J Oral Pathol Med (2014) 43: 344–349 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
doi: 10.1111/jop.12134
wileyonlinelibrary.com/journal/jop
Increased expression of MCM5 is significantly associated with aggressive progression and poor prognosis of oral squamous cell carcinoma Shang-Yang Yu1,2,3, Yi-Ping Wang1,2,3, Julia Yu-Fong Chang1,2,3,4, Wei-Ren Shen1,2,3, Hsin-Ming Chen1,2,3,5, Chun-Pin Chiang1,2,3,5 1
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan; 2School of Dentistry, National Taiwan University, Taipei, Taiwan; 3Department of Dentistry, National Taiwan University Hospital, College of Medicine, Taipei, Taiwan; 4 Department of Oral and Maxillofacial Surgery, Division of Oral Pathology, School of Dentistry, University of Washington, Seattle, WA, USA; 5Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan
BACKGROUND: Overexpression of MCM5 protein has been found to be significantly associated with the progression and prognosis of several human cancers. METHODS: This study used immunohistochemistry to examine the expression of MCM5 protein in 97 specimens of oral squamous cell carcinomas (OSCC), 80 specimens of oral epithelial dysplasia (OED, including 31 mild, 29 moderate, and 20 severe OED samples), and 20 specimens of normal oral mucosa (NOM). RESULTS: We found that the mean nuclear MCM5 labeling indices (LIs) increased significantly from NOM (15 6%), through mild (25 10%), moderate (34 9%), and severe OED (43 12%), to OSCC samples (61 16%, P < 0.001). A significant correlation was found between the higher mean nuclear MCM5 LI and OSCCs with site at the tongue (P = 0.046), larger tumor size (P = 0.032), positive lymph node metastasis (P = 0.003), more advanced clinical stage (P = 0.002), higher histological grade (P = 0.002), deeper invasion depth (P = 0.0001), and perineural invasion (P = 0.0047). Only nuclear MCM5 LI ≧ 60% was identified as independent unfavorable prognostic factor by multivariate regression analyses (P = 0.049). The Kaplan–Meier curve showed that patients with OSCC with a nuclear MCM5 LI ≧ 60% had a significantly poorer cumulative survival than those with a nuclear MCM5 LI < 60% (log-rank test, P = 0.0062). CONCLUSIONS: The increased expression of MCM5 protein begins at the oral pre-cancerous stage. The higher expression of MCM5 protein is significantly associated with the aggressive progression and poor prognosis of OSCC.
Correspondence: Chun-Pin Chiang, Department of Dentistry, National Taiwan University Hospital, No. 1, Chang-Te Street, Taipei 10048, Taiwan. Tel: +886 2 2312 3456 9 66855, Fax: +886 2 23893853, E-mail: [email protected] Accepted for publication October 10, 2013
J Oral Pathol Med (2014) 43: 344–349 Keywords: MCM5; oral epithelial dysplasia; oral squamous cell carcinoma; prognosis; progression
Introduction Oral and oropharyngeal cancer is the sixth most common cancer in the world (1). In Taiwan, oral squamous cell carcinomas (OSCCs) rank as the sixth most prevalent cancer in both sexes and account for the fourth most common cancers in males in 2011 (2). Despite improvement in treatment for oral cancers in the past few decades, the prognosis of oral cancer patients is still poor (3). It has been found that most OSCCs are preceded by a pre-cancerous lesion of oral epithelial dysplasia (OED) (4). For early diagnosis and treatment for OSCC, a biomarker that can reliably predict malignant transformation of OED as well as progression and prognosis of OSCC is needed. The gene for MCM5 protein is located on the chromosome 22q13.1. It is a member protein of the minichromosome maintenance (MCM) protein family. MCMs were first identified in the yeast Saccharomyces cerevisiae as mutants defective in the maintenance of minichromosomes, suggesting a role in plasmid replication and cell cycle progression (5). MCM proteins (MCM2-7) also bind to chromatin in a cell cycle-dependent manner, being tightly bound in late mitosis and G1 while being removed in S- and G2-phases, which is crucial to restrict the replication of the chromosome to only one round per cell cycle (6). The requirement for MCM proteins in cycling cells and their absence in quiescent cells support strong evidence for their potential clinical application as cell proliferation markers. Overexpression of MCM5 protein has been demonstrated in a variety of human cancers (7–12). In addition, higher expression of MCM5 has been found to be significantly associated with larger tumor size, positive lymph node
MCM5 expression in oral cancers Yu et al.
