ERCC1 mRNA Expression as a Postoperative ...

Ann Surg Oncol DOI 10.1245/s10434-014-3726-2

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

ERCC1 mRNA Expression as a Postoperative Prognostic Marker in Extrahepatic Bile Duct Cancer Shuichiro Uemura, MD1, Hidekazu Kuramochi, MD, PhD2, Ryota Higuchi, MD, PhD1, Go Nakajima, MD, PhD2, and Masakazu Yamamoto, MD, PhD1 Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Tokyo, Japan; 2Department of Chemotherapy and Palliative Care, Tokyo Women’s Medical University, Tokyo, Japan

1

ABSTRACT Background. No reliable biomarker for biliary tract cancer has yet been identified because of the varying composition of the cancer type, differences in tumor location, a mixture of curative and non-curative operations, and differences in operative methods. Methods. Fifty extrahepatic bile duct cancer patients, pathologically diagnosed with papillary or tubular adenocarcinoma who underwent a pancreatoduodenectomy with R0 resection, were included in the study. Messenger RNA (mRNA) expression levels were measured from formalinfixed, paraffin-embedded samples by real-time reversetranscription polymerase chain reaction. Results. The preliminary analysis selected ten patients who have survived more than 5 years (LS group) and ten who had a relapse within 2 years (SS group). mRNA expression of seven target genes was examined, but only excision repair cross-complementing 1 (ERCC1) mRNA levels showed a significant difference between the LS and SS groups (median ERCC1: LS 26.5 vs. SS 9.7; p = 0.0073). The median survival time of patients with high ERCC1 levels was significantly longer than in patients with low ERCC1 levels (p = 0.0105). Thirty more patients with identical backgrounds were added to the study, and ERCC1 mRNA levels were measured in all 50 patients. Those with high ERCC1 mRNA levels had a significantly greater overall survival (OS) time compared with those with low ERCC1 levels (MST: 174 vs. 86 M; p = 0.048). Multivariate analysis found that an absence of lymph node

Ó Society of Surgical Oncology 2014 First Received: 9 November 2013 H. Kuramochi, MD, PhD e-mail: [email protected]

metastases and high ERCC1 expression were significantly associated with improved OS. Conclusion. ERCC1 mRNA expression appears to be a useful prognostic biomarker for extrahepatic bile duct cancer with R0 resection.

The incidence of biliary tract cancer (BTC), a collective term that includes cancers of the gallbladder, bile duct, and ampullary carcinomas, is increasing consistently across many populations, including those of North America, Europe, Asia, and Australia.1 Although recent work is beginning to elucidate the molecular carcinogenic events that play a role in the development of BTC,2 many previous studies combined samples of gallbladder and bile duct cancers and ampullary carcinomas as BTC, as each of these cancers is relatively rare and it is difficult to obtain sufficient samples for each type. While these three malignancies are related anatomically and have similar metastatic patterns, each has distinct clinical presentations, molecular pathology, and prognosis.2 In addition, many of the previous studies did not take into account the type of operation performed when analyzing the relationship between molecular markers and prognosis of BTC. Even within bile duct cancers, operative methods differ between hilar carcinoma, and middle and lower carcinomas, leading to differences in prognosis. The prognosis of bile duct cancer remains poor even after curative resection, with the 5-year survival rate reported to be 27–62 % in distal bile duct cancer patients who underwent curative resection.3 There is no evidence that adjuvant therapy is effective in BTC, although this may be because of the lack of a large randomized clinical trial, and a mixture of cancer types. To identify useful adjuvant therapy for BTC, information about prognostic markers is clinically warranted for the benefit of the BTC

