves was tested by a log-rank test. In a stepwise forward fashion, all parameters were, in principle, entered into the Cox regression model to analyze their relative prognostic importance. For all analyses, 2-sided tests of significance were used with p < .05 considered significant. For the cell line experiments, the t test was used for the statistical analysis. Each experiment was carried out independently at least twice, with 3 repeats each. For mice experiments, tumor volumes were compared among the phosphate-buffered saline placebo group, and everolimus group, and docetaxel group, and docetaxel plus everolimus group using the 2-way analysis of variance followed by the Bonferroni post-hoc test.
showed N0 in 22 patients (21%), N1 in 11 patients (10%), N2a in 1 patient (1%), N2b in 36 patients (34%), N2c in 29 patients (27%), and N3 in 8 patients (7%), respectively. Additional analyses according to the AJCC staging system demonstrated a stage IVA tumor for 49 patients (46%) and stage IVB for 58 patients (54%). The primary tumor site was found in the hypopharynx in 8 patients (8%), the larynx in 11 patients (10%), the oropharynx in 39 patients (36%), and the oral cavity in 49 patients (46%). The overall response rate for the 107 patients was 66%. Seven patients (7%) achieved complete response (CR); 63 patients (59%) achieved partial response (PR); 25 patients (23%) had stable disease, and 12 patients (11%) showed progressive disease. At the time of the last analysis, patients had been followed for a minimum of 24 months. The median periods of follow-up were 28.9 months (range, 24.0–42.6 months) for the 50 survivors. The median PFS and OS were 15.6 and 26.4 months, respectively. The 2-year PFS and OS were 45% and 51%, respectively.
RESULTS
Phosphorylated-mammalian target of rapamycin and phosphorylated p70 ribosomal S6 protein kinase expressions in patients with locally advanced stage IV head and neck squamous cell carcinoma
Patient characteristics A total of 107 patients were collected in the study with a median age of 53 years (range, 29–72 years). The characteristics of 107 patients with locally advanced stage IV HNSCC receiving induction chemotherapy with TPF are summarized in Table 1. Among them, 101 were men and 6 were women. The analyses of T classification revealed T1 in 2 patients (2%), T2 in 11 patients (10%), T3 in 6 patients (6%), T4a in 34 patients (32%), and T4b in 54 patients (51%). Further analyses of N classification
Among the 107 patients, 61 patients (57%) showed “low expression” for p-mTOR expression and the other showed “overexpression” (Figure 1). Low expression and overexpression for p-p70S6K were observed in 63 patients (59%) and 44 patients (41%), respectively. The expression of p-mTOR or p-p70S6K was not associated with any clinicopathologic parameters, including age, sex, HEAD & NECK—DOI 10.1002/HED
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TABLE 2. Associations between phosphorylated mammalian target of rapamycin and phosphorylated p70 ribosomal S6 protein kinase expressions and clinicopathologic parameters in 107 patients with locally advanced stage IV head and neck squamous cell carcinoma receiving induction chemotherapy with docetaxel, cisplatin, and 5-fluorouracil. p-mTOR expression Parameters
Age, y
