Expression level of pre-B-cell leukemia transcription factor 2 (PBX2) as a prognostic marker for gingival squamous cell carcinoma

Expression level of pre-B-cell leukemia transcription factor 2 (PBX2) as a prognostic marker for gingival squamous cell carcinoma 1 first-author †E-mail:yingqiu2009@gmail.com †E-mail:yingqiu2009@gmail.com yingqiu2009@gmail.com 2 3 3 4 5 5 Department of Pathology, School of Medicine, Tsinghua University, Beijng 100084, China Department of Pathology, School of Medicine, Tsinghua University, Beijng 100084, China Department of Pediatric Dentistry, School of Stomatology, Tongji University, Shanghai 200072, China Department of Pediatric Dentistry, School of Stomatology, Tongji University, Shanghai 200072, China Department of Pathology, Tongji University School of Medicine, Shanghai 200092, China Department of Pathology, Tongji University School of Medicine, Shanghai 200092, China Department of Oral Pathology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Department of Oral Pathology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Department of Pathology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Department of Pathology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Objective: 2 In this study, we investigated the interrelationship between clinicopathologic findings and pre-B-cell leukemia transcription factor 2 (PBX2) expression in gingival squamous cell carcinoma (GSCC). Methods: 2 Expression level of PBX2 was immunohistochemically examined in 66 GSCC subjects (30 men and 36 women) with ages ranging from 42 to 85 (median 64.5) years, in which staining intensity in tumor cells was categorized as either weaker (level 1) or equal to/stronger (level 2) than that in the endothelial cells. Results: 2 PBX2 expression is correlated with valosin-containing protein (VCP) expression. Univariate and multivariate analyses revealed a high level of PBX2 expression to be a poor prognosticator for disease-free survival (DFS) and overall survival (OS), and PBX2 expression was an independent prognostic factor for both DFS and OS in GSCC. Conclusions: 2 PBX2 expression level in GSCC is prognostic. PBX2 may be a useful marker to identify the potential for progression in GSCC. Gingival squamous cell carcinoma Pre-B-cell leukemia transcription factor 2 (PBX2) Prognosis Introduction 1 Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the oral cavity (Randhawa et al., 2008 2008 ). Worldwide, the annual incidence of new cases exceeds 300 000 (Lippman and Hong, 2001 2001 ; Jemal et al., 2003 2003 ). Despite advances in diagnosis and treatments during the past 40 years, the overall 5-year survival rates for oral and oropharyngeal SCC have only slightly improved and remain around 50% (Levi et al., 2005 2005 ). For gingival squamous cell carcinoma (GSCC), several prognostic factors have been proposed. Among them, tumor size and lymph node metastases are the main prognostic factors (Yokoo et al., 2002 2002 ). Otherwise, tumor-node-metastasis (TNM) classification has been widely employed for the staging of GSCC (Sobin and Wittekind, 2002 2002 ), but does not always predict clinical outcome accurately. Therefore, identification of additional prognostic factors for GSCC is necessary to establish appropriate treatment modalities for patients with GSCC. We previously showed that a higher level expression of pre-B-cell leukemia transcription factor 2 (PBX2) is correlated with poor prognosis for patients with non-small cell lung cancer (NSCLC), gastric carcinoma (GC), and esophageal squamous cell carcinoma (ESCC) (Qiu et al., 2009 2009 ; 2010 2010 ). PBX2 expression was an independent prognostic indicator for GC and ESCC (Qiu et al., 2010 2010 ). PBX2 protein, a main member of PBX family, is ubiquitously expressed in various tissues. PBX2 works as a cofactor with other proteins to regulate the expression of numbers of genes, thus inducing the execution of various cellular functions, such as anti-apoptosis, and inhibition of cell differentiation and proliferation (van Dijk et al., 1995 1995 ). Experiments have shown that the knocked-down expression of PBX2 decreased in vitro colony formation and in vivo tumorigenic activities in cell lines of GC and ESCC. Increased apoptosis