Clin Transl Oncol DOI 10.1007/s12094-013-1136-8

RESEARCH ARTICLE

CHD1L is a novel independent prognostic factor for gastric cancer Z. Su • J. Zhao • G. Xian • W. Geng Z. Rong • Y. Wu • C. Qin

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Received: 23 September 2013 / Accepted: 11 November 2013 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2013

Abstract Background Chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) is involved in malignancies. However, the role of CHD1L in gastric cancer (GC) has not been elucidated. The aim of this study is to explore the clinical role of CHD1L in GC. Methods The gene and protein expression levels of CHD1L were detected by quantitative real-time PCR and Western blot analysis in fresh samples of GC and paired adjacent noncancerous tissue (n = 34). We evaluated the CHD1L expression by immunohistochemistry in a large number of GC patients (n = 616) and paired adjacent noncancerous tissues from December 1, 2004 to December 1, 2008. The correlations of CHD1L expression with clinicopathological features and clinical outcome were analyzed. Results The gene and protein expression levels of CHD1L were higher in fresh samples of GC than in paired adjacent noncancerous tissues as determined by quantitative real-time PCR and Western blot analysis. Immunohistochemical analysis showed that positive expression rates of CHD1L in GC and paired adjacent noncancerous tissues were 58.7 % (361/616) and 7.3 % (45/616), respectively. CHD1L positivity was significantly associated with clinical stage and distant metastasis. GC patients with positive CHD1L expression had shorter overall survival than those with negative CHD1L expression. Multivariate analysis showed that CHD1L was an independent prognostic marker for overall survival [Hazard Ratio

Z. Su (&)  J. Zhao  G. Xian  W. Geng  Z. Rong  Y. Wu  C. Qin Department of Hepatobiliary Surgery, Provincial Hospital Affiliated to Shandong University, 324 Jingwu Road, Jinan 250021, Shandong, China e-mail: [email protected]

(HR) = 5.952, 95 % confidence interval (CI) = 3.194– 11.187, P = 0.0043]. Conclusion These results indicated that CHD1L could serve as a prognostic marker for GC. Keywords Biomarker

Gastric cancer  CHD1L  Prognosis 

Introduction Gastric cancer (GC) is an aggressively invasive and metastatic tumor [1]. Worldwide, GC is one of the most common deadly cancers [2]. Although surgical resection can cure GC at the early stage [3], unfortunately, most patients are diagnosed at an advanced stage and good treatment options are unavailable [4]. Thus, exploring the potential novel biomarkers of GC will help to establish the early diagnosis, improve the treatment regimes and determine the prognosis for GC patients. Amplification of 1q21 is a frequent genetic alteration in GC and many other solid tumors [5]. Recently, one candidate oncogene, chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like gene (CHD1L), has been isolated from the 1q21 amplicon. CHD1L belongs to the SNF2 ATPase superfamily with a carboxy-terminal macrodomain, which is involved in DNA damage repair and cell cycle progression [6, 7]. It is reported that amplification of CHD1L at the genomic level and overexpression of CHD1L at the protein level were detected in 50.6 % (86/170) and 52.4 % (163/311) of informative hepatocellular carcinoma (HCC) tissues, respectively [8]. CHD1L-transfected cells possessed a strong oncogenic ability [8]. Furthermore, studies of HCC have shown that CHD1L expression was significantly associated with

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venous infiltration, microsatellite tumor nodule formation, advanced tumor stage and poor survival [9, 10]. However, the expression of CHD1L and its significance in GC has not been well documented and remains uncertain. In this study, we investigated the expression of CHD1L to evaluate the prognostic roles in a relatively large sample size of GC patients with long-term follow-up and extensive information on clinicopathological characteristics.

