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Cancer Biomarkers 15 (2015) 187–194 DOI 10.3233/CBM-140452 IOS Press
The prognostic role of TCF4 expression in locally advanced rectal cancer patients treated with neoadjuvant chemoradiotherapy Xue Doua , Renben Wanga,∗, Xiangjiao Menga, Hongjiang Yana , Shumei Jianga , Kunli Zhua, Xiaoqing Xua , Dong Chena, Xianrang Songb and Dianbin Muc
a Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China b Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China c Department of Pathology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China
Abstract. BACKGROUND: At present no useful factors to predict the sensitivity to neoadjuvant chemoradiotherapy (nCRT) have been established in patients with locally advanced rectal cancer (LARC). OBJECTIVE: The objective of this study was to explore the prognostic role of T cell factor 4 (TCF4) expression in predicting tumor response to nCRT and tumor outcomes for patients with LARC. METHODS: The study enrolled 96 patients who underwent nCRT followed by total mesorectal excision (TME). The TCF4 expression of all patients’ biopsies before nCRT was evaluated by Immunohistochemical staining method. RESULTS: After completion of nCRT, 5 cases (5.2%) achieved clinical complete response (cCR) thus the remaining 91 patients underwent a standardized total mesorectal excision (TME) procedure. There were 44 patients (45.8%) achieved good tumor response (including TRG 3-4 and 5 cCR patients) while poor response (TRG 0-2) was achieved in 52 patients (54.2%). Our results demonstrated that patients with low expression of TCF4 were more sensitive to nCRT than those with high TCF4 expression (P = 0.031). Low TCF4 expression before nCRT and good response were significantly associated with improved 5-year diseasefree survival and 5-year overall survival (P < 0.05). Multivariate analysis confirmed that the pretreatment TCF4 expression was an independent prognostic factor. CONCLUSIONs: Our data revealed that low TCF4 protein expression was a useful predictive factor of good tumor response to nCRT and good outcomes in patients with LARC. Keywords: T cell factor 4 (TCF4), rectal cancer, neoadjuvant chemoradiotherapy, tumor regression grade
1. Introduction Neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision (TME) has been the ∗ Corresponding author: Renben Wang, Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China. E-mail: [email protected].
standard treatment for locally advanced rectal cancer (LARC) patients. Previous studies have demonstrated that nCRT not only offers the lowest rates of local recurrence and patient toxicity [1], but also downstages locally advanced tumors and facilitate the process of surgical excision [2]. However, clinical response to chemoradiotherapy varies greatly and the complete pathologic response rate varies from 8% to 31% [1,3, 4]. This poses a considerable clinical dilemma as pa-
c 2015 – IOS Press and the authors. All rights reserved ISSN 1574-0153/15/$35.00
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X. Dou et al. / The prognostic role of TCF4 expression in locally advanced rectal cancer patients treated with nCRT
tients with poor response will be spared exposure to radiation or chemotherapeutic drugs, which is associated with substantial adverse effects. Accordingly, it is important to identify the clinical or molecular characteristics underlying this resistance. T cell factor 4 (TCF4), also known as TCF7L2, is a Wnt signaling pathway transcription factor involved in regulation of numerous Wnt targeted genes, playing a central role in the embryonic development and maintenance of tissue homeostasis [5]. It has been observed that dysregulation of different components of the Wnt signalling pathway could lead to constitutive activation of TCF-4/β-catenin driven transcription and thus promote variety of human cancers [6–10]. Despite the notoriety of Wnt signaling pathway, its role in the resistance to chemoradiotherapy has been a novel discovery. In neuroblastoma cell lines, Flahaut et al. reported the frizzled-1 Wnt receptor FZD1 could mediate chemoresistance [11]. For colorectal cancer, Spitzner et al. revealed that Wnt signaling pathway genes were significantly over-represented in an in vitro experiment for sensitivity of colorectal cancer cells lines to 5-FU-based chemoradiotherapy [12]. Ghadimi et al. found TCF4 to be significantly overexpressed in resistant tumors that treated with nCRT [13]. All findings promoted us to explore the relationship between TCF4 protein expression and tumor response to nCRT for patients with LARC.