rate and decreased Bcl-2 expression were found in the knocked-down expression of PBX2 (Qiu et al., 2010 2010 ). The previous study also showed that the level of valosin-containing protein (VCP) expression, which is involved in invasion and metastasis of cancers, is correlated with metastatic potential in tumor cells and poor prognosis in many kinds of cancers, including GSCC (Yamamoto et al., 2003 2003 ; 2004a 2004a ; 2004b 2004b ; 2004c 2004c ). The PBX2 expression is correlated with VCP expression, which has been revealed in NSCLC cell lines and in the clinical samples. It has been shown that PBX enhanced the promoter activity of VCP gene through their recognition sites (Qiu et al., 2007 2007 ). The PBX-VCP pathway may play an important role in regulating differentiation and proliferation of tumor cells (Qiu et al., 2009 2009 ). In the present study, the expression patterns of PBX2 and VCP proteins were evaluated by immunostaining in 66 subjects with GSCC who underwent curative surgery, and the association between VCP and PBX2, and its possible prognostic significance were analyzed in comparison with clinicopathological parameters and survival. Subjects and methods 1 The experiment is performed as described by Yamamoto et al. ( 2004a 2004a ). Subjects and tissue samples 2 Tissue samples were obtained from 66 subjects who underwent curative resections for GSCC at the Division of Oral and Maxillofacial Diseases, Osaka University Dental Hospital and the Division of Otorhinolaryngology, Osaka University Hospital, Suita, Japan, during the period from February 1989 to March 1997. There were 30 males and 36 females with ages ranging from 42 to 85 (median 64.5) years. Tumors were located in the maxillary gingiva in 21 subjects and mandibular gingiva in 45 subjects. Samples obtained from the gingival lesions and dissected lymph nodes were fixed in 10% formalin (0.1 g/ml) and routinely processed for paraffin-embedding. Histologic sections cut at 4 μm were stained with hematoxylin and eosin (H & E) and immunoperoxidase procedures (avidin-biotin complex (ABC) method). Stained sections were reviewed independently by two investigators (Ying QIU and Eiichi MORII) to determine histological subtype and lymph node metastasis. Tumor stages were defined based on the pathologic tumor-node-metastasis (pTNM) classification (Sobin and Wittekind, 2002 2002 ). After surgery, all subjects received laboratory examination such as routine peripheral blood cell counts at 1–6-month intervals and chest X-ray at 6–12-month intervals. Adjuvant therapy was performed in 56 subjects, chemotherapy alone in 22 (pre-operatively in 5, post-operatively in 3 and combined pre- and post-operatively in 14), radiotherapy alone in 11 (post-operatively in 2, and combined pre- and post-operatively in 9), and combined chemo- and radiotherapy in 23 (post-operatively in 2, and combined pre- and post-operatively in 21). Cis-platinum (CDDP) and fluorouracil (5-FU) were the main chemotherapeutic agents in the present study. Follow-up period for survivors ranged from 2.7 to 176.7 (median 75.6) months. Immunohistochemistry 2 For the detection of PBX2 and VCP, immunoperoxidase procedure (ABC method) was carried out on paraffin-embedded sections as previously described (Yamamoto et al., 2003 2003 ). Briefly, antigen retrieval was carried out by heating the sections in 10 mmol/L citrate buffer for 5 min. Primary antibodies used were rabbit polyclonal anti-human PBX2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-human VCP (1:3 000; Progen Biotechnik, Heidelberg, Germany). Sections were lightly counterstained by methyl green. Positive staining in endothelial cells was used as internal positive control. For negative controls, non-immunized mouse IgG serum (Vector Laboratories, Burlingame, CA, USA) was used as the primary antibody, and uniformly gave negative results. Stained sections were examined in a blind manner without any prior information of the clinical features of the subjects. Staining intensity in the cytoplasm of the tumor cells was evaluated in comparison to that of endothelial cells, and the staining intensity of tumor cells was categorized as equal to or stronger (level 2) and weaker (level 1) than that of endothelial cells. In total, there were four tumors with more than 50% representative section areas staining negative for PBX2. Thus, we also judged those samples as level 1 staining of PBX2. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of PBX2 2 Total RNA was extracted with GSCC using QuickExtract™ FFPE RNA extraction kit (Epicentre, Madison, USA) from slides in 20 GSCC cases of level 1 and level 2 expression PBX2, respectively, which was evaluated in immunohistochemistry staining. Complementary DNA (cDNA) was synthesized using oligo (dT) primers and SuperScript III reverse transcriptase (Invitrogen). Quantitative PCR was used to quantify mRNA expression of PBX2, using an ABI PRISM 7700 instrument (Applied Biosystems). RNA extracted from noncancerous sample in one subject was used as a standard. RT-PCR was carried out using TaqMan probe/primer sets specific for human PBX2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference for gene amplification (Applied Biosystems). Statistical analysis 2 Data for RT-PCR were expressed as mean±standard error (SE). The significance of differences of the mean values was determined by Student’s t -test. All experiments were performed in triplicate. Overall survival (OS) was measured from the date of surgery. Disease-free survival (DFS) was measured from the date of surgery to local recurrence of the disease, the occurrence of distant metastases, or death due to any cause. The data were analyzed using JMP software (SAS Institute Inc., Cary, NC, USA). The chi-square test and Fisher’s exact test were used to analyze the association between PBX2 expression measured by immunohistochemistry and clinicopathologic features of GSCC. Kaplan-Meier methods with the log-rank test were used to calculate survival rates and estimate differences in survival curves (Kaplan and Meier, 1958 1958 ). The Cox ( 1972 1972 ) proportional hazard regression model with a stepwise procedure was used to analyze the independent prognostic factors. P values of < 0.05 were considered statistically significant. Results 1 Histologic findings 2 Histologically, 36 tumors were classified as well-differentiated, 17 moderately-differentiated, and 13 poorly-differentiated GSCC. Tumor size was less than 2 cm (pT1) in 4 subjects, 2–4 cm (pT2) in 26 subjects, and more than 4 cm (pT3) in 19 subjects. Tumors in the remaining 17 subjects invaded into adjacent tissue (pT4). Dissected lymph nodes were histologically analyzed for the presence or absence of lymph node metastasis; 24 subjects had metastasis-positive nodes. Relationship between PBX2 expression and clinicopathologic factors in GSCC 2 Twenty nine (43.9%) and thirty seven (56.1%) subjects showed level 1 and level 2 PBX2 cytoplasm expressions, respectively (Fig. 1 1 ). In comparison to GSCC with level 1 PBX2 expression, level 2 GSCC showed significantly advanced rates of pT and stage factors of pTNM classification ( P < 0.05). Subjects with tumors of level 2 PBX2 expression showed the higher expression level of VCP (Table 1 1 ). (a, b) Cancer cells showing a high level expression of PBX2 and VCP in GSCC; (c, d) Cancer cells with a low level expression of PBX2 and VCP; (e, f) Cancer cells with a negative expression of PBX2 and VCP in GSCC n : number of patients (total number is 66); pTNM: pathologic tumor-node-metastasis; PBX2: pre-B-cell leukemia transcription factor 2; VCP: valosin-containing protein; NS: not significant Values are expressed as mean±SD or n (%) pT1–3 vs. pT4 Stages 1–2 vs. Stages 3–4 2 Clinicopathologic feature 2 n 2 Value # # 2 P PBX2 level 2 ( n =37) PBX2 level 1 ( n =29) Age (year) 64.6±9.9 64.5±9.6 NS Sex  Female 36 22 (61.1%) 14 (38.9%) NS  Male 30 15 (50.0%) 15 (50.0%) Tumor location  Mandibular gingival 45 24 (53.3%) 21 (46.7%) NS  Maxillary gingival 21 13 (61.9%) 8 (38.1%) 2 Lymph node metastasis  Present 24 11 (45.8%) 13 (54.2%) NS  Absent 42 26 (61.9%) 16 (38.1%) Lymphatic invasion  Present 58 33 (56.7%) 25 (43.3%) NS  Absent 8 4 (50.0%) 4 (50.0%) T(pTNM)  pT1 4 0 (0.0%) 4 (100.0%) < 0.05 * *  pT2 26 12 (46.2%) 14 (53.8%)  pT3 19 12 (63.2%) 7 (36.8%)  pT4 17 13 (76.5%) 4 (23.5%) 2 Histologic differentiation  Poorly 13 8 (61.5%) 5 (38.5%) NS  Moderately 17 10 (58.8%) 7 (41.2%)  Well 36 24 (66.7%) 12 (33.3%) Stage  Stage 1 