50 -GAGCCGGAG CCTTTTGAAGTT-30 , reverse primer 50 -TGCTAGTAGCTTATCCAGCAGG-30 ; b-actin: forward primer 50 -CATGTACGTTGCTATCCAGGC-30 , reverse primer 50 -CTCCTTAAT GTCACGCACGAT-30 . Data were analyzed according to the comparative Ct method and were normalized to b-actin expression in each sample. Western blot

Materials and methods Patient samples The fresh samples of GC (n = 34) and paired adjacent noncancerous tissue were collected for gene and protein expression levels of CHD1L detected by quantitative realtime PCR and Western blot analysis. Furthermore, a total of 616 patients who had surgery for GC between December 1, 2004 and December 1, 2008 at our hospital were included in this study. The group was composed of 371 men and 245 women with a mean age of 62.6 ± 17.4 (28–93) years. Among these patients, 157 had lymph node metastasis. Quantitative real-time PCR, Western blot and immunohistochemistry were performed to evaluate CHD1L expression in all patients. Correlations between CHD1L expression levels and survival were analyzed. This study was approved by the Ethics Committees of Provincial Hospital Affiliated to Shandong University and carried out according to the Declaration of Helsinki. All patients signed written consent prior to surgery to allow for this research.

Fresh GC samples were lysed in cell lysis buffer and the extracted proteins were quantified by the BCA assay kit (Millipore, USA). Total protein was separated by 10 % SDS-PAGE and electroblotted onto a polyvinylidene fluoride membrane (Millipore, USA). The membrane was immunoblotted overnight at 4 °C with primary antibodies against human CHD1L (1:2,000; Sigma-Aldrich, USA) or antibody against b-actin (1:2,000; Santa Cruz, USA). Following three washes with TBST, a horseradish peroxidaseconjugated secondary antibody IgG (1:2,000; Santa Cruz, USA) was incubated with the membrane for 2 h. Signals were detected with ECL detection reagent (Millipore, USA). The images were semiquantified by Quantity One (Bio-Rad, USA). All experiments were performed in triplicate. Immunohistochemical analysis

The 616 patients who underwent gastrectomy were subjected to clinical observation, including chest/abdominal/ pelvic computed tomography (CT) imaging and blood testing at 3-month intervals and annual gastroscopy. Follow-up was in line with National Comprehensive Cancer Network (NCCN) Practice Guidelines in GC. Median follow-up period was 41 (9–78) months for all patients. Overall survival was defined as the period from the time of surgery to clinically or radiologically proven recurrence or metastasis and death, respectively.

All histological sections were examined by two pathologists and representative tumor areas free from necrosis or hemorrhage were pre-marked in formalin-fixed paraffinembedded blocks. Sections were deparaffinized, hydrated and immersed in peroxidase-blocking solution (Dako, Denmark) to inhibit endogenous peroxidase. For antigen retrieval, microwave pretreatment was performed with a 0.01 mol/L citrate buffer (pH 6.0) for 30 min. Sections were incubated overnight at 4 °C with mouse monoclonal antibody to CHD1L (1:50, Abcam). Sections were then incubated in Dako-REAL EnVision/HRP rabbit/mouse detection reagent (Dako) for 20 min at room temperature. Staining was visualized using the diaminobenzidine color substrate. The slides were counterstained with Mayer’s hematoxylin. Absence of primary antibody served as negative control.

Quantitative real-time PCR (qRT-PCR)

Evaluation of immunohistochemical staining

Total RNA from fresh samples was extracted using RNA Kit according to the manufacturer’s instructions and reverse transcribed with the Reverse Transcription Kit (Invitrogen). Six nanograms of cDNA was used for quantification by SYBR Green PCR Kit (Invitrogen). The primers used were as follows: CHD1L: forward primer

To assess the positivity of immune staining, only nuclear staining was regarded as positive. We used a scoring method based on intensity and proportion of stained cells. The percentage of positive tumor cells was determined semiquantitatively and each sample was scored on a scale of 0–4 (0, \1 %; 1, 1–25 %; 2, 26–50 %; 3, 51–75 %;

Follow-up of patients

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4, 76–100 %). Staining intensity was determined as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The immunoreactive score of each tumor was calculated by the sum of these two parameters. The immunohistochemical results were graded as negative (total score; 0–1) or positive (total score; 2–7). All stained sections were assessed by two independent pathologists without knowledge of clinicopathological features, and any differences in interpretation were resolved by consensus.

gastric mucosa tissues. The results showed a CHD1L band, and the expression of CHD1L protein was further semiquantified by densitometry. Consistent with the qRT-PCR results, significantly increased CHD1L protein expression was observed in the GC tissues, when compared with the matched adjacent noncancerous tissues (P \ 0.01, Fig. 2).

