Vol. 117 No. 6 June 2014
Survivin and pAkt as potential prognostic markers in squamous cell carcinoma of the head and neck Anja Pickhard,a Simone Gröber,a Anna Katharina Haug,a Guido Piontek,a Markus Wirth,a Ulrich Straßen,a Martina Rudelius,b and Rudolf Reiterc Technical University of Munich, Munich, Germany; University of Ulm, Ulm, Germany
Objective. The purpose of our study was to investigate the expression patterns of cell cycle regulatory proteins and members of the epidermal growth factor receptor (EGFR) signaling pathway in squamous cell carcinoma of the head and neck (HNSCC). Study Design. The expression levels of survivin, Bub1 B (budding uninhibited by benzimidazoles 1 homolog beta), PLK-1 (polo-like kinase 1), Ki-67, cyclin D1, p53, EGFR, pMAPK (phosphorylated mitogen-activated protein kinase), pAkt (phosphorylated protein kinase B), and PTEN (phosphatase and tensin homolog) were studied in a series of 180 tumor samples obtained from HNSCC surgical resections, 50 metastatic lymph node samples, and 72 corresponding noncancerous epithelium samples. Protein expression analysis was performed by immunohistochemical staining. The results were correlated with clinicopathologic features and survival data. Results. Prognostic significance could be found only for the markers survivin and pAkt. Only the marker combination of cyclin D1 and p53 had positive prognosis potential regarding overall survival. Conclusions. Both pAkt and survivin show a positive correlation with distant metastases and may have utility as predictors of long-term outcomes for patients with HNSCC. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;117:733-742)
Head and neck squamous cell carcinoma (HNSCC) has a worldwide incidence of 500 000 cases per year and is the sixth most common cancer type.1,2 Despite improved diagnostic techniques and surgical, chemotherapeutic, and radiotherapeutic schemes for treatment, the 5-year survival rate is still estimated to be 30% to 40%. Thus, HNSCCs fall into the category of malignancies with a poor prognosis.3 An improved understanding of carcinogenesis on a molecular level improves the likelihood that new treatment options will be developed. For this reason, cell cycle regulation is at the center of current research. Survivin (baculoviral inhibitor apoptosis protein repeat-containing 5, BIRC5) is a cell cycle protein, and its expression increases during the G1 phase and reaches its maximum in the G2/M phase.4 To date, 2 major functions of survivin have been characterized: an antiapoptotic property, which is still controversial5,6 (Figure 1), and a role in cell cycle control.7 Bub1 B (budding uninhibited by benzimidazoles 1 homolog beta) belongs to the spindle checkpoint complex. According to current research, Bub1 B appears to have 2 roles within this complex. First, it directly binds to kinetochores and regulates the accurate attachment of chromosomes and the voltage of the microtubules of the
spindle apparatus in metaphase.8,9 Second, it allows for stable bonding to Cdc-20 owing to its catalase activity as part of the 4-membered mitotic checkpoint complex.10,11 PLK-1 (Polo-like kinase 1) is a key enzyme in the regulation of several steps in mitosis and cell proliferation.12 The main tasks of PLK-1 include the completion of mitosis,13 the preparation of a bond between the kinetochore and Mad2 to ensure the correct sequence of chromosome division,14 the phosphorylation of defective kinetochores,15 and participation in the S/G2 checkpoint.16 Ki-67 is exclusively expressed in proliferating cells (G1, S, G2, and M phase) and can be detected by various antibodies.17 Artificial blockage of Ki-67 halts cellular proliferation.18 Although it appears to play an important role in the cell cycle, the specific role of Ki-67 remains unclear, partially because no other comparable protein could be found in humans. Cyclin D1 is part of the cyclin family19 and functions as a key element in cell cycle regulation.20 In HNSCC, cyclin D1 overexpression and gene amplification were detected in 30% to 40% of cases.21,22 One of the most frequent changes in the genome of tumor cells is found
Statement of Clinical Relevance
a
Department of OtolaryngologyeHead and Neck Surgery, Technical University of Munich. b Department of Pathology, Technical University of Munich. c Department of OtolaryngologyeHead and Neck Surgery, Section of Phoniatrics and Pedaudiology, University of Ulm. Received for publication Aug 26, 2013; returned for revision Feb 7, 2014; accepted for publication Feb 10, 2014. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.02.005
We found prognostic significance for the markers survivin and pAkt. Furthermore, significant positive correlations with distant metastases were found. Therefore, we postulate that the markers pAkt and survivin may have utility as predictors of long-term outcomes for patients with head and neck squamous cell carcinoma. 733
ORAL AND MAXILLOFACIAL PATHOLOGY 734 Pickhard et al.