4 0 (0.0%) 4 (100.0%) < 0.05 ** **  Stage 2 22 10 (45.5%) 12 (54.5%)  Stage 3 23 14 (60.9%) 9 (39.1%)  Stage 4 17 4 (23.5%) 13 (76.5%) Adjuvant therapy  Not performed 10 6 (60.0%) 4 (40.0%) NS  Performed 56 31 (55.4%) 25 (44.6%) VCP expression  Level 2 37 26 (70.3%) 11 (29.7%) <0.05  Level 1 29 11 (37.9%) 18 (62.1%) PBX2 expression in GSCC 2 Quantitative RT-PCR analysis was performed in 20 GSCC with level 1 and level 2 expressions, respectively. Relative ratio of PBX2/β-actin expression in subjects with level 1 and level 2 expressions was 2.8±1.6 and 5.6±2.6 (mean±standard deviation (SD)), respectively ( P < 0.05; Fig. 2 2 ). Averages of mRNA expression with PBX2 level 1 showed ratios lower than those of level 2 ( P < 0.05). Values are expressed as mean±SD Uni- and multivariate analyses of prognostic factors in GSCC patients 2 Five-year DFS and OS rates were 74.8% and 90.3%, respectively. Tumor recurrence was found in 20 subjects, local recurrence in 14 subjects, metastasis to cervical node in five subjects, and metastasis to skin and liver in one subject. Among these, seven subjects died due to the tumor. Subjects with level 1 PBX2 GSCC showed a significantly better 5-year OS rate than those with level 2 PBX2 GSCC (5-year DFS: 88.9% vs. 63.7%, P < 0.05; 5-year OS: 100% vs. 82.1%, P < 0.01) (Table 2 2 and Fig. 3 3 ). Univariate analysis revealed that expression of PBX2 and VCP, pT(TNM), and stage of pTNM were significant factors for both DFS and OS. Lymph node metastasis significantly affected DFS but not OS. Histologic differentiation affected OS but not DFS (Table 2 2 ). Multivariate analysis with factors found to be significant in the univariate analysis revealed that PBX2 expression was an independent prognostic factor for both of OS and DFS. Lymph node metastasis and pT classification were equally significant for DFS, and histologic differentiation was significant for OS (Table 3 3 ). Significant difference was observed between the two groups n : number of patients (total number is 66); DFS: disease-free survival; OS: overall survival; pTNM: pathologic tumor-node-metastasis; PBX2: pre-B-cell leukemia transcription factor 2; VCP: valosin-containing protein; NS: not significant 2 Factor 2 n 2 5-year DFS rate 2 5-year OS rate Mean (%) P Mean (%) P PBX2 expression  Level 2 37 63.7 <0.05 82.1 <0.05  Level 1 29 88.9 100.0 Age  >65 years 33 71.6 NS 96.6 NS  ≤65 years 33 78.8 84.3 Sex  Female 36 71.1 NS 84.5 NS  Male 30 79.7 96.7 Tumor location  Mandibular gingival 45 79.2 NS 88.3 NS  Maxillary gingival 21 59.1 95.0 2 Lymph node metastasis  Present 24 50.9 <0.05 82.5 NS  Absent 42 85.0 94.7 Lymphatic invasion  Present 58 76.9 NS 90.9 NS  Absent 8 56.3 83.3 T(pTNM)  pT4 17 37.8 <0.0001 67.9 <0.05  pT1–3 49 87.5 97.8 2 Histologic differentiation  Moderately+poorly 30 66.7 NS 83.0 <0.05  Well 36 81.8 96.8 Stage  3+4 40 63.0 <0.05 83.6 <0.05  1+2 26 92.3 100.0 VCP expression  Level 2 37 58.9 <0.05 81.9 <0.05  Level 1 29 93.3 100.0 DFS: disease-free survival; OS: overall survival; pTNM: pathologic tumor-node-metastasis; PBX2: pre-B-cell leukemia transcription factor 2; VCP: valosin-containing protein; NS: not significant Factor χ 2 Relative risk P DFS  PBX2 expression   Level 1 vs. level 2 5.22 1.93 <0.05  T(pTNM)   pT1–3 vs. pT4 4.37 1.78 <0.05  Lymph node metastasis   Absent vs. present 9.00 2.15 <0.05  VCP expression   Level 1 vs. level 2 1.60 1.58 NS  Stage   1+2 vs. 3+4 0.38 1.27 NS 4 OS  PBX2 expression   Level 1 vs. level 2 5.35 3781.80 <0.05  T(pTNM)   pT1–3 vs. pT4 0.31 1.35 NS  VCP expression   Level 1 vs. level 2 2.93 3082.10 NS  Histologic differentiation   Well vs. moderately+poorly 4.89 2.79 <0.05  Stage   1+2 vs. 3+4 1.15 1713.50 NS Prognostic significance of PBX2 expression in grade 2 Prognostic significance of PBX2 expression was further analyzed according to the histologic differentiation. Subjects with good differentiation of GSCC showed favorable prognosis regardless of PBX2 expression status (Figs. 4a and 4b 4a and 4b ). However, at poor and moderate differentiations, subjects with level 1 PBX2 expression showed a better OS and DFS rates compared to level 2 patients (Figs. 4c and 4d 4c and 4d ; 5-year OS: 100% vs. 71.1%, P < 0.05; 5-year DFS: 100% vs. 44.4%, P < 0.001). (a, b) There