S. Uemura et al.

patient population with an unfavorable prognosis and who need adjuvant chemotherapy. The aim of the study was to investigate molecular prognostic markers for patients with middle and lower extrahepatic bile duct cancers who had received curative resection. In this study, we used specimens of middle and lower extrahepatic bile duct cancers taken from pancreatoduodenectomies, which is the standard operative method for middle and lower bile duct cancers. Seven candidate genes previously reported to be biomarkers of other cancer types were selected for this analysis. Epidermal growth factor receptor (EGFR) is involved in signaling pathways affecting cellular growth, differentiation, and proliferation,4 amphiregulin (AREG), and epiregulin (EREG) are EGFR ligands reported to be prognostic biomarkers in colorectal cancer,5 while matrix metalloproteinase-9 (MMP9) and E-cadherin (CDH1) are key genes of cell adhesion, which are associated with cancer metastases. Poly-ADP-ribose polymerase 1 (PARP1) is elevated in several cancer types and a PARP inhibitor is under clinical trial, while excision repair crosscomplementing 1 (ERCC1) plays an essential role in DNA nucleotide excision repair and is reported to be both a prognostic and predictive biomarker.6 In the first part of this study, we chose ten patients with good prognosis and ten patients with poor prognosis from our pooled samples. The messenger RNA (mRNA) expression levels of these seven genes were analyzed to identify a prognostic biomarker of extrahepatic bile duct cancer. Of these seven genes, only ERCC1 expression differed significantly between the two patient groups. Therefore, ERCC1 expression was analyzed in a further 30 samples to validate whether it could be a candidate prognostic biomarker of bile duct cancer.

MATERIALS AND METHODS Patient Samples Overall, 130 patients with middle or lower extrahepatic bile duct cancer underwent pancreatoduodenectomy at the Institute of Gastroenterology, Tokyo Women’s Medical University between 1990 and 2009. Of these, 95 received a curative resection (R0) and the pathological diagnosis of papillary or tubular adenocarcinoma. Patients with insufficient cancer tissue or those with an unknown prognosis were excluded from the analysis, leaving a total of 50 patients who were analyzed in this study. Fifteen of 50 patients (30 %) had received adjuvant fluoropyrimidinebased chemotherapy after surgery. This study was approved by the Ethics Committee of Tokyo Women’s Medical University, and was performed

in accordance with the Declaration of Helsinki. Patients gave their informed consent to participate in this study. Microdissection Formalin-fixed paraffin-embedded (FFPE) tumor specimens were cut into serial sections with a thickness of 10 lm. Manual microdissection was performed using a scalpel if the histology was homogeneous and the tissue contained more than 90 % cancer cells. For all other samples, laser-capture microdissection (P.A.L.M. Microlaser Technologies AG, Munich, Germany) was performed to ensure that only tumor cells were dissected. RNA Isolation and Complementary DNA (cDNA) Synthesis RNA isolation from FFPE specimens was performed using an RNeasy FFPE Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. From the total RNA yielded, complementary DNA (cDNA) was converted using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Tokyo, Japan). Reverse-Transcription Polymerase Chain Reaction cDNA was pre-amplified using a TaqMan PreAmp Master Mix Kit (Applied Biosystems) according to the manufacturer’s instructions. Quantification of target genes and the internal reference gene (b2-microglobulin) was performed using a fluorescence-based real-time detection method [StepOne real-time polymerase chain reaction (PCR) system, Applied Biosystems]. The primers and probes used were from TaqMan Gene Expression Assays (Applied Biosystems) with Assay IDs: Hs00157415_m1 for ERCC1, Hs00950669_m1 for AREG, Hs00914312_m1 for EREG, Hs00193306_m1 for EGFR, Hs00234579_m1 for MMP9, Hs01013953_m1 for CDH1, Hs00242302_m1 for PARP1, and Hs99999907_m1 for b2-microglobulin. The PCR reaction mixture consisted of 10 ll TaqMan Fast Universal PCR Master Mix, no UNG (Applied Biosystems), 5 ll preamplified cDNA sample, 1 ll TaqMan Gene Expression Assays primers and probe (209), and 3 ll nuclease-free water. Cycling conditions were 95 °C for 20 s, followed by 40 cycles at 95 °C for 1 s and 60 °C for 20 s. The threshold cycle (CT) value for each gene was determined by SDS software v1.2 (Applied Biosystems). The delta-CT (DCT) value, which is the difference between the CT value of the target gene and that of the endogenous control gene, was also calculated using the same software. Delta-DCT (DDCT), which is the difference in the DCT value for each sample and the highest DCT value as a calibrator, was also calculated.