Statistical analysis

To further investigate the clinicopathological significances of CHD1L expression, we performed immunohistochemical analysis of the 616 paraffin-embedded GC tissue blocks. The immunohistochemical results were graded as negative or positive. As shown in Fig. 3, in total, positive expression rate of CHD1L in GC samples was 58.7 (361/ 616). By contrast, only 7.3 % (45/616) of paired adjacent noncancerous tissues showed positive CHD1L expression. The correlations between the expression of CHD1L and various clinicopathological parameters are listed in Table 1. The results showed that increased expression of CHD1L was significantly correlated with the depth of tumor (T stage, P = 0.021), nodal involvement (P = 0.0013), distant metastasis (P = 0.011), vascular invasion (P = 0.032) and TNM stage (P = 0.014).

Statistical analyses were performed using SPSS16.0 software (SPSS Inc., USA). Chi-square and Fisher’s exact tests were used for comparisons of categorical variables. Kaplan–Meier plots and log-rank tests were used for survival analysis. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. P \ 0.05 was considered significant.

Results Increased CHD1L mRNA expression in human GC tissue by qRT-PCR

Immunohistochemical analysis of CHD1L expression and its relationship with clinicopathological parameters

The mRNA level of CHD1L was determined by qRT-PCR in 34 fresh GC specimens and paired corresponding, adjacent, noncancerous gastric mucosa tissues. The CHD1L gene expression level was significantly higher in the cancerous tissues, when compared with the adjacent noncancerous tissues (P \ 0.01, Fig. 1). Increased CHD1L protein expression by Western blot Western blot was also performed on the above 34 fresh GC specimens and paired corresponding, adjacent, noncancerous

Fig. 1 Increased mRNA expression of CHD1L in gastric cancer tissues as assessed by real-time quantitative RT-PCR (CHD1L/bactin, n = 34, #P \ 0.001). Horizontal lines represent the mean

Fig. 2 Increased protein expression of CHD1L in gastric cancer as assessed by Western blotting. a Representative result of CHD1L protein expression in 4 paired gastric tumorous and the matched adjacent non-tumorous tissues (A. adjacent matched noncancerous gastric mucosa, T. gastric cancer tissues). b Relative CHD1L protein expression levels in gastric cancer tissues and noncancerous tissues (CHD1L/b-actin, n = 34, #P \ 0.001). Bars represent the mean ± SD

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Clin Transl Oncol Fig. 3 Gastric cancer immunohistochemical score (9100). CHD1L expression by immunohistochemistry in gastric cancers. Shown are representative gastric cancer tissues with immunohistochemical scores of 0, 1, 2 and 3

Table 1 Correlation between clinical features and CHD1L positivity Clinical features

CHD1L Yes n = 361

Positivity No n = 255

p value

Gender Male

191

180

170

75

T1

70

76

T2

73

70

Female Tumor depth

T3

98

59

T4

120

50

Yes

209

104

No

152

151

Yes

228

125

No

133

130

Yes

191

165

No

165

279

N.S.

0.021

Nodal involvement 0.0013

Distant metastasis 0.011

Correlation of CHD1L expression with prognosis using Cox proportional hazard model Consistent with the qRT-PCR and Western blot results, the overall survival of the GC patients with positive CHD1L expression was shorter than that of those with negative CHD1L expression (P \ 0.001, log-rank test, Fig. 4). As shown by the univariate analysis in Table 2, over survival was significantly correlated with nodal status, distant metastasis, stage, vascular invasion and CHD1L positivity. Furthermore, the association between CHD1L positivity and survival was still significant after controlling other prognostic markers in multivariate analysis [Hazard Ratio (HR) = 5.952, 95 % confidence interval (CI) = 3.194–11.187, P = 0.0043] (Table 3). In addition, multivariate analysis showed nodal status, distant metastasis and vascular invasion were also independent prognostic factors.

Vascular invasion 0.032

Discussion

Stage I ? II III ? IV

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157 204

107 148

0.014

Despite improved diagnosis and therapeutic strategies, GC is one of the most deadly human malignancies [2, 11].