metastasis, more advanced pathological stage, and poorer prognosis of gastric adenocarcinoma (7); more advanced clinical stage, higher histological grade, and poorer prognosis of urinary bladder carcinoma (8) and ovarian adenocarcinoma (9); and higher histological grade of skin SCC (10). Higher positivity for MCM5 protein has been reported in four OSCCs transformed from proliferative verrucous hyperplasia (13). However, the influence of MCM5 protein expression on the progression and prognosis of a large series of OSCC cases has not been investigated. Therefore, this study used immunohistochemistry to examine the expression of MCM5 protein in 97 specimens of OSCC, 80 specimens of OED, and 20 specimens of normal oral mucosa (NOM) to assess mainly the correlation between the expression of MCM5 protein and clinicopathological parameters of OSCCs or survival of patients with OSCC.
Materials and methods Patients and specimens After approval by the Institutional Review Board, we obtained formalin-fixed, paraffin-embedded specimens from 97 patients (87 men and 10 women, mean age 53 years, range 28–80 years) with OSCC, 31 patients with mild OED, 29 patients with moderate OED, and 20 patients with severe OED. Diagnosis of OSCC and OED was based on histological examination of hematoxylin and eosin-stained tissue sections. All patients with OSCC received total surgical excision of their OSCCs and radical or selective neck dissection, and all patients with OED received either incisional biopsy or total excision of their OED lesions at the Department of Oral and Maxillofacial Surgery, National Taiwan University Hospital, Taipei, Taiwan, during the period from 2003 to 2008. If any cervical lymph nodes were diagnosed as having OSCC metastases, post-operative radiation and/or chemoradiation therapy was also included in the treatment protocol. None of these OSCC had received any form of tumor-specific therapy before total surgical excision of their cancers. Clinical staging and TNM status of OSCCs at initial presentation of the tumor were determined by clinical palpation, head-and-neck magnetic resonance imaging, chest X-ray, abdominal sonography, and the whole body bone scan according to the guidelines from AJCC (14). Of the 97 cases of OSCC, 68 (70%) were buccal mucosa and 29 (30%) tongue cancers. Histological features of OSCC were further classified into three different types (well-, moderately-, and poorly differentiated OSCC). Of the 97 OSCC cases, there were 78 (80%) well-, 16 (17%) moderately-, and 3 (3%) poorly differentiated OSCCs. OED lesions were further divided into mild, moderate, or severe dysplasia, when enough dysplastic cells were present in the basal one-third, in the basal two-thirds, or in more than the basal two-thirds but not the complete layer of the oral epithelium, respectively. Twenty biopsy specimens of NOM were obtained from 20 subjects without any oral habits during extraction of impacted permanent mandibular third molars after obtaining informed consent and used as the normal controls. Immunohistochemistry for MCM5 protein All the specimens for immunohistochemical staining were fixed in 10% neutral formalin, embedded in paraffin, and cut