2. Materials and methods 2.1. Patients Ninety-six (96) patients were entered into our study and all patients were diagnosed with primary locally advanced rectal adenocarcinoma. The routine examination included flexible endoscopy with rectal biopsy, complete blood count, and serum CEA and CA199 level tests. Chest X-ray, abdominal and pelvic computed tomography (CT), magnetic resonance imaging (MRI), endoscopic Ultrasonography (EUS) and/or PET-CT were also performed in order to exclude TNM stage I and IV tumors. The study was approved by the Ethics Committee of Shandong Cancer Hospital and Institute and informed consent was obtained from all patients. 2.2. Treatment All patients underwent the treatment of nCRT followed by TME. Preoperative radiotherapy was deliv-
ered with a dose of 45 Gy in 25 fractions, followed by a boost of 5.4 Gy in 3 fractions to the primary tumor over a period of 5.5 weeks. The application of chemotherapy was concurrent with radiotherapy and the chemotherapeutic regimens were not the same. In brief, 28 patients received continuous infusion of 5FU, 13 patients received 5-FU, oxaliplatin and leucovorin, 18 patients received capecitabine and oxaliplatin, and 37 patients received capecitabine. Five patients (5.2%) achieved clinical complete response (cCR) and thus the remaining 91 patients underwent the TME procedure. The interval between the completion of nCRT and surgery was 4–6 weeks. 2.3. Pathologic assessment All 96 patients’ biopsies before nCRT were available. Tumor response was evaluated according to a modification of the tumor regression grading (TRG) described by Dworak et al. [14]. The classification detailed as follows: grade 0, no regression; grade 1, minor regression (dominant tumor mass with obvious fibrosis in 25% or less of the tumor mass); grade 2, moderate regression (dominant tumor mass with obvious fibrosis in 26 to 50% of the tumor mass); grade 3, good regression (dominant fibrosis outgrowing the tumor mass; i.e., more than 50% tumor regression); and grade 4, total regression (no viable tumor cells, only fibrotic mass). We defined TRG 3 and 4 as “good response” while TRG 0, 1 and 2 as “poor response”. The 5 cCR patients were defined as “good response”. 2.4. Immunohistochemical assay of TCF4 The representative central tumor area was selected and biopsies were taken by two investigators. 4-μm sections were taken from the paraffin blocks and then deparaffinized in xylene and rehydrated with distilled water through a graded series of ethanol solutions. The process of antigen retrieval was performed by boiling sections under pressure for 2 min. After cool to room temperature, the sections were stained with primary polyclonal rabbit anti-human TCF4 antibody (ab76151) (Abcam, Cambridge, UK) with a diluted ratio of 1:100 and subsequently secondary goat antirabbit antibody (Beijing Zhongshan Golden Bridge Biotechnology Company, China). TCF4 expression was visualised using 3,3’-diaminobenzidine (DAB). Sections were counterstained in haematoxylin and then dehydrated through a graded series of alcohols and xylene before being mounted under a cover slip.
X. Dou et al. / The prognostic role of TCF4 expression in locally advanced rectal cancer patients treated with nCRT
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Table 1 Correlations between TCF4 expression and tumor response and pretreatment clinicopathological characteristics Clinicopathological characteristics Age < 50 50 Gender Male Female Tumor size(cm)
Cancer Biomarkers 15 (2015) 187–194 DOI 10.3233/CBM-140452 IOS Press
The prognostic role of TCF4 expression in locally advanced rectal cancer patients treated with neoadjuvant chemoradiotherapy Xue Doua , Renben Wanga,∗, Xiangjiao Menga, Hongjiang Yana , Shumei Jianga , Kunli Zhua, Xiaoqing Xua , Dong Chena, Xianrang Songb and Dianbin Muc
a Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China b Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China c Department of Pathology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China
Abstract. BACKGROUND: At present no useful factors to predict the sensitivity to neoadjuvant chemoradiotherapy (nCRT) have been established in patients with locally advanced rectal cancer (LARC). OBJECTIVE: The objective of this study was to explore the prognostic role of T cell factor 4 (TCF4) expression in predicting tumor response to nCRT and tumor outcomes for patients with LARC. METHODS: The study enrolled 96 patients who underwent nCRT followed by total mesorectal excision (TME). The TCF4 expression of all patients’ biopsies before nCRT was evaluated by Immunohistochemical staining method. RESULTS: After completion of nCRT, 5 cases (5.2%) achieved clinical complete response (cCR) thus the remaining 91 patients underwent a standardized total mesorectal excision (TME) procedure. There were 44 patients (45.8%) achieved good tumor response (including TRG 3-4 and 5 cCR patients) while poor response (TRG 0-2) was achieved in 52 patients (54.2%). Our results demonstrated that patients with low expression of TCF4 were more sensitive to nCRT than those with high TCF4 expression (P = 0.031). Low TCF4 expression before nCRT and good response were significantly associated with improved 5-year diseasefree survival and 5-year overall survival (P < 0.05). Multivariate analysis confirmed that the pretreatment TCF4 expression was an independent prognostic factor. CONCLUSIONs: Our data revealed that low TCF4 protein expression was a useful predictive factor of good tumor response to nCRT and good outcomes in patients with LARC. Keywords: T cell factor 4 (TCF4), rectal cancer, neoadjuvant chemoradiotherapy, tumor regression grade
1. Introduction Neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision (TME) has been the ∗ Corresponding author: Renben Wang, Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, University of Jinan, Jinan, Shandong, China. E-mail: [email protected].