Clin Transl Oncol Table 3 Multivariate analysis of prognosis in 616 gastric cancer patients Variables

HR (95 % CI)

P

Depth of invasion T1 ? T2

0.781 (0.132–2.033)

0.093

2.799 (1.064–7.273)

0.015

T3 ? T4 Nodal status N0 N1 ? N2 ? N3 Distant metastasis Absent

9.320 (3.117–19.344)

\0.001

0.602 (0.305–1.098)

0.76

1.604 (1.003–3.615)

0.020

5.952 (3.194–11.187)

0.0043

Present Stage I ? II III ? IV Fig. 4 Kaplan–Meier survival curves of gastric cancer patients (n = 616) after gastrectomy. The survival of patients in the CHD1L-high group was significantly shorter than that of patients in the CHD1L-low group (log-rank test, P \ 0.001)

Vascular invasion Absent Present CHD1L Positive

Table 2 Univariate analysis of prognosis in 616 gastric cancer patients Variables

HR (95 % CI)

P

0.984 (0.301–1.996)

0.086

3.112 (1.865–4.925)

0.021

T3 ? T4 Nodal status N0 N1 ? N2 ? N3 Distant metastasis Absent

6.292 (2.109–17.320)

\0.001

Present Stage I ? II

2.573 (1.410–5.694)

0.0019

III ? IV Vascular invasion Absent

3.112 (1.271–6.044)

\0.001

4.918 (1.979–9.208)

\0.001

Present CHD1L Positive

Statistical tests were two-tailed and the significance level was P \ 0.05 HR hazard ratio, CI confidence interval

Depth of invasion T1 ? T2

Negative

Negative Statistical tests were two-tailed and the significance level was P \ 0.05 HR hazard ratio, CI confidence interval

Therefore, identification of the GC-specific biomarkers is very important for diagnosis, therapy and prognostic prediction. In this study, we examined the CHD1L expression in 616 GC patients in relation to survival and clinicopathological features. Our data indicate that positive

CHD1L expression is found in approximately 58.7 % of GC cases. And we found significant correlations between CHD1L expression and poor clinical outcome, independent of other characteristics. Our results indicate that CHD1L expression is a novel potential prognostic marker for GC. Recent reports show that CHD1L, as a candidate oncogene isolated from 1q21 amplicon, has been found frequently amplified in HCC [8]. In addition, amplification of 1q21 has been associated with HCC metastasis [12]. Previous data demonstrated that CHD1L expression might be a prognostic marker of shorter disease-free survival in HCC patients after surgical resection [13]. Further investigation showed that protein expression of CHD1L is significantly correlated with the metastasis proceeding of ovarian carcinoma [14]. These results suggest that CHD1L might play an important role in promoting tumorigenesis or progression. However, to date, the expression and clinical significance of CHD1L in GC have not been explored. Therefore, in this present study, we aimed to analyze CHD1L protein expression in tumor tissue and to assess its prognostic significance for GC. In the present study, we investigated CHD1L mRNA expression in GC specimens by qRT-PCR, and protein expression in primary GC tissues by Western blot. Results showed that the CHD1L mRNA and protein levels were

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significantly increased in tumor tissue samples, respectively, when compared with those in the adjacent nontumor tissue samples. Moreover, immunohistochemical analysis demonstrated high CHD1L expression in 58.7 % (361/616) cases of GC patients. Moreover, in a relatively large series of GC patients (n = 616), we found that high expression of CHD1L was significantly correlated with T stage of GC, implying that an increase in CHD1L expression might promote tumor growth and invasion. These results suggest that CHD1L might play an important role in the tumorigenesis or progression of GC. In the Kaplan–Meier survival analysis, patients with positive CHD1L expression had significantly shorter overall survival than patients with negative CHD1L expression. The univariate analysis showed that increased expression of CHD1L in GC tissues was significantly correlated with overall survival. Cox hazard ratio regression analyses further demonstrated that CHD1L expression was an independent prognostic factor in GC patients. These results suggest that CHD1L might serve as a valuable prognostic biomarker for GC patients after surgery and as a potential target for gene therapy in the treatment of GC. Taken together, we have demonstrated that positive expression of CHD1L in GC is correlated with a more malignant phenotype and with a poor prognosis in a large number of clinical samples. Our data suggested that CHD1L may function as a valuable prognostic biomarker for GC. Clinical studies of CHD1L as a therapeutic target in GC are warranted. Conflict of interest

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None.

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