in serial sections of 5 lm. Immunohistochemical staining was performed using a super-sensitive polymer-horseradish peroxidase (HRP) technique. Briefly, tissues sections were deparaffinized and rehydrated. Then, sections were heated in a plastic slide holder (Dako, Copenhagen, Denmark) containing 0.01 M citrate buffer in a microwave oven for 15 min to retrieve antigenicity and treated with 3% H2O2 in methanol for 10 min to quench endogenous peroxidase activity. After washing in 10 mM TBS, pH 7.6, sections were incubated with 10% normal goat serum (BioGenex, San Ramon, CA, USA) to block non-specific binding. Sections were then incubated overnight at 4°C with 1:50 dilution of rabbit anti-human MCM5 monoclonal antibody (Epitomics, Burlingame, CA, USA). The BioGenex Super Sensitive TM detection systemsâ were used for detection of bound antibodies. After washing in TBS, sections were treated with super enhancer reagent for 10 min and subsequently treated with the polymer-HRP reagent for another 10 min. The 0.02% diaminobenzidine hydrochloride (DAB; Zymed Laboratories, San Francisco, CA, USA) containing 0.03% H2O2 was used as chromogen to visualize the peroxidase activity. The preparations were lightly counterstained with hematoxylin, mounted with Permount, and examined using light microscopy. Sections of human cervical squamous cell carcinomas that were previously shown to be positive for MCM5 were used as positive controls, and Tris-buffered saline (TBS) instead of primary antibody was used for negative controls. A prominent brown nuclear staining was considered as positive for MCM5 protein expression in our samples. The sections were initially scanned at low power. For sections that showed heterogeneous staining, the predominant pattern was taken into account for scoring. At least five high-power fields were chosen randomly, and the percentages of positively stained cells in 1000 benign or malignant epithelial cells were counted for each case. The MCM5 labeling indices (LIs) were counted as a ratio of immunostaining-positive cells to the total number of cells counted. An eyepiece graticule was used to ensure that all cells were evaluated once only. Each of these assessments was independently carried by two investigators. In this study, the interobserver reproducibility was 92%. The sections with an interobserver variation of more than 10% were reassessed using a double-headed light microscope to achieve consensus.
345
Statistical analysis All statistical analyses were performed by statistical software Stataâ (Stata Corp LP, College station, TX, USA). The mean nuclear MCM5 LIs for OSCC, OED (severe, moderate, or mild), and NOM samples were compared first among five groups by analysis of variance (ANOVA) and then between any two groups by Student’s ttest. The correlation between nuclear MCM5 LIs in OSCCs and clinicopathological parameters of patients with OSCC was analyzed by Student’s t-test or ANOVA, where appropriate. Cumulative survival was analyzed with the Kaplan–Meier method. Comparison of cumulative survival between groups was performed using the log-rank test. Univariate and multivariate survival analyses were performed with Cox proportional hazard regression model to J Oral Pathol Med
MCM5 expression in oral cancers Yu et al.
346
assess additional prognostic values of the different variables. A P-value of 50 Patients’ gender Male Female Site of cancer Tongue Buccal mucosa T status T1 + T2 T3 + T4 N status N0 N1 + N2 + N3 Clinical staging Stages 1 + 2 Stages 3 + 4 Recurrence Without With
347
LIs, labeling indices; OSCC, oral squamous cell carcinomas. Comparison between groups was performed by Student’s t-test or analysis of variance, where appropriate.