OOOO June 2014
Fig. 1. The pAkt and survivin pathway.
in the TP53 gene on chromosome 17 p13.1. Approximately 50% of tumors in humans have a missense mutation in this tumor suppressor gene.23,24 Early stage HNSCC has this mutation in 52% to 82% of cases.25 However, it is not known why the mutation also appears in normal mucosa without previous contact with carcinogens.26 Approximately 87% to 92% of squamous cell carcinoma cases have mRNA overexpression of the epidermal growth factor receptor (EGFR). Therefore, EGFR modulation appears to be one of the most promising approaches for the specific treatment of HNSCC.27,28 The EGFR-specific monoclonal antibody cetuximab is currently approved for the treatment of HNSCC.29 Additional monoclonal antibodies against members of the EGFR family, such as trastuzumab and pertuzumab, are currently in clinical phase II trials.30 The EGF receptor belongs to the ErbB/HER receptor tyrosine kinase family.31 Ligand binding is followed by a phosphorylation cascade of different elements, including mitogenactivated protein kinase (pMAPK/ERK), phosphoinositide-3-kinase (PI3 K), and the downstream protein kinase B (PKB/Akt). This cascade leads to modifications in the transcription of genes that are crucial for the control of cell proliferation, apoptosis, cellular migration, and angiogenesis.32,33 PTEN (phosphatase and tensin homolog) is the main antagonist of PI3 K and acts as an antagonist of the effects of PI3 K-Akt activation34 (see Figure 1).
The TNM classification is the best proxy for assessing prognosis at diagnosis of HNSCC thus far. However, this prediction can be difficult to apply to the individual patient. Despite adequate multimodal therapy, local recurrence or metastasis appears in 50% of patients with advanced HNSCC.35 To date, it remains unclear which parameters (apart from TNM classification) can be used to identify patients who are predisposed to tumor progression. Prognostic markers are therefore essential for enabling individual risk predictions and may allow individual treatment decisions in the future. The aim of this study was to identify proteins involved in the cell cycle and EGF receptor signaling pathways that are significantly associated with prognosis and overall survival in HNSCC. In addition, associations between clinical characteristics and protein expression patterns were sought, with the aim of separating HNSCCs into distinct subgroups and risk profiles.
MATERIALS AND METHODS Patient selection and tissue samples Paraffin-embedded tumor samples from 180 patients (mean age, 53 years; range, 35-72 years) with squamous cell carcinoma of the oral cavity (n ¼ 33), oropharynx (n ¼ 58), hypopharynx (n ¼ 33), and larynx (n ¼ 56) were included. The patients were treated by radical surgical resection between 1993 and 1997 at the Department of Head and Neck Surgery of the Technical University of Munich or the University of Regensburg;
OOOO Volume 117, Number 6
ORIGINAL ARTICLE Pickhard et al. 735
Table I. Clinical characteristics of patients included in the study Characteristics Age (y) Average Range Sex Male/female Tobacco consumption Smoker/nonsmoker Not specified Alcohol consumption Regularly/not regularly Not specified TNM classification pT classification pT1 pT2 pT3 pT4 pN classification pN0/pNþ pN1 pN2a pN2b pN2c pN2 all pN3 cM classification cM0 cM1 Grading G1/G2/G3 Anatomic location Oral cavity Oropharynx Hypopharynx Larynx
We aimed to obtain at least 3 tissue cylinders per tumor with a diameter of 0.6 mm in each bioptic specimen.