standard treatment for locally advanced rectal cancer (LARC) patients. Previous studies have demonstrated that nCRT not only offers the lowest rates of local recurrence and patient toxicity [1], but also downstages locally advanced tumors and facilitate the process of surgical excision [2]. However, clinical response to chemoradiotherapy varies greatly and the complete pathologic response rate varies from 8% to 31% [1,3, 4]. This poses a considerable clinical dilemma as pa-
c 2015 – IOS Press and the authors. All rights reserved ISSN 1574-0153/15/$35.00
188
X. Dou et al. / The prognostic role of TCF4 expression in locally advanced rectal cancer patients treated with nCRT
tients with poor response will be spared exposure to radiation or chemotherapeutic drugs, which is associated with substantial adverse effects. Accordingly, it is important to identify the clinical or molecular characteristics underlying this resistance. T cell factor 4 (TCF4), also known as TCF7L2, is a Wnt signaling pathway transcription factor involved in regulation of numerous Wnt targeted genes, playing a central role in the embryonic development and maintenance of tissue homeostasis [5]. It has been observed that dysregulation of different components of the Wnt signalling pathway could lead to constitutive activation of TCF-4/β-catenin driven transcription and thus promote variety of human cancers [6–10]. Despite the notoriety of Wnt signaling pathway, its role in the resistance to chemoradiotherapy has been a novel discovery. In neuroblastoma cell lines, Flahaut et al. reported the frizzled-1 Wnt receptor FZD1 could mediate chemoresistance [11]. For colorectal cancer, Spitzner et al. revealed that Wnt signaling pathway genes were significantly over-represented in an in vitro experiment for sensitivity of colorectal cancer cells lines to 5-FU-based chemoradiotherapy [12]. Ghadimi et al. found TCF4 to be significantly overexpressed in resistant tumors that treated with nCRT [13]. All findings promoted us to explore the relationship between TCF4 protein expression and tumor response to nCRT for patients with LARC.
2. Materials and methods 2.1. Patients Ninety-six (96) patients were entered into our study and all patients were diagnosed with primary locally advanced rectal adenocarcinoma. The routine examination included flexible endoscopy with rectal biopsy, complete blood count, and serum CEA and CA199 level tests. Chest X-ray, abdominal and pelvic computed tomography (CT), magnetic resonance imaging (MRI), endoscopic Ultrasonography (EUS) and/or PET-CT were also performed in order to exclude TNM stage I and IV tumors. The study was approved by the Ethics Committee of Shandong Cancer Hospital and Institute and informed consent was obtained from all patients. 2.2. Treatment All patients underwent the treatment of nCRT followed by TME. Preoperative radiotherapy was deliv-
ered with a dose of 45 Gy in 25 fractions, followed by a boost of 5.4 Gy in 3 fractions to the primary tumor over a period of 5.5 weeks. The application of chemotherapy was concurrent with radiotherapy and the chemotherapeutic regimens were not the same. In brief, 28 patients received continuous infusion of 5FU, 13 patients received 5-FU, oxaliplatin and leucovorin, 18 patients received capecitabine and oxaliplatin, and 37 patients received capecitabine. Five patients (5.2%) achieved clinical complete response (cCR) and thus the remaining 91 patients underwent the TME procedure. The interval between the completion of nCRT and surgery was 4–6 weeks. 2.3. Pathologic assessment All 96 patients’ biopsies before nCRT were available. Tumor response was evaluated according to a modification of the tumor regression grading (TRG) described by Dworak et al. [14]. The classification detailed as follows: grade 0, no regression; grade 1, minor regression (dominant tumor mass with obvious fibrosis in 25% or less of the tumor mass); grade 2, moderate regression (dominant tumor mass with obvious fibrosis in 26 to 50% of the tumor mass); grade 3, good regression (dominant fibrosis outgrowing the tumor mass; i.e., more than 50% tumor regression); and grade 4, total regression (no viable tumor cells, only fibrotic mass). We defined TRG 3 and 4 as “good response” while TRG 0, 1 and 2 as “poor response”. The 5 cCR patients were defined as “good response”. 2.4. Immunohistochemical assay of TCF4 The representative central tumor area was selected and biopsies were taken by two investigators. 4-μm sections were taken from the paraffin blocks and then deparaffinized in xylene and rehydrated with distilled water through a graded series of ethanol solutions. The process of antigen retrieval was performed by boiling sections under pressure for 2 min. After cool to room temperature, the sections were stained with primary polyclonal rabbit anti-human TCF4 antibody (ab76151) (Abcam, Cambridge, UK) with a diluted ratio of 1:100 and subsequently secondary goat antirabbit antibody (Beijing Zhongshan Golden Bridge Biotechnology Company, China). TCF4 expression was visualised using 3,3’-diaminobenzidine (DAB). Sections were counterstained in haematoxylin and then dehydrated through a graded series of alcohols and xylene before being mounted under a cover slip.
X. Dou et al. / The prognostic role of TCF4 expression in locally advanced rectal cancer patients treated with nCRT
189
Table 1 Correlations between TCF4 expression and tumor response and pretreatment clinicopathological characteristics Clinicopathological characteristics Age < 50 50 Gender Male Female Tumor size(cm)