MCM5 LI ≧ 60% (P = 0.0017) as correlating with poor survival. However, only the nuclear MCM5 LI ≧ 60% (P = 0.049) was identified as independent unfavorable prognosis factor by multivariate analyses with Cox proportional hazard regression model (Table 4). Kaplan–Meier curve showed that patients with OSCC with a nuclear MCM5 LI ≧ 60% had a significantly poorer cumulative Table 3 Correlation between the nuclear MCM5 LIs in OSCC samples and histopathological factors of patients with OSCC Mean nuclear MCM5 LI SD (%)
P-value
78 19
59 15 69 18
0.002
63 34
56 16 69 14
0.0001
59 38
60 17 62 15
0.512
90 7
60 17 67 12
0.319
88 9
60 16 67 18
0.2166
76 21
58 17 70 12
0.0047
Case number Histological grade Well Moderately/poorly Invasion depth
doi: 10.1111/jop.12134
wileyonlinelibrary.com/journal/jop
Increased expression of MCM5 is significantly associated with aggressive progression and poor prognosis of oral squamous cell carcinoma Shang-Yang Yu1,2,3, Yi-Ping Wang1,2,3, Julia Yu-Fong Chang1,2,3,4, Wei-Ren Shen1,2,3, Hsin-Ming Chen1,2,3,5, Chun-Pin Chiang1,2,3,5 1
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan; 2School of Dentistry, National Taiwan University, Taipei, Taiwan; 3Department of Dentistry, National Taiwan University Hospital, College of Medicine, Taipei, Taiwan; 4 Department of Oral and Maxillofacial Surgery, Division of Oral Pathology, School of Dentistry, University of Washington, Seattle, WA, USA; 5Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan
BACKGROUND: Overexpression of MCM5 protein has been found to be significantly associated with the progression and prognosis of several human cancers. METHODS: This study used immunohistochemistry to examine the expression of MCM5 protein in 97 specimens of oral squamous cell carcinomas (OSCC), 80 specimens of oral epithelial dysplasia (OED, including 31 mild, 29 moderate, and 20 severe OED samples), and 20 specimens of normal oral mucosa (NOM). RESULTS: We found that the mean nuclear MCM5 labeling indices (LIs) increased significantly from NOM (15 6%), through mild (25 10%), moderate (34 9%), and severe OED (43 12%), to OSCC samples (61 16%, P < 0.001). A significant correlation was found between the higher mean nuclear MCM5 LI and OSCCs with site at the tongue (P = 0.046), larger tumor size (P = 0.032), positive lymph node metastasis (P = 0.003), more advanced clinical stage (P = 0.002), higher histological grade (P = 0.002), deeper invasion depth (P = 0.0001), and perineural invasion (P = 0.0047). Only nuclear MCM5 LI ≧ 60% was identified as independent unfavorable prognostic factor by multivariate regression analyses (P = 0.049). The Kaplan–Meier curve showed that patients with OSCC with a nuclear MCM5 LI ≧ 60% had a significantly poorer cumulative survival than those with a nuclear MCM5 LI < 60% (log-rank test, P = 0.0062). CONCLUSIONS: The increased expression of MCM5 protein begins at the oral pre-cancerous stage. The higher expression of MCM5 protein is significantly associated with the aggressive progression and poor prognosis of OSCC.
Correspondence: Chun-Pin Chiang, Department of Dentistry, National Taiwan University Hospital, No. 1, Chang-Te Street, Taipei 10048, Taiwan. Tel: +886 2 2312 3456 9 66855, Fax: +886 2 23893853, E-mail: [email protected] Accepted for publication October 10, 2013
J Oral Pathol Med (2014) 43: 344–349 Keywords: MCM5; oral epithelial dysplasia; oral squamous cell carcinoma; prognosis; progression
Introduction Oral and oropharyngeal cancer is the sixth most common cancer in the world (1). In Taiwan, oral squamous cell carcinomas (OSCCs) rank as the sixth most prevalent cancer in both sexes and account for the fourth most common cancers in males in 2011 (2). Despite improvement in treatment for oral cancers in the past few decades, the prognosis of oral cancer patients is still poor (3). It has been found that most OSCCs are preceded by a pre-cancerous lesion of oral epithelial dysplasia (OED) (4). For early diagnosis and treatment for OSCC, a biomarker that can reliably predict malignant transformation of OED as well as progression and prognosis of OSCC is needed. The gene for MCM5 protein is located on the chromosome 22q13.1. It is a member protein of the minichromosome maintenance (MCM) protein family. MCMs were first identified in the yeast Saccharomyces cerevisiae as mutants defective in the maintenance of minichromosomes, suggesting a role in plasmid replication and cell cycle progression (5). MCM proteins (MCM2-7) also bind to chromatin in a cell cycle-dependent manner, being tightly bound in late mitosis and G1 while being removed in S- and G2-phases, which is crucial to restrict the replication of the chromosome to only one round per cell cycle (6). The requirement for MCM proteins in cycling cells and their absence in quiescent cells support strong evidence for their potential clinical application as cell proliferation markers. Overexpression of MCM5 protein has been demonstrated in a variety of human cancers (7–12). In addition, higher expression of MCM5 has been found to be significantly associated with larger tumor size, positive lymph node
MCM5 expression in oral cancers Yu et al.