Data 69 45-87 165/15
91.67%/8.33%
109/40 31
60.56%/22.22% 17.22%
92/37 51
51.11%/20.56% 28.33%
25 66 48 41
13.89% 36.67% 26.67% 22.78%
94/86 23 2 39 20 41 0
52.22%/47.78% 12.78% 1.11% 21.67% 11.11% 22.78% 0%
180 0
100% 0%
10/110/60
5.56%/61.11%/33.34%
33 58 33 56
18.33% 32.22% 18.33% 31.11%
75% of the patients received adjuvant radiotherapy. The pT and pN categories of the tumor were determined according to the current TNM classification.36 For all tumors, histopathologic and clinical follow-up data were available for a period of 5 to 13.5 years. These findings were correlated with the expression levels of the markers survivin, Bub1 B, PLK-1, Ki-67, cyclin D1, p53, EGFR, pMAPK, pAkt, and PTEN. The study was approved by the Medical Ethics Committee of the Technical University of Munich. Detailed patient characteristics and histomorphologic features are provided in Table I. Tissue microarray preparation For each of the 180 HNSCC samples, 1 paraffin block was selected from the archives. An experienced pathologist marked the viable, representative areas of the tumor specimens. Core needle biopsy specimens were retrieved from the original tumor blocks using a manual arrayer (Beecher Instruments, Sun Prairie, WI, USA) and positioned in a recipient paraffin array block.
Tissue microarrays and protein analyses For analysis of protein marker expression, an HNSCC tissue microarray (TMA) containing tumor tissues, lymph node metastasis tissues, and noncancerous mucosa from margins of the resection was used. The TMA was sectioned, placed on coated glass slides, and deparaffinized for the subsequent procedures. The thickness of the sections used for immunohistochemistry was 1.5 mm. Immunohistochemical study Fresh 1.5-mm sections from TMA blocks were transferred to glass slides, dewaxed, and rehydrated. An antigen retrieval method (heating in citrate-buffered saline in a microwave oven) was applied according the manufacturer’s recommendations. The TMA slides were cooled and incubated with the primary antibody. Table II shows the antibodies used in this study. The reaction was developed with the labeled streptavidinbiotin-alkaline phosphatase system using fast red as a reaction indicator. After counterstaining with hematoxylin, the slides were dehydrated using an ethanol series and mounted. Tissue samples known to express the respective antigen were used as a positive control. Antibodies of irrelevant specificity with an immunoglobulin isotype identical to that of the primary antibody were used as a negative control. Scoring system for protein expression in immunohistochemical staining A scoring system was used to describe the expression levels of the different proteins. Staining intensity was graded from 0 to 3 points (0 points, no staining; 1 point, low staining intensity; 2 points, moderate staining intensity; 3 points, strong staining intensity). The proportion of stained cells was estimated and graded from 0 to 4 points (0 points, 0% of the tumor cells; 1 point,
Survivin and pAkt as potential prognostic markers in squamous cell carcinoma of the head and neck Anja Pickhard,a Simone Gröber,a Anna Katharina Haug,a Guido Piontek,a Markus Wirth,a Ulrich Straßen,a Martina Rudelius,b and Rudolf Reiterc Technical University of Munich, Munich, Germany; University of Ulm, Ulm, Germany
Objective. The purpose of our study was to investigate the expression patterns of cell cycle regulatory proteins and members of the epidermal growth factor receptor (EGFR) signaling pathway in squamous cell carcinoma of the head and neck (HNSCC). Study Design. The expression levels of survivin, Bub1 B (budding uninhibited by benzimidazoles 1 homolog beta), PLK-1 (polo-like kinase 1), Ki-67, cyclin D1, p53, EGFR, pMAPK (phosphorylated mitogen-activated protein kinase), pAkt (phosphorylated protein kinase B), and PTEN (phosphatase and tensin homolog) were studied in a series of 180 tumor samples obtained from HNSCC surgical resections, 50 metastatic lymph node samples, and 72 corresponding noncancerous epithelium samples. Protein expression analysis was performed by immunohistochemical staining. The results were correlated with clinicopathologic features and survival data. Results. Prognostic significance could be found only for the markers survivin and pAkt. Only the marker combination of cyclin D1 and p53 had positive prognosis potential regarding overall survival. Conclusions. Both pAkt and survivin show a positive correlation with distant metastases and may have utility as predictors of long-term outcomes for patients with HNSCC. (Oral Surg Oral Med Oral Pathol Oral Radiol 2014;117:733-742)