ERCC1 in Extrahepatic Bile Duct Cancer

The 2-DDCT number was used for relative mRNA quantification. Statistical Analysis Clinicopathological background comparisons were assessed using the chi square test. The comparisons of median mRNA levels of target genes between long-term survivors and patients with early recurrence were assessed using the Mann–Whitney U test. The Kaplan–Meier method was used for survival curves, and the log-rank test was used for statistical analysis. Overall survival (OS) was defined as the time from the day of operation to death from any cause. The cutoff value of ERCC1 was defined as the 75th percentile. Statistical significance was recognized at p values \0.05. All values were two-sided.

seven, only ERCC1 showed statistically significant differences in expression levels between the two groups (p = 0.0073): ERCC1 expression levels were significantly higher in the LS group (median 26.5) than the SS group (median 9.7). Excision Repair Cross-Complementing 1 (ERCC1) Messenger RNA (mRNA) Level and Prognosis The median OS time for these 20 patients was 53.5 months (range 1.8–180 months). Patients with high ERCC1 mRNA expression levels (n = 5; cutoff: 75th percentile) had a significantly longer OS than those with low levels (n = 15; median survival: not reached vs. 13.4 months; p = 0.0105). Validation of ERCC1 mRNA Expression as a Prognostic Marker in Extrahepatic Bile Duct Cancer

RESULTS Gene Expression in Long-Term Survivors and Patients with Early Recurrence In the initial analysis, ten long-term survivors, who had survived more than 5 years without recurrence (LS group), and ten patients with early recurrence, who had recurred and died within 2 years of surgery (SS group), were selected from the group of 50 samples. mRNA levels of EGFR, AREG, EREG, MMP9, CDH1, PARP1, and ERCC1 were measured in both groups. The characteristics of the 20 patients are shown in Table 1. Age, sex, histopathology, and recurrent site did not differ between the two groups. Expression levels between the samples from the LS and SS groups for these seven genes are shown in Fig. 1. Of the TABLE 1 Characteristics of the ten patients in the LS group and the ten in the SS group

Based on this preliminary experiment, we hypothesized that ERCC1 mRNA expression is a potentially useful biomarker for predicting survival time after curative resection in extrahepatic bile duct cancer. Thus, the remaining 30 patients from the 50 initially identified with sufficient tumor samples and known prognosis were also examined for ERCC1 mRNA expression. Characteristics of these 50 patients (including 20 from the preliminary experiment) are shown in Table 2. One patient had received cisplatin via hepatic artery infusion in the adjuvant setting after surgery, but none of the others had received platinum-based chemotherapy either before or after surgery, or upon recurrence. Survival SS group (n = 10)

LS group (n = 10)

p value

Age [years; mean (range)]

67.5 (54–84)

60.4 (48–80)

0.93

Sex (male/female)

6/4

8/2

0.33

Tumor location (middle/lower)

1/9

2/8

0.53

Histopathology (pap/tub1/tub2/tub3)

1/2/5/2

1/1/6/2

0.94

pT (1/2/3/4)

2/0/6/2

2/1/5/2

0.78

pN (0/1)

3/7

6/4

0.37

Stage (IA/IB/IIA/IIB/III)

2/0/1/4/3

2/0/1/5/2

0.96

Local

1

0

Liver

4

0

Lymph nodes Others

2 2

1 0

Total

9

1

14

174

Recurrent site

LS group long-term survival group, SS group early relapse group

Median survival time (months)

\0.001

6 4 2 0

Relative mRNA expression

SS group

60 50 40 30 20 10 0

SS group

1000 800 600 400 200 0 -200

LS group

AREG (p=0.2703)

LS group

MMP9 (p=0.0604)

SS group

200

400 350 300 250 200 150 100 50 0 LS group

ERCC1 (p=0.0073)

150 100 50 0

LS group

EREG (p=0.5186)

SS group


8

1200

SS group


10


EGFR (p=0.9698) 12



S. Uemura et al.

150

LS group

PARP1 (p=0.9490)

125 100 75 50 25 0

SS group

LS group


CDH1 (p=0.4274) 15

10

5

0 SS group

LS group

FIG. 1 mRNA expression of seven target genes in the LS and SS groups. Only ERCC1 showed a significant difference. mRNA messenger RNA, LS group long-term survival group, SS group early

relapse group, MMP9 matrix metalloproteinase-9, ERCC1 excision repair cross-complementing 1, AREG amphiregulin, EREG epiregulin, PARP1 poly-ADP-ribose polymerase 1, CDH1 E-cadherin