metastasis, more advanced pathological stage, and poorer prognosis of gastric adenocarcinoma (7); more advanced clinical stage, higher histological grade, and poorer prognosis of urinary bladder carcinoma (8) and ovarian adenocarcinoma (9); and higher histological grade of skin SCC (10). Higher positivity for MCM5 protein has been reported in four OSCCs transformed from proliferative verrucous hyperplasia (13). However, the influence of MCM5 protein expression on the progression and prognosis of a large series of OSCC cases has not been investigated. Therefore, this study used immunohistochemistry to examine the expression of MCM5 protein in 97 specimens of OSCC, 80 specimens of OED, and 20 specimens of normal oral mucosa (NOM) to assess mainly the correlation between the expression of MCM5 protein and clinicopathological parameters of OSCCs or survival of patients with OSCC.
Materials and methods Patients and specimens After approval by the Institutional Review Board, we obtained formalin-fixed, paraffin-embedded specimens from 97 patients (87 men and 10 women, mean age 53 years, range 28–80 years) with OSCC, 31 patients with mild OED, 29 patients with moderate OED, and 20 patients with severe OED. Diagnosis of OSCC and OED was based on histological examination of hematoxylin and eosin-stained tissue sections. All patients with OSCC received total surgical excision of their OSCCs and radical or selective neck dissection, and all patients with OED received either incisional biopsy or total excision of their OED lesions at the Department of Oral and Maxillofacial Surgery, National Taiwan University Hospital, Taipei, Taiwan, during the period from 2003 to 2008. If any cervical lymph nodes were diagnosed as having OSCC metastases, post-operative radiation and/or chemoradiation therapy was also included in the treatment protocol. None of these OSCC had received any form of tumor-specific therapy before total surgical excision of their cancers. Clinical staging and TNM status of OSCCs at initial presentation of the tumor were determined by clinical palpation, head-and-neck magnetic resonance imaging, chest X-ray, abdominal sonography, and the whole body bone scan according to the guidelines from AJCC (14). Of the 97 cases of OSCC, 68 (70%) were buccal mucosa and 29 (30%) tongue cancers. Histological features of OSCC were further classified into three different types (well-, moderately-, and poorly differentiated OSCC). Of the 97 OSCC cases, there were 78 (80%) well-, 16 (17%) moderately-, and 3 (3%) poorly differentiated OSCCs. OED lesions were further divided into mild, moderate, or severe dysplasia, when enough dysplastic cells were present in the basal one-third, in the basal two-thirds, or in more than the basal two-thirds but not the complete layer of the oral epithelium, respectively. Twenty biopsy specimens of NOM were obtained from 20 subjects without any oral habits during extraction of impacted permanent mandibular third molars after obtaining informed consent and used as the normal controls. Immunohistochemistry for MCM5 protein All the specimens for immunohistochemical staining were fixed in 10% neutral formalin, embedded in paraffin, and cut
in serial sections of 5 lm. Immunohistochemical staining was performed using a super-sensitive polymer-horseradish peroxidase (HRP) technique. Briefly, tissues sections were deparaffinized and rehydrated. Then, sections were heated in a plastic slide holder (Dako, Copenhagen, Denmark) containing 0.01 M citrate buffer in a microwave oven for 15 min to retrieve antigenicity and treated with 3% H2O2 in methanol for 10 min to quench endogenous peroxidase activity. After washing in 10 mM TBS, pH 7.6, sections were incubated with 10% normal goat serum (BioGenex, San Ramon, CA, USA) to block non-specific binding. Sections were