Head and neck squamous cell carcinoma (HNSCC) has a worldwide incidence of 500 000 cases per year and is the sixth most common cancer type.1,2 Despite improved diagnostic techniques and surgical, chemotherapeutic, and radiotherapeutic schemes for treatment, the 5-year survival rate is still estimated to be 30% to 40%. Thus, HNSCCs fall into the category of malignancies with a poor prognosis.3 An improved understanding of carcinogenesis on a molecular level improves the likelihood that new treatment options will be developed. For this reason, cell cycle regulation is at the center of current research. Survivin (baculoviral inhibitor apoptosis protein repeat-containing 5, BIRC5) is a cell cycle protein, and its expression increases during the G1 phase and reaches its maximum in the G2/M phase.4 To date, 2 major functions of survivin have been characterized: an antiapoptotic property, which is still controversial5,6 (Figure 1), and a role in cell cycle control.7 Bub1 B (budding uninhibited by benzimidazoles 1 homolog beta) belongs to the spindle checkpoint complex. According to current research, Bub1 B appears to have 2 roles within this complex. First, it directly binds to kinetochores and regulates the accurate attachment of chromosomes and the voltage of the microtubules of the
spindle apparatus in metaphase.8,9 Second, it allows for stable bonding to Cdc-20 owing to its catalase activity as part of the 4-membered mitotic checkpoint complex.10,11 PLK-1 (Polo-like kinase 1) is a key enzyme in the regulation of several steps in mitosis and cell proliferation.12 The main tasks of PLK-1 include the completion of mitosis,13 the preparation of a bond between the kinetochore and Mad2 to ensure the correct sequence of chromosome division,14 the phosphorylation of defective kinetochores,15 and participation in the S/G2 checkpoint.16 Ki-67 is exclusively expressed in proliferating cells (G1, S, G2, and M phase) and can be detected by various antibodies.17 Artificial blockage of Ki-67 halts cellular proliferation.18 Although it appears to play an important role in the cell cycle, the specific role of Ki-67 remains unclear, partially because no other comparable protein could be found in humans. Cyclin D1 is part of the cyclin family19 and functions as a key element in cell cycle regulation.20 In HNSCC, cyclin D1 overexpression and gene amplification were detected in 30% to 40% of cases.21,22 One of the most frequent changes in the genome of tumor cells is found
Statement of Clinical Relevance
a
Department of OtolaryngologyeHead and Neck Surgery, Technical University of Munich. b Department of Pathology, Technical University of Munich. c Department of OtolaryngologyeHead and Neck Surgery, Section of Phoniatrics and Pedaudiology, University of Ulm. Received for publication Aug 26, 2013; returned for revision Feb 7, 2014; accepted for publication Feb 10, 2014. Ó 2014 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2014.02.005
We found prognostic significance for the markers survivin and pAkt. Furthermore, significant positive correlations with distant metastases were found. Therefore, we postulate that the markers pAkt and survivin may have utility as predictors of long-term outcomes for patients with head and neck squamous cell carcinoma. 733
ORAL AND MAXILLOFACIAL PATHOLOGY 734 Pickhard et al.
OOOO June 2014
Fig. 1. The pAkt and survivin pathway.
in the TP53 gene on chromosome 17 p13.1. Approximately 50% of tumors in humans have a missense mutation in this tumor suppressor gene.23,24 Early stage HNSCC has this mutation in 52% to 82% of cases.25 However, it is not known why the mutation also appears in normal mucosa without previous contact with carcinogens.26 Approximately 87% to 92% of squamous cell carcinoma cases have mRNA overexpression of the epidermal growth factor receptor (EGFR). Therefore, EGFR modulation appears to be one of the most promising approaches for the specific treatment of HNSCC.27,28 The EGFR-specific monoclonal antibody cetuximab is currently approved for the treatment of HNSCC.29 Additional monoclonal antibodies against members of the EGFR family, such as trastuzumab and pertuzumab, are currently in clinical phase II trials.30 The EGF receptor belongs to the ErbB/HER receptor tyrosine kinase family.31 Ligand binding is followed by a phosphorylation cascade of different elements, including mitogenactivated protein kinase (pMAPK/ERK), phosphoinositide-3-kinase (PI3 K), and the downstream protein kinase B (PKB/Akt). This cascade leads to modifications in the transcription of genes that are crucial for the control of cell proliferation, apoptosis, cellular migration, and angiogenesis.32,33 PTEN (phosphatase and tensin homolog) is the main antagonist of PI3 K and acts as an antagonist of the effects of PI3 K-Akt activation34 (see Figure 1).
The TNM classification is the best proxy for assessing prognosis at diagnosis of HNSCC thus far. However, this prediction can be difficult to apply to the individual patient. Despite adequate multimodal therapy, local recurrence or metastasis appears in 50% of patients with advanced HNSCC.35 To date, it remains unclear which parameters (apart from TNM classification) can be used to identify patients who are predisposed to tumor progression. Prognostic markers are therefore essential for enabling individual risk predictions and may allow individual treatment decisions in the future. The aim of this study was to identify proteins involved in the cell cycle and EGF receptor signaling pathways that are significantly associated with prognosis and overall survival in HNSCC. In addition, associations between clinical characteristics and protein expression patterns were sought, with the aim of separating HNSCCs into distinct subgroups and risk profiles.