The median survival time of these 50 patients was 174 months (range 5–240 months). ERCC1 mRNA expression was measurable in all 50 patients, and median levels were 9.12 (range 1.00–181.0). When the 75th percentile (16.6) was used as a cutoff level, 13 patients were categorized in the high ERCC1 expression group and 37 in the low expression group. Patients with high ERCC1 mRNA levels had significantly improved OS than those with low levels (median survival 174 vs. 86 months; 5 year survival rate: 92 vs. 52 %; p = 0.048; Fig. 2). Multivariate analysis (Table 3) showed that lymph node metastasis (p = 0.0002) and ERCC1 mRNA expression (p = 0.01) were significant independent prognostic markers after the curative resection of extrahepatic bile duct cancer.

for extrahepatic bile duct cancer. ERCC1, an excision nuclease located on chromosome 19q13, plays an essential role in nucleotide excision repair of DNA, and has been reported to be involved in the response to platinum-based chemotherapies such as cisplatin and oxaliplatin. ERCC1 forms a heterodimer with xeroderma pigmentosum F (XPF) and executes a 50 incision into the damaged DNA strand, a process that is the last of several steps to remove the platinum-damaged DNA lesion.7–9 Therefore, high levels of intratumoral ERCC1 mRNA are expected to be associated with resistance to platinum-based chemotherapies. As expected, several studies demonstrated that this is indeed the case for higher ERCC1 levels in colorectal, gastric, non-small cell lung cancer (NSCLC), and ovarian cancers10–13 in a metastatic setting. The consensus therefore appears to be that higher ERCC1 mRNA expression is related to poor outcome in advanced cancer patients treated with platinum-based chemotherapy. On the other hand, the role of ERCC1 in cancer patients undergoing curative resection remains controversial. Several previous studies demonstrated that high ERCC1

DISCUSSION In the present study, ERCC1 mRNA expression was screened as a prognostic marker and showed significant differentiation in patient survival after curative resection

ERCC1 in Extrahepatic Bile Duct Cancer TABLE 2 Characteristics of all 50 patients according to ERCC1 mRNA expression levels

ERCC1 expression levels High (n = 13)

Low (n = 37)

p value 0.94

Age [years; mean (range)]

66.3 (46–84)

61.5 (30–80)

Sex (male/female)

7/6

10/27

0.079

Tumor location (middle/lower)

0/13

9/28

0.050

Histopathology (pap/tub1/tub2/tub3)

2/3/6/2

6/7/18/6

0.99

pT (1/2/3/4)

3/1/7/2

7/3/23/4

0.95

pN (0/1)

6/7

20/14

0.43

Stage (IA/IB/IIA/IIB/III)

3/1/1/5/3

7/2/7/17/4

0.72

Adjuvant chemotherapy (yes/no)

2/11

15/22

0.084

Local

0

2

Liver Lymph nodes

3 1

6 7

Others

1

3

Total

5

16

Recurrence site

ERCC1 excision repair crosscomplementing 1, mRNA messenger RNA

proportion of survival

1.0

0.8

0.6

0.4

0.2

ERCC1 high (n=13) ERCC1 low (n=37)

0.0 0

50

100

150

200

months

FIG. 2 Kaplan–Meier curves of OS by ERCC1 mRNA expression. Patients with high ERCC1 mRNA levels showed significantly longer survival times than those with low levels. ERCC1 excision repair cross-complementing 1, mRNA messenger RNA

expression was associated with favorable survival after curative resection in NSCLC and gallbladder cancer,6,14–16 while another reported that high ERCC1 expression was associated with reduced survival time in pancreas adenocarcinoma.17 Squires et al.18 found that high ERCC1 expression was associated with decreased OS in the subset of patients who received adjuvant chemotherapy in resected gastric adenocarcinoma, while, conversely, in patients who underwent resection only, high ERCC1 expression demonstrated a trend toward improved OS. Olaussen et al.14 reported similar results in which adjuvant cisplatinbased chemotherapy significantly prolonged survival among patients with ERCC1-negative tumors; however, among patients who did not receive adjuvant chemotherapy, those with ERCC1-positive tumors survived for longer than those with ERCC1-negative tumors in completely