then incubated overnight at 4°C with 1:50 dilution of rabbit anti-human MCM5 monoclonal antibody (Epitomics, Burlingame, CA, USA). The BioGenex Super Sensitive TM detection systemsâ were used for detection of bound antibodies. After washing in TBS, sections were treated with super enhancer reagent for 10 min and subsequently treated with the polymer-HRP reagent for another 10 min. The 0.02% diaminobenzidine hydrochloride (DAB; Zymed Laboratories, San Francisco, CA, USA) containing 0.03% H2O2 was used as chromogen to visualize the peroxidase activity. The preparations were lightly counterstained with hematoxylin, mounted with Permount, and examined using light microscopy. Sections of human cervical squamous cell carcinomas that were previously shown to be positive for MCM5 were used as positive controls, and Tris-buffered saline (TBS) instead of primary antibody was used for negative controls. A prominent brown nuclear staining was considered as positive for MCM5 protein expression in our samples. The sections were initially scanned at low power. For sections that showed heterogeneous staining, the predominant pattern was taken into account for scoring. At least five high-power fields were chosen randomly, and the percentages of positively stained cells in 1000 benign or malignant epithelial cells were counted for each case. The MCM5 labeling indices (LIs) were counted as a ratio of immunostaining-positive cells to the total number of cells counted. An eyepiece graticule was used to ensure that all cells were evaluated once only. Each of these assessments was independently carried by two investigators. In this study, the interobserver reproducibility was 92%. The sections with an interobserver variation of more than 10% were reassessed using a double-headed light microscope to achieve consensus.
345
Statistical analysis All statistical analyses were performed by statistical software Stataâ (Stata Corp LP, College station, TX, USA). The mean nuclear MCM5 LIs for OSCC, OED (severe, moderate, or mild), and NOM samples were compared first among five groups by analysis of variance (ANOVA) and then between any two groups by Student’s ttest. The correlation between nuclear MCM5 LIs in OSCCs and clinicopathological parameters of patients with OSCC was analyzed by Student’s t-test or ANOVA, where appropriate. Cumulative survival was analyzed with the Kaplan–Meier method. Comparison of cumulative survival between groups was performed using the log-rank test. Univariate and multivariate survival analyses were performed with Cox proportional hazard regression model to J Oral Pathol Med
MCM5 expression in oral cancers Yu et al.
346
assess additional prognostic values of the different variables. A P-value of 50 Patients’ gender Male Female Site of cancer Tongue Buccal mucosa T status T1 + T2 T3 + T4 N status N0 N1 + N2 + N3 Clinical staging Stages 1 + 2 Stages 3 + 4 Recurrence Without With
347
LIs, labeling indices; OSCC, oral squamous cell carcinomas. Comparison between groups was performed by Student’s t-test or analysis of variance, where appropriate.
MCM5 LI ≧ 60% (P = 0.0017) as correlating with poor survival. However, only the nuclear MCM5 LI ≧ 60% (P = 0.049) was identified as independent unfavorable prognosis factor by multivariate analyses with Cox proportional hazard regression model (Table 4). Kaplan–Meier curve showed that patients with OSCC with a nuclear MCM5 LI ≧ 60% had a significantly poorer cumulative Table 3 Correlation between the nuclear MCM5 LIs in OSCC samples and histopathological factors of patients with OSCC Mean nuclear MCM5 LI SD (%)
P-value
78 19
59 15 69 18
0.002
63 34
56 16 69 14
0.0001
59 38
60 17 62 15
0.512
90 7
60 17 67 12
0.319
88 9
60 16 67 18
0.2166
76 21
58 17 70 12
0.0047
Case number Histological grade Well Moderately/poorly Invasion depth