was no significant difference between PBX2 level 1 and level 2 subjects at good differentiation of GSCC; (c, d) Significant difference was observed between PBX2 level 1 and level 2 subjects at poor and moderate differentiations of GSCC Discussion 1 GSCC grows very rapidly and typically invades nearby bone and tissue. The 5-year survival rate of GSCC patients is only 26% (Soo et al., 1988 1988 ; Eicher et al., 1996 1996 ). Standard treatment is still based on surgery and radiation therapy (Byers et al., 1981 1981 ; Soo et al., 1988 1988 ), but it does not change the low survival rates of GSCC. Treatment failures can be attributed to multiple factors but remain difficult to predict because no reliable molecular marker is currently available for early detection or as an indicator of prognosis (Shaw et al., 2009 2009 ). TNM staging is used as a standard method for prediction of prognosis of GSCC; however, prognosis is inconsistent even among patients in the same stage (Gomez et al., 2000 2000 ). Therefore, there is an urgent need to seek an accurate marker of prognosis that might predict aggressive tumors. In a previous study, the correlation between the high expression of PBX2 and poor prognosis was reported for several cancer tissues, including the lung, stomach, and esophagus (Qiu et al., 2009 2009 ; 2010 2010 ). PBX genes encode a family of three-amino-acid loop extension (TALE) homeodomain proteins that function as transcriptional regulators in numerous cell types (Mann and Affolter, 1998 1998 ). PBX2 protein, a main member of the PBX family, works as a cofactor with other proteins, such as homeobox (HOX), and forms dimeric complexes with increasing DNA binding affinity and specificity (Longobardi and Blasi, 2003 2003 ), thus, regulating proliferation and differentiation of normal and cancer cells with those proteins (Kamps et al., 1991 1991 ; Phelan et al., 1995 1995 ). In our present study, to clarify whether PBX2 expression level could be used as a reliable prognostic factor for GSCC, the clinical implications of PBX2 immunostaining with prognosis were assessed. PBX2 expression level was examined in 20 subjects by combined quantitative RT-PCR and immunohistochemical analyses and in 46 subjects by immunohistochemistry alone. In the 20 subjects, there was a clear correlation in PBX2 expression between the protein (immunohistochemistry) and mRNA (RT-PCR) levels, which indicates the reliability of immunohistochemistry for evaluation of PBX2 expression. Among the clinicopathologic factors examined, high PBX2 expression was associated with increased pT classification and stage of TNM. pT and stage were prognostic for aggressive character of tumor growth and invasiveness of tumor. The correlation between PBX2 and pT/stage demonstrated that PBX2 may play a significant role in the development and invasion of GSCC. In addition, high PBX2 expression was associated with high VCP expression. VCP is involved in the regulation of activation of nuclear factor kappa B (NFκB) (Naora et al., 2001 2001 ; Takahashi et al., 2007 2007 ), which is a transcription factor correlated with various cellular activities such as anti-apoptosis, and cell proliferation and invasion (Plowright et al., 2009 2009 ). Univariate analysis revealed that expressions of VCP and PBX2 were significant factors for both DFS and OS. However, multivariate analysis showed that PBX2 expression, not VCP, was an independent prognostic factor for both of OS and DFS (Tables 2 2 and 3 3 ). It has been shown that VCP was an independent prognostic factor for both of OS and DFS by removing PBX2 factor (data not shown). This agrees with Yamamoto et al. ( 2004a 2004a )’s study. Therefore, PBX2 is a stronger prognostic factor than VCP in GSCC. In our previous study, it was shown that PBX2 works as a transcription factor enhancing VCP expression. PBX2 transactivated VCP promoter in NSCLC (Yamamoto et al., 2004b 2004b ). Prognostic significance of PBX2 expression was further analyzed according to the histological differentiation. At poor and moderate differentiations of GSCC, subjects with low PBX2 expression showed a better OS and DFS rates compared to subjects with high expression. 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