resected NSCLC. It has been suggested that in patients who have not received chemotherapy, high ERCC1 expression reflects an intact DNA repair mechanism that may reduce the accumulation of genetic aberrations, and contribute to a decrease in malignant potential and relapse after curative resection.19 In the studies that previously showed favorable outcomes associated with high ERCC1, the percentages of patients who received adjuvant chemotherapy were very low, at 0 %11,16 and 1.9 %.6 However, in the study of Maithel et al.17 which showed a poor outcome in pancreatic cancer patients with high ERCC1 expression after curative resection, this percentage was 77 %. This supports the finding that high ERCC1 expression leads to a favorable outcome in patients who have never received adjuvant chemotherapy after curative resection. In our study, 17 patients (34 %) received adjuvant chemotherapy after curative resection. However, none received platinum-based therapy, instead being administered oral fluoropyrimidine only. This therefore seems to have little effect on ERCC1 behavior. Few studies have reported an association between bile duct cancer and ERCC1. As bile duct cancer is a relatively rare malignancy, gallbladder cancers, bile duct cancers (intrahepatic, extrahepatic, and hilar), and ampullary carcinomas are often combined and analyzed together as BTC.20–23 However, even within bile duct cancers, intrahepatic and extrahepatic types require different operative methods, so prognoses are different. Some reports also combined operable and inoperable patients, but these too have different prognoses.21 Thus, we postulate that each malignancy should be analyzed separately against a uniform patient background to determine an association between molecular marker and prognosis. In the present

S. Uemura et al. TABLE 3 Multivariate analysis using the Cox proportional hazard method Factor

Cutoff

No. of patients

Univariate analysis HR (95 % CI)

Age (years) Sex pN pT Adjuvant chemotherapy ERCC1

B65

24

1.00

[65

26

1.66 (0.65–4.36)

Male

34

1.00

Female

16

0.89 (0.28–2.37)

0

26

1.00

1

24

7.55 (2.42–33.4)

T1, T2 T3, T4

14 36

1.00 2.17 (0.71–9.42)

Multivariate analysis p value

HR (95 % CI)

p value

0.28 0.83 1.00

Yes

17

1.00

No

33

0.74 (0.29–2.05)

High

13

1.00

Low

37

4.22 (1.17–27.00)

0.0002

13.1 (2.98–101.8)

0.0002

0.19

1.00 2.56 (0.40–20.8)

0.31

0.55 1.00 0.025

6.55 (1.46–60.1)

0.01

HR hazard ratio, CI confidence interval, ERCC1 excision repair cross-complementing 1

study, all patients had the same cancer type, the same tumor location, underwent the same operative method, and had the same curability. To the best of our knowledge, this is the only study to show an association between gene expression levels and prognosis in such a uniform group of patients with extrahepatic bile duct cancer. Real-time reverse transcription-PCR was used to measure mRNA expression levels in this study, compared with the immunohistochemistry staining used in previous studies that showed an association between ERCC1 and BTC.20–23 The results of immunohistochemistry staining are often affected by differences in the antibodies used, laboratory and sample conditions, and the subjective opinion of each pathologist. The rates of positive ERCC1 staining in previous work were 4.8 %,22 49.5 %,20 and 34.5 %.21 Quantitation of mRNA expression by real-time PCR is an objective method, but the determination of the cutoff line is subjective. In this study, the 75th percentile of ERCC1 mRNA expression levels was chosen as a cutoff, as this maximized the prognosis differences between high and low expression of ERCC1. CONCLUSIONS High ERCC1 mRNA expression was associated with favorable prognosis after curative resection for extrahepatic bile duct cancer, which is consistent with a previous report of other malignancies.10–13 We therefore suggest that ERCC1 can be used as a prognostic biomarker in patients who undergo curative resection for extrahepatic bile duct cancer.

ACKNOWLEDGMENT The authors wish to thank Yasuto Sato, PhD, for his advice on statistics. This study was supported by departmental funds from Tokyo Women’s Medical University. DISCLOSURES

None.

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