MATERIALS AND METHODS Patient selection and tissue samples Paraffin-embedded tumor samples from 180 patients (mean age, 53 years; range, 35-72 years) with squamous cell carcinoma of the oral cavity (n ¼ 33), oropharynx (n ¼ 58), hypopharynx (n ¼ 33), and larynx (n ¼ 56) were included. The patients were treated by radical surgical resection between 1993 and 1997 at the Department of Head and Neck Surgery of the Technical University of Munich or the University of Regensburg;
OOOO Volume 117, Number 6
ORIGINAL ARTICLE Pickhard et al. 735
Table I. Clinical characteristics of patients included in the study Characteristics Age (y) Average Range Sex Male/female Tobacco consumption Smoker/nonsmoker Not specified Alcohol consumption Regularly/not regularly Not specified TNM classification pT classification pT1 pT2 pT3 pT4 pN classification pN0/pNþ pN1 pN2a pN2b pN2c pN2 all pN3 cM classification cM0 cM1 Grading G1/G2/G3 Anatomic location Oral cavity Oropharynx Hypopharynx Larynx
We aimed to obtain at least 3 tissue cylinders per tumor with a diameter of 0.6 mm in each bioptic specimen.
Data 69 45-87 165/15
91.67%/8.33%
109/40 31
60.56%/22.22% 17.22%
92/37 51
51.11%/20.56% 28.33%
25 66 48 41
13.89% 36.67% 26.67% 22.78%
94/86 23 2 39 20 41 0
52.22%/47.78% 12.78% 1.11% 21.67% 11.11% 22.78% 0%
180 0
100% 0%
10/110/60
5.56%/61.11%/33.34%
33 58 33 56
18.33% 32.22% 18.33% 31.11%
75% of the patients received adjuvant radiotherapy. The pT and pN categories of the tumor were determined according to the current TNM classification.36 For all tumors, histopathologic and clinical follow-up data were available for a period of 5 to 13.5 years. These findings were correlated with the expression levels of the markers survivin, Bub1 B, PLK-1, Ki-67, cyclin D1, p53, EGFR, pMAPK, pAkt, and PTEN. The study was approved by the Medical Ethics Committee of the Technical University of Munich. Detailed patient characteristics and histomorphologic features are provided in Table I. Tissue microarray preparation For each of the 180 HNSCC samples, 1 paraffin block was selected from the archives. An experienced pathologist marked the viable, representative areas of the tumor specimens. Core needle biopsy specimens were retrieved from the original tumor blocks using a manual arrayer (Beecher Instruments, Sun Prairie, WI, USA) and positioned in a recipient paraffin array block.
Tissue microarrays and protein analyses For analysis of protein marker expression, an HNSCC tissue microarray (TMA) containing tumor tissues, lymph node metastasis tissues, and noncancerous mucosa from margins of the resection was used. The TMA was sectioned, placed on coated glass slides, and deparaffinized for the subsequent procedures. The thickness of the sections used for immunohistochemistry was 1.5 mm. Immunohistochemical study Fresh 1.5-mm sections from TMA blocks were transferred to glass slides, dewaxed, and rehydrated. An antigen retrieval method (heating in citrate-buffered saline in a microwave oven) was applied according the manufacturer’s recommendations. The TMA slides were cooled and incubated with the primary antibody. Table II shows the antibodies used in this study. The reaction was developed with the labeled streptavidinbiotin-alkaline phosphatase system using fast red as a reaction indicator. After counterstaining with hematoxylin, the slides were dehydrated using an ethanol series and mounted. Tissue samples known to express the respective antigen were used as a positive control. Antibodies of irrelevant specificity with an immunoglobulin isotype identical to that of the primary antibody were used as a negative control. Scoring system for protein expression in immunohistochemical staining A scoring system was used to describe the expression levels of the different proteins. Staining intensity was graded from 0 to 3 points (0 points, no staining; 1 point, low staining intensity; 2 points, moderate staining intensity; 3 points, strong staining intensity). The proportion of stained cells was estimated and graded from 0 to 4 points (0 points, 0% of the tumor cells; 1 point,
